MX2008008365A

MX2008008365A - Placental stem cell populations

Info

Publication number: MX2008008365A
Application number: MXMX/A/2008/008365A
Authority: MX
Inventors: J Hariri Robert; Ye Qian; Faleck Herbert; Wang Jialun; W Edinger James; Dawn Abramson Sascha; S Labazzo Kristen; Periera Marian
Original assignee: Dawn Abramson Sascha; Anthrogenesis Corporation; W Edinger James; Faleck Herbert; J Hariri Robert; S Labazzo Kristen; Periera Marian; Wang Jialun; Ye Qian
Priority date: 2005-12-29
Filing date: 2008-06-25
Publication date: 2008-09-26

Abstract

The present invention provides placental stem cells and placental stem cell populations, and methods of culturing, proliferating and expanding the same. The invention also provides methods of differentiating the placental stem cells. The invention further provides methods of using the placental stem cells in assays and for transplanting.

Description

STEM CELL POPULATIONS OF PLACENTA This application claims benefit of the provisional application of E.U.A. No. 60 / 754,968, filed on December 29, 2005; and claims benefit of the provisional application of E.U.A. No. 60 / 846,641, filed on September 22, 2006. 1. FIELD OF THE INVENTION The present invention provides isolated placental stem cells, populations of stem cells from the placenta, compositions comprising the stem cells, and methods of obtaining the stem cells. 2. BACKGROUND OF THE INVENTION Human stem cells are totipotent or pluripotent precursor cells capable of generating a variety of mature human cell lines. There is evidence to show that stem cells can be used to repopulate many, if not all, tissues and restore physiological and anatomical functionality. Many different types of mammalian stem cells have been characterized. See, for example, Captan et al., Patent of E.U.A. No. 5,486,359 (human mesentery stem cells); Boyse and others, patent of E.U.A. No. 5,004,681 (fetal and neonatal hematopoietic stem and progenitor cells); Boyse et al., Patent of E.U.A. No. 5,192,553 (equal); Beltrami et al., Cell 114 (6): 763-766 (2003) (cardiac stem cells); Forbes et al., J. Pathol. 197 (4): 510-518 (2002) (hepatic stem cells). Umbilical cord blood and total nucleated cells derived from cord blood have been used in transplants to restore, partially or completely, hematopoietic function in patients who have undergone ablative therapy. 3. BRIEF DESCRIPTION OF THE INVENTION The present invention provides isolated placental stem cells, populations of stem cells from the placenta, compositions comprising the stem cells, and cell populations comprising said stem cells, wherein the stem cells are present in, and can be isolated from, tissue. of the placenta (for example, cotyledons of amnion, chorion, placenta, etc.). Placental stem cells exhibit one or more characteristics of a stem cell (e.g., exhibit markers associated with stem cells, replicate at least 10-20 fold in culture in an undifferentiated state, differentiate into adult cells representative of all three layers of germ, etc.), and can be adhered to a tissue culture substrate (e.g., tissue culture plastic such as the surface of a culture dish). tissue or multiple well plate). In one embodiment, the invention provides an isolated placental stem cell that is CD200 + or HLA-G +. In a specific embodiment, said cell is CD200 + and HLA-G +. In a specific embodiment, said stem cell is CD73 + and CD105 +. In another specific embodiment, said stem cell is CD34", CD38 'or CD45". In another specific embodiment, said stem cell is CD34", CD38 and CD45". In another specific embodiment, said stem cell is CD34", CD38", CD45", CD73 + and CD105 + .In another specific embodiment, said stem cell facilitates the formation of one or more embryoid-like bodies from a population of isolated placental cells that they comprise placental stem cells when said population is cultured under conditions that allow the formation of embryoid-like bodies In another embodiment, the invention provides a population of isolated placental cells comprising, for example, enriched for human stem cells. CD200 +, HLA-G + In various modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more of said isolated placental cells are CD200 + stem cells, HLA-G + In a specific embodiment, said stem cells are CD73 + and CD105 + In another specific modality, said cells dre are CD34", CD38" or CD45". In a more specific embodiment, said stem cells are CD34", CD38", CD45", CD73 + and CD105 *. In another specific embodiment, said population has been expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times. times. In another specific embodiment, said population forms one or more embryoid type bodies when they are cultured under conditions that allow the formation of embryoid type bodies. In another embodiment, the invention provides an isolated stem cell that is CD73 \ CD105 + and CD200 +. In a specific embodiment, said stem cell is HLA-G +. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38"and CD45 \ In a more specific embodiment, said stem cell is CD34", CD38" , CD45"and HLA-G +. In another specific embodiment, said stem cell facilitates the development of one or more embryoid-like bodies from a population of isolated placental cells comprising the stem cell when said population is cultured under conditions that allow the formation of embryoid-like bodies. .

In another embodiment, the invention provides a population of isolated placental cells comprising, for example, enriched for, stem cells of CD73 +, CD105 +, CD200 +. In various modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, at least 90%, or at least 95% of said placental cells isolated are stem cells of CD73 +, CD105 +, CD200 +. In a specific modality of said populations, said stem cells are HLA-G +. In another specific embodiment, said stem cells are CD34", CD38", or CD45. "In another specific embodiment, said stem cells are CD34", CD38"and CD45". In a more specific embodiment, said stem cells are CD34", CD38", CD45 'and HLA-G +. In other specific embodiments, said population has been expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times. times. In another specific embodiment, said population forms one or more embryoid type bodies in culture under conditions that allow the formation of embryoid-like bodies. The invention also provides an isolated stem cell which is CD200 + and OCT-4 +. In a specific embodiment, said stem cell is HLA-G +. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38"and CD45". In a more specific embodiment, said stem cell is CD34", CD38", CD45-, CD73 +, CD105 + and HLA-G +. In another specific embodiment, said stem cell facilitates the formation of one or more embryoid-like bodies from a population of isolated placental cells comprising placental stem cells when said population is cultured under conditions that allow the formation of bodies of type embryoid In another embodiment, the invention provides a population of isolated cells comprising, for example, enriched for, stem cells of CD200 +, OCT-4 +. In various modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, at least 90%, or at least 95% of said isolated placental cells are CD200 +, OCT-4 + stem cells. In a specific embodiment of the above populations, said stem cells are HLA-G +. In another specific embodiment, said stem cells are CD34", CD38", and CD45. "In another more specific embodiment, said stem cells are CD34", CD38", CD45", CD73 +, CD105 * and HLA-G +. In other specific embodiments, said population has been expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times. times. In another specific modality, said population forms one or more embryoid type bodies in culture under conditions that allow the formation of embryoid type bodies. In another embodiment, the invention provides an isolated stem cell which is CD73 + and CD105 + and which facilitates the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when said population is cultured under conditions that they allow the formation of embryoid type bodies. In a specific embodiment, said stem cell is CD34 ', CD38"or CD45". In another specific embodiment, said stem cell is CD34", CD38" and CD45. "In another specific embodiment, said stem cell is OCT4 + .In a more specific embodiment, said stem cell is OCT4 \ CD34", CD38" and CD45. "The invention also provides a population of isolated placental cells comprising, for example, enriched for, CD73 +, CD105 + stem cells, wherein said population forms one or more embryoid-like bodies under conditions that allow the formation of embryoid-like bodies In various modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said isolated placental cells are CD73 +, CD105 + stem cells. of the above populations, said stem cells are CD34", CD38" or CD45". In another specific embodiment, said stem cells are CD34", CD38" and CD45. "In another specific embodiment, said stem cells are OCT-4 * .In a more specific embodiment, said stem cells are OCT-4 +, CD34", CD38"and CD45". In other specific embodiments, said population has been expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times. times. The invention also provides an isolated stem cell that is CD73 +, CD105 + and HLA-G +. In a specific embodiment, said stem cell is CD34", CD38 'and CD45". In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is OCT-4 + In a more specific embodiment, said stem cell is CD200 *. specifically, said stem cell facilitates the formation of one or more embryoid-like bodies from a population of isolated placental cells comprising placental stem cells in culture under conditions that allow the formation of embryoid-like bodies. The invention further provides a population of isolated placental cells comprising, for example, enriched for, stem cells of CD73 +, CD105 + and HLA-G +. In various modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, at least 90%, or at least 95% of said isolated placental cells are stem cells of CD73 +, CD105 + and HLA-G *. In a specific embodiment of the above populations, said stem cells are CD34", CD38" or CD45. "In another specific embodiment, said stem cells are CD34", CD38"and CD45". In another specific embodiment, said stem cells are OCT-4 +. In another specific embodiment, said stem cells are CD200 +. In a more specific embodiment, said stem cells are CD34-, CD38-, CD45", OCT-4 + and CD200 + In another specific embodiment, said population has been expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.In another specific modality, said population forms embryoid-like bodies when they are cultivated under conditions that allow the formation of embryoid type bodies.

The invention further provides an isolated stem cell which is OCT-4 + and which facilitates the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when grown under conditions that allow the formation of embryoid type bodies. In a specific embodiment, said stem cell is CD73 + and CD105 +. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD200 + In a more specific embodiment, said stem cell is CD73 +, CD105 +, CD200 +, CD34" , CD38"and CD45" The invention also provides a population of isolated cells comprising, for example, enriched for, placental stem cells of OCT-4 +, wherein said population forms one or more embryoid-like bodies when It grows under conditions that allow the formation of embryoid-like bodies., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said isolated placental cells are CD73 * and CD105 *. In another specific embodiment, said stem cells are CD34", CD38" or CD45. In another specific embodiment, said stem cells are CD200 +. In a more specific embodiment, said stem cells are CD73 +, CD105 +, CD200 +, CD34", CD38" and CD45. "In another specific embodiment, said population has been expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times. The invention further provides a population isolated from the placental stem cells described herein that is produced in accordance with a method comprising pumping a mammalian placenta that has been drained of umbilical cord blood and pumped to remove residual blood; pump said placenta with a perfusion solution; and collecting said perfusion solution, wherein said infusion solution after perfusion comprises a population of placental cells comprising placental stem cells; and isolating a plurality of said placental stem cells from said population of cells. In a specific modality, the perfusion solution is passed through the umbilical vein and umbilical arteries and collected after it is exuded from the placenta. In another specific modality, the perfusion solution is passed through the umbilical vein and collected from the umbilical arteries, or passed through the umbilical arteries and collected from the umbilical vein. The invention further provides an isolated population of the placental stem cells described herein that is produced in accordance with a method comprising digesting placental tissue with a tissue destabilizing enzyme to obtain a population of placental cells comprising stem cells of the placenta, and isolate a plurality of placental stem cells from the rest of said placental cells. In modalities Specific, said placental tissue is an entire placenta, an amniotic membrane, chorion, a combination of amnion and chorion, or a combination of any of the above. In another specific embodiment, the tissue destabilizing enzyme is trypsin or collagenase. In more specific embodiments, the invention provides any of the above isolated stem cells, wherein said stem cell expresses one or more genes at a detectably higher level than a mesentery stem cell derived from the spinal cord, wherein said one or more genes are selected from the group consisting of ACTG2, ADARB1, AMIGO2, ART-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, CPA4, DMD, DSC3, DSG2, EOLVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLMIM3, PJP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, and wherein said stem cell derived from the spinal cord has passed through a number of passages in culture equivalent to the number of passages by which said placental stem cell has passed. The sequences corresponding to these genes are found in Affymetrix GENECHIP® orders. These genes can also be found in GenBank access numbers: NM_001615 (ACTG2), BC065545 (ADARB1), (NM_181847 (AMIGO2), AY358590 (ARTS-1), BC074884 (B4GALT6), BC00896 (BCHE), BC020196 (C11orf9), BC031103 (CD200), NM_001845 (COL4A1), NM_001846 (COL4A2), BC052289 (CPA4), BC094758 (DMD), AF293359 (DSC3), NM_001943 (DSG2), AF338241 (ELOVL2), AY336105 (F2RL1), NM_018215 (FLJ10781), AY416799 (GATA6), BC075798 (GPR126), NM_016235 (GPRC6B), AF340038 (ICAM1), BC000844 (IER3), BC0066339 (IGFBP7), BC013142 (IL1A), BT019749 (IL6), BC007461 (IL18), (BC072017 KRT18, BC075839 (KRT8), BC060825 (LIPG), BC065240 (LRAP), BC010444 (MATN2), BC011908 (MEST), BC068455 (NFE2L3), NM_014840 (NUAK1), AB006755 (PCDH7), NM_014476 (PDLIM3), BC126199 (PKP-2), BC090862 (RTN1) , BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201 (ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21), BCO96235 (TGFB2), BC005046 (VTN), and BC005001 (ZC3H12A) since December 2006. In a more specific modality, said stem cell expresses ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A at a detectably higher level than a derived mesenteric stem cell of the spinal cord. In more specific embodiments, the invention also provides any of the above isolated stem cell populations, wherein said stem cells express one or more genes at a level detectably higher than a population of mesentery stem cells derived from the spinal cord, wherein said one or more genes are selected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200 , COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2 , MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, and wherein said population of stem cells derived from the spinal cord has passed through a number of passages in culture equivalent to the number of passages through which said placental stem cell has passed, and wherein said population of mesentery stem cells derived from the spinal cord has a number of cells equivalent to said population of isolated stem cells. In a more specific embodiment, the population of isolated stem cells expresses ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6 , GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5 , SLC12A8, TCF21, TGFB2, VTN and ZC3H12A at a level detectably higher than said population of mesentery stem cells derived from the spinal cord.

In more specific embodiments of methods of selecting cell populations, the invention also provides methods of selecting one of the cell populations mentioned above, comprising selecting cells expressing one or more genes at a detectably higher level than a mesentery stem cell derived from the spinal cord, wherein said one or more genes are selected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTSrl, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1 , FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9 , ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, and wherein said stem cell derived from the spinal cord has passed through a number of passages in culture equivalent to the number of passages through which said placental stem cell has passed. . In a more specific embodiment, said selection comprises selecting cells expressing ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6. , GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5 , SLC12A8, TCF21, TGFB2, VTN and ZC3H12A at a detectably higher level than a mesentery stem cell derived from the spinal cord.

The invention also provides compositions comprising one or more of the stem cells of the invention, wherein the stem cell has been isolated from the placenta. In this way, the invention further provides a composition comprising a stem cell, wherein said stem cell is CD200 + and HLA-G +. In a specific embodiment, said stem cell is CD73 + and CD105 +. In another specific embodiment, said stem cell is CD34", CD38" and CD45. "In a more specific embodiment, said stem cell is CD34", CD38, CD45-, CD73 +, CD105 +, CD200 + and HLA-G \ In another embodiment, The invention provides a composition comprising a stem cell, wherein said stem cell is CD73 +, CD105 + and CD200 +. In a specific embodiment, said stem cell is HLA-G +. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38"and CD45". In another specific embodiment, said stem cell is CD34", CD38" and HLA-G *. In another specific embodiment, the invention provides a composition comprising a stem cell, wherein said stem cell is CD200 + and OCT-4 +. In a specific embodiment, said stem cell is CD73 + and CD105 +. In another specific embodiment, said stem cell is HLA-G +. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38"and CD45". In another specific embodiment, said stem cell is CD34", CD38", CD45", CD73 \ CD105 + and HLA-G +.

In another embodiment, the invention provides a composition comprising a stem cell that is CD73 + and CD105 +, wherein said stem cell facilitates the formation of an embryoid-like body in a population of isolated placental cells comprising said stem cell under conditions that they allow the formation of an embryoid type body. In a specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is OCT-4 + In another specific embodiment, said stem cell is CD200 + In another specific embodiment, said cell mother is OCT-4 +, CD200 +, CD34 ', CD38' and CD45 '. Still in another embodiment, the invention provides a composition comprising a stem cell which is CD73 +, CD105 + and HLA-G +. stem cell is CD34", CD38" or CD45". In another specific embodiment, said stem cell is OCT-4 +. In another specific embodiment, said stem cell is CD200 +. In another specific embodiment, said mother cell is OCT-4 +, CD200 +, CD34", CD38" and CD45. In another embodiment, the invention provides a composition comprising a mother cell that is OCT-4 +, wherein said mother cell facilitates the formation of an embryoid-like body in a population of isolated placental cells comprising said stem cell under conditions that allow the formation of an embryoid-like body. In a specific embodiment, said stem cell is CD73 + and CD105 +. In another specific embodiment, said stem cell is CD34", CD38" and CD45. "In another specific embodiment, said mother cell is CD200 +. In another specific embodiment, said stem cell is CD73 +, CD105 +, CD200 +, CD34", CD38" and CD45. "In more specific embodiments of the above compositions, said stem cell expresses one or more genes at a detectably higher level than a mesentery stem cell derived from the spinal cord, wherein said one or more genes are selected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, and wherein said stem cell derived from the spinal cord has passed through a number of passages in culture equivalent to the number of passages per which has passed said mother cell of the placenta In a more specific modality of the compositions above, said stem cells express ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA- G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A at a detectably higher level than a population of mesentery stem cell derived from isolated spinal cord, wherein said mare cell population and said population of mesentery cells derived from the spinal cord have equivalent numbers of cells. In another specific embodiment, any of the above compositions comprises a matrix. In a more specific embodiment, said matrix is a three-dimensional shell. In another more specific embodiment, said matrix comprises collagen, gelatin, laminin, fibronectin, pectin, ornithine or vitronectin. In another more specific modality, the matrix is an amniotic membrane or a biomaterial derived from amniotic membrane. In another more specific embodiment, said matrix comprises an extracellular membrane protein. In another more specific embodiment, said matrix comprises a synthetic compound. In another more specific embodiment, said matrix comprises a bioactive compound. In another more specific embodiment, said bioactive compound is a growth factor, cytokine, antibody or organic molecule of less than 5,000 daltons. In another embodiment, the invention further provides a composition comprising medium conditioned by any of the above stem cells, or any of the above stem cell populations. In a specific embodiment, any composition as such comprises a stem cell that is not derived from a placenta. In a more specific embodiment, said stem cell is an embryonic stem cell. In another more specific embodiment, said stem cell is a mesentery stem cell.

In another more specific embodiment, said stem cell is a stem cell derived from the spinal cord. In another more specific embodiment, said stem cell is a hematopoietic progenitor cell. In another more specific embodiment, said stem cell is a somatic stem cell. In an even more specific embodiment, said somatic stem cell is a neural stem cell, a liver stem cell, a pancreatic stem cell, an endothelial stem cell, a cardiac stem cell or a muscle stem cell. The invention also provides methods for producing stem cell populations derived from mammalian placenta. In one embodiment, for example, the invention provides a method of producing a cell population comprising selecting cells that (a) adhere to a substrate, and (b) expressing CD200 and HLA-G; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate, and (b) expressing CD73, CD105 and CD200; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate and (b) express CD200 and OCT-4; and isolating said cells from other cells to form a cell population. In yet another embodiment, the invention provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate, (b) express CD73 and CD105, and (c) facilitate the formation of one or more embryoid-like bodies when cultured with a population of placental cells under conditions that allow the formation of embryoid type bodies; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate, and (b) expressing CD73, CD105 and HLA-G; and isolating said cells from other cells to form a cell population. The invention also provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate, (b) expressing OCT-4, and (c) facilitating the formation of one or more embryoid-like bodies when they are cultured with a population of placental cells under conditions that allow the formation of embryoid-like bodies; and isolating said cells from other cells to form a cell population. In a specific embodiment of any of the above methods, said substrate comprises fibronectin. In another specific embodiment, the methods comprise selecting cells that express ABC-p. In another specific embodiment, the methods comprise selecting cells that exhibit at least one characteristic specific to a mesentery stem cell. In a more specific embodiment, said characteristic specific to a mesentery stem cell is expression of CD29, expression of CD44, expression of CD90, or expression of a combination of the previous In another specific embodiment of the methods, said selection is achieved using an antibody. In another specific embodiment, said selection is achieved using flow cytometry. In another specific embodiment, said selection is achieved using magnetic beads. In another specific embodiment, said selection is achieved by classifying fluorescence activated cell. In another specific embodiment of the above methods, said cell population expands. The invention also provides a method of producing a stem cell line, comprising transforming a stem cell with a DNA sequence encoding a growth promoter protein; and exposing said stem cell to conditions that promote the production of said growth promoter protein. In a specific embodiment, said growth promoter protein is v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1a adenovirus or E7 protein of human papillomavirus. In a more specific embodiment, said DNA sequence can be regulated. In a more specific embodiment, said DNA sequence can be regulated by tetracycline. In another specific embodiment, said growth promoter protein has an activity that can be regulated. In another specific embodiment, said growth promoter protein is a temperature sensitive mutant. The invention also provides cryoconserved stem cell populations. For example, the invention provides a population of CD200 +, HLA-G + stem cells, wherein said cells have been cryopreserved, and wherein said population is contained within a container. The invention also provides a population of CD73 +, CD105 +, CD200 + stem cells, wherein said stem cells have been cryopreserved, and wherein said population is contained within a container. The invention also provides a population of CD200 +, OCT-4 + stem cells, wherein said stem cells have been cryopreserved, and wherein said population is contained within a container. The invention also provides a population of CD73 +, CD105 + stem cells, wherein said cells have been cryopreserved, and wherein said population is contained within a container, and wherein said stem cells facilitate the formation of one or more embryoid-like bodies. when they are grown with a population of placental cells under conditions that allow the formation of embryoid-like bodies. The invention further provides a population of CD73 +, CD105 +, HLA-G + stem cells, wherein said cells have been cryopreserved, and wherein said population is contained within a container. The invention also provides a population of OCT-4 + stem cells, wherein said cells have been cryopreserved, wherein said population is contained within a container, and wherein said stem cells facilitate the formation of one or more embryoid-like bodies. when they are grown with a population of placental cells under conditions that allow the formation of embryoid-like bodies. In a specific modality of any of the Previous cryopreserved populations, said container is a bag. In several specific embodiments, said population comprises, at least, at least 1 x 106 said stem cells, 5 x 106 said stem cells, 1 x 107 said stem cells, 5 x 107 said stem cells, 1 x 108 said cells mother, 5 x 108 said stem cells, 1 x 109 said stem cells, 5 x 10? said stem cells, or 1 x 1010 said stem cells. In other specific embodiments of any of the above cryopreserved populations, said stem cells have been passed at least, at least, or no more than 5 times, no more than 10 times, no more than 15 times, or no more than 20 times. In another specific embodiment of any of the above cryopreserved populations, said stem cells have expanded within said container. 3. 1 DEFINITIONS As used herein, the term "SH2" refers to an antibody that binds an epitope on the CD105 tag. In this way, the cells that are referred to as SH2 + are CD105 +. As used herein, the terms "SH3" and "SH4" refer to antibodies that bind epitopes present in the marker CD73. In this way, the cells that are referred to as SH3 + and / or SH4 + are CD73 +. As used herein, the term "isolated stem cell" means a stem cell that is substantially separated from another, non-stem cell of the tissue, for example, placenta, from which the stem cell is derived. A stem cell is "isolated" if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the non-stem cells with which the stem cell is naturally associated , or stem cells that exhibit a different marker profile, are removed from the stem cell, for example, during harvesting and / or culture of the stem cell. As used herein, the term "population of isolated cells" means a population of cells that is substantially separated from other cells of the tissue, for example, placenta, from which the population of cells is derived. A stem cell is "isolated" if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with which the population of cells, or cells of which the population of cells is derived, it is associated in a natural way, that is, stem cells that exhibit a different marker profile, are eliminated from the mother cell, for example, during the collection and / or culture of the mother cell. As used herein, the term "placental stem cell" refers to a stem cell or progenitor cell that is derived from a mammalian placenta, regardless of morphology, cell surface markers, or number of passages after of a primary culture. The term "placental stem cell" as used herein, however, does not refer to a trophoblast. A cell is considered a "stem cell" if the cell retains minus one attribute of a stem cell, for example, a marker or gene expression profile associated with one or more types of stem cells; the ability to replicate at least 10-40 times in culture, the ability to differentiate into cells from all 3 layers of germs; the lack of adult (ie, differentiated) cellular characteristics, or the like. The terms "placental stem cell" and "placental derived stem cell" can be used interchangeably. As used herein, a stem cell is "positive" for a particular marker when that marker can be detected in the background. For example, a placental stem cell is positive, for example, for CD73 since CD73 can be detected in placental stem cells in an amount detectably greater than the background (compared to, for example, an isotype control) . A cell is also positive for a marker when that marker can be used to distinguish the cell from at least one other cell type, or it can be used to select or isolate the cell when it is present or expressed by the cell. In the context of, for example, antibody-mediated detection, "positive", as an indication that a particular cell surface marker is present, it means that the label is detectable using an antibody, for example, a fluorescently labeled antibody. , specific to that marker; "positive" also means that a cell carries that marker in an amount that produces a signal, for example, in a cytometer, which is detectably on the background. For example, a cell is "CD200 +" wherein the cell is detectably labeled with a specific antibody to CD200, and the antibody signal is detectably greater than a control (e.g., background). Conversely, "negative" in the same context means that the cell surface marker is not detectable using an antibody specific for that marker compared to the background. For example, a cell is "CD34" wherein the cell is not detectably labeled with a specific antibody to CD34. Unless stated otherwise herein, the grouping of differentiation markers ("CD") is detected using antibodies. OCT-4 is determined to be present, and a cell is "OCT-4 +" if OCT-4 is detectable using RT-PCR. 4. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Viability of perfusion placenta stem cells (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E). The numbers on the X axis designate placenta from which stem cells were obtained. Figure 2: Percentage of HLA ABC7CD457CD437CD133 + perfusion cells (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E) as determined by FACSCalibur. The numbers on the X axis designate placenta from which stem cells were obtained.

Figure 3: Percentage of HLA ABC 7CD457CD34 CD133 + perfusion cells (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E) as determined by FACS Aria. The numbers on the X axis designate placenta from which stem cells were obtained. Figure 4: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in stem cells derived from placental perfusate. Figure 5: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in amnion-derived stem cells. Figure 6: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in chorion-derived stem cells. Figure 7: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in mother cells derived from amnion-chorion plate. Figure 8: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in stem cells derived from umbilical cord. Figure 9: Average expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in perfusion-derived stem cells (A), amnion (B), chorion (C), amnion plate -corion (D) or umbilical cord (E). Figure 10: Culture time courses for cell lines of amnion / chorion (AC), umbilical cord (UC), stem cell derived from the spinal cord (BM-MSC) and human dermal fibroblast (NHDF) used in this study. All crops grew and propagated using the same seed and passage densities. The circles indicate which cultures were used for RNA isolation. The last crops were harvested just before senescence. Two UC cultures were harvested at 38 duplications (UC-38) to compare the effect of trypsinization on gene expression. All other cultures were used directly in their culture flasks before RNA isolation. Figure 11: Line diagram of relative expression levels of 8215 genes in amnion-chorion (AC), umbilical cord (UC), stem cell derived from the spinal cord (BM-MSC) and human dermal fibroblast cells (DF). The number associated with each cell line designation on the X axis indicates the number of days that the cell line was cultured before the evaluation of gene expression levels. The graph was generated from the RNA expression data analyzed by the GeneSpring software. AC-03 was used as the selected condition. Figure 12: Subseries of the whole list of genes showing over-expressed genes = fold in AC-03 for amnion / chorion (AC), umbilical cord (UC), stem cell derived from the spinal cord (BM-MSC) and cells of the human dermal fibroblast (DF). The number associated with each cell line designation on the X axis indicates the number of days that the cell line was cultured before the evaluation of gene expression levels. The graph was generated from the RNA expression data analyzed by the GeneSpring software. AC-03 was used as the selected condition. Figure 13: Placental stem cell specific or umbilical cord stem cell-specific genes found by fold change filtration for amnion / chorion (AC), umbilical cord (UC), stem cell derived from the spinal cord (BM- MSC) and human dermal fibroblast cells (DF). The number associated with each cell line designation on the X axis indicates the number of days that the cell line was cultured before the evaluation of gene expression levels. The graph was generated from the RNA expression data analyzed by the GeneSpring software. AC-03 was used as the selected condition.

. DETAILED DESCRIPTION OF THE INVENTION . 1 PLACENTA MOTHER CELLS AND PLACENTA MOTHER CELL POPULATIONS Placental stem cells are stem cells, obtainable from a placenta or part thereof, that adhere to a tissue culture substrate and have the ability to differentiate into non-placental cell types. Placental stem cells may be of fetal or maternal origin (that is, they may have the genotype of the fetus or mother, respectively). Preferably, the stem cells of the placenta and stem cell populations of the placenta of the invention are fetal in origin. Placental stem cell populations, or populations of cells comprising placental stem cells, may comprise placental stem cells that are solely fetal or maternal in origin, or may comprise a mixed population of placental stem cells. fetal and maternal origin. The placenta stem cells, and placental stem cell populations, can be identified and selected by the morphological, marker, and culture characteristic discussed below. . 1.1 Physical and morphological characteristics The placental stem cells of the present invention, when cultured in primary cultures or in cell culture, adhere to the tissue culture substrate, for example, tissue culture containing surface (e.g., tissue culture plastic) . Placental stem cells in culture assume a generally fibroblastoid, stellate appearance, with a number of cytoplasmic processes that extend from the central cell body. However, placental stem cells are morphologically differentiable from fibroblasts grown under the same conditions, since placental stem cells exhibit a greater number of such processes than fibroblasts. Morphologically, the placental stem cells are also Differentiating hematopoietic stem cells, which generally assume a more rounded, or cobbled, morphology in culture. . 1.2 Cell, molecular and genetic markers The placental stem cells of the present invention, and populations of stem cells from the placenta, express a plurality of markers that can be used to identify and / or isolate the stem cells, or populations of cells comprising the stem cells. Placental stem cells, and stem cell populations of the invention (ie, two or more placental stem cells) include stem cells and cell populations containing stem cells obtained directly from the placenta, or any part thereof (for example, amnion, chorion, placenta cotyledons, and the like). The stem cell populations of the placenta also include populations of (i.e., two or more) placental stem cells in culture, and a population in a container, e.g., a bag. However, the placental stem cells are not trophoblasts. The placental stem cells of the invention generally express the markers CD73, CD105, CD200, HLA-G and / or OCT-4, and do not express CD34, CD38 or CD45. Placental stem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify placental stem cells, and to distinguish placental stem cells from others mother cell types. Since the placental stem cells can express CD73 and CD105, they can have characteristics of the mesentery stem cell type. However, since the placental stem cells can express CD200 and HLA-G, a specific fetal marker, they can be distinguished from mesentery stem cells, for example, mesentery stem cells derived from the spinal cord, which do not express CD200 nor HLA-G. In the same way, the lack of expression of CD34, CD38 and / or CD45 identifies the placental stem cells as non-hematopoietic stem cells. Thus, in one embodiment, the invention provides an isolated host cell that is CD200 + or HLA-G *. In a specific embodiment, said stem cell is a mother cell of the placenta. In a specific modality, the stem cell is CD200 + and HLA-G *. In a specific embodiment, said stem cell is CD73 + and CD105 *. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38"and CD45". In another specific embodiment, said stem cell is CD34", CD38", CD45", CD73 + and CD105 *. In another specific embodiment, said stem cell CD200 * or HLA-G * facilitate the formation of one or more bodies of embryoid type in a population of placental cells comprising the stem cells, under conditions that allow the formation of embryoid-like bodies In another specific embodiment, said placental stem cell is isolated away from placental cells that are not stem cells. specific mode, said placental stem cell is isolated away from placental stem cells that do not exhibit these markers. In another embodiment, the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a placental cell CD200 * or HLA-G *, whereby said cell is a stem cell of the placenta. In a specific embodiment, said selection comprises selecting a placental cell that is also CD73 * and CD105 *. In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38" or CD45. "In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38"and CD45". In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38", CD45", CD73 * and CD105." In another specific embodiment, said selection comprises selecting a placental cell which also facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising the stem cells, under conditions that allow the formation of embryoid type bodies. In another embodiment, the invention provides an isolated cell population comprising, for example, enriched for, stem cells CD200 *, HLA-G *. In a specific embodiment, said population is a population of placental cells. In several embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of said cells are stem cells CD200 *, HLA-G *. Preferably, at least about 70% of said cells are CD200 *, HLA-G * stem cells. More preferably, at least about 90%, 95% or 99% of said cells are CD200 *, HLA-G * stem cells. In a specific modality of the isolated populations, said stem cells are also CD73 * and CD105 *. In another specific embodiment, said stem cells are also CD34", CD38" or CD45. "In a more specific embodiment, said stem cells are also CD34", CD38", CD45", CD73 * and CD105 *. In another embodiment, said isolated population produces one or more embryoid type bodies when cultured under conditions that allow the formation of embryoid type bodies. In another specific embodiment, said population of placental stem cells is isolated away from placental cells that are not stem cells. In another specific embodiment, said population of placental stem cells is isolated away from placental stem cells that do not exhibit these markers. In another embodiment, the invention also provides a method of selecting a population of placental stem cells from a plurality of placental cells, comprising selecting a population of placental cells where at least about 10%, less approximately 20%, so less about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 90%, or at least about 95% by weight of said cells are CD200 *, HLA-G * stem cells. In a specific embodiment, said selection comprises selecting stem cells that are also CD73 * and CD105 *. In another specific embodiment, said selection comprises selecting stem cells that are also CD34", CD38" or CD45. "In another specific embodiment, said selection comprises selecting stem cells that are also CD34-, CD38-CD45-, CD73 * and CD105 * In another specific embodiment, said selection also comprises selecting a population of placental stem cells that form one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies. an isolated stem cell which is CD73 *, CD105 * and CD200 * In a specific embodiment, said isolated stem cell is an isolated placental stem cell In another specific embodiment, said stem cell is HLA-G *. specific, said stem cell is CD34", CD38" or CD45". In another specific embodiment, said stem cell is CD34 ', CD38"and CD45." In a more specific embodiment, said stem cell is CD34", CD38", CD45' and HLA-G * In another specific embodiment, the stem cell CD73 *, CD105 * and CD200 * they facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising the stem cell, when the population is cultured under conditions that allow the formation of embryoid-like bodies. In another specific embodiment, said placental stem cell is isolated away from placental cells that are not stem cells. In another specific embodiment, said placental stem cell is isolated away from placental stem cells that do not exhibit these markers. In another embodiment, the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a placental cell CD73 *, CD105 * and CD200 *, whereby said cell is a mother cell of the placenta. In a specific embodiment, said selection comprises selecting a placental cell that is also HLA-G *. In another specific modality, said selection comprises selecting a placental cell which is also CD34", CD38" or CD45. "In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38 'and CD45'. In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38", CD45"and HLA-G *. In another specific embodiment, said selection additionally comprises selecting a CD73 *, CD105 * and CD200 stem cell. * that facilitate the formation of one or more embryoid-like bodies in a population of placental cells that comprise the stem cell, when the population is grown under conditions that allow the formation of embryoid type bodies. In another embodiment, the invention provides an isolated cell population comprising, for example, enriched for, CD73 *, CD105 *, CD200 * stem cells. In a specific embodiment, said stem cells are stem cells of the placenta. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of said cells are stem cells CD73 *, CD105 *. CD200 *. In another embodiment, at least about 70% of said cells in said cell population are CD73 *, CD105 *, CD200 * stem cells. In another embodiment, at least about 90%, 95% or 99% of said cells in said cell population are CD73 *, CD105 *, CD200 * stem cells. In a specific modality of said populations, said stem cells are HLA-G *. In another specific embodiment, said stem cells are CD34", CD38" or CD45. "In another specific embodiment, said stem cells are CD34", CD38"and CD45.In a more specific embodiment, said stem cells are CD34 ', CD38" , CD45"and HLA-G * In another specific embodiment, said population of cells produces one or more embryoid-like bodies when they are cultured under conditions that allow the formation of embryoid-like bodies In another specific embodiment, said population of cells mother of the placenta is isolated away from placental cells that are not stem cells, in another specific modality, said population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention also provides a method of selecting a population of placental stem cells from a plurality of placental cells, comprising selecting a population of placental cells in which at least about 10%, therefore less about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of said cells are CD73 *, CD105 *, CD200 * stem cells. In a specific embodiment, said selection comprises selecting stem cells that are also HLA-G *, CD34", CD38" or CD45. "In another specific embodiment, said selection comprises selecting stem cells that are also CD34", CD38"and CD45" . In another specific embodiment, said selection comprises selecting stem cells that are also CD34", CD38", CD45 'and HLA-G *. In another specific embodiment, said selection further comprises selecting a population of placental cells that produces one or more embryoid-like bodies when the population is cultured under conditions that allow the formation of embryoid-like bodies. The invention also provides an isolated stem cell which is CD200 * and OCT-4 *. In a specific modality, the stem cell is CD73 * and CD105 *. In a specific embodiment, the stem cell is a placental stem cell. In another specific embodiment, said stem cell is HLA-G *. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38 'and CD45. "In a more specific embodiment, said stem cell is CD34", CD38. ", CD45", CD73 *, CD105 * and HLA-G *. In another specific embodiment, the stem cell facilitates the production of one or more embryoid-like bodies by a population of placental cells comprising the stem cell, when the population is cultured under conditions that allow the formation of embryoid-like bodies. In another specific embodiment, said placental stem cell is isolated away from placental cells that are not stem cells. In another specific embodiment, said placental stem cell is isolated away from placental stem cells that do not exhibit these markers. In another modality, the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a placental cell CD200 * and OCT-4 *, whereby said cell is a stem cell of the placenta In a specific embodiment, said selection comprises selecting a placental cell that is also HLA-G *. In another specific embodiment, said selection comprises selecting a placental cell which is also CD34, CD38 or CD45. "In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38"and CD45. "In another specific embodiment, said selection comprises selecting a placental cell that is also CD34", CD38", CD45", CD73 *, CD105 * and HLA-G *. In another specific embodiment, said selection comprises selecting a placental stem cell that also facilitates the production of one or more embryoid-like bodies by a population of placental cells comprising the stem cell, when the population is cultured under conditions that allow the formation of embryoid type bodies. The invention also provides an isolated population of cells comprising, for example, enriched for, stem cells CD200 *, OCT-4 *. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of said cells stem cells CD200 *, OCT-4 *. In another embodiment, at least about 70% of said cells said CD200 *, OCT-4 * stem cells. In another embodiment, at least about 90%, 95% or 99% of said cells said CD200 *, OCT-4 * stem cells. In a specific modality of the isolated populations, said stem cells CD73 * and CD105 *. In another specific embodiment, said stem cells HLA-G *. In another specific embodiment, said stem cells CD34, CD38"and CD45." In a more specific embodiment, said stem cells CD34", CD38", CD45", CD73 *, CD105 * and HLA-G *. In another specific modality, the population produces one or more embryoid-like bodies when grown under conditions that allow the formation of embryoid-like bodies. In another specific embodiment, said population of placental cells is isolated away from placental cells that not stem cells. In another specific embodiment, said population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention also provides a method of selecting a population of placental stem cells from a plurality of placental cells, comprising selecting a population of placental cells where at least about 10%, less about 20%, at least about 30%, at least about 40%, at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least approximately 95% of said cells CD200 *, OCT-4 * stem cells. In a specific embodiment, said selection comprises selecting stem cells that also CD73 * and CD105 *. In another specific embodiment, said selection comprises selecting stem cells that also HLA-G *. In another specific embodiment, said selection comprises selecting stem cells which also CD34", CD38" and CD45. "In another specific embodiment, said stem cells also CD34", CD38", CD45 \ CD73 *, CD105 * and HLA-G The invention also provides an isolated stem cell which is CD73 *, CD105 * and HLA-G *. In a specific embodiment, the stem cell is a mother cell of the placenta. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD34", CD38"and CD45". In another specific embodiment, said stem cell is OCT-4 *. In another specific embodiment, said stem cell is CD200 *. In a more specific embodiment, said stem cell is CD34", CD38", CD45", OCT-4 * and CD200 *. In another specific embodiment, said stem cell facilitates the formation of embryoid-like bodies in a population of placental cells. which comprises said stem cell, when the population is cultured under conditions that allow the formation of embryoid-like bodies In another specific embodiment, said placental stem cell is isolated away from placental cells that not stem cells. specific, said placental stem cell is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a placental cell CD73 *, CD105 * and HLA-G *, whereby said cell is a mother cell of the placenta. In a specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38" or CD45. "In another specific embodiment, said selection comprises selecting a placental cell which is also CD34 ', CD38' and CD45". In another specific modality, said selection comprises selecting a placental cell which is also OCT-4 *. In another specific embodiment, said selection comprises selecting a placental cell that is also CD200 *. In another specific embodiment, said selection comprises selecting a placental cell which is also CD34", CD38", CD45", OCT-4 * and CD200 *. In another specific embodiment, said selection comprises selecting a placental cell which also facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell, when said population is cultured under conditions that allow the formation of embryoid-like bodies The invention also provides an isolated population of cells comprising , for example, which is enriched for stem cells CD73 *, CD105 * and HLA-G * In a specific embodiment, said stem cells are placental stem cells In various modalities, at least about 10%, so less about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of said cells on stem cells CD73 *. CD105 * and HLA-G *. In another embodiment, at least about 70% of said cells are CD73 *, CD105 * and HLA-G *. In another embodiment, at least about 90%, 95% or 99% of said cells are CD73 *, CD105 * and HLA-G * stem cells. In a specific modality of the previous populations, said stem cells are CD34", CD38" or CD45. "In another modality specifically, said stem cells are CD34", CD38" and CD45. "In another specific embodiment, said stem cells are OCT-4 * .In another specific embodiment, said stem cells are CD200 * .In a more specific embodiment, said stem cells are they are CD34", CD38", CD45", OCT-4 * and CD200 *. In another specific embodiment, said population of placental stem cells is isolated from placental cells that are not stem cells. In another specific embodiment, said population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention also provides a method of selecting a population of placental stem cells from a plurality of placental cells, comprising selecting a population of placental cells where a majority of said cells are CD73 *, CD105 * and HLA-G *. In a specific embodiment, said majority of cells are also CD34", CD38 'and / or CD45". In another specific embodiment, said majority of cells are also CD200 *. In another specific embodiment, said majority of cells are also CD34", CD38", CD45", OCT-4 * and CD200 *. In another embodiment, the invention provides an isolated stem cell which is CD73 * and CD105 * and which facilitates the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of embryoid-like bodies In a specific embodiment, said stem cell is CD34" , CD38"or CD45". In another specific embodiment, said stem cell is CD34", CD38 'and CD45". In another specific embodiment, said stem cell is OCT-4 *. In a more specific embodiment, said stem cell is OCT-4 *, CD34", CD38" and CD45. "In another specific embodiment, said placental stem cell is isolated away from placental cells that are not stem cells. specific embodiment, said placental stem cell is isolated away from placental stem cells that do not exhibit these characteristics.The invention also provides a population of isolated placental cells comprising, for example, enriched for, stem cells CD73 * , CD105 *, wherein said population forms one or more embryoid-like bodies under conditions that allow the formation of embryoid-like bodies In several modalities, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of said isolated placental cells are CD73 *, CD105 * stem cells. In a specific embodiment of the above populations, said stem cells are CD34", CD38" or CD45. "In another specific embodiment, said stem cells are CD34", CD38"and CD45". In another specific embodiment, said stem cells are OCT-4 *. In a more specific modality, said stem cells are OCT-4 *. CD34", CD38" and CD45".

In other specific modalities, said population has expanded, for example, it has been passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least minus 20 times In another specific embodiment, said population of placental stem cells is isolated away from placental cells that are not stem cells. In another specific embodiment, said population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. The invention also provides an isolated stem cell that is OCT-4 * and which facilitates the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when cultured under conditions that allow the formation of embryoid-like bodies. In a specific embodiment, said stem cell is CD73 * and CD105 *. In another specific embodiment, said stem cell is CD34", CD38" or CD45. "In another specific embodiment, said stem cell is CD200 * In a more specific embodiment, said stem cell is CD73 *, CD105 *, CD200 *. \ CD38"and CD45 \ In another specific embodiment, said placental stem cell is isolated away from placental cells that are not stem cells. In another specific embodiment, said placental stem cell is isolated away from placental stem cells that do not exhibit these characteristics. The invention also provides a population of isolated cells comprising, for example, enriched for, cells mother OCT-4 *, where said population forms one or more embryoid-like bodies when they are cultured under conditions that allow the formation of embryoid-like bodies. In various embodiments, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70 %, at least about 80%, at least about 90%, or at least about 95% of said isolated placental cells are OCT-4 * stem cells. In a specific modality of the above populations, said stem cells are CD73 * and CD105 *. In another specific embodiment, said stem cells are CD34", CD38 * or CD45". In another specific embodiment, said stem cells are CD200 *. In a more specific modality, said stem cells are CD73 *. CD105 *, CD200 *, CD34", CD38" and CD45. "In another specific modality, said population has expanded, for example, passed at least once, at least three times, at least five times, at least 10 times at least 15 times, or at least 20 times In another specific embodiment, said population of placental stem cells is isolated away from placental cells that are not stem cells In another specific embodiment, said population of placental stem cells is isolates away from placental stem cells that do not exhibit these characteristics.In another embodiment, the invention also provides an isolated placental stem cell that is CD10 *, CD34", CD105 * and CD200 *. The invention further provides an isolated population of placental stem cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of said placental stem cells are CD10 *, CD34", CD105 *, CD200 *. In a specific embodiment of the above embodiments, said stem cells are additionally CD90 * and CD45". In a specific embodiment, said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells. In another specific embodiment, said stem cell or population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another specific embodiment, said isolated placental stem cell is non-maternal in origin. In another specific modality, at least about 90%, at least 95% or at least about 99% of said cells in said isolated population of placental stem cells, are non-maternal in origin. In another embodiment, the invention provides an isolated placental stem cell that is HLA-A, B, C ", CD45", CD133"and CD34". The invention further provides an isolated population of placental stem cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of said placental stem cells are HLA-A, B, C ", CD45", CD133"and CD34". In a specific embodiment, said stem cell or population of placental stem cells is isolated from placental cells that are not stem cells. In another specific embodiment, said population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another specific embodiment, said isolated placental stem cell is non-maternal in origin. In another specific embodiment, at least about 90%, at least about 95% or at least about 99% of said cells in said isolated population of placental stem cells are non-maternal in origin. In another embodiment, the invention provides a method of obtaining a placental stem cell that is HLA-A, B, C ", CD45", CD133"and CD34" which comprises isolating said placental perfusate cell. In another embodiment, the invention provides an isolated placental stem cell that is CD10 *, CD13 *, CD33 *, CD45 ', CD117"and CD133". The invention further provides an isolated population of placental stem cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of said placental stem cells are CD10 *, CD13 *, CD33 *, CD45 \ CD117"and CD133 \ In a specific embodiment, said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells. In another specific modality, said isolated placental mother cell is non-maternal in origin. specific, at least about 90%, at least about 95% or at least about 99% of said cells in said isolated population of placental stem cells, are non-maternal in origin. In another specific embodiment, said stem cell or population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention provides a method of obtaining a placental stem cell that is CD10 *, CD13 *, CD33 *, CD45", CD117" and CD133 * which comprises isolating said placental perfusate cell. In another embodiment, the invention provides an isolated placental stem cell that is CD10 *, CD33", CD44 *, CD45" and CD117. "The invention further provides an isolated population of placental stem cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of said placental stem cells are CD10", CD33", CD44 *, CD45"and CD117 In a specific embodiment, said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.In another specific embodiment, said isolated placental stem cell is non-maternal in origin. specific modality, at least about 90%, at least about 95% or at least about 99% of said cells in said isolated population of placental stem cells, are non-maternal in origin. specific, said stem cell or population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention provides a method of obtaining a placental stem cell that is CD10", CD33", CD44 *, CD45", CD117" which comprises isolating said placental perfusate cell. In another embodiment, the invention provides an isolated placental stem cell that is CD10", CD13", CD33", CD45" and CD117. "The invention further provides an isolated population of placental stem cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of said placental stem cells are CD10", CD13", CD33", CD45 'and CD117 ' In a specific embodiment, said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells. In another specific modality, said stem cell of the isolated placenta is non-maternal in origin. In another specific embodiment, at least about 90%, at least about 95% or at least about 99% of said cells in said isolated population of placental stem cells are non-maternal in origin. In another specific embodiment, said stem cell or population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention provides a method of obtaining a placental stem cell that is CD10", CD13", CD33", CD45"and CD117" which comprises isolating said placental perfusate cell. In another embodiment, the invention provides an isolated placental stem cell which is HLA-A, B, C ", CD45", CD34", CD133", positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and / or HLA-G, and / or negative for CD117. The invention further provides an isolated population of placental stem cells, wherein said stem cells are HLA A, B, C ", CD45", CD34", CD133", and at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or approximately 99% of the stem cells in the population are positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and / or HLA-G, and / or negative for CD117. In a specific embodiment, said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells. In another specific embodiment, said isolated placental stem cell is non-maternal in origin. In another specific embodiment, at least about 90%, at least about 95% or at least about 99% of said cells in said isolated population of placental stem cells are non-maternal in origin. In another specific embodiment, said stem cell or population of placental stem cells is isolated away from placental stem cells that do not exhibit these characteristics. In another embodiment, the invention provides a method of obtaining a placental stem cell that is HLA A, B, C ", CD45", CD34-, CD133- and positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and / or HLA-G, and / or negative for CD117, which comprises isolating said placental perfusate cell. In another embodiment, the invention provides a placental stem cell that is CD200 * and CD10 *, as determined by antibody binding, and CD117", as determined by antibody binding and RT-PCR. invention provides a placental stem cell which is CD10 *, CD29", CD54 *, CD200 *, HLA-G *, HLA class I" and β-2-microglobulin ". In another embodiment, the invention provides placental stem cells, wherein the expression of at least one marker is at least two times greater than for a mesentery stem cell (e.g., a mesentery stem cell derived from the spinal cord. ). In another specific embodiment, said isolated placental stem cell is non-maternal in origin. In another specific embodiment, at least about 90%, at least about 95%, or at least about 99%, of said cells in said isolated population of placental stem cells are non-maternal in origin. In another embodiment, the invention provides an isolated population of placental stem cells, wherein a plurality of said placental stem cells are positive for aldehyde dehydrogenase (ALDH), as evaluated by an aldehyde dehydrogenase activity assay. Such assays are known in the art (see, for example, Bostian and Betts, Biochem. J., 173, 787, (1978)). In a specific embodiment, said ALDH assay uses ALDEFLUOR® (Aldagen, Inc., Ashland, Oregon) as a marker of aldehyde dehydrogenase activity. In a specific embodiment, said plurality is between about 3% and about 25% of cells in said population of cells. In another embodiment, the invention provides a population of umbilical cord stem cells, wherein a plurality of said umbilical cord stem cells are positive for aldehyde dehydrogenase, as evaluated by an aldehyde dehydrogenase activity assay using ALDEFLUOR® as a activity indicator of aldehyde dehydrogenase. In a specific embodiment, said plurality is between about 3% and about 25% of cells in said population of cells. In another embodiment, said population of placental stem cells or umbilical cord stem cells shows at least three times, or at least five times, higher ALDH activity than a population of mesentery stem cells derived from the spinal cord having the same number of cells and cultivated under the same conditions. The invention provides any of the previous placental stem cells, or populations of stem cells from the placenta, where the mother cell or population of stem cells from the placenta have passed through at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 , 14, 16, 18 or 20 times, or more, or expanded by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 population doublings, or more. In a specific modality of any of the previous placental stem cells or cell populations, the karyotype of the cells, or at least about 95% or about 99% of the cells in said population, is normal. In another specific embodiment of any of the cells from the above placenta or cell populations, the cells, or cells in the cell population, are non-maternal in origin. Placental stem cells isolated, or isolated populations of placental stem cells, carrying any of the above combinations of markers, can be combined in any ratio. The invention also provides for the isolation of, or enrichment for, any of two or more of the populations of stem cells from the previous placenta to form a population of placental stem cells. For example, the invention provides an isolated population of placental stem cells comprising a first population of placental stem cells defined by one of the marker combinations described above and a second population of placental stem cells defined by another of the marker combinations described above, wherein said first and second populations are combined in a ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95: 5, 96: 4, 97: 3, 98: 2 or approximately 99: 1. Similarly, any three, four, five or more of the placental stem cells described above or populations of stem cells from the placenta can be combined. The invention also provides placental stem cells that they are obtained by disruption of placental tissue, with or without enzymatic digestion, followed by culture (see section 5.2.3) or perfusion (see section 5.2.4). For example, the invention provides an isolated population of placental stem cells that is produced according to a method comprising pumping a mammalian placenta that has been drained of cord blood and pumping to remove residual blood; pump said placenta with a perfusion solution; and collecting said perfusion solution, wherein said infusion solution after perfusion comprises a population of placental cells comprising placental stem cells; and isolating a plurality of said placental stem cells from said population of cells. In a specific modality, the perfusion solution is passed through the umbilical vein and umbilical arteries and collected after it exudes from the placenta. Placental stem cell populations produced by this method typically comprise a mixture of fetal and maternal cells. In another specific modality, the perfusion solution is passed through the umbilical vein and collected from the umbilical arteries, or passed through the umbilical arteries and collected from the umbilical vein. The populations of placental stem cells produced by this method are typically substantially exclusively fetal in origin; that is, for example, greater than 90%, 95%, 99% or 99% of the placental stem cells in the population are fetal in origin.

In several modalities, placental stem cells, contained within a population of cells obtained from perfusion of a placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or by at least 99.5% of said population of placental cells. In another specific embodiment, placental stem cells collected by perfusion comprise fetal and maternal cells. In another specific embodiment, the placental stem cells harvested by perfusion are at least 50%, 60%, 70%, 80%, 90%, 95% or at least 99.5% of fetal cells. In another specific embodiment, the invention provides a composition comprising a population of isolated placental stem cells harvested by perfusion, wherein said composition comprises at least a portion of the perfusion solution used to collect the placental stem cells. The invention further provides an isolated population of placental stem cells described herein that is produced in accordance with a method comprising digesting placental tissue with a tissue destabilizing enzyme to obtain a population of placental cells comprising human stem cells. the placenta, a plurality of placental stem cells from the rest of said placental cells. All, or any part of, the placenta can be digested to obtain stem cells from the placenta. In specific embodiments, for example, said placental tissue is an entire placenta, an amniotic membrane, chorion, a combination of amnion and chorion, or a combination of any of the previous ones. In another specific embodiment, the tissue destabilizing enzyme is trypsin or collagenase. In several modalities, the placental stem cells, contained within a population of cells obtained from digesting a placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or so less 99.5% of said population of placental cells. Gene profiling confirms that isolated placental stem cells, and isolated placental stem cell populations, can be distinguished from other cells, for example, mesentery stem cells, e.g. stem cells derived from the spinal cord. The placental stem cells described herein, can be distinguished from mesentery stem cells on the basis of the expression of one or more genes, the expression of which is specific to placental stem cells or umbilical cord stem cells compared with mesentery stem cells derived from the spinal cord. In particular, the placental stem cells can be distinguished from mesentery stem cells on the basis of the expression of one or more genes, the expression of which is significantly higher (ie, at least twice as high) in cells mother of the placenta than in mesentery stem cells, where the one or more genes is (are) ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, ZC3H12A, or a combination of any of the foregoing, wherein the expression of these genes is higher in placental stem cells or cells umbilical cord stem cells in stem cells derived from the spinal cord, when the stem cells are grown under equivalent conditions. In a specific embodiment, the specific gene of placental stem cell or umbilical cord stem cell specific is CD200. The level of expression of these genes can be used to confirm the identity of a population of placental cells, to identify a population of cells as comprising at least a plurality of placental stem cells, or the like, the population of stem cells of the placenta, the identity of which is confirmed, can be clonal, for example, a population of stem cells from the expanded placenta forms an individual placental stem cell, or a mixed population of stem cells, for example, a population of cells comprising only placental stem cells that expand from multiple placental stem cells, or a population of cells comprising placental stem cells and at least one other type of cell. The level of expression of these genes can be used to select populations of stem cells from the placenta. For example, a population of cells, for example, clonally expanded cells, is selected if the expression of one or more of these genes is significantly higher in a sample from the cell population than in an equivalent population of mesentery stem cells. Said selection may be from a population from a plurality of populations of stem cells of the placenta, from a plurality of cell populations, the identity of which is not known, etc. The placental stem cells can be selected on the basis of the level of expression of one or more said genes compared to the level of expression in said one or more genes in a mesentery stem cell control. In one embodiment, the level of expression of said one or more genes in a sample comprising an equivalent number of mesentery stem cells is used as a control. In another embodiment, the control, for placental stem cells tested under certain conditions, is a numerical value representing the level of expression of said one or more genes in mesentery stem cells under said conditions. The stem cells of the placenta of the invention exhibit the above characteristics (e.g., combinations of cell surface markers and / or gene expression profiles) in primary culture, or during proliferation in medium comprising 60% of DMEM-LG (Gibco), 40% of MCDB-201 (Sigma), 2% of Fetal bovine serum (FCS) (Hyclone Laboratories), 1x insulin-transferrin-selenium (STI), 1x bovine serum albumin of linolenic acid (LA-BSA), 10'9 M dexamethasone (Sigma), 10 * 4 M ascorbic acid 2-phosphate (Sigma), epidermal growth factor (EGF) 10ng / ml (R & amp; amp;; D Systems), 10 ng / ml platelet-derived growth factor (PDGF-BB) (R & D Systems), and 100U penicillin / 1000U streptomycin. The isolated populations of placental stem cells described above, and populations of stem cells from the placenta in general, may comprise at least, or not more than, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more placental stem cells. . 1.3 Growth in cultivation The growth of the placental stem cells described above, as well as for any mammalian cell, depends in part on the particular medium selected for growth. Under optimal conditions, the placental stem cells typically double in number in 3-5 days. During culture, the placental stem cells of the invention adhere to a substrate in culture, for example, the surface of a tissue culture container (eg, plastic tissue culture dish, plastic coated with fibronectin, and the like) and form a monolayer. The populations of isolated placental cells comprising the placental stem cells of the invention, when cultured under appropriate conditions, form embryoid-like bodies, ie, three-dimensional clusters of cells grow on top of the adherent stem cell layer. The cells inside Embryoid-like bodies express markers associated with very early stem cells, for example, OCT-4, Nanog, SSEA3 and SSEA4. Cells within embryoid-like bodies are typically not adherent to the culture substrate, and are the placental stem cells described herein, but remain fixed to the adherent cells during culture. The cells of embryoid-like bodies are dependent on adherent placental stem cells for viability, since embryoid-like bodies are not formed in the absence of adherent stem cells. The placental stem cells adherent in this manner facilitate the growth of one or more embryoid-like bodies in a population of placental cells comprising the adherent placental stem cells. Without wishing to be bound by theory, cells of embryoid-like bodies are believed to grow in adherent placental stem cells as much as embryonic stem cells grow in a cell-feeder layer. Mesenteric stem cells, for example, stem cells from the mesentery derived from the spinal cord, do not develop embryoid-like bodies in culture. . 2 METHODS OF OBTAINING STEM CELLS FROM THE PLACENTA . 2.1 Composition of stem cell collection The present invention also provides methods of collecting and Isolate stem cells from the placenta. Generally, stem cells are obtained from a mammalian placenta using a physiologically acceptable solution, for example, a stem cell harvesting composition. A stem cell harvesting composition is described in detail in the related provisional application of E.U.A. No. 60 / 754,969, entitled "Improved Medium for Collecting Placental Stem Cells and Preserving Organs," filed December 29, 2005. The stem cell harvesting composition may comprise any physiologically acceptable solution suitable for the collection and / or culture of stem cells, for example, a saline solution (eg, phosphate buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl, etc.), a culture medium (eg, DMEM, H. DMEM, etc.), and the like. The stem cell harvesting composition may comprise one or more components that tend to retain placental stem cells, that is, prevent placental stem cells from drying out, or delay the death of the placental stem cells, reduce the number of placental stem cells in a population of cells that die, or the like, from the time of collection to the time of culture. Such components may be, for example, an apoptosis inhibitor (eg, a caspase inhibitor or JNK inhibitor); a vasodilator (eg, magnesium sulfate, an anhypertensive drug, natriuretic peptide atrial (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.); a necrosis inhibitor (eg, 2- (1 H-indol-3-yl) -3-pentylamino-maleimide, pyrrolidone dithiocarbamate, or clonazepam); a TNF-a inhibitor; and / or a perfluorocarbon carrying oxygen (eg, perfluorooctyl bromide, perfluorodecyl bromide, etc.). The stem cell harvesting composition may comprise one or more tissue degrading enzymes, for example, metalloprotease, a serine protease, a neutral protease, an RNase, or a DNase, or the like. Such enzymes include, but are not limited to, collagenases (for example, collagenase I, II, III or IV, or Collagenase from Clostridium histolyticum, etc.); shoot, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like. The stem cell harvesting composition may comprise a bacteriocidal or bacteriostatically effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime, or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g. , penicillin V) or a quinolone (for example, ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is active against Gram (+) and / or Gram (- ), for example, Pseudomonas aeruginosa, Staphylococcus aureus, and the like. The stem cell harvesting composition may also comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (approximately 20 mM to around 100 mM); Magnesium ions (about 1 M to about 50 mM); a macromolecule of molecular weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cell viability (eg, a synthetic colloid or occurring naturally, a polysaccharide such as dextran or a polyethylene glycol present at about 25 g / l about 100 g / l, or about 40 g / l about 60 g / l); an antioxidant (eg, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 μM to about 100 μM); an agent that prevents entry of calcium into cells (for example, verapamil present at about 2 μM to about 25 μM); nitroglycerin (e.g., about 0.05 g / L to about 0.2 g / L); an anticoagulant, in one embodiment, present in an amount sufficient to help prevent coagulation of residual blood (eg, heparin or hirudin present at a concentration of about 1000 units / l to about 100,000 units / l); or an amiloride containing compound (eg, amiloride, ethyl isopropyl amiloride, hexamethylene) amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 μM to about 5 μM). . 2.2 Collection and management of placenta Usually, a human placenta is recovered soon after its expulsion after delivery. In a preferred embodiment, the placenta is retrieved from a patient after informed consent and after making a complete medical history of the patient and is associated with the placenta. Preferably, the medical history continues after giving birth. Said medical record can be used to coordinate later use of the placenta or the stem cells harvested therefrom. For example, stem cells from the human placenta may be used, in view of medical history, for personalized medicine for the infant associated with the placenta, or for parents, siblings or other relatives of the infant. Before the placental stem cells are recovered, blood is removed from the umbilical cord and blood from the placenta. In certain modalities, after delivery, cord blood is recovered in the placenta. The placenta can be subjected to a conventional cord blood recovery process. Typically, a needle or cannula is used, with the help of gravity, to leave the placenta without blood (see, for example, Anderson, U.S. Patent No. 5,372,581; Hessel et al., Patent of E.U.A. No. 5,415,665). The needle or cannula is usually placed in the umbilical vein and the Placenta can be gently massaged to help drain cord blood from the placenta. Said cord blood recovery can be performed commercially, for example, LifeBank USA, Cedar Knolls, N. J., ViaCord, Cord Blood Registry and Cryocell. Preferably, the placenta is drained by gravity without further manipulation to minimize tissue disruption during cord blood recovery. Typically, a placenta is transported from the delivery room or labor to another location, for example, a laboratory, to retrieve cord blood and collect stem cells, for example, by perfusion or dissociation of tissue. The placenta is preferably transported in a thermally insulated, sterile transport device (maintaining the placental temperature between 20-28 ° C), for example, by placing the placenta, with the attached umbilical cord attached, in a plastic bag Sterile closure, which is then placed in an insulated container. In another embodiment, the placenta is transported in a cord blood collection equipment substantially as described in the E patent. U .A. pending No. 7, 147, 626. Preferably, the placenta is delivered to the laboratory four to twenty-four hours after delivery. In certain embodiments, the proximal umbilical cord is secured, preferably within 4-5 cm (centimeter) of the insertion into the placental disc before cord blood recovery. In other modalities, the nearby umbilical cord is attached after retrieving cord blood but before additional processing of the placenta. The placenta, before harvesting the stem cell, can be stored under sterile conditions and either at room temperature or at a temperature of 5 to 25 ° C (centigrade). The placenta can be stored for a period of four to twenty-four hours, up to forty-eight hours, or more than forty-eight hours, before pumping the placenta to remove any residual cord blood. In one embodiment, the placenta is harvested from about zero hours to about two hours after the expulsion. The placenta is preferably stored in an anticoagulant solution at a temperature of 5 to 25 ° C (centigrade). Suitable anticoagulant solutions are well known in the art. For example, a sodium solution of heparin or warfarin may be used. In a preferred embodiment, the anticoagulant solution comprises a heparin solution (eg, 1% w / w in 1: 1000 solution). The bled placenta is preferably stored for no more than 36 hours before collecting the placental stem cells. The mammalian placenta or part thereof, once harvested and prepared generally as before, can be treated in any manner known in the art, for example, it can be pumped or damaged, eg, digested with one or more enzymes destabilizing tissue, to obtain stem cells. . 2.3 Physical disruption and enzymatic digestion of placental tissue In one embodiment, stem cells from a mammalian placenta are harvested by physical disruption of part or all of the organ. For example, the placenta, or a portion of it, for example, can be crushed, chopped, minced, chopped into pieces, diced, marinated or the like. The tissue can then be cultured to obtain a population of stem cells. Typically, the placental tissue is destabilized using, for example, a stem cell harvesting composition (section 5.2.1 and below). The placenta can be dissected into components prior to physical disruption and / or enzymatic digestion and stem cell recovery. Stem cells from the placenta can be obtained from all or a portion of the amniotic membrane, chorion, umbilical cord, cotyledons of placenta, or any combination thereof, including an entire placenta. Preferably, placental stem cells are obtained from placental tissue comprising amnion and chorion. Typically, placental stem cells can be obtained by disrupting a small block of placental tissue, for example, a block of placental tissue that is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or about 1000 cubic millimeters in volume. Any method of physical disruption can be used, as long as the disruption method leaves a plurality, more preferably a majority, and more preferably at least 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in said viable organ, as determined, for example, by exclusion of blue guts. Generally, stem cells from a placenta, or portion thereof, can be harvested at any time within about the f three days after the expulsion, but preferably between about 8 hours and about 18 hours after the expulsion. In a specific embodiment, the destabilized tissue is cultured in tissue culture medium suitable for the proliferation of placental stem cells (see, for example, section 5.3, below, describing the culture of placental stem cells). In another specific embodiment, stem cells are harvested by physical disruption of placental tissue, where physical disruption includes enzymatic digestion, which can be achieved by the use of one or more enzymes that digest tissue. The placenta, or a portion thereof, can also be physically destabilized and digested with one or more enzymes, and the resulting material then submerged in, or mixed in, a stem cell collection composition. A preferred stem cell harvesting composition comprises one or more tissue destabilizing enzyme (s). The enzymatic digestion preferably uses a combination of enzymes, for example, a combination of a matrix metalloprotease and a neutral protease, for example, a combination of collagenase and dispase. In one embodiment, the enzymatic digestion of placental tissue uses a combination of a matrix metalloprotease, a neutral protease, and a mucolytic enzyme for digestion of hyaluronic acid, such as a combination of collagenase, triggers and hyaluronidase or a combination of LIBERASE ( Boehringer Mannheim Corp., Indianapolis, Ind.) And hyaluronidase. Other enzymes that can be used to destabilize placental tissue include papain, deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, or elastase. Serine proteases can be inhibited by alpha 2 microglobulin in serum and therefore the medium used for digestion is usually free of serum. EDTA and DNase are commonly used in enzyme digestion procedures to increase the efficiency of cell recovery. The digestate is preferably diluted in order to avoid entrapment of stem cells within the viscous digestion. Any combination of tissue digestion enzymes can be used. Typical concentrations for tissue digestion enzymes include, for example, 50-200 U / mL for collagenase I and collagenase IV, 1-10 U / mL for triggers, and 10-100 U / mL for elastase. Proteases can be used in combination, ie, two or more proteases in the same digestion reaction, or they can be used in sequence in order to release placental stem cells. For example, in one embodiment, a placenta, or part thereof, is f digested with an appropriate amount of collagenase I at about 1 to about 2 mg / ml, for example, during 30 minutes. minutes, followed by digestion with trypsin, at a concentration of approximately 0.25%, for example, for 10 minutes, at 37 ° C. Serine proteases are preferably used consecutively following the use of other enzymes. In another embodiment, the tissue can be destabilized by the addition of a chelator, for example, bi (2-aminoethyl ether) -NNN'N'-tetraacetic acid ethylene glycol (EGTA) or ethylenediaminetetraacetic acid (EDTA) to the composition of stem cell collection comprising the stem cells, or to a solution wherein the tissue is destabilized and / or digested prior to isolation of the stem cells with the stem cell harvesting composition. In one embodiment, a digestion can proceed as follows. Approximately one gram of placental tissue is obtained and itches. The tissue is digested in 10 mL of a solution comprising approximately 1 mg / mL of collagenase 1A and about 0.25% of trypsin at 37 ° C in a shaker at approximately 100 RPM. The digestate is washed three times with culture medium, and the washed cells are seeded in 2 bottles T-75. The cells are then isolated by differential adhesion, and are characterized, for example, by cell surface markers, differentiation, and the like. It will be appreciated that where an entire placenta, or portion of a placenta comprising fetal and maternal cells (eg, where the portion of the placenta comprises the chorion or cotyledons), the placental stem cells harvested they will comprise a mixture of placental stem cells derived from fetal and maternal sources. Where a portion of the placenta comprises, or an insignificant number of, no maternal cell (eg, amnion), the collected placental stem cells will almost exclusively comprise fetal placental stem cells. Stem cells from destabilized tissue can be isolated by differential trypsinization (see section 5.2.5, below) followed by culture in one or more containers of new culture in fresh proliferation medium, optionally followed by a second step of differential trypsinization. . 2.4 Perfusion of the placenta Stem cells from the placenta can also be obtained by perfusion of the mammalian placenta. Methods of pumping mammalian placenta to obtain stem cells are described, for example, in Hariri, application publication of E.U.A. No. 2002/0123141, and in the provisional application of E.U.A. No. 60 / 754,969, entitled "Improved Medium for Collecting Placental Stem Cells and Preserving Organs", filed December 29, 2005. Placental stem cells can be collected by perfusion, for example, through the vasculature of the placenta , using, for example, a cell collection composition mother as an infusion solution. In one embodiment, a mammalian placenta is pumped by passage of perfusion solution through one or both of the umbilical artery and umbilical vein. The flow of perfusion solution through the placenta can be achieved using, for example, gravity in the placenta. Preferably, the perfusion solution is forced through the placenta using a pump, for example, a peristaltic pump. The umbilical vein can be cannulated, for example, with a cannula, for example, a TEFLON® cannula or plastic, which is connected to a sterile connecting apparatus, such as a sterile tube. The sterile connecting apparatus is connected to a perfusion manifold. In the preparation for perfusion, the placenta is preferably oriented (eg, suspended) in such a way that the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be pumped by passing a perfusion fluid through the vasculature of the placenta and surrounding tissue. The placenta can also be pumped by passing a perfusion fluid into the umbilical vein and collecting the umbilical arteries, or passing a perfusion fluid into the umbilical arteries and collecting it from the umbilical vein. In one embodiment, for example, the umbilical artery and the umbilical vein are connected simultaneously, for example, to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The infusion solution is passed into the vein and umbilical artery. The perfusion solution exudes from and / or passes through the walls of the blood vessels in the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was fixed to the mother's uterus during pregnancy. The perfusion solution can also be introduced through the opening of the umbilical cord and allowed to flow or filter out of the openings in the wall of the placenta that interconnect with the maternal uterine wall. Placental cells that are harvested by this method, which can be referred to as a "bread" method, are typically a mixture of fetal and maternal cells. In another modality, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or passed through the umbilical artery and collected from the umbilical veins. Placental cells collected by this method, which can be referred to as a "closed loop" method, are typically almost exclusively fetal. It will be appreciated that perfusion using the "bread" method, ie, by which perfusate is collected after it has exuded from the maternal side of the placenta, results in a mixture of fetal and maternal cells. As a result, the cells harvested by this method comprise a mixed population of placental stem cells of both fetal and maternal origin. In contrast, perfusion only through the placental vasculature in the closed loop method, so perfusion fluid passes through one or two vessels of the placenta and is collected only through the remaining vessel (s), results in the collection of a population of stem cells from the placenta almost exclusively of fetal origin. The closed circuit perfusion method, in one modality, can be performed in the following manner. A placenta after childbirth is obtained within approximately 48 hours after delivery. The umbilical cord is attached and cut above the clamp. The umbilical cord is discarded, or it can be processed to recover, for example, stem cells from the umbilical cord, and / or to process the umbilical cord membrane for the production of a biomaterial. The amniotic membrane can be retained during perfusion, or it can be separated from the chorion, for example, by using a short needle dissection with the fingers. If the amniotic membrane is separated from the chorion before the perfusion, for example, it can be discarded, or processed, for example, to obtain stem cells by enzymatic digestion, or to produce, for example, an amniotic membrane biomaterial, for example, the biomaterial described in the US application publication No. 2004/0048796. After clearing the placenta of all visible blood clots and residual blood, for example, using sterile gauze, the umbilical cord vessels are exposed, for example, by partially cutting the umbilical cord membrane to expose a cord cross section. The vessels are identified, and open, for example, by advancing a clamp of closed teeth through the cutting end of each vessel. The apparatus, for example, plastic tube connected to a device Perfusion or peristaltic pump, then it is inserted into each of the arteries of the placenta. The pump can be any pump suitable for the purpose, for example, a peristaltic pump. The plastic tube, connected to a sterile collection reservoir, for example, a blood bag such as a 250 mL collection bag, is then inserted into the vein of the placenta. Alternatively, the tube connected to the pump is inserted into the vein of the placenta, and tubes to a collection reservoir (s) are inserted into one or both of the arteries of the placenta. The placenta is then pumped with a volume of perfusion solution, for example, approximately 750 ml of perfusion solution. Then cells are collected in the perfusate, for example, by centrifugation. In one embodiment, the proximal umbilical cord is held during perfusion, and more preferably, is held within 4-5 cm (centimeter) of the cord insertion in the placental disc.

The first collection of perfusion fluid from a mammalian placenta during the bleeding process is usually colored with residual red blood cells from the cord blood and / or blood from the placenta. The perfusion fluid becomes more colorless as the perfusion proceeds and the residual cord blood is washed out of the placenta. In general, 30 to 100 ml (millimeter) of perfusion fluid is adequate to bleed the placenta to the beginning, but more or less perfusion fluid can be used depending on the results observed. The volume of perfusion fluid to collect cells The mother of the placenta can vary depending on the number of stem cells to be harvested, the size of the placenta, the number of collections to be made from an individual placenta, etc. In various modalities, the volume of perfusion fluid can be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is pumped with 700-800 mL of perfusion fluid after bleeding. The placenta can be pumped a plurality of times over the course of several hours or several days. Where the placenta is to be pumped a plurality of times, it can be maintained or cultured under aseptic conditions in a container or other suitable vessel, and pumped with the stem cell harvesting composition, or a standard perfusion solution (e.g. a normal saline solution such as phosphate buffered saline ("PBS") with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bihydroxycoumarin), and / or with or without an antimicrobial agent (e.g., β -mercaptoethanol (0.1 mM); antibiotics such as streptomycin (for example, at 40-100 μg / ml), penicillin (for example, at 40U / ml), amphotericin B (for example, at 0.5 μg / ml). In one embodiment, an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, so that the placenta is maintained or cultured by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or 2 or 3 or more days before perfusion and perfusate collection. The placenta of perfusate can be maintained for one or more additional time (s), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24 or more hours, and pump a second time with, for example, 700-800 mL of perfusion fluid. The placenta can be pumped 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, perfusion of the placenta and collection of perfusion solution, eg, stem cell harvesting composition, is repeated until the number of nucleated cells recovered falls below 100 cells / ml. The perfusates at different time points can be processed more individually to recover cell-dependent populations of time, e.g., stem cells. You can also gather perfumes from different time points. In a preferred embodiment, stem cells are collected at one time or times between about 8 hours and about 18 hours after ejection. Without wishing to limit the theory, after bleeding and a sufficient time of perfusion of the placenta, it is believed that placental stem cells migrate in the bled and pumped microcirculation of the placenta where, according to the methods of the invention , are collected, preferably by washing in a collection vessel by perfusion. The perfusion of the isolated placenta not only serves to remove blood from the residual cord, but also to provide the placenta with the appropriate nutrients, including oxygen. The placenta can be cultured and pumped with a similar solution that was used to remove residual bead globules, preferably, without the addition of anticoagulant agents. Perfusion according to the methods of the invention results in the collection of significantly more placental stem cells than the number obtainable from a mammalian placenta not pumped with said solution, and otherwise not treated to obtain stem cells (e.g. , for tissue destabilization, for example, enzymatic digestion). In this context, "significantly more" means at least 10% more. Perfusion according to the methods of the invention produces significantly more placental stem cells than, for example, the number of placental stem cells obtainable from culture medium where a placenta, or portion thereof, has been cultured . Placental stem cells can be isolated by perfusion with a solution comprising one or more proteases or other tissue destabilizing enzymes. In a specific embodiment, a placenta or portion thereof (eg, amniotic membrane, amnion and chorion, lobe of placenta or cotyledon, umbilical cord, or combination of any of the above) is brought to 25-37 ° C, and incubated with one or more tissue destabilizing enzymes in 200 mL of a culture medium for 30 minutes. Perfusate cells are collected, taken at 4 ° C, and washed with a cold inhibitor mixture comprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem cells are washed after several minutes with a cold stem cell harvesting composition (e.g., 4 ° C). . 2.5 Isolation, classification and characterization of placental stem cells Mammalian placental stem cells, whether obtained by perfusion or enzymatic digestion, can be purified at the start of (ie, isolate from) other cells by Ficoll gradient centrifugation. Said centrifugation can follow any standard protocol for centrifugation speed, etc. In one embodiment, for example, cells harvested from the placenta are recovered from perfusate by centrifugation at 5000 x g for 1 5 minutes at room temperature, which separates cells from, for example, contaminating debris and platelets. In another embodiment, the placental perfusate is concentrated to approximately 200 ml, gently covered by Ficoll, and centrifuged at approximately 1 100 xg for 20 minutes at 22 ° C, and the low cell density interface layer is harvested for processing additional. Cell agglomerates can be resuspended in fresh stem cell harvesting composition, or a medium suitable for stem cell maintenance, for example, MDM serum free medium containing 2U / ml heparin and 2mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction can be isolated, for example, using Lymphoprep (Nycomed Pharma, Oslo, Norway) according to the manufacturer's recommended procedure. As used herein, "isolating" placental stem cells means eliminating at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells with which the stem cells are normally associated in the intact mammalian placenta. A stem cell of an organ is "isolated" when present in a population of cells comprising less than 50% of the cells whereby the stem cell is normally associated in the intact organ. Placental cells obtained by perfusion or digestion, for example, in addition, or at the outset, can be isolated by differential trypsinization using, for example, a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis, MO). Differential trypsinization is possible since the placental stem cells typically detach from the plastic surfaces in approximately five minutes while other adherent populations typically require more than 20-30 minutes of incubation. The detached placental stem cells can be harvested after trypsinization and neutralization of trypsin, using, for example, Trypsin Neutralizing Solution (TNS, Cambrex). In an adherent cell isolation mode, aliquots of, for example, approximately 5-10 x 106 cells are placed in each of several T-75 bottles, preferably flasks.

T75 coated with fibronectin. In such modality, the cells can be cultured with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex), and placed in a tissue culture incubator (37 ° C, 5% CO2). After 10 to 15 days, non-adherent cells are removed from the flasks when washing with PBS. The PBS is then replaced by MSCGM. The bottles are preferably examined daily for the presence of various adherent cell types and in particular, for the identification and expansion of groups of fibroblast cells. The number and type of cells collected from a mammalian placenta can be monitored, for example, by measuring changes in cell surface markers and morphology using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., spotting with tissue-specific or cell-marker specific antibodies), fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), by examination of cell morphology using light or microscopy with the same focus, and / or by measuring changes in gene expression using techniques well known in the art, such as PCR and profiling of gene expression. These techniques can also be used to identify cells that are positive for one or more particular markers. For example, using antibodies to CD34, one can determine, using the prior art, whether a cell comprises a detectable amount of CD34; if so, the cell is CD34 *. Likewise, if a cell produces enough OCT-4 RNA to be detectable by RT-PCR, or significantly more OCT-4 RNA than an adult cell, the cell is OCT-4 *. Antibodies to cell surface markers (eg, CD markers such as CD34) and the stem cell-specific gene sequence, such as OCT-4, are well known in the art. Placental cells, in particular cells that have been isolated by Ficoll separation, differential adhesion, or a combination of both, can be stored using a fluorescence activated cell sorter (FACS). Fluorescence activated cell sorting (FACS) is a well-known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151: 150-165). The laser excitation of fluorescent portions in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture. In a modality, antibodies or specific ligands of cell surface marker are marked with different fluorescent labels. The cells are processed through the cell sorter, allowing separation of cells based on their ability to bind the antibodies used. The particles classified with FACS can be deposited directly in individual wells of 96 well or 384 well plates to facilitate separation and cloning. In a classification scheme, the placental stem cells they are stored in the base of expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4 and / or HLA-G. This can be achieved in connection with procedures for selecting stem cells on the basis of their adhesion properties in culture. For example, an adhesion selection stem cell can be achieved before or after sorting on the basis of marker expression. In one embodiment, for example, cells are first classified on the basis of their CD34 expression; CD34 'cells are retained, and cells that are CD200 * HLA-G *, are separated from the other CD34 cells. "In another embodiment, placental cells rely on their expression of CD200 and / or HLA-G markers for example, cells that display any of these markers are isolated for further use Cells expressing, for example, CD200 and / or HLA-G, in a specific embodiment, can be further classified based on their expression of CD73 and / or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45 For example, in one embodiment, placental cells are classified by expression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38, and CD45, and placental cells that are CD200 *, HLA-G *, CD73 *, CD105 *, CD34 ', CD38"and CD45" are isolated from other cells of placenta for aional use With respect to antibody-mediated detection and classification of placental stem cells, any antibody is For a particular marker, it can be used, in combination with any fluorophore or other suitable mark for the detection and classification of cells (for example, cell sorting activated with fluorescence). Combinations of antibody / fluorophore to specific markers include, but are not limited to, fluorescein-isothiocyanate-conjugated monoclonal antibodies (FITC) against HLA-G (available from Serotec, Raleigh, NC), CD10 (available from BD Immunocytometry Systems, San Jose, California), CD44 (available from BD Biosciences Pharmingen, San Jose, California), and CD105 (available from R &D Systems Inc., Minneapolis, Minnesota); monoclonal antibodies conjugated with phycoerythrin (PE) against CD44, CD200, CD117 and CD13 (BD Biosciences Pharmingen); monoclonal antibodies conjugated with phycoerythrin-Cy7 (PE Cy7) against CD33 and CD10 (BD Biosciences Pharmingen); monoclonal and streptavidin antibodies conjugated to allophicocyanin (APC) against CD38 (BD Biosciences Pharmingen); and biotinylated CD90 (BD Biosciences Pharmingen). Other antibodies that can be used include, but are not limited to, CD133-APC (Miltenyl), KDR-Biotin (CD309, Abcam), CytokeratinK-Fitc (Sigma or Dako), HLA ABC-Fitc (BD), HLA DRDQDP- PE (BD), β-2-microglobulin-PE (BD), CD80-PE (BD) and CD86-APC (BD). Other antibody / tag combinations that can be used include, but are not limited to, CD45-PerCP (peridin chlorophyll protein); CD44-PE; CD19-PE; CD10-F (fluorescein); HLA-G-F and 7-amino-actinomycin-D (7-AAD); HLA-ABC-F; and similar. Placental stem cells can be tested for CD117 or CD133 using, for example, conjugated monoclonal antibodies with streptavidin and biotin conjugated with phycoerythrin-Cy5 (PE Cy5) against CD117 or CD133; however, using this system, the cells may appear positive for CD117 or CD133, respectively, due to a relatively high background. Placental stem cells can be labeled with an antibody to an individual marker and detected and / or classified. Placental stem cells can also be labeled simultaneously with multiple antibodies to different markers. In another embodiment, magnetic beads can be used to separate cells. The cells can be classified using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (0.5-100 μm diameter). A variety of useful modifications can be made in the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten. The beads are then mixed with the cells to allow binding. The cells then pass through a magnetic field to separate the cells that have the specific cell surface marker. In one embodiment, these cells can then be isolated and remixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells pass once more through a magnetic field, isolating cells that bind the antibodies. Said cells can then be diluted in separate plates, such as microtiter plates for clonal isolation.

Placental stem cells can also be characterized and / or classified based on morphology and cell growth characteristics. For example, placental stem cells can be characterized as having, and / or selecting on the basis of, for example, a fibroblast-like appearance in culture. Placental stem cells can also be characterized as having, and / or selecting, based on their ability to form embryoid-like bodies. In one embodiment, for example, placental cells that are fibroblast-like in shape, express CD73 and CD105, and produce one or more embryoid-like bodies in culture are isolated from other placental cells. In another embodiment, placental OCT-4 * cells that produce one or more embryoid-like bodies in culture are isolated from other placental cells. In another embodiment, the placental stem cells can be identified and characterized by a colony forming unit assay. Colony forming unit assays are commonly known in the art, such as MESEN CULT ™ medium (Stem Cell Technologies, Inc., Vancouver British Columbia). Placental stem cells can be evaluated for viability, proliferation potential, and longevity using standard techniques known in the art, such as trypan blue exclusion assay, fluorescein diacetate absorption assay, propium iodide absorption assay ( to evaluate viability); and thymidine uptake assay, MTT cell proliferation assay (to evaluate proliferation). Longevity can be determined by methods well known in the art, such as in determining the maximum number of population doubling in an extended culture.

The placental stem cells can also be separated from other placental cells using other techniques known in the art, for example, selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based on differential cell agglutination ability in the mixed population, for example, as with soy agglutinin; freeze-thaw procedures; filtration; conventional and zonal centrifugation; centrifugal distillation (jet-count centrifugation); unit severity separation; countercurrent distribution; electrophoresis; and similar. . 3 CULTIVATION OF CELLS MOTHER OF PLACENTA . 3.1 Culture medium Isolated placental stem cells, or population of placental stem cells, or placental cells or tissue from which placental stem cells grow, can be used to initiate, or seed, cell cultures. The cells are usually transferred to sterile tissue culture vessels either uncoated or coated with extracellular matrix or ligands such as laminin, collagen (eg, native or denatured), gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane protein (e.g., MATRIGEL® (BD Discovery Labware, Bedford, Mass.)). Placental stem cells can be cultured in any medium, and under any conditions, recognized in the art as acceptable for stem cell culture. Preferably, the culture medium comprises serum. Placental stem cells can be cultured in, for example, DMEM-LG (Dulbecco's Modified Essential Medium, low glucose) / MCDB 201 (basal chicken fibroblast medium) containing ITS (insulin-transferrin-selenium), LA + BSA (linoleic acid-bovine serum albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGFt1, and penicillin / streptomycin; DMEM-HG (high glucose) comprising 10% fetal bovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM (modified Dulbecco's medium from Iscove) comprising 10% FBS, 10% horse serum, and hydrocortisone; M199 comprising 10% FBS, EGF, a heparin; α-MEM (minimum essential medium) comprising 10% FBS, GLUTAMAX ™ and gentamicin; DMEM comprising 10% FBS, GLUTAMAX ™ and gentamicin, etc. A preferred medium is DMEM-LG / MCDB-201 comprising 2% FBS, ITS, LA + BSA, dextrose, L-ascorbic acid, PDGF, EGF, and penicillin / streptomycin. Other means that can be used to culture placental stem cells include DMEM (high or low glucose), Eagle's basal medium, Ham's F10 medium (F10), Ham's F012 medium (F12), Dulbecco modified from Iscove, growth medium from mesentery stem cell (MSCGM), L-15 medium from Liebovitz, MCDB, DMEM / F12, RPMI 1640, advanced DMEM (Gibco), DMEM / MCDB201 (Sigma), and CELL-GRO FREE The culture medium can be supplemented with one or more components including, for example, serum (eg, fetal bovine serum (FBS), preferably about 2-15% (v / v); horse serum (horse)); human serum (HS)); betamercaptoethanol (BME), preferably about 0.001% (v / v); one or more growth factors, eg, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-1 growth factor (IGF-1) ), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO); amino acids, including L-valine; and one or more antibiotic and / or antifungal agents to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination. The placenta stem cells can be cultured under standard tissue culture conditions, for example, in tissue culture dishes or multi-well plates. Placental stem cells can also be cultured using a hanging drop method. In this method, the placental stem cells are suspended at approximately 1 x 104 cells per mL in approximately 5 mL of medium, and one or more drops of the medium are placed inside the lid of a tissue culture container, for example, a 100 mL Petri dish. The drops can be, for example, individual drops, or multiple drops of, for example, a multichannel pipette. The lid is inverted and placed carefully above the bottom of the dish, which contains a volume of liquid, eg, sterile PBS sufficient to maintain the moisture content in the atmosphere of the dish, and stem cells are grown. In one embodiment, the placental stem cells are cultured in the presence of a compound that acts to maintain an undifferentiated phenotype in the placental stem cell. In a specific embodiment, the compound is a substituted 3,4-dihydropyridimol [4,5-d] pyrimidine. In a more specific embodiment, the compound is a compound that has the following chemical structure: The compound can be contacted with a placental stem cell, or population of stem cells from the placenta, at a concentration of, for example, between about 1 μM to about 10 μM. . 3.2 Expansion and proliferation of placental stem cells Once an isolated placental stem cell, or population isolated from placental stem cells (eg, a stem cell or stem cell population separated from at least 50% of the placental cells with which the stem cell or stem cell population is normally associated in vivo), the stem cell or stem cell population can be proliferated and expanded in vivo. For example, a population of placental stem cells can be cultured in tissue culture containers, e.g., dishes, flasks, multi-well plates, or the like, for a sufficient time for the stem cells to proliferate at 70-90% of confluence, that is, until the stem cells and their progeny occupy 70-90% of the surface area of culture of the tissue culture container. Placental stem cells can be planted in culture vessels at a density that allows cell growth. For example, cells can be seeded at low density (eg, about 1,000 to about 5,000 cells / cm 2) at high density (eg, about 50,000 or more cells / cm 2). In a preferred embodiment, the cells are cultured at about 0 to about 5 volume percent CO2 in air. In some preferred embodiments, the cells are cultured at about 25 to about 25 percent O2 in air, preferably about 5 to about 20 percent O2. in air. Cells are preferably cultured at about 25 ° C to about 40 ° C, preferably 37 ° C. The cells are preferably cultured in an incubator. The culture medium can be static or stirred, for example, using a bioreactor. The placental stem cells preferably grow under low oxidative stress (for example, with the addition of glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, or the like). Once 70% -90% confluence is obtained, the cells can be passed. For example, cells can be treated enzymatically, for example, trypsinize, using techniques well known in the art, to separate them from the tissue culture surface. After removing the cells by pipetting and counting the cells, approximately 20,000-100,000 stem cells, preferably approximately 50,000 stem cells, are transferred to a new culture container containing fresh culture medium. Typically, the new medium is the same type of medium from which the stem cells are removed. The invention encompasses populations of placental stem cells that have been passed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 12, 14, 16, 18 or 20 times, or more . . 3.3 Populations of stem cells from the placenta The invention provides populations of placental stem cells. The population of placental stem cells can be isolated directly from one or more placentas; that is, the population of Placental stem cells can be a population of placental cells comprising placental stem cells obtained from, or contained within, perfusate, or obtained from, or contained within, destabilized placental tissue, eg, tissue digestate of placenta (that is, the collection of cells obtained by enzymatic digestion of a placenta or part of it). The isolated placental stem cells of the invention can also be cultured and expanded to produce populations of placental stem cells. Placental cell populations that comprise placental stem cells can also be cultured and expanded to produce populations of placental stem cells. The stem cell populations of the placenta of the invention comprise placental stem cells, for example, placental stem cells as described herein. In various modalities, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells in a population of stem cells of the Isolated placenta are stem cells from the placenta. That is, a population of placental stem cells can comprise, for example, as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 % of non-stem cells. The invention provides methods of producing population of placental stem cells isolated, for example, by selecting placental stem cells, either derived from enzymatic digestion or perfusion, that express particular markers and / or characteristics of cultivation or particular morphological. In one embodiment, for example, the invention provides a method of producing a cell population comprising selecting placental cells that (a) adhere to a substrate, and (b) expressing CD200 and HLA-G; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population that comprises identifying placental cells expressing CD200 and HLA-G, and isolating said cells from other cells to form a cell population. In another embodiment, the method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, and (b) expressing CD73, CD105 and CD200; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population that comprises identifying placental cells expressing CD73, CD105 and CD200, and isolating said cells from other cells to form a cell population. In another embodiment, the method of producing a cell population comprises selecting placental cells which (a) adhere to a substrate and (b) express CD200 and OCT-4.; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population that comprises identifying placental cells expressing CD200 and OCT-4, and isolating said cells from other cells to form a cell population. In another embodiment, the method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, (b) express CD73 and CD105, and (c) facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of an embryoid type body; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a ceiular population that comprises identifying placental cells expressing CD73 and CD105, and facilitating the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of an embryoid type body, and isolate said cells from other cells to form a cell population. In another embodiment, the method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, and (b) expressing CD73, CD105 and HLA-G; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population that comprises identifying placental cells expressing CD73, CD105 and HLA-G, and isolating said cells from other cells to form a cell population. In another embodiment, the method of producing a cell population comprises selecting placental cells which (a) adhere to a substrate, (b) express OCT-4, and (c) facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of an embryoid type body; and isolating said cells from other cells to form a cell population. In another embodiment, the invention provides a method of producing a cell population that comprises identifying placental cells expressing OCT-4, and facilitating the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell. when said population is cultured under conditions that allow the formation of an embryoid type body, and isolate said cells to form other cells to form a cell population. Said cell populations may be used to treat any of the diseases and conditions listed hereunder. Such cell populations can be used to evaluate populations of placental stem cells, for example, as part of a quality control method. In any of the above embodiments, the method may further comprise selecting placental cells expressing ABC-p (a placental-specific ABC transport protein, see, eg, Allikmets et al., Cancer Res. 58 (23): 5337- 9 (1998)). The method may also comprise selecting cells that exhibit at least one characteristic specific to, for example, a mesentery stem cell, eg, expression of CD29, expression of CD44, expression of CD90, or expression of a combination of the foregoing.

In the above embodiments, the substrate can be any surface where cell culture and / or selection can be achieved, for example, placental stem cells. Typically, the substrate is plastic, for example, tissue culture dish or multi-well plastic dish. The tissue culture plastic can be coated with a biomolecule, for example, laminin or fibronectin. Cells, for example, placental stem cells, can be selected for a population of placental stem cells by any means known in the cell selection art. For example, cells can be selected using an antibody or antibodies to one or more cell surface markers, for example, in flow cytometry or FACS. The selection can be achieved using antibodies in conjunction with magnetic beads. Antibodies that are specific for certain markers related to stem cell are known in the art. For example, antibodies to OCT-4 (Abcam, Cambridge, MA), CD200 (Abcam), HLA-G (Abcam), CD73 (BD Biosciences Pharmingen, San Diego, CA), CD105 (Abcam; BioDesign International, Saco, ME), etc. Antibodies to other labels are also commercially available, for example, CD34, CD38 and CD45 are available from, for example, StemCell Technologies or BioDesign International. The population of isolated placental stem cells may comprise placental cells that are not stem cells, or cells that are not placental cells. Isolated stem cell populations of the placenta are they can combine with one or more populations of non-stem cells or non-placental cells. For example, an isolated population of placental stem cells can be combined with blood (for example, placental blood or umbilical cord blood), blood stem cells (for example, stem cells derived from blood from the placenta or umbilical cord blood), umbilical cord stem cells, populations of nucleated cells derived from blood, mesentery cells derived from the spinal cord, cell populations derived from bone, raw spinal cord, adult stem cells (somatic), stem cell populations contained within tissue, cultured stem cells, completely differentiated cell populations (e.g., condor, fibroblasts, amniotic cells, osteoblasts, muscle cells, cardiac cells, etc.) and the like. In a specific embodiment, the invention provides a population of stem cells comprising placental stem cells and umbilical cord stem cells. Cells in a population of isolated placental stem cells can be combined with a plurality of other cells at ratios of approximately 100,000,000: 1, 50,000,000: 1, 20,000,000: 1, 10,000,000: 1, 5,000,000: 1, 2,000,000: 1 , 1,000,000: 1, 500,000: 1, 200,000: 1, 100,000: 1, 50,000: 1, 20,000: 1, 10,000: 1, 5,000: 1, 2,000: 1, 1,000: 1, 500: 1, 200: 1, 100 : 1, 50: 1, 20: 1, 10: 1, 5: 1, 2: 1, 1: 1; 1: 2; 1: 5; 1:10; 1: 100; 1: 200; 1: 500; 1: 1,000; 1: 2,000; 1: 5,000; 1: 10,000; 1: 20,000; 1: 50,000; 1: 100,000; 1: 500,000; 1: 1,000,000; 1: 2,000,000; 1: 5,000,000; 1: 10,000,000; 1: 20,000,000; 1: 50,000,000; or approximately 1: 100,000,000, comparing numbers of total nucleated cells in each population. Cells in a population of isolated placental stem cells can be combined with a plurality of cells of a plurality of cell types, too. In one, an isolated population of placental stem cells is combined with a plurality of hematopoietic stem cells. Said hematopoietic stem cells, for example, may be inside unprocessed placental blood, umbilical cord or peripheral blood; in total nucleated cells from placental blood, umbilical cord blood or peripheral blood; in an isolated population of CD34 * cells from placental blood, umbilical cord blood or peripheral blood; in spinal cord not processed; in total nucleated cells of the spinal cord; in an isolated population of CD34 * spinal cord cells, or the like. . 4 PRODUCTION OF A BANK OF CELLS MOTHER OF PLACENTA Post-partum placental stem cells can be cultured in a number of different ways to produce a series of batches, for example, a series of individually administrable, stem-cell doses of the placenta. Such batches, for example, may be obtained from placental perfusate stem cells or from enzyme-digested placenta tissue. The series of batches of placental stem cells, obtained from a plurality of placentas, can be placed in a bank of placental stem cells, for example, for long-term storage. Typically, adherent stem cells are obtained from an initial culture of placental material to form a seed culture, which expands under controlled conditions to form cell populations of approximately equivalent number of duplications. The lots are preferably derived from the tissue of a single placenta, but can be derived from the tissue of a plurality of placentas. In one embodiment, batches of the stem cell are obtained in the following manner. The tissue of the placenta is first destabilized, for example, by itching, digested with a suitable enzyme, for example, collagenase (see section 5.2.3 above). The tissue of the placenta preferably comprises, for example, the whole amnion or chorion. The digested tissue is grown, for example, for approximately 1-3 weeks, preferably about 2 weeks. After removing non-adherent cells, high density colonies are collected which are formed, for example, by trypsinization. These cells are harvested and resuspended in a convenient volume of culture medium, and are defined as 0 passage cells. Passage cells are then used to grow expansion cultures. The expansion cultures can be any arrangement of separate cell culture apparatus, for example, a Cell Factory by NUNC ™. The cells in the passage culture 0 were they can subdivide to any degree in order to plant expansion cultures with, for example, stem cells 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, or 10 x 104. Preferably, around 2 x 104 to about 3 x 104 passage cells 0 are used to seed each expansion culture. The number of expansion cultures may depend on the number of passage cells 0, and may be greater or lesser in number depending on the particular placenta (s) from which the stem cells are obtained. Expansion cultures are grown until the density of cells in culture reaches a certain value, for example about 1 x 10 5 cells / cm 2. The cells can be harvested and cryopreserved at this point, or passed into new expansion cultures as described above. Cells may be passed, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times before being used. A record of the cumulative number of population duplications is preferably maintained during expansion culture (s). The cells of a passage culture 0 can be expanded by 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 duplications, or up to 60 duplications. However, preferably, the number of population duplications, before dividing the cell population into individual doses, is between about 15 and about 30, preferably about 20 duplications. The cells they can be continuously grown throughout the expansion process, or they can be frozen at one or more points during the expansion. The cells to be used for individual doses can be frozen, for example, cryopreserved for later use. The individual doses may comprise, for example, from about 1 million to about 100 million cells per ml. and may comprise between about 1 O6 and about 109 cells in total. In a specific embodiment of the method, passageway 0 cells are cultured by a first number of duplications, eg, about 4 duplications, then frozen in a first cell bank. The cells of the first cell bank are frozen and used to seed a second cell bank, the cells of which expand by a second number of duplications, for example, approximately another eight duplications. Cells in this stage are harvested and frozen and used to plant new expansion cultures that are allowed to proceed for a third number of duplications, eg, approximately eight additional duplications, bringing the cumulative number of cell duplications to approximately 20. Cells in intermediate points in passage can be frozen in units of about 100,000 to about 10 million cells per ml, preferably about 1 million cells per ml to be used in culture of subsequent expansion. The cells at approximately 20 duplications can be frozen in individual doses of between about 1 million to about 100 million cells per ml for administration or use in making a composition containing stem cell. Therefore, in one embodiment, the invention provides a method of making a bank of placental stem cells, comprising: expanding primary culture placenta stem cells from a postpartum human placenta for a first plurality of population duplications; cryopreserve said stem cells from the placenta to form a Master Ceil Bank (master ceiular bank); expanding a plurality of placental stem cells from the Master Cell Bank for a second plurality of population duplications; to preserve said stem cells from the placenta to form a Working Cell Bank (functioning cellular bank); expand a plurality of placental stem cells from Working Cell Bank for a third plurality of population duplications; and cryopreservating said placental stem cells in individual doses, wherein said individual doses collectively make up a bank of placental stem cells. In a specific embodiment, the total number of population duplications is approximately 20. In another specific embodiment, said first plurality of population duplications is approximately four population duplications; said second plurality of population duplications is approximately eight population doublings; and said third plurality of duplications of population is approximately eight population doublings. In another specific embodiment, said primary cultured placental stem cells comprise placental stem cells from digested placental tissue. In another specific embodiment, said primary cultured placental stem cells comprise placental stem cells from placental perfusate and digested placenta tissue. In another specific embodiment, all of said placental stem cells in said primary culture of placental stem cells are from the same placenta. In another specific embodiment, the method further comprises the step of selecting placental stem cells CD200 * and HLA-G * from said plurality of said placental stem cells of said Working Cell Bank to form individual doses. In another specific embodiment, said individual doses comprise from about 104 to about 105 of placental stem cells. In another specific embodiment, said individual doses comprise from about 105 to about 106 of placental stem cells. In another specific embodiment, said individual doses comprise from about 106 to about 107 of placental stem cells. In another specific embodiment, said individual doses comprise from about 107 to about 108 of placental stem cells. In a preferred embodiment, the donor from which the placenta is obtained (eg, the mother) is tested for at least one pathogen. If the mother tests positive for a tested pathogen, the Whole batch of the placenta is discarded. Such a test may be performed at any time during the production of batches of placental stem cells, including before or after establishment of 0 passage cells, or during expansion culture. Pathogens for which the presence is tested may include, without limitation, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human immunodeficiency virus (types I and II), cytomegalovirus, herpes virus, and the like. . 5 DIFFERENTIATION OF STEM CELLS OF THE PLACENTA . 5.1 Induction of differentiation in neuronal or neurogenic cells Neuronal differentiation of placental stem cells can be achieved, for example, by placing placental stem cells under cell culture conditions that induce differentiation in neurons. In an exemplary method, a neurogenic medium comprises DMEM / 20% FBS and 1 mM beta-mercaptoethanol; said medium can be replaced after cultivation for about 24 hours with medium consisting of DMEM and 1-10 mM of betamercaptoethanol. In another embodiment, cells are contacted with DMEM / 2% DMSO / 200 μM butylated hydroxyanisole. In a specific embodiment, the differentiation medium comprises serum-free DMEMIF-12, butylated hydroxyanisole, potassium chloride, insulin, forskolin, valproic acid and hydrocortisone. In another embodiment, neuronal differentiation is achieved by plating placental stem cells on laminin coated plates in Neurobasal-A medium (Invitrogen, Carlsbad CA) containing supplement B27 and L-glutamine, optionally supplemented with bFGF and / or EGF. Placental stem cells can also be induced to neural differentiation by co-cultivation with neural cells, or culture in neuron-conditioned media. Neuronal differentiation can be evaluated, for example, by detection of neuron-type morphology (for example, bipolar cells comprising extended processes), detection of the expression of, for example, nerve growth factor receptor and heavy chain genes of neurofilament by RT-PCR; or detection of electrical activity, for example, by patch-clamp. A placental stem cell is considered to have differentiated into a neuronal cell when the cell exhibits one or more of these characteristics. . 5.2 Induction of differentiation in adipogenic cells Adipogenic differentiation of placental stem cells can be achieved, for example, by placing placental stem cells under cell culture conditions that induce differentiation into adipocytes. A preferred adipogenic medium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cord serum umbilical. In one embodiment, placental stem cells are fed Adipogenesis Induction Medium (Cambrex) (induction medium of adipogenesis) and cultured for 3 days (at 37 ° C, 5% CO2), followed by 1-3 days of culture in Adipogenesis Maintenance Medium (Cambrex) (adipogenesis maintenance medium). After 3 complete induction / maintenance cycles, the cells are cultured for an additional 7 days in the maintenance medium of adipogenesis, replacing the medium every 2-3 days. In another embodiment, placental stem cells are grown in medium comprising 1 μM of dexamethasone, 0.2 mM of indomethacin, 0.01 mg / ml of insulin, 0.5 mM of IBMX, DMEM-high glucose, FBS and antibiotics. Placental stem cells can also be induced to adipogenesis by culture in medium comprising one or more glucocorticoids (e.g., dexamethasone, indomethasone, hydrocortisone, cortisone), insulin, a compound that elevates intracellular levels of cAMP (e.g., dibutyl- cAMP; 8-CPT-cAMP (8- (4) chlorophenylthio) -adenosine, 3 ', 5' cyclic monophosphate); 8-bromo-cAMP; dioctanoyl-cAMP; forscholine) and / or a compound that inhibits the degradation of cAMP (for example, a phosphodiesterase inhibitor such as isobutylmethylxanthine (IBMX), methyl isobutylxanthine, theophylline, caffeine, indomethacin). A hallmark of adipogenesis is the development of multiple intracitiplasmic lipid blisters that can be easily observed using red O lipophilic spot oil. The expression of lipase and / or fatty acid binding protein genes is confirmed by RT / PCR in placental stem cells that have started to differentiate into adipocytes. A placental stem cell is considered to have differentiated into an adipocyte cell when the cell exhibits one or more of these characteristics. . 5.3 Induction of differentiation in chondrocytic cells Chondrogenic differentiation of placental stem cells can be achieved, for example, by placing placental stem cells under cell culture conditions that induce differentiation into chondrocytes. A preferred chondrocyte medium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum. In one embodiment, the placental stem cells are aliquoted into a sterile polypropylene tube, centrifuged (eg, at 150 x g for 5 minutes), and washed twice in Incomplete Chondrogenesis Medium (Cambrex) (incomplete chondrogenesis medium). The cells are resuspended in Complete Chondrogenesis Medium (Cambrex) (complete chondrogenesis medium) containing 0.01 μg / ml of TFG-beta-3 at a concentration of approximately 1-20 x 10 5 cells / ml. In other embodiments, the placental stem cells are contacted with exogenous growth factors, for example, GDF-5 or transforming growth factor beta3 (TGF-beta3), with or without ascorbate. The chondrogenic medium can be supplemented with amino acids including proline and glutamine, sodium pyruvate, dexamethasone, Ascorbic acid, and insulin / transferrin / Selenium. The chondrogenic medium can be supplemented with sodium hydroxide and / or collagen. Placental stem cells can be cultured at high or low density. The cells are preferably cultured in the absence of serum. Chondrogenesis can be evaluated, for example, by observation of production of eisonophilic soil substance, safranin-O staining for glycosaminoglycan expression; staining of hematoxylin / eosin, evaluating cell morphology, and / or confirming RT / PCR of collagen 2 gene expression and collagen 9. Chondrogenesis can also be observed by growing the stem cells in an agglomerate, formed, for example, by gently centrifuging mother cells in suspension (eg, at about 800 g for about 5 minutes). After approximately 1-28 days, the agglomerate of stem cells begins to form a hard matrix and demonstrates a structural integrity not found in uninduced cell lines, or non-chondrogenic, agglomerated ones of which tend to disintegrate when challenged. Chondrogenesis can also be demonstrated, for example, in such cell agglomerates, by staining with a stain that stains collagen, for example, Sirius Red (Syrian red), and / or a spot staining glycosaminoglycans (GAGs), such as, by example, Alcian Blue (Alcian blue). A placental stem cell is considered to have differentiated into a chondrocytic cell when the cell exhibits one or more of these characteristics. . 5.4 Differentiation induction in osteocytic cells Osteogenic differentiation of placental stem cells can be achieved, for example, by placing placental stem cells under cell culture conditions that induce osteocyte differentiation. A preferred osteocytic medium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum, followed by Osteogenic I nduction Medium (Cambrex) (osteogenic induction medium) containing 0.1 μM dexamethasone, 0.05 mM ascorbic acid- 2-phosphate, 10 mM beta glycerophosphate. In another embodiment, placental stem cells are cultured in medium (e.g., DMEM-low glucose) containing from about 10"7 to about 10" 9 M dexamethasone, about 10-50 μM phosphate salt of ascorbate (eg, ascorbate-2-phosphate) and from about 10 nM to about 10 mM of β-glycerophosphate. The osteogenic medium can also include serum, one or more antibiotic / antimyotonic agents, transforming growth factor-beta (eg, TGF-β1) and / or bone morphogenic protein (eg, BMP-2, BMP- 4, or a combination thereof). Differentiation can be evaluated using a specific calcium spot, eg, von Kossa staining, and RT / PCR detection of, for example, alkaline phosphatase, osteocalcin, bone sialoprotein and / or osteopontin gene expression. A placental stem cell is considered to have Differentiated in an osteocytic cell when the cell exhibits one or more of these characteristics. . 5.5 Induction of differentiation in pancreatic cells The differentiation of placental stem cells into pancreatic cells producing insulin can be achieved, for example, by placing placental stem cells under cell culture conditions that induce differentiation in pancreatic cells. An example of a pancreagenic medium comprises DMEM / 20% of CBS, supplemented with basic fibroblast growth factor, 10 ng / ml; and transforming growth factor beta-1, 2 ng / ml. This medium is combined with conditioned medium of nestin-positive neuronal cell cultures at 50/50 v / v. Can be used Knockout Serum Replacement (serum replacement) can be used instead of CBS. Cells are cultured for 14-28 days, feeding every 3-4 days. Differentiation can be confirmed by testing, for example, by insulin protein production, or insulin gene expression by RT / PCR. A placental stem cell is considered to have differentiated into a pancreatic cell when the cell exhibits a? more of these characteristics. . 5.6 I nduction of differentiation in cardiac cells The myogenic (cardiogenic) differentiation of placental stem cells can be achieved, for example, by placing placental stem cells under cell culture conditions that induce cardiomyocyte differentiation. A preferred cardiomyocyte medium comprises DMEM / 20% CBS supplemented with retinoic acid, 1 μM; basic fibroblast growth factor, 10 ng / ml; and transforming growth factor beta-1, 2 ng / ml; and epidermal growth factor, 100 ng / ml. Knockout Serum Replacement (I nvitrogen, Carsbad, California) can be used instead of CBS. Alternatively, placental stem cells are cultured in DMEM / 20% CBS supplemented with 50 ng / ml Cartiotropin-1 for 24 hours. In another modality, placental stem cells may be grown 10-14 days in protein-free medium for 5-7 days, then stimulated with human myocardium extract, for example, produced by homogenizing human myocardium in 1% of H EPES regulator supplemented with 1% cord blood serum. Differentiation can be confirmed by demonstration of cardiac actin gene expression, for example, by RT / PCR, or by visible cell beating. A placental stem cell is considered to have differentiated into a cardiac cell when the cell exhibits one or more of these characteristics. . 6 CONSERVATION OF STEM CELLS OF THE PLACENTA Placental stem cells can be conserved, that is, placed under conditions that allow long-term storage, or conditions that inhibit cell death, for example, by apoptosis or necrosis. Placental stem cells can be preserved using, for example, a composition comprising an apoptosis inhibitor, necrosis inhibitor and / or an oxygen carrying perfluorocarbon, as described in the provisional application of E.U.A. No. 60 / 754,969, entitled "Improved Medium for Collecting Placental Stem Cells and Preserving Organs," filed December 25, 2005. In one embodiment, the invention provides a method of preserving a population of stem cells comprising said population of stem cells with a stem cell harvesting composition comprising an apoptosis inhibitor and a perfluorocarbide carrying oxygen, wherein said apoptosis inhibitor is present in an amount and for a time sufficient to reduce or prevent apoptosis in the stem cell population, compared to a population of stem cells not contacted with the apoptosis inhibitor. In a specific embodiment, said inhibitor of apoptosis is a caspase inhibitor. In another specific embodiment, said inhibitor of apoptosis is a JNK inhibitor. In a more specific embodiment, said JNK inhibitor does not modulate differentiation or proliferation of said cells mother. In another embodiment, said stem cell harvesting composition comprises said apoptosis inhibitor and said perfiuorocarbon carrying oxygen in separate phases. In another embodiment, said stem cell harvesting composition comprises said apoptosis inhibitor and said perfluorocarbon carrying oxygen in an emulsion. In another embodiment, the mother cell harvest composition further comprises an emulsifier, for example, lecithin. In another embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 0 ° C and about 25 ° C at the time of contacting the stem cells. In another more specific embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 2 ° C and 1 0 ° C, or between about 2 ° C and about 5 ° C, at the time of contacting the stem cells. In another more specific embodiment, said contact is made during the transport of said population of stem cells. In another more specific embodiment, said contact is made during the freezing and thawing of said population of stem cells. In another embodiment, the invention provides a method of preserving a population of placental stem cells comprising said population of stem cells with an apoptosis inhibitor and an organ preservative compound, wherein said inhibitor of apoptosis is present in an amount and for a sufficient time to reduce or prevent apoptosis in the stem cell population, compared to a population of stem cells not contacted with the apoptosis inhibitor. In a specific embodiment, the organ preservative compound is UW solution (described in U.S. Patent No. 4,798,824; also known as ViaSpan; see also Southard et al., Transplantation 49 (2): 251-257 (1990)) or a solution described in Stern et al., US patent No. 5,552,267. In another embodiment, said organ-conserving compound is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof. In another embodiment, the mother cell harvesting composition additionally comprises an oxygen carrying perfluorocarbon, either in two phases or as an emulsion. In another embodiment of the method, placental stem cells are contacted with a stem cell harvesting composition comprising an apoptosis and perfluorocarbon inhibitor carrying oxygen, oxygen preserving compound, or combination thereof, during perfusion. In another modality, said stem cells are contacted during a process of tissue disruption, for example, enzymatic digestion. In another embodiment, placental stem cells are contacted with said stem cell harvesting compound after harvesting by perfusion, or after harvesting by disruption of tissue, eg, enzymatic digestion. Typically, during collection, enrichment and cellular isolation of the placenta, it is preferable to minimize or eliminate cellular stress due to hypoxia and mechanical stress. In other method mode, therefore, a stem cell, or population of stem cells, is exposed to a hypoxic condition during collection, enrichment or isolation for less than six hours during such storage, where a hypoxic condition is an oxygen concentration that it is less than the normal blood oxygen concentration. In a more specific embodiment, said population of stem cells is exposed to said hypoxic condition for less than two hours during said storage. In another more specific embodiment, said population of stem cells is exposed to said hypoxic condition for less than one hour, or less than thirty minutes, or is not exposed to a hypoxic condition, during collection, enrichment or isolation. In another specific embodiment, said population of stem cells is not exposed to shear stress during collection, enrichment or isolation. The stem cells of the placenta of the invention can be cryopreserved, for example, in cryopreservation medium in small containers, for example, ampoules. Suitable cryopreservation medium includes, but is not limited to, culture medium including, for example, growth medium, or cell freezing medium, for example, commercially available cell freezing medium, for example, C2695, C2639 or C6039 ( Sigma). The cryopreservation medium preferably comprises DMSO (dimethyl sulfoxide), at a concentration of, for example, about 10% (v / v). The means of cryopreservation can comprising additional agents, for example, methylcellulose and / or glycerol. Placental stem cells are preferably cooled to approximately 1 ° C / min during cryopreservation. A preferred cryopreservation temperature is from about -80 ° C to about -180 ° C, preferably from about -125 ° C to about -140 ° C. The cryopreserved cells can be transferred to liquid nitrogen before thawing to be used. In some embodiments, for example, once the ampoules have reached approximately -90 ° C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be done using a controlled speed freezer. The cryopreserved cells are preferably thawed at a temperature of about 25 ° C to about 40 ° C, preferably at a temperature of about 37 ° C. . 7 USES OF PLACENTA MOTHER CELLS . 7.1 Placental stem cell populations Placental stem cell populations can be used to treat any disease, disorder or condition that is given to treatment by the administration of a population of stem cells. As used herein, "treating" encompasses the cure of, remedy for, improvement of, decrease in the severity of, or reduction in the course of, a disease, disorder or condition, or any parameter or symptom of it. Placental stem cells, and populations of stem cells from the placenta, can be induced to differentiate into a particular cell type, either ex vivo or in vivo, in preparation for administration to an individual in need of stem cells, or differentiated cells of stem cells. For example, placental stem cells can be injected into a damaged organ, and for organ neogenesis and damage repair in vivo. Such damage may be due to such conditions and disorders including, but not limited to, myocardial infarction, seizure disorder, multiple sclerosis, embolism, hypotension, cardiac arrest, ischemia, inflammation, thyroiditis, loss of age-related cognitive function, radiation damage, cerebral palsy, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Leigh's disease, AIDS, dementia, memory loss, amyotrophic lateral sclerosis, muscular dystrophy, ischemic kidney disease, brain or spinal trauma, bypass heart-lung, glaucoma, rheumatoid arthritis, and the like. Placental stem cells can be used to treat autoimmune conditions such as juvenile diabetes, lupus, muscular dystrophy, rheumatoid arthritis and the like. Isolated populations of placental stem cells may be used, in specific modalities, in autologous or heterologous enzyme replacement therapy to treat specific diseases or conditions, including, but not limited to, lysosomal storage diseases, such as Tay-Sachs, Niemann-Pick, Fabry, Gaucher diseases (eg, glucocerbrosidase deficiency), Hunter and Hurler syndromes, Maroteaux-Lamy syndrome, fucosidosis (fucosidase deficiency), Batten (CLN3), as well as other gangliosidoses, mucopolysaccharides and glycogenases. Isolated populations of placental stem cells, alone or in combination with populations of stem or progenitor cells, can be used alone, or as carriers of autologous or heterologous transgenes in gene therapy, to correct congenital errors of metabolism, cystic fibrosis, adenoleukodystrophy (eg, co-A ligase deficiency), metachromatic leukodystrophy (arylsulfatase A deficiency) (eg, symptomatic or presymptomatic late infant or juvenile forms), globoid cell leukodystrophy (Krabee's disease, galactocerebrosidase deficiency), deficiency of acid lipase (Wolman's disease), glycogen storage disease, hypothyroidism, anemia (for example, aplastic anemia, sickle cell anemia, etc.), Pearson's syndrome, Pompe's disease, phenylketonuria (PKU), porphyrias, Maple honey urine, homocystinuria, mucopolysaccharidenosis, chronic granulomatous disease and tyrosinemia and Tay-Sachs or to treat cancer (for example, a hematologic malignancy), tumors or other pathological conditions. Placental stem cells can be used to treat skeletal dysplasia. In a embodiment, placental stem cells transformed to express tissue plasminogen activator (tPA) can be administered to an individual to treat thrombus. In other embodiments, isolated populations of placental stem cells in autologous or heterologous tissue regeneration or replacement therapies or protocols may be used, including, but not limited to, treatment of corneal epithelial defects, treatment of osteogenesis imperfecta, repair of cartilage, removal of the facial dermis, mucous membranes, tympanic membranes, intestinal lining, neurological structures (for example, retina, auditory neurons in basilar membrane, olfactory neurons in olfactory epithelium), burn repair and wound for traumatic skin damage , or for reconstruction of other damaged or diseased organs or tissues. In a preferred embodiment, an isolated population of placental stem cells is used in hematopoietic reconstitution in an individual who has suffered a partial or total loss of hematopoietic stem cells, for example, individuals exposed to lethal or sub-lethal doses of radiation ( whether industrial, medical or military); individuals who have undergone myeloablation as part of, for example, cancer therapy, and the like, in the treatment of, for example, a haematological malignancy. Placental stem cells can be used in hematopoietic reconstitution in individuals having anemia (eg, aplastic anemia, sickle cell anemia, etc.). Preferably, the placental stem cells are administer such individuals with a population of hematopoietic stem cells. Isolated populations of stem cells derived from the placenta can be used instead of, or to supplement, spinal cord or stem cell populations derived from the spinal cord. Typically, about 1 x 108 to 2 x 108 spinal cord mononuclear cells per kilogram of patient weight are infused to graft into a spinal cord transplant (i.e., about 70 ml of marrow for a 70 kg donor). To obtain 70 ml, an intensive donation and significant blood loss from the donor is required in the donation process. An isolated population of placental stem cells for haematopoietic reconstitution may comprise, in various embodiments, at least, or not more than 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more placental stem cells.

Therefore, in one embodiment, placental stem cells can be used to treat patients having blood cancer, such as a lymphoma, leukemia (such as chronic or acute myelogenous leukemia, acute lymphocytic leukemia, Hodgkin's disease, etc.). , myelodysplasia, myelodysplastic syndrome, and the like. In another embodiment, the disease, disorder or condition is chronic granulomatous disease. Since hematopoietic reconstitution can be used in the treatment of anemias, the present invention also encompasses the treatment of an individual with a combination of the invention, wherein the individual has an anemia or disorder of blood hemoglobin. The anemia or disorder can be natural (for example, caused by genetics or disease), or it can be induced artificially (for example, by accidental or deliberate poisoning, chemotherapy, and the like). In another embodiment, the disease or disorder is marrow failure syndrome (e.g., aplastic anemia, Kostmann's syndrome, Diamond-Blackfan anemia, amegacariocytic thrombocytopenia, and the like), a spinal cord disorder or a hemat disorder or disease. ? poyético. Placental stem cells can also be used to treat severe combined immunodeficiency disease, including, but not limited to, combined immunodeficiency disease (eg, Wiskott-Aldrich syndrome, severe DiGeorge syndrome, and the like). The stem cells of the placenta of the invention, alone or in combination with other populations of stem cell or progenitor cell, can be used in the manufacture of a tissue or organ in vivo. The methods of the invention encompass using cells obtained from the placenta, e.g., stem cells or progenitor cells, to seed a matrix and cultured under the appropriate conditions to allow the cells to differentiate and flush the matrix. The tissues and organs obtained by the methods of the invention can be used for a variety of purposes, including research and therapeutic purposes.

In a preferred embodiment of the invention, placental stem cells and placental stem cell populations can be used for autologous and allogeneic transplants, including matched and unmatched HLA-type hematopoietic transplants. In one embodiment of the use of placental stem cells as allogeneic hematopoietic transplants, the host is treated to reduce immunological rejection of the donor cells, or to create immunotolerance (see, for example, U.S. Patent Nos. 5,800,539 and 5,806,529). In another embodiment, the host is not treated to reduce immune rejection or to create immunotolerance. Placental stem cells, either alone or in combination with one or more other stem cell populations, can be used in therapeutic transplant protocols, for example, to augment or replace stem or progenitor cells of the liver, pancreas, kidney, lung, nervous system, muscular system, bones, spinal cord, thymus, spleen, mucous tissue, gonads or hair. Additionally, placental stem cells may be used instead of specific classes of progenitor cells (e.g., condorcytes, hepatocytes, hematopoietic cells, pancreatic basic tissue cells, neuroblasts, muscle progenitor cells, etc.) in therapeutic or research protocols. where progenitor cells would typically be used. In one embodiment, the invention provides for the use of placental stem cells, particularly placental stem cells CD200 *, as an adjunct to hair replacement therapy. For example in In one embodiment, the placental stem cells, for example, placental stem cells CD200 *, are injected subcutaneously or intradermally at a site where hair growth or regrowth is desired. The number of injected stem cells can be, for example, between about 100 and about 10, 000 per injection, in a volume of about 0.1 to about 1.0 μL, although more or less cells can also be used in a larger or smaller volume. The administration of placental stem cells to facilitate hair regrowth may comprise an individual injection or multiple injections in, for example, a regular or random pattern in an area where hair regrowth is desired. Known hair regrowth therapies can be used in conjunction with the placental stem cells, for example, topical minoxidil. Hair loss that can be treated using placental stem cells may occur naturally (eg, male pattern baldness) or induced (eg, resulting from toxic chemical exposure). Placental stem cells and placental stem cell populations can be used to augment, repair or replace cartilage, tendon or ligament. For example, in certain embodiments, prostheses (e.g., hip prostheses) may be coated with replacement cartilage tissue constructs grown from stem cells of the placenta of the invention. In other modalities, joints can be reconstructed (for example, knee) with Constructions of cartilage tissue grown from placental stem cells. Cartilage tissue constructions can also be used in major reconstructive surgery for different types of joints (see, for example, Resnick &Niwayama, eds., 1988, Diagnosis of Bone and Joint Disorders, 2nd ed., WB Saunders Co. .).

The stem cells of the placenta of the invention can be used to repair damage to tissues and organs resulting from, for example, trauma, metabolic disorders, or disease. The trauma can be, for example, trauma of surgery, for example, cosmetic surgery. In such a modality, a patient can be administered placental stem cells, alone or combined with other populations of stem or progenitor cells, to regenerate or restore tissues or organs that have been damaged as a result of a disease. . 7.2 Compositions that comprise placental stem cells The present invention provides compositions comprising placental stem cells, or biomolecules thereof. The placental stem cells of the present invention can be combined with any physiologically or medically acceptable compound, composition or device for use in, for example, research or therapeutics. . 7.2.1 Cryopreserved placental stem cells The stem cell populations of the placenta of the invention can be conserved, for example, cryopreserved for later use. Methods of cryopreservation of cells, such as stem cells, are well known in the art. Placental stem cell populations can be prepared in a form that can be easily administered to an individual. For example, the invention provides a population of placental stem cells that is contained within a container that is suitable for medical use. Such a container can be, for example, a sterile plastic bag, vial, bottle, or other container from which the population of placental stem cells can be easily dispensed. For example, the container may be a blood bag or other medically acceptable plastic bag suitable for intravenous administration of a liquid to a container. The container is preferably one that allows the cryopreservation of the population of combined stem cells. The population of cryopreserved placental stem cells may comprise placental stem cells derived from a single donor, or from multiple donors. The placental stem cell population can be completely HLA-matched to a target recipient, or partially or completely unequal to HLA. Thus, in one embodiment, the invention provides a composition comprising a population of stem cells of the placenta in a container. In a specific embodiment, the stem cell population is cryopreserved. In another specific embodiment, the container is a bag, vial or flask. In a more specific embodiment, said bag is a sterile plastic bag. In a more specific embodiment, said bag is suitable for, allows or facilitates the intravenous administration of said population of placental stem cells. The bag may comprise multiple luminous flux units or compartments that interconnect to allow mixing of the placental stem cells and one or more other solutions, eg, a drug, before, or during, the administration. In another specific embodiment, the composition comprises one or more compounds that facilitate the cryopreservation of the population of combined stem cells. In another specific embodiment, said population of placental stem cells is contained within a physiologically acceptable aqueous solution. In a more specific embodiment, said physiologically acceptable aqueous solution is a solution of 0.9% NaCl. In another specific embodiment, said population of placental stem cells comprises placental cells that are matched with HLA to a recipient of said population of stem cells. In another specific embodiment, said population of combined stem cells comprises placental cells that are at least partially HLA-mislabeled to a recipient of said population of stem cells. In another specific embodiment, said placental stem cells are derived from a plurality of donors . 7.2.2 Pharmaceutical compositions Placental stem cell populations, or populations of cells comprising placental stem cells, can be formulated into pharmaceutical compositions for use in vivo. Said pharmaceutical compositions comprise a population of placental stem cells, or a population of cells comprising placental stem cells, in a pharmaceutically acceptable carrier, for example, a saline or other physiologically acceptable solution accepted for in vivo administration. The pharmaceutical compositions of the invention may comprise any of the populations of placental stem cells, or types of placental stem cells, described herein. The pharmaceutical compositions may comprise fetal, maternal, or fetal or maternal placental stem cells. The pharmaceutical compositions of the invention may further comprise placental stem cells obtained from an individual or placenta, or from a plurality of individuals or placentas. The pharmaceutical compositions of the invention can comprise any number of placental stem cells. For example, a single unit dose of placental stem cells can comprise, in several embodiments, at least, or not more than 1 x 105, 5 x 105, 1 x 10e, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more placental stem cells. The pharmaceutical compositions of the invention comprise populations of cells comprising 50% viable cells or more (ie, at least 50% of the cells in the population are functional or living). Preferably, at least 60% of the cells in the population are viable. More preferably, at least 70%, 80%, 90%, 95% or 99% of the cells in the population in the pharmaceutical composition are viable. The pharmaceutical compositions of the invention may comprise one or more compounds that, for example, facilitate grafting (eg, anti-T-cell receptor antibodies, an immunosuppressant, or the like); stabilizers such as albumin, dextrin 40, gelatin, hydroxyethyl starch, and the like. When formulated as an injectable solution, in one embodiment, the pharmaceutical composition of the invention comprises approximately 1.25% HSA and approximately 2.5% dextrin. Other injectable formulations suitable for the administration of cellular products can be used. In one embodiment, the composition of the invention comprises placental stem cells that are substantially, or completely, non-maternal in origin. For example, the invention provides in one embodiment a composition comprising a population of placental stem cells which are CD200 * and HLA-G *; CD73 *, CD105 * and CD200 *; CD200 * and OCT-4 *; CD73 *, CD105 * and HLA-G *; CD73 * and CD105 * and facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising said population of placental stem cells when said population of placental cells is cultured under conditions that allow the formation of an embryoid type body; or OCT-4 * and facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising said population of placental stem cells when said population of placental cells is cultured under conditions that allow the formation of an embryoid type body; or a combination of the above, wherein at least 70%, 80%, 90%, 95% or 99% of said placental stem cells are non-maternal in origin. In a specific embodiment, the composition additionally comprises a stem cell that is not obtained from a placenta. . 7.2.3 Conditioned medium of the mother cell of the placenta The placental stem cells of the invention can be used to produce conditioned medium, i.e., medium comprising one or more biomolecules secreted or excreted by the stem cells. In various embodiments, the conditioned medium comprises medium in which placental stem cells have been grown by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the conditioned medium comprises medium where placental stem cells have grown at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100% confluence. Said conditioned medium can be used to support the culture of a separate population of placental stem cells, or stem cells of another class. In another embodiment, the conditioned medium comprises a medium in which the placental stem cells have been differentiated into an adult cell type. In another embodiment, the conditioned medium of the invention comprises medium in which the placental stem cells and non-placental stem cells have been cultured. . 7.2.4 Matrices that comprise placental stem cells The invention further comprises matrices, hydrogels, shells, and the like comprising a stem cell of the placenta, or a population of stem cells of the placenta. Placental stem cells of the invention can be planted in a natural matrix, for example, a placental biomaterial such as an amniotic membrane material. Said amniotic membrane material can be, for example, amniotic membrane directly dissected from a mammalian placenta; fixed or heat-treated amniotic membrane, substantially dry amniotic membrane (ie, <20% H2O), chorionic membrane, and the like. The preferred placental biomaterials where can plant placental stem cells are described in Hariri, application publication of E.U.A. No. 2004/0048796. Stem cells from the placenta of the invention can be suspended in a suitable hydrogel solution, for example, for injection. Suitable hydrogels for such compositions include self-assembly peptides, such as RAD16. In one embodiment, a hydrogel solution comprising the cells can be allowed to harden, for example in a mold, to form a matrix having cells dispersed there for implantation. The placental stem cells in said matrix can also be cultured so that the cells expand mitotically before implantation. The hydrogel, for example, is an organic polymer (natural or synthetic) that is entangled via covalent, ionic or hydrogen bonds to create a three-dimensional open network structure that traps water molecules to form a gel. Hydrogel forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are ionically crosslinked, or block polymers such as polyethylene-polypropylene glycol oxide block copolymers which are interlaced by temperature or pH, respectively. In some embodiments, the hydrogel or matrix of the invention is biodegradable. In some embodiments of the invention, the formulation comprises a polymerizable in situ gel (see, e.g., U.S. Patent Application Publication No. 2002/0022676; Anseth et al.

J. Control Reléase, 78 (1-3): 199-209 (2002); Wang et al., Biomaterials, 24 (22): 3969-80 (2003). In some embodiments, the polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, which have loaded side groups, or a monovalent ion salt thereof. Examples of polymers having acid side groups that can be reacted with cations are poly (phosphazenes), poly (acrylic acids), poly (methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate), and sulfonated polymers , such as sulfonated polystyrene. Copolymers having acidic side groups formed by the reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used. Examples of acid groups are carboxylic acid groups, sulfonic acid groups, halogenated alcohol groups (preferably fluorinated), phenolic OH groups, and acid OH groups.

The stem cells of the placenta of the invention or co-cultures thereof can be seeded in a three-dimensional structure or shell and implanted in vivo. Said structure can be implanted in combination with any one or more growth factors, cells, drugs or other components that stimulate tissue formation or otherwise enhance or improve the practice of the invention.

Examples of housings that can be used in the present invention include non-woven mats, porous foams, or self-assembly peptides. Non-woven mats can be formed using fibers comprised of a synthetic absorbable copolymer and lactic acids (eg, PGA / PLA) (VIC RYL, Ethicon, I nc., Somerville, N.J.). Foams, composed of, for example, poly (e-caprolactone) / poly (glycolic acid) (PCL / PGA) copolymer, formed by processes such as freeze drying, or lyophilization (see, for example, patent) can also be used. from E. U.A. No. 6,355,699) as carcasses. Placental stem cells of the invention can also be planted in, or contacted with, a physiologically acceptable ceramic material including, but not limited to, mono-, di-, tri-, alpha-tri, beta- phosphate. tri and tetra-calcium, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, calcium magnesium phosphates, biologically active glasses such as BIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materials currently commercially available include SU RBIGON E® (CanMedica Corp., Canada), EN DOBON® (Merck Biomaterial France, France), CEROS® (Mathys, AG, Bettlach, Switzerland), and products of mineralized collagen bone graft such as HEALOS® (Dupuy, Inc., Raynham, MA) and VITOSS®, RHAKOSS ™ and CORTOSS® (Orthovita, Malvern, Pa.). The structure can be a mixture, combination or composite of natural and / or synthetic materials. In another embodiment, placental stem cells may be planted in, or contacted with, a felt, for example, which may be composed of a multifilament yarn made of a material bioabsorbable such as copolymers or mixtures of PGA, PLA, PCL, or hyaluronic acid. The stem cells of the placenta of the invention, in another embodiment, can be sown in foam shells that can be mixed structures. Said foam shells can be molded into a useful shape, such as that of a portion of a specific structure in the body to be repaired, replaced or enlarged. In some embodiments, the structure is treated, for example, with 0.1 M acetic acid followed by incubation in polylysine, PBS, and / or collagen, before inoculation of the cells of the invention in order to enhance cellular fixation. The outer surfaces of a matrix can be modified to improve cell attachment or growth and tissue differentiation, such as by plasma coating the matrix, or adding one or more proteins (e.g., collagens, elastic fibers, reticular fibers) , glycoprotein, glycosaminoglycans (eg, heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), a cell matrix and / or other materials such as, but not limited to, gelatin, alginates, agar, agarose and plant gums, and the like. In some embodiments, the housing comprises, or is treated with, materials that make it non-thrombogenic. These treatments and materials can also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include, but are not limited to, natural materials such as base membrane proteins such as laminin and type IV collagen, synthetic materials such as EPTFE, and segmented polyurethaneurea silicones, such as PURSPAN ™ (The Polymer Technology Group, I nc., Berkeley, Calif.). The housing may also comprise anti-thrombotic agents such as heparin; carcasses can also be treated to alter the surface charge (eg, plasma coating) before seeding with placental stem cells. . 7.3 Immortalized placental stem cell lines Mammalian placental cells can be immortalized conditionally by transfection with any suitable vector containing a growth promoting gene., ie, a gene encoding a protein that, under appropriate conditions, promotes growth of the transfected cell, so that the production and / or activity of the growth promoter protein can be regulated by an external factor. In a preferred embodiment, the growth promoter gene is a shrinkage such as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T antigen, large polyoma T antigen, E 1 to adenovirus or E7 protein of human papillomavirus.

External regulation of the growth promoter protein can be achieved by placing the growth promoter gene under the control of an externally regulatable promoter, eg, a promoter from which activity can be controlled, example, by modifying the temperature of the transfected cells or the composition of the medium in contact with the cells. In one embodiment, a tetracycline-controlled gene (tet) expression system can be employed (see Gossen et al., Proc. Natl. Acad. Sci. USA 89: 5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad Sci. USA 93: 1518-1523, 1996). In the absence of tet, a tet-controlled transactivator (tTA) within this vector strongly activates transcription from phCMv * - ?, a minimal human cytomegalovirus promoter fused to tet operator sequences. tTA is a repressor fusion protein (tetR) of the tet resistance operon derived from transposon 10 of Escherichia coli and the acid domain of VP16 of herpes simplex virus. Non-toxic, low concentrations of tet (for example, 0.01-1.0 μg / mL) almost completely cancel transactivation by tTA. In one embodiment, the vector further contains a gene encoding a selectable marker, for example, a protein that confers drug resistance. The bacterial neomycin resistance gene (neo ") is such a marker that it can be used within the present invention. NeoR bearing cells can be selected by means known to those skilled in the art, such as the addition of, for example. , 100-200 μg / mL G418 to growth medium Transfection can be achieved by any variety of means known to those skilled in the art including, but not limited to, retroviral infection. it can be transfected by incubation with a mixture of conditioned medium collected from the producer cell line for the vector and DMEM / F12 containing N2 supplements. For example, a placental cell culture prepared as described above can then be infected, for example, five days in vitro by incubation for about 20 hours in a volume of conditioned medium and two volumes of DMEM / F12 containing N2 supplements. The transfected cells carrying a selectable marker can then be selected as described above. After transfection, cultures are grown on a surface that allows proliferation, for example, allowing at least 30% of the cells to be duplicated in a 24-hour period. Preferably, the substrate is a polyornithine / laminin substrate, which consists of tissue culture plastic coated with polyornithine (10 μg / mL) and / or laminin (10 μg / mL), a polylysine / laminin substrate or a treated surface with fibronectin. Then cultures are fed every 3-4 days with growth medium, which may or may not be supplemented with one or more factors that enhance proliferation. Factors that enhance proliferation can be added to the growth medium when the crops are less than 50% confluent. Conditionally immortalized placental stem cell lines, which may or may not be clonal, can usually be induced to differentiate by suppressing the production and / or activity of the low growth promoter protein. culture conditions that facilitate differentiation. For example, if the gene encoding the growth promoter protein is under the control of an externally regulatable promoter, the conditions, eg, temperature or composition of medium, can be modified to suppress transcription of the growth promoter gene. . For the tetracycline-controlled gene expression system discussed above, differentiation can be achieved by the addition of tetracycline to suppress the transcription of the growth promoter gene. In general, 1 μg / mL of tetracycline for 4-5 days is sufficient to initiate differentiation. To promote further differentiation, additional genes can be included in the growth medium. . 7.4 Tests Placental stem cells for the present invention can be used in assays to determine the influence of culture conditions, environmental factors, molecules (e.g., biomolecules, small inorganic molecules, etc.) and the like in proliferation, expansion and / or stem cell differentiation, compared to placental stem cells not exposed to these conditions. In a preferred embodiment, the placental stem cells of the present invention are tested for changes in proliferation, expansion or differentiation upon contacting a molecule. In one embodiment, for example, the invention provides a method of identifying a compound that modulates the proliferation of a plurality of placental stem cells, which comprises contacting said plurality of stem cells with said compound under conditions that allow proliferation, wherein said compound causes a detectable change in proliferation of said plurality of stem cells in comparison with a plurality of stem cells not contacted with said compound, said compound is identified as a compound that modulates the proliferation of placental stem cells. In a specific embodiment, said compound is identified as a proliferation inhibitor. In another specific embodiment, said compound is identified as a proliferation enhancer. In another embodiment, the invention provides a method of identifying a compound that modulates the expansion of a plurality of placental stem cells, which comprises contacting said plurality of stem cells with said compound under conditions that allow expansion, wherein said compound causes a detectable change in expansion of said plurality of stem cells compared to a plurality of stem cells not contacted with said compound, said compound is identified as a compound that modulates the expansion of placental stem cells. In a specific embodiment, said compound is identified as an expansion inhibitor. In another specific embodiment, said compound is identified as an expansion enhancer.

In another embodiment, the invention provides a method of identifying a compound that modulates the differentiation of a mother cell from the placenta, which comprises contacting said stem cells with said compound under conditions that allow differentiation, wherein said compound causes a detectable change in differentiation of said stem cells compared to a mother cell not contacted with said compound, said compound is identified as a compound that modulates the proliferation of placental stem cells. In a specific embodiment, said compound is identified as a differentiation inhibitor. In another specific embodiment, said compound is identified as a differentiation inhibitor. In another specific embodiment, said compound is identified as a differentiation enhancer. 6. EXAM PLOS 6. 1 EX EMPLOYMENT 1: CULTIVATION OF STEM CELLS OF PLACENTA Stem cells are obtained from the placenta of a post-partum mammary placenta either by perfusion or by physical disruption, eg, enzymatic digestion. The cells are cultured in a culture medium comprising 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma), 2% fetal bovine serum (FCS) (Huclone Laboratories), 1 x insulin-transferrin -selenium (ITS), 1 x acid linolenic-bovine serum albumin (LA-BSA), 10"9 M ascorbic acid 2-phosphate (Sigma), epidermal growth factor (EGF) 10ng / ml (R & D Systems), platelet-derived growth factor ( PDGF-BB) 10ng / ml (R & D Systems), and 100U of penicillin / 1000U of streptomycin The culture flask in which the cells are grown is prepared as follows: Flasks T75 are coated with fibronectin (FN), by adding 5 ml of PBS containing 5ng / ml of human FN (Sigma F0895) to the bottle.The bottles with FN solution are left at 37 ° C for 30 minutes.The FN solution is then removed before cell culture. The bottles need to be dried after treatment, alternatively, the bottles are left in contact with the FN solution at 4 ° C overnight or longer, before the culture, the bottles are heated and the FN solution is eliminated.

Placental stem cells isolated by perfusion Placental perfusate placental stem cells are established in the following manner. Ficoll gradient cells are seeded in T75 flasks coated with FN, prepared as above, at 50-100 × 10 6 cells / vial in 15 ml of culture medium. Typically, 5 to 10 bottles are sown. The flasks are incubated at 37 ° C for 12-18 hours to allow attachment of adherent cells. 10 ml of warm PBS is added to each bottle to remove suspended cells, and mixed gently. 15 mL of the medium is then removed and replaced with ml of fresh culture medium. The entire medium is changed 3-4 days after the start of cultivation. Subsequent culture medium changes are made, during which 50% of 7.5 ml of the medium is removed. Beginning approximately on day 12, the culture is reviewed under a microscope to examine the growth of adherent cell colonies. When cell cultures become approximately 80% confluent, typically between day 13 to day 18 after starting the culture, adherent cells are harvested by trypsin digestion. The cells harvested from these primary cultures are designated 0 (zero) passage.

Isolated cells of the placenta isolated by physical disruption and enzymatic digestion Placental stem cell cultures of digested placenta tissue are established in the following manner. The perfusion placenta is placed on a sterile sheet of paper with the maternal side up. Approximately 0.5 cm of the surface layer on the maternal side of the placenta is scraped with a blade, and the blade is used to remove a block of tissue from the placenta by measuring approximately 1 x 2 x 1 cm. This placental tissue is then chopped into pieces of approximately 1 mm3. These pieces are collected in a 50 ml Falcon tube and digested with collagenase IA (2mg / ml, Sigma) for 30 minutes, followed by trypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at 37 ° C in bath of water. The The resulting solution is centrifuged at 400g for 10 minutes at room temperature. The tissue / cell agglomerate is resuspended in 130 mL of culture medium, and the cells are seeded at 13 ml per T75 flask coated with fibronectin. Cells are incubated at 37 ° C with a humidified atmosphere with 5% CO2. Optionally, placental stem cells are cryopreserved in this stage.

Subculture and expansion of placental stem cells Frozen cells are rapidly thawed in a 37 ° C water bath. Immediately, placental cells are removed from the cryoprobe with 10m of warm medium and transferred to a sterile 15ml tube. The cells are centrifuged at 400g for 10 minutes at room temperature. The cells are gently resuspended in 10m of warm culture medium when pipetting, and viable cell counts are determined by exclusion of Trypan blue. Cells are then seeded at approximately 6000-7000 cells per cm2 in flasks coated with FN, prepared as before (approximately 5x105 cells per flask T-75). The cells are incubated at 37 ° C, 5% CO2 and 90% humidity. When the cells reach 75-85% confluence, all spent media are aseptically removed from the bottles and discarded. 3ml of 0.25% trypsin / EDTA (w / v) solution is added to cover the cell layer, and the cells are incubated at 37 ° C, 5% CO2 and 90% humidity for 5 minutes. The bottle is capped once or twice to expedite cell detachment. Once > 95% of the cells are round and detached, 7ml of warm culture medium is added to each T-75 bottle, and the solution is dispersed by pipetting on the cell layer surface several times. After counting the cells and determining viability as before, the cells are centrifuged at 1000 RPM for 5 minutes at room temperature. The cells are passed by gently resuspending the cell pellet from a T-75 flask with culture medium, and evenly stapling the cells in two T-75 flasks coated with FN. Using the above methods, exemplary populations of adherent placental stem cells expressing CD105, CD33, CD73, CD29, CD44, CD10 and CD90 markers are identified. These cell populations typically do not express CD34, CD45, CD117 or CD133. Some, but not all, cultures of these placental stem cells expressed HLA-ABC and / or HLA-DR. 6. 2 EXAMPLE 2: INSULATION OF STEM CELLS OF THE PLACENTA OF PLACENTA STRUCTURES 6. 2.1 Materials & methods 6. 2.1.1 Isolation of placental cell populations comprising placental stem cells Different populations of placental cells were obtained from the placentas of normal full-term pregnancies. All donors provided written consent for the use of their placentas for research purposes. Placental stem cells were obtained from the following sources: (1) placental perfusate (perfusion of the placental vasculature); and enzymatic digestions of (2) amnios, (3) chorion, (4) amnion-chorion plaque, and (5) umbilical cord. The various placental tissues were cleaned in sterile PBS (Gibco-Invitrogen Corporation, Carlsbad, CA) and placed in separate sterile Petri dishes. The various tissues were stitched using a surgical scalpel and placed in 50 mL Falcon conical tubes. The chopped tissues were digested with 1X collagenase (Sigma-Aldrich, St. Louis, MO) for 20 minutes in a water bath at 37 ° C, centrifuged, and then digested with 0.25% trypsin-EDTA (Gibco-Invitrogen Corp) for 10 minutes in a water bath at 37 ° C. The various tissues were centrifuged after digestion and rinsed once with sterile PBS (Gibco-Invitrogen Corp.). The reconstituted cells were then filtered twice, once with 100 μm cell strainers and once with 30 μm separation filters, to remove any residual extracellular matrix or cell debris. 6. 2.1.6 Evaluation of cell viability and cell counts The manual exclusion method of trypan blue was used after digestion to calculate cell counts and evaluate cell viability. Cells were mixed with blue trypan dye (Sigma-Aldrich) at a ratio of 1: 1, and the cells were read in hemacytometer. 6. 2.1.3 Characterization of cell surface marker Cells that were HLA ABC 7CD457CD34"/ CD 133 * were selected for characterization, cells were identified, quantified and characterized by two flow cytometers Becton-Dickinson, FACSCalibur and FACS Aria (Becton-Dickinson, San José, CA , USA) The various placental cells were stained, at a ratio of approximately 10 μL of antibody per 1 million cells, for 30 minutes at room temperature on a shaker The following anti-human antibodies were used: conjugated monoclonal antibodies fluorescein isothiocyanate (FITC) against HLA-G (Serotec, Raleigh, NC), CD 10 (BD Immunocytometry Systems, San Jose, CA), CD44 (BD Biosciences Pharmigen, San Jose, CA) and CD 105 (R &D Systems I nc., Minneapolis, MN), monoclonal antibodies conjugated with phycoerythrin (PE) against CD44, CD200, CD117 and CD13 (BD Biosciences Pharmingen); monoclonal and streptavidin antibodies conjugated with phycoerythrin-Cy5 (PE Cy5) against CD117 (BD Biosciences Pharmingen); monoclonal antibodies conjugated with picoerythrin-Cy7 (PE Cy7) against CD33 and CD10 (BD Biosciences); monoclonal and streptavidin antibodies conjugated to allophicocyanin (APC) against CD38 (BD Biosciences Pharmingen); and biotinylated CD90 (BD Biosciences Pharmingen). After incubation, the cells were rinsed once to remove unbound antibodies and fixed overnight with 4% paraformaldehyde (USB, Cleveland, OH) at 4 ° C. The next day, the cells were rinsed twice, filtered through a 30 μm separation filter, and tested on the flow cytometer (s). Samples that were stained with anti-mouse IgG antibodies (BD Biosciences Pharmingen) were used as negative controls and were used to adjust Photo Multiplier photo tubes (PMTs). Samples that were stained with anti-human antibodies were used as positive controls and were used to adjust overlaps / spectral offsets. 6. 2.1.4 Classification and cell culture A series of placental cells (from perfusate, amnion or chorion), before any culture, was stained with 7-amino-actinomycin D (7AAD, BD Biosciences Pharmingen) and antibodies monoclonal antibodies specific to the phenotype of interest. The cells were stained at a ratio of 10 μL of antibody per 1 million cells, and incubated for 30 minutes at room temperature on a shaker. These cells were then positively classified for living cells expressing the phenotype of interest in the BD FACS Aria and plated in culture. Placental cell populations classified (population of interest) and "all" (unclassified) were plated for comparison. The cells were plated in a 96-well plate coated with fibronectin (Sigma-Aldrich) at the cell densities listed in Table 1 (cells / cm2). The cell density, and if the cell type was plated in duplicate or triplicate, was determined and governed by the number of cells expressing the phenotype of interest.

Table 1: Cell plating densities Complete medium (60% of DMEM-LG (Gibco) and 40% of MCDB-201 (Sigma), 2% of fetal bovine serum (Hyclone Labs.), 1x insulin-transferrin-selenium (ITS), 1x linoleic acid-albumin of bovine serum (LA-BSA), 10"9 M of dexamethasone (Sigma), 10" 4 M of ascorbic acid 2-phosphate (Sigma), epidermal growth factor 10 ng / mL (R & D Systems); of platelet-derived growth (PDGF-BB) 10 ng / mL (R &D Systems)) was added to each well of the 96-well plate and the plate was placed in a 5% CO2 / 37 ° C incubator. On day 7, 100 μL of complete medium was added to each of the wells. The 96-well plate was monitored for approximately two weeks and a final evaluation of the culture was completed on day 12. This is very early in the culture of placental stem cells, and represents 0 passageway cells. 6. 2.1.5 Data analysis FACSCalibur data was analyzed in FlowJo (Tree star, Inc.) using standard periodic activation techniques. BD data FACS Aria were analyzed using the FACSDiva software (Becton- Dickinson). The FACS Aria data were analyzed using periodic activation of pair discrimination to minimize the pairs, as well as standard periodic activation techniques. All the results were compiled in Microsoft Excel and all the values, in the present, are represented as mean ± standard deviation (number, standard average error). 6. 2.2 Results 6. 2.2.1 Cell viability Post-digestion viability was evaluated using the manual trypan blue exclusion method (Figure 1). The average cell viability obtained from most of the digested tissue (from amnion, chorion or amnion-chorion plaque) was around 70%. The amnion had an average viability of 74.35% ± 10.31% (n = 6, SEM = 4.21), corion had an average viability of 78.18% ± 12.65% (n = 4, SEM = 6.32), plate of amnion-corion had a average viability of 69.05% ± 10.80% (n = 4, SEM = 5.40), and the umbilical cord had an average viability of 63.30% ± 20.13% (n = 4, SEM = 10.06). The perfusion cells, which did not undergo digestion, retained the highest average viability, 89.98% ± 6.39% (n = 5, SEM = 2.86). 6. 2.2.2 Cellular quantification The populations of placental cells and umbilical cord cells were analyzed to determine the numbers of HLA ABC 7CD457CD347CD133 * cells. From the data analysis of BD FACSCalibur, it was observed that the amnion, perfusate and chorion contained the highest total number of these cells, 30.72 ± 21.80 cells (n = 4, SEM = 10.90), 26.92 ± 22.56 cells ( n = 3, sem = 1 3.02), and 1 8.39 ± 6.44 cells (n = 2, SEM = 4.55) respectively (data not shown). The amnion-chorion plate and umbilical cord contained the lowest total number of cells expressing the phenotype of interest, 4.72 ± 4.16 cells (n = 3, SEM = 2.40) and 3.94 ± 2.58 cells (n = 3, SEM = 1.49). ) respectively (data not shown). In a similar way, when the percentage of total cells expressing the phenotype of interest was analyzed, it was observed that amnios and placental perfusate contained the highest percentages of cells expressing this phenotype (0.0319% ± 0.0202% (n = 4, SEM = 0.0101) and 0.0269% ± 0.0226% (n = 3, SEM = 0.0130) respectively (Figure 2) Although the umbilical cord contained a small number of cells expressing the phenotype of interest (figure 2), it contained the third highest percentage of cells expressing the phenotype of interest, 0.020 ± 0.0226% (n = 3, SEM = 0.0131) (Figure 2) Corium and plaque of amnion-chorion contained the lowest percentages of cells expressing the phenotype of interest, 0.01 84 ± 0.0064% (n = 2, SEM = 0.0046) and 0.0177 ± 0.0173% (n = 3, SEM = 0.010) respectively (figure 2). Consistent with the results of BD FACSCalibur analysis, data from BD FACS Aria also identified amnion, perfusate and chorion as providing larger numbers of HLA ABC cells "/ CD457CD347CD133 * than the remaining sources.The total average number of cells expressing the phenotype of interest between amnion, perfusate and chorion was 126.47 ± 55.61 cells (n = 15, SEM = 14.36), 81.65 ± 34.64 cells (n = 20, SEM = 7.75), and 51.47 ± 32.41 cells (n = 15, SEM = 8.37) , respectively (data not shown) The amnion-chorion plate and umbilical cord contained the lowest total number of cells expressing the phenotype of interest, 44.89 ± 37.43 cells (n = 9, SEM = 12.48) and 11.00 ± 4.03 cells (n = 9, SEM = 1.34) respectively (data not shown) Data from BD FACS Aria revealed that the perfusate and amnion produced the highest percentages of HLA cells ABC7CD45"/ CD347CD133 *, 0.1523 ± 0.0227% (n = 15, SEM = 0.0059) and 0.0929 ± 0.0419% (n = 20, SEM = 0.0094) respectively (figure 3). The amnion-chorion plate contained the third highest percentage of cells expressing the phenotype of interest, 0.0632 ± 0.0333% (n = 9, SEM = 0.0111) (figure 3). The chorion and umbilical cord contained the lowest percentages of cells expressing the phenotype of interest, 0.0623 ± 0.0249% (n = 15, SEM = 0.0064) and 0.0457 ± 0.0055% (n = 9, SEM = 0.0018) respectively (figure 3) . After the HLA ABC 7CD457CD347CD133 * cells were identified and quantified from each cell source, their cells were further analyzed and characterized by their expression of cell surface markers HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 and CD105. 6. 2.2.2 Cells derived from placental perfusate The perfusate-derived cells were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105 and CD13 (FIG. 4). The average expression of each marker for perfusate-derived cells was as follows: 37.15% ± 38.55% (n = 4, SEM = 19.28) of the cells expressed HLA-G; 36.37% ± 21.98% (n = 7, SEM = 8.31) of the cells expressed CD33; 39.39% ± 39.91% (n = 4, SEM = 19.96) of the cells expressed CD117; 54.97% ± 33.08% (n = 4, SEM = 16.54) of the cells expressed CD10; 36.79% ± 11.42% (n = 4, SEM = 5.71) of the cells expressed CD44; 41.83% ± 19.42% (n = 3, SEM = 11.21) of the cells expressed CD200; 74.25% ± 26.74% (n = 3, SEM = 15.44) of the cells expressed CD90; 35.10% ± 23.10% (n = 3, SEM = 13.34) of the cells expressed CD38; 22.87% ± 6.87% (n = 3, SEM = 3.97) of the cells expressed CD105; and 25.49% ± 9.84% (n = 3, SEM = 5.68) of the cells expressed CD13. 6. 2.2.3 Cells derived from amnion The cells derived from amnios were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105 and CD13 (figure 5). The average expression of each marker for amnion derivatives was as follows: 57.27% ± 41.11% (n = 3, SEM = 23.73) of the cells expressed HLA-G; 16.23% ± 15.81% (n = 6, SEM = 6.46) of the cells expressed CD33; 62.32% ± 37.89% (n = 3, SEM = 21.87) of the cells expressed CD117; 9.71% ± 13.73% (n = 3, SEM = 7.92) of the cells expressed CD10; 27.03% ± 22.65% (n = 3, SEM = 13.08) of the cells expressed CD44; 6.42% ± 0.88% (n = 2, SEM = 0.62) of the cells expressed CD200; 57.61% ± 22.10% (n = 2, SEM = 15.63) of the cells expressed CD90; 63.76% ± 4.40% (n = 2, SEM = 3.11) of the cells expressed CD38; 20.27% ± 5.88% (n = 2, SEM = 4.16) of the cells expressed CD105; and 54.37% ± 13.29% (n = 2, SEM = 9.40) of the cells expressed CD13. 6. 2.2.4 Cells derived from chorion Corium-derived cells were consistently positive for HLA-G, CD117, CD10, CD44, CD200, CD90, CD38 and CD13, while the expression of CD33 and CD105 varied (Figure 6). The average expression of each marker for chorion derivatives was as follows: 53.25% ± 32.87% (n = 3, SEM = 18.98) of the cells expressed HLA-G; 15.44% ± 11.17% (n = 6, SEM = 4.56) of the cells expressed CD33; 70.76% ± 11.87% (n = 3, SEM = 6.86) of the cells expressed CD117; 35.84% ± 25.96% (n = 3, SEM = 14.99) of the cells expressed CD10; 28.76% ± 6.09% (n = 3, SEM = 3.52) of the cells expressed CD44; 29.20% ± 9.47% (n = 2, SEM = 6.70) of the cells expressed CD200; 54.88% ± 0.17% (n = 2, SEM = 0.12) of the cells expressed CD90; 68.63% ± 44.37% (n = 2, SEM = 31.37) of the cells expressed CD38; 23.81% ± 33.67% (n = 2, SEM = 23.81) of the cells expressed CD105; and 53.16% ± 62.70% (n = 2, SEM = 44.34) of the cells expressed CD13. 6. 2.2.5 Cells derived from amnio-chorion plate The amnion-chorion plate cells were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105 and CD13 (FIG. 75). The average expression of each marker for cells derived from amnion-chorion plate was as follows: 78.52% ± 13.13% (n = 2, SEM = 9.29) of the cells expressed HLA-G; 38.33% ± 15.74% (n = 5, SEM = 7.04) of the cells expressed CD33; 69.56% ± 26.41% (n = 2, SEM = 18.67) of the cells expressed CD117; 42.44% ± 53.12% (n = 2, SEM = 37.56) of the cells expressed CD10; 32.47% ± 31.78% (n = 2, SEM = 22.47) of the cells expressed CD44; 5.56% (n = 1) of the cells expressed CD200; 83.33% (n = 1) of the cells expressed CD90; 83.52% (n = 1) of the cells expressed CD38; 7.25% (n = 1) of the cells expressed CD105; and 81.16% (n = 1) of the cells expressed CD13. 6. 2.2.6 Cells derived from umbilical cord Cells derived from umbilical cord were consistently positive for HLA-G, CD33, CD90, CD38, CD105 and CD13, while the expression of CD117, CD10, CD44 and CD200 varied (figure 8). The average expression of each marker for cells derived from umbilical cord was as follows: 62.50% ± 53.03% (n = 2, SEM = 37.50) of the cells expressed HLA-G; 25.67% ± 11.28% (n = 5, SEM = 5.04) of the cells expressed CD33; 44.45% ± 62.85% (n = 2, SEM = 44.45) of the cells expressed CD117; 8.33% ± 11.79% (n = 2, SEM = 8.33) of the cells expressed CD10; 21.43% ± 30.30% (n = 2, SEM = 21.43) of the cells expressed CD44; 0.0% (n = 1) of the cells expressed CD200; 81.25% (n = 1) of the cells expressed CD90; 64.29% (n = 1) of the cells expressed CD38; 6.25% (n = 1) of the cells expressed CD105; and 50.0% (n = 1) of the cells expressed CD13. A summary of all the marker expression averages is shown in Figure 9. 6. 2.2.7 BD FACS classification report Aria The three distinct populations of placental cells that expressed the highest percentages of HLA ABC, CD45, CD34 and CD133 (cells derived from perfusate, amnion and chorion) were stained with 7AAD and the antibodies for these markers. The three populations were positively classified for living cells expressing the phenotype of interest. The results of the BD FACS Aria classification are listed in table 2.

Table 2: The three different populations of positively classified ("classified") cells and their corresponding unclassified cells were plated and the culture results were evaluated on day 12 (Table 3). The perfusate-derived cells sorted, plated at a cell density of 40,600 / cm 2, resulted in small, round, non-adherent cells. Two of the three series of unclassified perfusate-derived cells, each plated at a cell density of 40,600 / cm 2, resulted in the majority of small, round, nonadherent cells with several adherent cells located around the periphery of the well. Unperforated perfusate-derived cells, plated at a cell density of 93,800 / cm2, resulted mostly in small, round, nonadherent cells with several adherent cells around the peripheries of the well. Cells derived from classified amnions, plated at a density of 6,300 / cm2, resulted in small, round, non-adherent cells. Cells derived from unclassified amnions, plated at a density of 6,300 / cm2, resulted in small, round, non-adherent cells. Cells derived from unclassified amnion plated at a density of 62, 500 cm / 2 resulted in small, round, non-adherent cells. Chorion derived cells classified, plated at a density of 6,300 / cm2, resulted in small, round, non-adherent cells. Uncharted corium derived cells, plated at a density of 6,300 / cm2, resulted in small, round, nonadherent cells. The unclassified chorion-derived cells plated at a density of 62,500 cm / 2 resulted in small, round, non-adherent cells. Following the performance of the above-mentioned experiments, and additional culture of the placental stem cells, it was determined that the labeling of the antibodies for CD117 and CD133, where an antibody conjugated with streptavidin was marked with phycoerythrin conjugated with biotin (PE) , produced significant enough background to seem a positive reading. This background had initially resulted in the placental stem cells being considered as positive for both markers. When a different brand, APC or PerCP, was used, the background was reduced, and the placental stem cells were correctly determined as negative for CD117 and CD133. 6. 3 EXAMPLE 3: CHARACTERIZATION OF PLACENTA STEM CELLS AND UMBILICAL CORD STEM CELLS This example demonstrates an exemplary cell surface marker profile of placental stem cells. Placental stem cells and umbilical cord stem cells, obtained by enzymatic digestion, in culture medium were washed once by adding 2 mL of 2% FBS-PBS and centrifuging at 400g for 5 minutes. The supernatant was decanted, and the agglomerate was resuspended in 100-200 μL of 2% FBS-PBS. Four tubes were prepared with BD ™ CompBeads (Cat # 552843) by adding 100 μl of 2% FBS-PBS to each tube, adding 1 full drop (approximately 60 μl) of BD ™ CompBeads Negative Control and 1 drop of BD ™ beads CompBeads Anti-Mouse to each tube, and make swirl. To the 4 tubes of BD ™ CompBeads, the following antibodies were added: Control tubes were prepared as follows: The following antibodies were added to the sample tubes; The control and sample tubes were incubated in the dark at room temperature for 30 minutes. After incubation, the tubes were washed by adding 2mL of 2% FBS-PBS and centrifuged at 400g for 5 minutes. The supernatant was decanted, and the agglomerate was resuspended in 100-200 μl of 2% FBS-PBS and acquired in a flow cytometer. All other antibodies were used after this procedure. Placental stem cells matched with membrane Amniotic and umbilical cord stem cells were analyzed using fluorescently labeled antibodies and flow cytometry to identify surface markers that were present or absent. The markers analyzed included CD105 (specific endothelial marker related to proliferation); CD200 (marker associated with regulatory function); CD34 (expressed in endothelial cells and in hematopoietic stem cells); CD10 (marker of stem cell / precursor cell); CD45 (lineage marker); CD133 (marker for hematopoietic progenitor cells); CD117 (stem cell factor (c-Kit)); CD90 (expressed in primitive hematopoietic stem cells in normal spinal cord, umbilical cord and fetal liver cells); HLA ABC (bread MHC I, antigen presentation, immunogenicity); β-2-microglobulin (associates with MHC I, antigen presentation, immunogenicity); HLA DR, DQ, DP (bread MHC II, antigen presentation, immunogenicity); and CD80 / 86 (co-stimulatory molecules for antigen presentation). The results of the flow cytometry showed that for the placenta stem cells that were tested, 93.83% of the cells were CD105 *, 90.76% of the cells were CD200 *, and 86.93% of the cells were CD105 * and CD200 * . 99.97% of the cells were CD10 *, 99.15% of the cells were CD34", and 99.13% of the cells were CD10 * and CD34". 98.71% of the cells were cytokeratin positive, 99.95% of the cells were CD44 *, and 98.71% of the cells were positive for cytokeratin and CD44. 99. 51% of the cells were CD45, and 99.78% of the cells were negative for CD133, and 99.39% of the cells were negative for CD45 and CD133. 99.31% of the cells were CD90 positive, 99.7% were negative for CD117, and 99.01% were positive for CD90 and negative for CD117.95.7% of the cells were negative for CD80 and CD86. The results of flow cytometry for umbilical cord stem cells showed that 95.95% of the cells were CD200 *, 94.71% were CD105 * and 92.69% were CD105 * and CD200 * 99.93% of the cells were CD10 *, 99.99% of the cells were CD34, and 99.6% of the cells were CD10 * and CD34". 99.45% of the cells were cytokeratin positive, 99.78% of the cells were CD44 * and 99.3% of the cells were positive for cytokeratin and CD44. 99.33% of the cells were CD45 ', 99.74% were CD133"and 99.15% of the cells were CD45" and CD133'. 99.84% of the cells were CD117", 98.78% of the cells were CD90 *, and 98.64% of the cells were CD90 * and CD117 * .A phenotype (CD200 *. CD105 *, CD10 *, CD34) seems to be consistent over numerous such analyzes.This phenotype is additionally positive for CD90, CD44, HLA ABC (weak), β-2-microglobulin (weak) and cytokeratin K, and negative for HLA DR.DQ.DP, CD117, CD133 and CD45. 6. 4 EXAMPLE 4: DETERMINATION OF ACTIVITY OF ALDEHYDE DEHYDROGENASE IN CELLS MOTHER OF PLACENTA The activity level of aldehyde dehydrogenase (ALDH), a potential marker of stem cell graft capacity, was determined using an ALDEFLUOR® Assay Kit from Stem Cell Technologies, Inc. Typically, more primitive, undifferentiated stem cells demonstrate less activity of ALDH than more differentiated cells. The assay uses ALDEFLUOR®, a fluorescent ALDH substrate (Aldagen, Inc., Durham, North Calorina). The manufacturer's protocol was followed. The dry ALDEFLUOR® reagent was provided in a stable, inactive form. ALDEFLUOR® was activated by dissolving the dry compound in dimethylsulfoxide (DMSO) and adding 2N of HCl, and was immediately added to the cells. A control tube was also established by combining the cells with ALDEFLUOR® plus DEAB, a specific inhibitor of ALDH. The cells analyzed included four umbilical cord stem cell lines and three lines of amnion-chorion plate placental stem cells, a line of mesentery stem cells derived from the spinal cord (BM-MSC), a cell line adipose derived stem (ADSC), a human villous trophoblast cell line (HVT), and umbilical cord CD34 * stem cells. The trial proceeded as follows. The concentration of the sample at 1X106 cells / ml with assay buffer provided with the ALDEFLUOR® Assay Kit. 1 mL of cell suspension adjusted in experimental and control tube for each of the tested cell lines, and 5 μl of DEAB was added in addition to the control tube marked as control. ALDEFLUOR® substrate was activated by adding 25 μl DMSO to the dry ALDEFLUOR® reagent, and allowing it to stand for 1 minute at room temperature. 25 μl of 2N HCL was added and mixed well. This mixture was incubated for 15 minutes at room temperature. 360 μl of ALDEFLUOR® assay regulator was added to the flask and mixed. The resulting mixture was stored at 2-8 ° C during use. 5 μl of the activated ALDEFLUOR® reagent per 1 milliliter of sample was added to the experimental tubes, and 0.5 ml of this mixture was immediately transferred into the control tubes. The experimental and control tubes for each cell line were incubated for 30 minutes at 37 ° C. After incubation, the tubes were centrifuged at 400 x g, and the supernatant was discarded. The cells in the resulting pellet were resuspended in 0.5 ml of assay buffer and analyzed by flow cytometry. The data was analyzed using FLOWJO ™ software (Tree Star, Ashland, Oregon). Charts of SSC vs FSC and SSC vs FL1 were created in the FLOWJO ™ workspace. Control and experimental data files were opened for each sample, and appropriate grids were determined based on control samples. They were calculated positive cells as a percentage of ALDEFLUOR® positive of the total number of events counted. The stem cell lines of the placenta demonstrated ALDH activity from about 3% to about 25% (3.53%, 8.76%, and 25.26%). The umbilical cord stem cell lines demonstrated ALDH activity from about 16% to about 20% (16.59%, 17.01%, 18.44% and 19.83%). In contrast, BM-MSC and HVT were negative and 1.5% respectively for ALDH, but the adipose-derived MSC is close to 30% of ALDH *. CD34 * cells from positive control of umbilical cord blood were, as expected, highly positive (75%) for ALDH. 6. 5 EXAMPLE 5: COLLECTION OF STEM CELLS OF THE PLACENTA BY PERFUSION OF CLOSED CIRCUIT This example demonstrates a method of collecting placental stem cells by perfusion. A placenta after delivery is obtained within 24 hours after delivery. The umbilical cord is held with an umbilical cord clamp about 3 to 4 inches above the placental disc, and the cord is cut above the forceps. The umbilical cord is discarded, or processed to retrieve, for example, stem cells from the umbilical cord and / or to process the umbilical cord membrane for the production of a biomaterial. The excess of Amniotic membrane and chorion is cut from the placenta, leaving approximately 1/4 inch around the edge of the placenta. The cut material is discarded. Starting from the edge of the placental membrane, the amniotic membrane separates from the chorion using a short needle dissection with the fingers. When the amniotic membrane is completely separated from the chorion, the amniotic membrane is cut around the base of the umbilical cord with scissors, and detached from the disc of the placenta. The amniotic membrane can be discarded, or processed, for example, to obtain stem cells by enzymatic digestion, or to produce, for example, an amniotic membrane biomaterial. The fetal side of the remaining placenta material is cleaned of all visible blood clots and residual blood using sterile gauze, and then sterilized by wiping with an iodide cotton with a cotton ball of alcohol. The umbilical cord is then held crosswise with a sterile hemostat below the umbilical cord clamp, and the hemostat is rotated, pulling the cord over the forceps to create a fold. The cord is then partially cut under the hemostat to expose a cross section of the cord supported by the clip. Alternatively, the cord is fastened with a sterile hemostat. The cord is then placed in sterile gauze and held with the hemostat to provide tension. The cord is then cut straight down directly under the hemostat, and the edge of the cord near the vessel is reattached.

The vessels exposed as described above, usually a vein and two arteries, are identified and opened in the following manner. A closed tooth clip is advanced through the cut end of each beaker, being careful not to puncture the clamp through the vessel wall. The insertion stops when the tip of the clamp is slightly above the base of the umbilical cord. The clamp then opens slightly, and is removed slowly from the vessel to dilate the vessel. Plastic tube, connected to an infusion device or peristaltic pump, is inserted into each of the arteries of the placenta. The plastic tube, connected to a 250 mL collection bag, is inserted into the vein of the placenta. The tube is stuck in place. A small volume of sterile injection grade 0.9% of NaCl solution verifies leaks. If there are no leaks, the speed of the pump increases, and approximately 750 mL of the 0.9% grade injection of NaCl solution is pumped through the vasculature of the placenta. The perfusion can be helped by gently massaging the placental disc from the outer edges of the cord. When a collection bag is full, the bag is removed from the coupler that connects the tube to the bag, and a new bag is connected to the tube. When harvesting is complete, the collection bags are weighed and balanced for centrifugation. After centrifugation, each bag is placed inside an extractor plasma without disturbing the agglomerate of cells. The supernatant within the bags is then removed and discarded. The bag is then gently massaged to resuspend the cells in the remaining supernatant. Using a sterile 1mL syringe, approximately 300-500 μL of cells are removed from the collection bag, via a sample site coupler, and transferred to a 1.5 mL centrifuge tube. The weight and volume of the remaining perfusate are determined, and 1/3 volume of hetastarch is added to the perfusate and mixed thoroughly. The number of cells per mL is determined. Red blood cells are removed from the perfusate using a plasma extractor. Placental cells are then cultured immediately to isolate placental stem cells, or cryopreserved for later use. 6. 6 EXAMPLE 6: DIFFERENTIATION OF STEM CELLS OF THE PLACENTA 6. 6.1 Induction of differentiation in neurons Neuronal differentiation of placental stem cells can also be achieved in the following way: 1. Placental stem cells are grown for 24 hours in preinduction medium consisting of DMEM / 20% FBS and 1 mM beta-mercaptoethanol. 2. The preinduction medium is removed and the cells are washed with PBS. 3. Neuronal induction medium consisting of DMEM and 1-10 mM of betamercaptoethanol is added to the cells. Alternatively, induction media consisting of DMEM / 2% DMSO / 200 μM butylated hydroxyanisole can be used. 4. In certain embodiments, morphological and molecular changes may occur as early as 60 minutes after exposure to serum-free medium and betamercaptoethanol. RT / PCR can be used to evaluate the expression of, for example, nerve growth factor receptor and neurofilament heavy chain genes. 6. 6.2 Induction of differentiation in adipocytes Several cultures of placental stem cells derived from enzymatic digestion of amnios, at 50-70% confluence, were induced in medium comprising (1) DMEM / MCDB-201 with 2% FCS, 0.5% hydrocortisone, 0.5 mM isobutylmethylxanthine ( IBMX), 60 μm indomethacin; or (2) DMEM / MCDB-201 with 2% FCS and 0.5% linoleic acid. Cells were examined for morphological changes; after 3-7 days, oil drops appeared. Quantitative real-time PCR differentiation was also evaluated to examine the expression of specific genes associated with adipogenesis, ie, PPAR-? 2, aP-2, lipoprotein lipase, and osteopontin. Two cultures of placental stem cells showed an increase of 6.5 times and 24.3 fold in the expression of adipocyte-specific genes, respectively. Four other cultures showed a moderate increase (1.5-2.0 fold) in the expression of PPAR-? 2 after the induction of adipogenesis. In another experiment, placental stem cells obtained from perfusate were cultured in DMEM / MCDB-201 (basal chicken fibroblast medium) with 2% FCS. The cells were trypsinized and centrifuged. The cells were resuspended in adipo induction medium (AIM) 1 or 2. AIM1 comprised MesenCult basal medium for human mesentery stem cells (StemCell Technologies) supplemented with adipogenic mesentery stem cell supplements (StemCell Technologies). AIM2 comprised DMEM / MCDB-201 with 2% FCS and LA-BSA (1%). Approximately 1.25 x 105 of placental stem cells grew in 5 mL of AIM1 or AIM2 in T-25 bottles. The cells were cultured in incubators for 7-21 days. The cells developed oil drop vacuoles in the cytoplasm, as confirmed by red oil staining, suggesting the differentiation of the stem cells into adipocytes. Adipogenic differentiation of placental stem cells can also be achieved in the following way: Placental stem cells were grown in MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum. Three cycles of induction / maintenance are used. Each cycle consists of feeding the placental stem cells with adipogenesis induction medium (Cambrex) and culturing the cells for 3 days (at 37 ° C, 5% CO2), followed by 1-3 days of culture in medium. maintenance of adipogenesis (Cambrex). An alternative induction medium that can be used contains 1 μM of dexamethasone, 0.2 M of indomethacin, 0.01 mg / ml of insulin, 0.5 mM of IBMX, high DMEM-glucose, FBS, and antibiotics. After 3 complete induction / maintenance cycles, the cells are cultured for an additional 7 days in the maintenance medium of adipogenesis, replacing the medium every 2-3 days. One hallmark of adipogenesis is the development of multiple intracytoplasmic lipid vesicles that can be easily observed using the red O oil of lipophilic stain. The expression of lipase-binding protein genes and / or fatty acid is configured by RT / PCR in placental stem cells that have begun to differentiate into adipocytes. 6. 6.3 Induction of differentiation in osteocytes An osteogenic medium of 185 mL of basal media of Cambrex-osteogenic differentiation and SingleQuots (one each of dexamethasone, 1-glutamine) was prepared., ascorbate, pen / strep, MCGS and ß-glycerophosphate). Plaque stem cells from the perfusate placenta were plated at approximately 3 x 10 3 cells per cm 2 of tissue culture surface area in 0.2-0.3 mL of MSCGM per cm 2 of tissue culture area. Typically, all cells adhered to the culture surface for 4-24 hours in MSCGM at 37 ° in 5% CO2. Osteogenic differentiation was induced by replacing the medium with osteogenic differentiation medium. The cell morphology began to change from the spindle-like appearance typical of adherent placental stem cells to an appearance like a bucket, accompanied by mineralization. Some cells delaminated from the tissue culture surface during differentiation. Osteogenic differentiation can also be achieved in the following way: Adherent cultures of placental stem cells are grown in MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum. 2. Cultures are grown for 24 hours in tissue culture flasks. 3. Osteogenic differentiation is induced by replacing MSCGM with osteogenic induction medium (Cambrex) containing 0.1 μM of dexamethasone, 0.05 mM of ascorbic acid-2-phosphate, 10 mM of beta-glycerophosphate. 4. Cells are fed every 3-4 days for 2-3 weeks with osteogenic induction medium. 5. Differentiation was tested using a specific calcium spot and RT / PCR for alkaline phosphatase and alkaline gene expression. 6. 6.4 Induction of differentiation in pancreatic cells Pancreatic differentiation is achieved as follows: 1. Placental stem cells are cultured in DMEM / 20% CBS, supplemented with basic fibroblast growth factor, 10 ng / ml; and transforming growth factor beta-1, 2 ng / ml. Knockout serum replacement can be used instead of CBS. 2. Conditioned media of cell cultures are added to the nestin-positive neurons in a 50/50 concentration. 3. Cells are cultured for 14-28 days, feeding every 3-4 days. 4. Differentiation is characterized by testing expression of insulin protein or insulin gene by RT / PCR. 6. 6.5 Induction of differentiation in cardiac cells Myogenic (cardiogenic) differentiation is achieved as follows: 1. Placental stem cells are cultured in DMEM / 20% CBS, supplemented with 1 μM retinoic acid; basic fibroblast growth factor, 10 ng / ml; and transforming growth factor beta-1, 2 ng / ml; and epidermal growth factor, 100 ng / ml. Knockout serum replacement (Invitrogen, Carlsbad, California) can be used instead of CBS. 2. Alternatively, placental stem cells are cultured in DMEM / 20% CBS supplemented with 50 ng / ml cardipin-1 for 24 hours. 3. Alternatively, placental stem cells are maintained in protein-free medium for 5-7 days, then stimulated with human myocardium extract (scale dose analysis). Myocardial extract is produced by homogenizing 1 gm of human myocardium in 1% of HEPES regulator supplemented with 1% cord blood serum. The suspension is incubated for 60 minutes, then centrifuged and the supernatant is collect 4. Cells are cultured for 10-1.4 days, feeding every 3-4 days. 5. The differentiation of cardiac actin gene expression by RT / PCR is confirmed. 6. 6.6 Induction of differentiation in condorcitos 6. 6.6.1 General method The chondrogenic differentiation of placental stem cells is usually as follows: 1. Placental stem cells are maintained in MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum. 2. Placental stem cells are aliquoted in a sterile polypropylene tube. The cells are centrifuged (150 x g for 5 minutes) and washed twice in incomplete chondrogenesis medium (Cambrex). 3. After the last wash, the cells are resuspended in complete chondrogenesis medium (Cambrex) containing 0.01 μg / ml of TGF-beta-3 at a concentration of 5 x 10 (5) cells / ml. 4. 0.5 ml of cells are aliquoted into a 15-well culture tube. ml of polypropylene. The cells are agglomerated at 150 x g for 5 minutes. The agglomerate is left intact in the medium. Tubes are incubated poorly covered at 37 ° C, 5% CO2 for 24 hours. The cellular agglomerates are fed every 2-3 days with freshly prepared complete chondrogenesis medium. Agglomerates suspended in medium are maintained by daily agitation using a low speed vortex. Chondrogenic cell agglomerates were harvested after 14-28 days in culture. Chondrogenesis is characterized, for example, by observation of esoinophilic soil substance production, by assaying cell morphology, and / or confirmation of RT / PC R expression of collagen 2 and / or collagen 9 gene and / or production of acidic mucopolysaccharides of cartilage matrix, as confirmed by histochemical staining of Alcian blue. 6. 6.6.2 Differentiation of placental and umbilical cord stem cells in chondrogenic cells mplo demonstrates the differentiation of stem cells from placenta in chondrogenic cells and the development of cartilage-like tissue of said cells. Cartilage is an avascular, alimatic tissue that lacks a nerve supply. The cartilage has a low condorcytic density (<5%), however, these cells are surprisingly efficient in maintaining the extracellular matrix around them. Three main types of cartilage exist in the body: (1) articular cartilage, which facilitates the lubrication of joint in joints; (2) fibrocartilage, which provides shock absorption, for example, in the meniscus and the intervertebral disc; and (3) elastic cartilage, which provides anatomical structure, for example, in the nose and ears. All three types of cartilage are similar in biochemical structure. Joint pain is a leading cause of disability and provides an unfulfilled need for relief in the area of orthopedics. Primary osteoarthritis (which can cause degeneration of the joints), and trauma are two common causes of pain. Approximately 9% of the US population has hip or knee osteoarthritis, and more than 2 million knee surgeries are performed per year. Unfortunately, current treatments are more focused on treating symptoms instead of repairing cartilage. Natural repair occurs when fibroblast-like cells invade the area and fill it with fibrous tissue that is not elastic like normal tissue, thus causing more damage. Treatment options historically included tissue grafts, subcartilage perforation, or replacement of total conjuncture. However, the most recent treatments include CARTICEL®, an autologous condor injection; SYNVISC® and ORTHOVISC®, which are injections of hyaluronic acid for temporary relief of pain; and CHONDROGEN ™, an adult mesentery stem cell injection for meniscus repair. In general, the trend seems to be more towards cellular therapies and / or products made of tissue involving condorcytes or stem cells.

Materials and methods Two lines of stem cells from the placenta, designated AC61665, P3 (passage 3) and AC63919, P5, and two umbilical cord stem cell lines, designated UC67249, P2 and UC67477, P3 were used in the more detailed studies ahead. Positive adult controls were used, and an osteosarcoma cell line, MC3T3, and human dermal fibroblasts (HDF) were used as negative controls. Placental and umbilical cord stem cells were isolated and purified from full-term human placenta by enzymatic stion. MSC cells and Cambrex HDF cells were purchased, and MC3T3 cells were purchased from the American Type Culture Collection. All the cell lines used were centrifuged in pellets in polypropylene centrifuge tubes at 800 RPM for 5 minutes and were grown in chondrogenic induction medium (Cambrex) and non-inducing basal MSC medium.

(Cambrex). Agglomerates were harvested and analyzed histologically at 7, 14, 21 and 28 days by staining for glycosaminoglycans (GAGs) with Alcian blue, and / or for collagens with Syrian red. The type of collagen with immunoblot was further tested. RNA analysis was performed for specific cartilage genes at 7 and 14 days.

Results Experiment 1: Chondrogenesis studies were designed to achieve three main objectives: (1) to demonstrate that placental and umbilical cord stem cells can differentiate and form cartilage tissue; (2) demonstrate that placental and umbilical cord stem cells can differentiate functionally into chondrocytes; and (3) validate the results obtained from the stem cells when evaluating control cell lines. For objective 1, in a preliminary study, a line of stem cells from the placenta was cultured in chondrogenic induction medium in the form of cell agglomerates, either with or without bone morphogenic protein (BMP) at a final concentration of 500 ng / mL. Agglomerates were evaluated for evidence of chondrogenic induction each week for 4 weeks. The results indicated that the agglomerates did not increase in size over time. Without bitterness, no visual differences were noted between the BMP + and BMP- samples. The agglomerates for GAG's, an indicator of cartilage tissue, were also analyzed histologically. stain with Alcian blue. BMP + cells generally appeared more metabolically active with pale vacuoles while BMP- cells were smaller with dense stained nuclei and less cytoplasm (reflecting low metabolic activity). At 7 days, the BMP + cells had stained blue heavily, while BMP- had stained only slightly. For the 28 days of induction, both BMP + and BMP-cells were almost equally stained with Alcian blue. Overall, the cell density decreased over time, and the matrix reached the agglomerate. In contrast, the negative cell line of MC3T3 showed no presence of GAG when stained with Alcian blue. Experiment 2: Based on the results of experiment 1, a more detailed study was designed to evaluate the potential chondrogenic differentiation of two placental stem cell lines and umbilical cord stem cells. In addition to the histology of Alcian blue, the cells were also stained with Syrian red, which is specific for type II collagen. Multiple agglomerates were made for each cell line, with or without induction medium. The agglomerated cell lines, cultured first, were evaluated by gross observation for macroscopic generation of cartilage. Overall, the stem cell lines were observed to make agglomerates as early as day 1. These agglomerates grew over time and formed a matrix hard, looking white, shining and like cartilage, and they became mechanically hard. By visual inspection, the agglomerates of placental stem cells or umbilical cord stem cells were much larger than the MSC controls. The control agglomerates in non-induction medium began to disintegrate by day 1 1, and were much smaller at 28 days than the agglomerates developed by cells cultured in chondrogenic induction medium. Visually, there were no differences between the agglomerates formed by placental or umbilical cord stem cells. However, the UC67249 stem cell line, which was initiated in dexamethasone-free medium, formed larger agglomerates. However, the negative control MC3T3 cells did not form agglomerates; H DFs if they formed agglomerates. The representative agglomerates of all the test groups were then subjected to histological analysis for GAG's and collagen. In general, the agglomerates formed by the stem cells under induction conditions were much larger and remained intact better than the agglomerates formed under non-inducing conditions. The agglomerates formed under induction conditions showed production of GAGs and collagen content increasing over time, and as early as seven days, while the agglomerates formed under non-induction conditions showed little or no collagen production , as is proved by spotting of weak Alcian blue. In general, placental stem cells and cord stem cells umbilical appeared, by visual inspection, to produce harder, larger agglomerates, and appeared to be producing more collagen, over time, than the hMSCs. In addition, during the course of the study, the collagen appeared to thicken, and the type of collagen appeared to change, as is proven by changes in fiber colors under polarized light (colors correlate to fiber thickness which may be indicative of type of collagen). The uninduced placental stem cells produced much less type II collagen, if any, compared to the induced stem cells. Over the 28-day period, the cell density decreased as the matrix production increased, a characteristic of cartilage tissue. These studies confirm that placental and umbilical cord stem cells can be differentiated over a chondrogenic pathway, and can easily be induced to form cartilage tissue. Initial observations that such stem cells may be preferred to MSCs for the formation of cartilage tissue. 6. 7 EXAMPLE 7: CROPPING DROP OF CELLS MOTHER OF PLACENTA Placental stem cells adhering to the placenta were trypsinized 37 ° C for about 5 minutes, and they were loosened from the culture dish by tapping. 10% FBS was added to the culture to stop the trypsinization. The cells are diluted to approximately 1 x 104 cells per mL in approximately 5 mL of medium. The drops (either a single drop or drops from a multi-channel micropipette) are placed inside the foot of a 100 mL Petri dish. The lid is gently inverted and placed on top of the bottom of the plate, which contains approximately 25 ml of sterile PBS to maintain the moisture content in the atmosphere of the dish. Cells are grown for 6-7 days. 6. 8 EXAMPLE 8: PLACENTA TISSUE DIGESTION TO OBTAIN STEM CELLS FROM PLACENTA This example demonstrates scale isolation of placental stem cells by enzymatic digestion. Approximately 10 grams of placental tissue (amnion and chorion) is obtained, macerated and digested using equal volumes of collagenase A (1 mg / ml) (Sigma) and Trypsin-EDTA (0.25%) (Gibco-BRL) in a total volume of about 30 ml for about 30 minutes at 37 ° C. The cells released by digestion are washed 3X with culture medium, distributed in four bottles T-25 and cultured as described in example 1. The performance of the placental stem cells is between approximately 4 x 108 and 5. x 108 cells per 10g of starting material. The cells, characterized in passage 3, are predominantly CD10 *, CD90 *, CD105 *. CD200 *, CD34"and CD45 \ 6. 9 EXAMPLE 9: PRODUCT PRODUCTION OF CRIOCONSERVED STEM CELLS AND BANK OF STEM CELLS This example demonstrates the isolation of placental stem cells and the production of a product based on frozen stem cells. Abstract: Placental tissue is dissected and digested, followed by primary cultures and expansion to achieve an expanded cellular product that produces many cellular doses. Cells are stored in a 2-cell cell bank and distributed as a frozen cell product. All cellular doses derived from an individual donor placenta are defined as a batch, and a batch of placenta is processed one at a time using sterile technique in a dedicated room and Class 100 laminar flow hood. The cellular product is defined as being CD105 +, CD200 +, CD10 + and CD34-, having a normal karyotype and no or substantially no maternal cellular content. 6. 9.1 Obtaining stem cells Dissection and digestion of tissue: A placenta is obtained less than 24 hours after the expulsion. The tissue of the placenta is obtained from amnion, a combination of amnion and chorion, or chorion. The fabric is chopped into small pieces, approximately 1mm in size. Digestion of pledged tissue in 1mg / ml of collagenase 1A during 1 hour at 37 ° C followed by trypsin-EDTA for 30 minutes at 37 ° C. After three washes in 5% FBS in PBS, the tissue is resuspended in culture medium. Primary culture: The purpose of the primary culture is to establish tissue cells from digested placenta. The digested tissue is suspended in culture medium and placed in Corning T-bottles, which are incubated in a humidified chamber maintained at 37 ° C with 5% CO2. Half of the medium is filled after 5 days of culture. Colonies of high density cells are formed at 2 weeks of culture. Colonies are harvested with trypsin-EDTA, which is then quenched with 2% FBS in PBS. The cells are centrifuged and resuspended in culture medium to seed expansion cultures. These cells are defined as 0 passageway cells having 0 times doubled. Expansion culture: Cells harvested from primary culture, harvested from expansion culture, or thawed from the cell bank are used to plant expansion cultures. Cell factories (NU NC ™) are treated with 5% CO2 in air at 50 ml / min / tray for 10 minutes through a sterile filter and heated in a humidified incubator maintained at 37 ° C with 5% CO2. The cell seeds are counted in a hemacitometer with trypan blue, and the cell number, viability, number of passage, and the cumulative number of duplications are recorded. The cells are suspended in culture medium at approximately 2.3 x 10 4 cells / ml and 100 ml / tray are seeded in cell factories. After 3-4 days and once again after 5-6 days of culture, the culture medium is removed and replaced with fresh medium, followed by another treatment with 5% CO2 in air. When the cells reach approximately 105 cells / cm 2, the cells are harvested with trypsin-EDTA, followed by quenching with 2% FBS in PBS. The cells are then centrifuged and resuspended in culture medium.

Cryopreservation: The cells to be frozen are harvested from culture with trypsin-EDTA, quenched with 2% FBS in PBS, and counted in a hemacytometer. After centrifugation, cells are resuspended with 10% DMSO in FBS at a concentration of about 1 million cells / ml for cells to be used for assembly of a cell bank, and 10 million cells / ml for individual frozen cell doses . The cell solution is transferred to a frozen container, which is placed in a bath of isopropyl alcohol in a freezer at -80 ° C. The following days, cells are transferred to liquid nitrogen. 6. 9.2 Design of a stem cell bank A "lot" is defined as all cellular doses derived from a placenta from a single donor. The cells maintained normal growth, karyotype and cell surface marker phenotype for more than 8 passages and 30 duplications during the expansion culture. Given this limitation, the doses comprise cells of 5 passages and approximately 20 duplications. To generate a supply of equivalent cells, a single batch is expanded in culture and stored in a two-level cell bank and frozen doses. In particular, cells harvested from the primary culture, which are defined as passage cells 0 having passed through 0 duplications, are used to initiate an expansion culture. After the first passage, approximately 4 duplications occur, and cells are frozen in a master cell bank (MCB). MCB bottles are used to seed additional expansion cultures. After two additional passages of thawed cells of the MCB, the cells are frozen in a working cell bank (WCB), approximately 12 cumulative duplications. The WCB bottles are used to seed an expansion culture for another 2 passages, resulting in passage cells 5 to approximately 20 duplications that are frozen in individual doses. 6. 9.3 Defrosting cells for culture Frozen cell containers are placed in a sealed plastic bag and immersed in a 37 ° C water bath. The containers are swirled gently until all the contents melt except for a small piece of ice. The containers are removed from the sealed plastic bag and a volume of 10X culture medium is added slowly to the cells with gentle mixing. A sample is counted in the hemacitometer and it is sown in expansion cultures. 6. 9.4 Defrosting cells for injection The frozen cell containers are transferred to the administration site in an exporter of dry nitrogen. Before administration, the containers are placed in a sealed plastic bag and immersed in a water bath at 37 ° C. The containers swirl gently until all the contents are melted except for a small piece of ice. The containers are removed from the sealed plastic bag and an equal volume of 2.5% HSA / 5% dextran is added. The cells are injected with no additional washing. 6. 9.5 Test v specifications A sample of maternal blood accompanies all donor placentas. The sample is filtered for hepatitis B core antibody and surface antigen, hepatitis C virus antibody and nucleic acid, and HIV I and II antibody and nucleic acid. Placental processing and primary culture begins before receiving test results, but continues only for placentas associated with maternal blood samples testing negative for all viruses. A lot is rejected if the donor is positive for any pathogen. In addition, the tests described in table 3 are perform in the MCB, the WCB, and a sample of the cellular dose material derived from a WCB bottle. A batch is released only when all specifications are met.

Table 3: Cellular test and specifications * For the product designed to be 40 μl of frozen cells / dose and a maximum of 5 EU / ml, the cell product is below the upper limit of 5EU / kg / dose for containers over 40kg in body weight. 6. 9.6 Surface marker phenotype analysis Cells are placed in 1% paraformaldehyde (PFA) in PBS for 20 minutes and stored in a refrigerator until stained (up to a week). The cells are washed with 2% FBS, 0.05% sodium azide in PBS (spotting buffer) and then resuspended in spotting buffer. The cells are stained with the following antibody conjugates: CD105-FITC, CD200-PE, CD34-PECy7, CD10-APC. The cells were also stained with isotype controls. After 30 minutes of incubation, the cells were washed and resuspended with spotting buffer, followed by analysis on a flow cytometer. Cells having an increased fluorescence compared to isotype controls are counted as positive for a marker. 6. 10 EXAMPLE 10: IDENTIFICATION OF SPECIFIC GENES OF STEM CELLS OF THE PLACENTA The expression patterns of amnion-chorion (CA) and umbilical cord (UC) placental stem cell genes were compared with the expression patterns of mesentery stem cell genes derived from multipotent spinal cord (BM) and dermal fibroblasts (DF), the last of which are considered to be terminally differentiated. Cells grew for a single passage, an intermediate number of passages, and large numbers of passages (including until senescence). The results indicate that the number of population duplications has a greater impact on gene expression. A series of genes was identified that are up-regulation in AC and UC, and down-regulation or absent in BM and DF, and that they are expressed independent of passage number. This series of specific genes from placental stem cells and umbilical cord stem cells encode a number of cytoskeletal and cell-to-cell adhesion proteins associated with epithelial cells and a surface protein such as immunoglobulin, CD200, implicated in maternal immune tolerance. fetal. The placental stem cells and umbilical cord stem cells will be collectively referred to hereinafter as AC / UQ stem cells. 6. 10.1 Methods and materials 6. 10.1.1 Cells and cell culture BM (Cat # PT-2501) and DF (Cat # CC-251 1) were purchased from Cambrex. AC and UC originated from tissue culture vials of passage 0. AC and UC in the flasks were obtained by digestion of a donor placenta designated 2063919. T-25 culture flasks were seeded at 6000 cells / cm 2 and the cells passed when they became confluent. Population duplications of trypan blue cell counts were calculated. The crops were evaluated for Gene expression after 3, 11-14 and 24-28 population duplications. 6. 10.1.2 RNA, microdispositions and analysis Cells were used directly in their tissue culture flasks, with the exception of a culture that was trypsinized before lysis. Total RNA was isolated with the RNeasy equipment from QIAGEN. The integrity and concentrations of RNA were determined with an Agilent 2100 bioanalyzer. Ten micrograms of total RNA from each culture were hybridized on an Affymetrix GENECHIP® platform. The total RNA was converted to labeled cRNAs and hybridized to human genome arrays of oligonucleotide U133A 2.0 according to the manufacturer's methods. Image files were processed with Affymetrix MAS 5.0 software, and normalized and analyzed with Agilent GeneSpring 7.3 software. 6. 10.2 Results 6. 10.2.1 Selection of BM-MSC, AC / UC stem cell and DF culture time points for microdisposition analysis To establish a unique gene expression pattern for AC / UC stem cells, two stem cell lines, AC (6) and UC (6), were grown in parallel with BM-MSC and DF. To maximize the identification of a gene expression profile attributable to cellular origin and minimize exogenous influences, all cells grew in the same medium, were sown, and sub-cultivated using the same criteria. The cells were harvested after 3 population doublings, 11-14 duplications, or 35 duplications or senescence, whichever occurred first. Genes whose expression in AC / UC stem cells are unchanged by time-in-culture and up-regulation relative to BM and DF are candidates for specific AC / UC stem cell genes. Figure 10 shows growth profiles for the four cell lines in the study; the circles indicate which cultures were harvested for RNA isolation. In total, twelve samples were collected. BM, AC (6) and UC (6) were harvested after three population doublings; These samples are seen as being in culture for a "short" period of time. A short-term sample of DF was not collected. Intermediate length cultures, 11 to 14 duplications, were collected for all cell types. Long-term cultures of all cell lines were collected at approximately 35 population doublings or just before senescence, whichever occurred first. Senescence occurred before 15 duplications for BM and 25 duplications for DF. The acquired BM and DF cells expanded many times before gene analysis, and can not be considered early stage. However, operationally, grown in BM for three Duplications (BM-03) are considered a short-term crop. Likewise, BM-11 is operationally referred to as a culture of intermediate length, but because senescence occurred at 14 duplications, BM-11 is very likely a biologically long-term culture. 6. 10.2.2 The hierarchical grouping shows relationship between BM, AC / UC and DF stem cells The microdissection analysis identifies gene expression patterns, and the hierarchical grouping (HC) tries to find similarities in the context of two dimensions - genes in the first dimension and different conditions (different RNA samples) in the second. The GeneChips used in this experiment contained 22,000 probe sets (referred to as the "list of all genes"), but many of these series interrogate genes that are not expressed under any conditions. To reduce the list of all genes, genes not expressed or expressed at low levels (crude values below 250) in all samples were removed to produce a list of 8,215 genes. 6. 10.2.3 Analysis of gene expression using the line graph view The gene expression patterns of the 8215 genes are displayed using the line graph view in GeneSpring (figure 11). The x-axis shows the twelve experimental conditions and the y-axis shows the normalized probe series expression values on a logarithm scale. The y-axis covers a range of 10,000 times, and genes that are not expressed or expressed at very low levels are set to a value of 0.01. By default, the normalized value is set to 1. Each line represents a single gene (actually a probe series, some genes have multiple probe sets) and runs over the twelve conditions as a single color. The colors illustrate relative expression levels, as described for heat maps ("heatmaps"), but the color pattern is determined by selecting a condition. AC-03 is the condition selected in Figure 11. Up-regulation genes relative to the normalized value are displayed by the software as red, and those that are down-regulated, are displayed as blue. The spikes pointing up and down obvious in AC-03 through UC-11 indicate that many genes are differentially expressed on these conditions. The similarity of the spikes in the color patterns between AC-03 and UC-03 shows that many of the same genes are up-regulation or down-regulation in these two samples. The horizontal line segments indicate that a level of gene expression is unchanged over a number of conditions. This is more noticeable when comparing UC-36, UC-38 and UC-38-T. There are no obvious spikes, but there is a subtle tendency in when a number of red lines between UC-36 and UC-38-T are below the normalized value of 1. This indicates that these genes, which are up-regulation in AC-03 and UC-93, are down-regulation in subsequent cultures. The fact that the expression patterns between UC-38 and UC-38-T are so similar indicates that trypsinizing cells just before RNA isolation has little effect on gene expression. In addition to the computationally intensive HC method, by visual inspection the two BM samples are more similar to each other than to the other conditions. The same is true for the two DF crops. And, despite the large number of differentially expressed genes present in BM and DF samples, the overall appearance suggests that two BMs and the two DFs are more similar to each other than to AC / UC stem cells. This is confirmed by the HC results described above. When the above process is applied using AC-11 as the selected condition, it is clear that AC-11 and UC-11 share many of the same differentially expressed genes, but the total number of genes in common between these two conditions appears less than number of differentially expressed genes shared by AC-03 and UC-03. Figure 12 shows differentially over-expressed genes, six times or more relative to the baseline, in AC-03. The majority of up-regulation genes in AC-03 are also up-regulation in UC-03, and more divergent in BM and DF. 6. 10.2.4 Filtration methods used to identify specific genes of AC / UC stem cells Genes that remain constant over all AC / UC samples, and are down-regulated in BM and DF, are considered specific for AC / UC stem cells. Two filtration methods are combined to create a list of 58 genes specific for AC / UC stem cells (Table 4).

Table 4: 58 specific genes from placental stem cells and umbilical cord stem cells First, 58 genes were identified by selecting those overexpressed genes = three times in at least seven of eight conditions of AC / UC stem cells relative to all samples of BM and DF (Figure 13). Filtering in eight of the eight AC / UC stem cell conditions produced a similar list. The second filtering method used "absent" and "present" calls provided by the Affymetrix MAS 5.0 software. A list was created by identifying missing genes in all BM and DF conditions and present in AC-03, AC-11, UC-03 and UC-11. Gene calls in the latter conditions of AC / UC stem cells were not stipulated. The two lists overlapped significantly and were combined. The combined list was cut by eliminating (1) several genes expressed at very low levels in most or all conditions of AC / UC stem cells, and (2) genes carried on the Y chromosome. The AC and UC cells used in this study it was confirmed that they are male by FISH analysis, and BM and DF were derived from a female donor. The resulting list of 46 specific AC / UC stem cell genes is shown in Table 5.

Table 5. Specific AC / UC genes listed by ontology This list of 46 genes encodes a collection of proteins presenting a number of ontology groups. The most highly expressed group, cell adhesion, contains eight genes. No gene codes for proteins involved in DNA replication or cell division. Sixteen genes with specific references to epithelium were also listed. 6. 10.3 Discussion A specific expression pattern was identified for placental stem cells, and distinguishable from mesentery cells derived from the spinal cord. Operationally, this pattern includes 46 genes that are overexpressed in all samples of stem cells from the placenta relative to all samples of BM and DF. The experimental design compared cells grown for short periods of time, medium and long in culture. For AC and UC cells, each culture period has a characteristic series of differentially expressed genes. During the short term or early phase (AC-03 and UC-03) two hundred genes with ascending regulation return to the average after eight population doublings. Without wishing to be bound by theory, it is likely that this early stage gene expression pattern resembles the expression profile of AC and UC while in the natural placental environment. In the placenta, these cells do not actively divide, they are metabolizing nutrients, pointing to each other, and ensuring their location by remodeling the extracellular surroundings. The expression of genes by intermediate length cultures is defined by rapid cell division and differentially expressed genes at this time are quite different from those differentially expressed during the early phase. Many of the up-regulation genes in AC-11 and UC-11, together with BM-03 and DF-14, are involved in chromosome replication and cellular division. Based on gene expression, BM-03 appears biologically as a medium-term culture. In this cell-mediated stage-specific gene expression, it is shaded by cell proliferation. In addition, almost every gene overexpressed in short-term AC or UC cultures is down-regulated in the middle and later stage conditions. 143 genes were up-regulation = 5 times during this highly proliferating phase, constituting approximately 1.7% of the expressed genes. Long-term crops represent the final or senescent phase. In this phase, the cells have extinguished their ability to divide, and, especially for AC and UC, the absolute number of differentially expressed genes is markedly reduced. This may be the result of cells being completely adapted to their culture environment and a consequently reduced load to biosynthesize. Surprisingly, subsequent BM and DF cultures do not exhibit the same behavior; a large number of genes are differentially expressed in BM-11 and DF-24 relative to AC and UC and the normalized value of 1. AC and UC are distinguishable from BM and DF most notably in long-term cultures. The list of specific genes of placental stem cells described here is diverse. COL4A1 and COL4A1 are regulated in a coordinated manner, and KRT18 and KRT8 also appear to be co-expressed. Eight of the genes encode proteins involved in cell-to-cell contact, three of which (DSC3, DSG2 and PKP2) are localized to desmosomes, intercellular contact points anchored to intermediate filament cytoskeleton proteins such as keratin 18 and keratin 8. Fair cell-to-cell contact is characteristic of epithelial and endothelial cells and is not typically associated with fibroblasts. Table 3 lists 16 genes, out of 46 in all, characteristic of epithelial cells. Placental stem cells are usually described as small fibroblast spindle-shaped cells. This morphology is typically different from BM and DF, especially at lower cell densities. Also noteworthy is the expression pattern of CD200, which is present in AC / UC stem cells and absent in all BM and DF samples. In addition, CD200 has been shown to be associated with immune tolerance in the placenta during fetal development (see, for example, Clark et al., Am. J. Reprod. Immunol .50 (3): 187-195 (2003)). This gene subset of 46 genes constitutes a series of molecular biomarkers that distinguish AC / UC stem cells from mesentery stem cells derived from the spinal cord or fibroblasts

Claims

CLAIMS 1. - A method of producing a cell population that comprises identifying placental cells that adhere to a substrate and: express CD100 and HLA-G; express CD73, CD105 and CD200; express CD200 and OCT-4; express CD73, CD105 and HLA-G; express CD73 and CD105, and facilitate the formation of one or more embryoid-like bodies in a population of placental cells when said population is cultured under conditions that allow the formation of embryoid-like bodies; or express OCT-4, and (c) facilitate the formation of one or more embryoid-like bodies in a population of placental cells when said population is cultured under conditions that allow the formation of embryoid-like bodies; and isolating said cells from other cells to form a cell population.
2. The method according to claim 1, wherein said substrate comprises fibronectin.
3. The method according to claim 1, wherein said identification is achieved using an antibody.
4. The method according to claim 1, wherein said identification is achieved using flow cytometry.
5. The method according to claim 1, wherein said isolation is achieved using magnetic beads.
6. The method according to claim 1, wherein said isolation is achieved by fluorescence-activated cell sorting.
7. The method according to claim 1, wherein said cell population expands after said isolation.
8. A method of producing a stem cell cell line, which comprises transforming a stem cell with a sequence of DNA encoding a growth promoter protein; and exposing said stem cell to conditions that promote the production of said growth promoter protein.
9. The method according to claim 8, wherein said growth promoter protein is v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1a adenovirus or E7 protein of human papilloma virus.
10. The method according to claim 8, wherein said DNA sequence can be regulated.
11. The method according to claim 8, wherein said DNA sequence can be regulated by tetracycline.
12. The method according to claim 8, wherein said growth promoter protein has an activity that can be regulated.
13. The method according to claim 8, wherein said growth promoter protein is a temperature sensitive mutant. 14. - A method of producing a population of stem cells, comprising identifying adherent placental stem cells expressing one or more genes, wherein said genes are ACTG2, ADARB1, AMIGO2, ART-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, CPA4, DMD, DSC3, DSG2, EOLVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLMIM3, PJP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, at a detectably higher level than a mesentery stem cell derived from spinal cord that has passed by the same number of passages in culture as said placental stem cell, and isolating said placental cells. 15. The method according to claim 14, wherein said adherent placental cell expresses five or more of said genes at a detectably higher level than a mesentery stem cell derived from spinal cord that has passed through the same number of passages. in culture as said mother cell of the placenta. 16. The method according to claim 14, wherein said adherent placental cell expresses ten or more of said genes at a detectably higher level than a mesentery stem cell derived from spinal cord that has passed through the same number of passages. in culture as said mother cell of the placenta. 17. - The method according to claim 14, wherein said adherent placental cell expresses twenty or more genes at a detectably higher level than a mesentery stem cell derived from spinal cord that has passed through the same number of passages in culture as said mother cell of the placenta. 18. The method according to claim 14, wherein said adherent placental cell expresses each of said genes at a detectable level higher than a mesentery stem cell derived from spinal cord that has passed through the same number of passages in culture as said mother cell of the placenta. 19. A method of making a stem cell bank of the placenta, comprising: expanding stem cells from the primary placenta culture of a placenta subsequent to human birth for a first plurality of population duplications; cryopreserve said stem cells from the placenta to form a master cell bank; expanding a plurality of placental stem cells from the master cell bank for a second plurality of population duplications; cryopreserve said stem cells from the placenta to form a working cell bank; expand a plurality of placental stem cells from the cellular work bank for a third plurality of population duplications; and cryopreservating said placental stem cells in individual doses, wherein said individual doses collectively make up a bank of placental stem cells. 20. The method according to claim 19, wherein the total number of population duplications is approximately 20. 21. The method according to claim 19, wherein said first plurality of population duplications is approximately four duplications. of population; said second plurality of population duplications is approximately eight population doublings; and said third plurality of population duplications is approximately eight population doublings. 22. The method according to claim 19, wherein said primary cultured placental stem cells comprise placental stem cells from placental perfusate. 23. The method according to claim 19, wherein said primary cultured placental stem cells comprise placental stem cells from digested placenta tissue. 24. The method according to claim 19, wherein said stem cells of the primary culture placenta they comprise placental stem cells from placental perfusate and from digested placenta tissue. 25. The method according to claim 19, wherein all of said placental stem cells in said primary culture of placental stem cells are from the same placenta. 26. The method according to claim 19, further comprising the step of selecting placental stem cells CD200 * or HLA-G * of said plurality of said placental stem cells from said working cell bank to form dose individual 27. The method according to claim 19, wherein said individual doses comprise from about 104 to about 105 of placental stem cells. 28. The method according to claim 19, wherein said individual doses comprise from about 105 to about 10β of placental stem cells. 29. The method according to claim 19, wherein said individual doses comprise from about 106 to about 107 of placental stem cells. 30. The method according to claim 19, wherein said individual doses comprise from about 107 to about 108 of placental stem cells. 31.- A composition comprising conditioned medium, wherein said medium is conditioned by placental stem cells that are: CD200 * and HLA-G *; CD73 *, CD 105 * and CD200 *; CD200 * and OCT-4 *; CD73 *, CD105 * and HLA-G *; CD73 * and CD105 * and facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of embryoid-like bodies; or OCT-4 * and facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising the stem cell when said population is cultured under conditions that allow the formation of embryoid-like bodies. 32. The composition according to claim 31, wherein said placental stem cells have been grown in said medium for at least one day. 33. The composition according to claim 31, wherein said placental stem cells have been grown in said medium for at least three days. 34.- A composition comprising a population of placental stem cells that are: CD200 * and HLA-G *; CD73 *, CD105 * and CD200 *; CD200 * and OCT-4 *; C D73 *, CD 105 * and HLA-G *; CD73 * and CD105 * and facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of embryoid-like bodies; or OCT-4 * and facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising the stem cell when said population is cultured under conditions that allow the formation of embryoid-like bodies; or a combination of the above; and a stem cell that is not obtained from a placenta, where at least 70% of said placental stem cells are non-maternal in origin. 35. The composition according to claim 34, wherein said stem cell not obtained from a placenta is a brionic stem cell. 36. The composition according to claim 34, wherein said stem cell not obtained from a placenta is a mesentery stem cell. 37. The composition according to claim 34, wherein said stem cell not derived from a placenta is a stem cell derived from spinal cord. 38.- The composition according to claim 34, wherein said stem cell not obtained from a placenta is a hematopoietic progenitor cell. 39.- The composition according to claim 34, wherein said stem cell not obtained from a placenta is a somatic stem cell. The composition according to claim 39, wherein said somatic stem cell is a neural stem cell, a liver stem cell, a pancreatic stem cell, an endothelial stem cell, a cardiac stem cell, or a muscle stem cell. 41. A method of isolating a population of placental stem cells, comprising culturing a portion of a placenta for a sufficient time for a plurality of placental stem cells to proliferate outside said placenta, and isolating said stem cells from the placenta of said tissue. 42. The method according to claim 41, wherein said tissue is amniotic membrane, chorion, a combination of amnion and chorion, or a portion of any thereof, or a combination of any of the foregoing. 43. An isolated adherent placental stem cell, wherein said stem cell is: CD200 * and HLA-G *; CD73 *, CD105 * and CD200 *; CD200 * and OCT-4 *; CD73 *, CD105 * and HLA-G *; CD73 * and CD105 * and facilitate the formation of one or more brioid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow the formation of brioid-like bodies; or OCT-4 * and facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising the stem cell when said population is cultured under conditions that allow the formation of brioid-like bodies; or a combination of the above; and wherein said mother cell of the placenta is non-maternal in origin. 44. - A population of isolated adherent placental stem cells, wherein said stem cells are: CD200 * and H LA-G *; CD73 *, CD 1 05 * and CD200 *; CD200 * and OCT-4 *; CD73 *, CD 1 05 * and H LA-G *; CD73 * and CD 1 05 * and facilitate the formation of one or more bryoid-like bodies in a placental cell population comprising said stem cell when said population is cultured under conditions that allow the formation of brioid-like bodies.; or OCT-4 * and facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising the stem cell when said population is cultured under conditions that allow the formation of embryoid type bodies; or a combination of the above; and wherein at least 70% of said placental stem cells are non-maternal in origin. 45.- The population in accordance with claim 44, where at least 90% of said placental stem cells are non-maternal in origin. 46. The population according to claim 44, wherein at least 99% of said placental stem cells are non-maternal in origin. 47. A composition comprising the isolated placenta stem cell according to claim 43. 48. A composition comprising the population of the isolated placental stem cell according to claim 44. 49. The composition of according to claim 47 which is in a form suitable for intravenous administration. 50.- The composition according to claim 48 which is in a form suitable for intravenous administration. 51 The composition according to claim 47, wherein said composition is in a container suitable for intravenous administration of said composition. 52. The composition according to claim 48, wherein said composition is in a container suitable for intravenous administration of said composition. 53. - A method of collecting stem cells from the placenta, comprising: perfusing a mammalian placenta that has been drained of umbilical cord and prefused to remove residual blood; prefuse said placenta with a perfusion solution through placental vasculature; collecting said perfusion solution only from said vasculature, wherein said perfusion solution after perfusion comprises a population of cells comprising placental stem cells, wherein at least 95% of said placental stem cells are fetal in origin; and isolating a plurality of said placental stem cells from said population of cells. 54. The method according to claim 53, wherein the perfusion solution is passed through the umbilical vein and collected from the umbilical arteries. 55. The method according to claim 53, wherein the perfusion solution is passed through the umbilical arteries and collected from the umbilical vein. 56.- An isolated population of adherent placental stem cells harvested by the method according to claim 53, wherein at least 95% of said isolated population of placental stem cells is fetal in origin. 57. - The isolated population according to claim 51, wherein more than 99% of said placental stem cells are fetal in origin. 58.- The isolated population according to claim 51, wherein the placental stem cells are at least 50% of said population of placental cells. 59. - The isolated population according to claim 51, wherein the placental stem cells are at least 95% of said population of placental cells.