US20080032401A1 - Placental stem cell populations - Google Patents

Placental stem cell populations Download PDF

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Publication number
US20080032401A1
US20080032401A1 US11/648,804 US64880406A US2008032401A1 US 20080032401 A1 US20080032401 A1 US 20080032401A1 US 64880406 A US64880406 A US 64880406A US 2008032401 A1 US2008032401 A1 US 2008032401A1
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Prior art keywords
cells
placental
stem cells
population
cell
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US11/648,804
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Inventor
James Edinger
Qian Ye
Jia-Lun Wang
Robert Hariri
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Celularity Inc
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Individual
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Priority to US11/648,804 priority Critical patent/US20080032401A1/en
Application filed by Individual filed Critical Individual
Publication of US20080032401A1 publication Critical patent/US20080032401A1/en
Assigned to ANTHROGENESIS CORPORATION reassignment ANTHROGENESIS CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YE, QIAN, WANG, JIA-LUN, EDINGER, JAMES, HARIRI, ROBERT J.
Priority to US12/848,007 priority patent/US8202703B2/en
Priority to US13/107,727 priority patent/US20110280845A1/en
Priority to US13/473,509 priority patent/US8591883B2/en
Priority to US13/480,370 priority patent/US8691217B2/en
Priority to US14/181,159 priority patent/US9078898B2/en
Priority to US14/735,727 priority patent/US10383897B2/en
Assigned to CLARITY ACQUISITION II LLC reassignment CLARITY ACQUISITION II LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: ANTHROGENESIS CORPORATION
Assigned to Celularity, Inc. reassignment Celularity, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CLARITY ACQUISITION II LLC
Abandoned legal-status Critical Current

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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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Definitions

  • the present invention provides isolated placental stem cells, populations of placental stem cells, compositions comprising the stem cells, and methods of obtaining the stem cells.
  • Human stem cells are totipotential or pluripotential precursor cells capable of generating a variety of mature human cell lineages. Evidence exists that demonstrates that stem cells can be employed to repopulate many, if not all, tissues and restore physiologic and anatomic functionality.
  • the present invention provides isolated placental stem cells, populations of placental stem cells, compositions comprising the stem cells, and methods of obtaining the stem cells.
  • the invention first provides isolated stem cells, and cell populations comprising such stem cells, wherein the stem cells are present in, and isolatable from placental tissue (e.g., amnion, chorion, placental cotyledons, etc.)
  • placental tissue e.g., amnion, chorion, placental cotyledons, etc.
  • the 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 times in culture in an undifferentiated state, differentiate into adult cells representative of the three germ layers, etc.), and can adhere to a tissue culture substrate (e.g., tissue culture plastic such as the surface of a tissue culture dish or multiwell plate).
  • tissue culture substrate e.g., tissue culture plastic such as the surface of a tissue culture dish or multiwell plate.
  • the invention provides an isolated placental stem cell that is CD200 + or HLA-G + .
  • said cell is CD200 + and HLA-G + .
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + and CD105 + .
  • 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 formation of embryoid-like bodies.
  • the invention provides a population of isolated placental cells comprising, e.g., that is enriched for, CD200 + , HLA-G + stem cells.
  • 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 + , HLA-G + stem cells.
  • said stem cells are CD73 + and CD105 + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + and CD105 + .
  • said population has been expanded, e.g., passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
  • said population forms one or more embryoid-like bodies when cultured under conditions that allow formation of embryoid-like bodies.
  • the invention provides an isolated stem cell that is CD73 + , CD105 + , and CD200 + .
  • said stem cell is HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + .
  • said stem cell facilitates 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 formation of embryoid-like bodies.
  • the invention provides a population of isolated placental cells comprising, e.g., that is enriched for, CD73 + , CD105 + , CD200 + stem cells.
  • 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 + , CD200 + stem cells.
  • said stem cells are HLA-G + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + .
  • said population has been expanded, for example, passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
  • said population forms one or more embryoid-like bodies in culture under conditions that allow formation of embryoid-like bodies.
  • the invention also provides an isolated stem cell that is CD200 + and OCT-4 + .
  • the stem cell is CD73 + and CD105 + .
  • said stem cell is HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + and HLA-G + .
  • 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 formation of embryoid-like bodies.
  • the invention provides a population of isolated cells comprising, e.g., that is enriched for, CD200 + , OCT-4 + stem cells.
  • 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.
  • said stem cells are CD73 + and CD105 + .
  • said stem cells are HLA-G + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + and HLA-G + .
  • said population has been expanded, for example, has been passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
  • said population forms one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • the invention provides an isolated stem cell that 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 formation of embryoid-like bodies.
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is OCT4 + .
  • said stem cell is OCT4 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • the invention further provides a population of isolated placental cells comprising, e.g., that is enriched for, CD73 + , CD105 + stem cells, wherein said population forms one or more embryoid-like bodies under conditions that allow 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 + , CD105 + stem cells.
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are OCT-4 + .
  • said stem cells are OCT-4 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said population has been expanded, for example, has been passaged 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 an isolated stem cell that is CD73 + , CD105 + and HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is OCT-4 + .
  • said stem cell is CD200 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , OCT-4 + and CD200 + .
  • 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 formation of embryoid-like bodies.
  • the invention further provides a population of isolated placental cells comprising, e.g., that is enriched for, CD73 + , CD105 + and HLA-G + stem cells.
  • a population of isolated placental cells comprising, e.g., that is enriched for, CD73 + , CD105 + and HLA-G + stem cells.
  • 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 + and HLA-G + stem cells.
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are OCT-4 + .
  • said stem cells are CD200 + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , OCT-4 + and CD200 + .
  • said population has been expanded, for example, has been passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
  • said population forms embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • the invention further provides an isolated stem cell that is OCT-4 + and which facilitates 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 formation of embryoid-like bodies.
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , or CD45 ⁇ .
  • said stem cell is CD200 + .
  • said stem cell is CD73 + , CD105 + , CD200 + , CD34 ⁇ , CD38 ⁇ , and CD45 ⁇ .
  • the invention also provides a population of isolated cells comprising, e.g., that is enriched for, OCT-4 + placental stem cells, wherein said population forms one or more embryoid-like bodies when cultured 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 OCT4 + placental stem cells.
  • said stem cells are CD73 + and CD105 + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , or CD45 ⁇ .
  • said stem cells are CD200 + .
  • said stem cells are CD73 + , CD105 + , CD200 + , CD34 ⁇ , CD38 ⁇ , and CD45 ⁇ .
  • said population has been expanded, for example, passaged 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 an isolated population of the placental stem cells described herein that is produced according to a method comprising perfusing a mammalian placenta that has been drained of cord blood and perfused to remove residual blood; perfusing said placenta with a perfusion solution; and collecting said perfusion solution, wherein said perfusion solution after perfusion comprises a population of placental cells that comprises placental stem cells; and isolating a plurality of said placental stem cells from said population of cells.
  • the perfusion solution is passed through both the umbilical vein and umbilical arteries and collected after it exudes from the placenta.
  • 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 according to a method comprising digesting placental tissue with a tissue-disrupting enzyme to obtain a population of placental cells comprising placental stem cells, and isolating a plurality of placental stem cells from the remainder of said placental cells.
  • said placental tissue is a whole placenta, an amniotic membrane, chorion, a combination of amnion and chorion, or a combination of any of the foregoing.
  • the tissue-disrupting enzyme is trypsin or collagenase.
  • the invention provides any of the isolated stem cells above, wherein said stem cell expresses one or more genes at a detectably higher level than a bone marrow-derived mesenchymal stem cell, 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, PJP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN
  • 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 bone marrow-derived mesenchymal stem cell.
  • the invention also provides any of the populations of isolated stem cells above, wherein said stem cells express one or more genes at a detectably higher level than a population of bone marrow-derived mesenchymal stem cells, 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, TGFB
  • 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 detectably higher level than said population of isolated bone marrow-derived mesenchymal stem cells.
  • the invention also provides methods of selecting one of the above-mentioned cell populations, comprising selecting cells that express one or more genes at a detectably higher level than a bone marrow-derived mesenchymal stem cell, 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, SLC12
  • said selecting comprises selecting cells that 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 bone marrow-derived mesenchymal stem cell.
  • the invention also provides compositions that comprise one or more of the stem cells of the invention, wherein the stem cell has been isolated from the placenta.
  • the invention further provides a composition comprising a stem cell, wherein said stem cell is CD200 + and HLA-G + .
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + , CD200 + and HLA-G + .
  • the invention provides a composition comprising a stem cell, wherein said stem cell is CD73 + , CD105 + and CD200 + .
  • said stem cell is HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + .
  • the invention provides a composition comprising a stem cell, wherein said stem cell is CD200 + and OCT-4 + .
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + , and HLA-G + .
  • the invention provides a composition comprising a stem cell that is CD73 + and CD105 + , wherein said stem cell facilitates 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.
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is OCT-4 + .
  • said stem cell is CD200 + .
  • said stem cell is OCT-4 + , CD200 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • the invention provides a composition comprising a stem cell that is CD73 + , CD105 + and HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is OCT-4 + .
  • said stem cell is CD200 + .
  • said stem cell is OCT-4+, CD200 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • the invention provides a composition comprising a stem cell that is OCT-4 + , wherein said stem cell facilitates 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.
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD200 + .
  • said stem cell is CD73 + , CD105 + , CD200 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell expresses one or more genes at a detectably higher level than a bone marrow-derived mesenchymal stem cell, 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
  • 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 isolated bone marrow-derived mesenchymal stem cell, wherein said population of stem cells and said population of bone marrow-derived mesenchymal stem cell, wherein said
  • any of the foregoing compositions comprises a matrix.
  • said matrix is a three-dimensional scaffold.
  • said matrix comprises collagen, gelatin, laminin, fibronectin, pectin, ornithine, or vitronectin.
  • the matrix is an amniotic membrane or an amniotic membrane-derived biomaterial.
  • said matrix comprises an extracellular membrane protein.
  • said matrix comprises a synthetic compound.
  • said matrix comprises a bioactive compound.
  • said bioactive compound is a growth factor, cytokine, antibody, or organic molecule of less than 5,000 daltons.
  • the invention further provides a composition comprising medium conditioned by any of the foregoing stem cells, or any of the foregoing stem cell populations.
  • any such composition comprises a stem cell that is not derived from a placenta.
  • said stem cell is an embryonic stem cell.
  • said stem cell is a mesenchymal stem cell.
  • said stem cell is a bone marrow-derived stem cell.
  • said stem cell is a hematopoietic progenitor cell.
  • said stem cell is a somatic stem cell.
  • said somatic stem cell is a neural stem cell, a hepatic 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 populations of stem cells derived from mammalian placenta.
  • the invention provides a method of producing a cell population comprising selecting cells that (a) adhere to a substrate, and (b) express CD200 and HLA-G; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate, and (b) express CD73, CD105, and CD200; and isolating said cells from other cells to form a cell population.
  • 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.
  • 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 for the formation of embryoid-like bodies; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population, comprising selecting cells that (a) adhere to a substrate, and (b) express 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) express OCT-4, and (c) facilitate the formation of one or more embryoid-like bodies when cultured with a population of placental cells under conditions that allow for the formation of embryoid-like bodies; and isolating said cells from other cells to form a cell population.
  • said substrate comprises fibronectin.
  • the methods comprise selecting cells that express ABC-p. In another specific embodiment, the methods comprise selecting cells exhibiting at least one characteristic specific to a mesenchymal stem cell. In a more specific embodiment, said characteristic specific to a mesenchymal stem cell is expression of CD29, expression of CD44, expression of CD90, or expression of a combination of the foregoing. In another specific embodiment of the methods, said selecting is accomplished using an antibody. In another specific embodiment, said selecting is accomplished using flow cytometry. In another specific embodiment, said selecting is accomplished using magnetic beads. In another specific embodiment, said selecting is accomplished by fluorescence-activated cell sorting. In another specific embodiment of the above methods, said cell population is expanded.
  • the invention also provides a method of producing a stem cell line, comprising transforming a stem cell with a DNA sequence that encodes a growth-promoting protein; and exposing said stem cell to conditions that promote production of said growth-promoting protein.
  • said growth-promoting protein is v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1a adenovirus or human papillomavirus E7 protein.
  • said DNA sequence is regulatable.
  • said DNA sequence is regulatable by tetracycline.
  • said growth-promoting protein has a regulatable activity.
  • said growth-promoting protein is a temperature-sensitive mutant.
  • the invention further provides cryopreserved stem cell populations.
  • 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 cultured with a population of placental cells under conditions that allow for 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 cultured with a population of placental cells under conditions that allow for the formation of embryoid-like bodies.
  • said container is a bag.
  • said population comprises about, at least, or at most 1 ⁇ 10 6 said stem cells, 5 ⁇ 10 6 said stem cells, 1 ⁇ 10 7 said stem cells, 5 ⁇ 10 7 said stem cells, 1 ⁇ 10 8 said stem cells, 5 ⁇ 10 8 said stem cells, 1 ⁇ 10 9 said stem cells, 5 ⁇ 10 9 said stem cells, or 1 ⁇ 10 10 said stem cells.
  • said stem cells have been passaged about, at least, or no more than 5 times, no more than 10 times, no more than 15 times, or no more than 20 times.
  • said stem cells have been expanded within said container.
  • SH2 refers to an antibody that binds an epitope on the marker CD105.
  • cells that are referred to as SH2 + are CD105 + .
  • SH3 and SH4 refer to antibodies that bind epitopes present on the marker CD73.
  • cells that are referred to as SH3 + and/or SH4 + are CD73 + .
  • isolated stem cell means a stem cell that is substantially separated from other, non-stem cells of the tissue, e.g., 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 displaying a different marker profile, are removed from the stem cell, e.g., during collection and/or culture of the stem cell.
  • the term “population of isolated cells” means a population of cells that is substantially separated from other cells of the tissue, e.g., 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 from which the population of cells is derived, is naturally associated, i.e., stem cells displaying a different marker profile, are removed from the stem cell, e.g., during collection and/or culture of the stem cell.
  • 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 the number of passages after a primary culture.
  • placental stem cell does not, however, refer to a trophoblast.
  • a cell is considered a “stem cell” if the cell retains at least one attribute of a stem cell, e.g., 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 of all three germ layers; the lack of adult (i.e., differentiated) cell characteristics, or the like.
  • the terms “placental stem cell” and “placenta-derived stem cell” may be used interchangeably.
  • a stem cell is “positive” for a particular marker when that marker is detectable above background.
  • a placental stem cell is positive for, e.g., CD73 because CD73 is detectable on placental stem cells in an amount detectably greater than background (in comparison to, e.g., 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 can be used to select or isolate the cell when present or expressed by the cell.
  • “positive,” as an indication a particular cell surface marker is present, means that the marker is detectable using an antibody, e.g., a fluorescently-labeled antibody, specific for that marker; “positive” also means that a cell bears that marker in a amount that produces a signal, e.g., in a cytometer, that is detectably above background.
  • a cell is “CD200 + ” where the cell is detectably labeled with an antibody specific to CD200, and the signal from the antibody is detectably higher than a control (e.g., background).
  • “negative” in the same context means that the cell surface marker is not detectable using an antibody specific for that marker compared to background.
  • a cell is “CD34 ⁇ ” where the cell is not detectably labeled with an antibody specific to CD34.
  • cluster of differentiation (“CD”) markers are detected using antibodies.
  • OCT-4 is determined to be present, and a cell is “OCT-4 + ” if OCT-4 is detectable using RT-PCR.
  • FIG. 1 Viability of placental stem cells from perfusion (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E). Numbers on X-axis designate placenta from which stem cells were obtained.
  • FIG. 2 Percent HLA ABC ⁇ /CD45 ⁇ /CD34 ⁇ /CD133 + cells from perfusion (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E) as determined by FACSCalibur. Numbers on X-axis designate placenta from which stem cells were obtained.
  • FIG. 3 Percent HLA ABC ⁇ /CD45 ⁇ /CD34 ⁇ /CD133 + cells from perfusion (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E), as determined by FACS Aria. Numbers on X-axis designate placenta from which stem cells were obtained.
  • FIG. 4 HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 expression in stem cells derived from placental perfusate.
  • FIG. 5 HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 expression in stem cells derived from amnion.
  • FIG. 6 HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 expression in stem cells derived from chorion.
  • FIG. 7 HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 expression in stem cells derived from amnion-chorion plate.
  • FIG. 8 HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 expression in stem cells derived from umbilical cord.
  • FIG. 9 Average expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 expression in stem cells derived from perfusion (A), amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E).
  • FIG. 10 Culture time courses for amnion/chorion (AC), umbilical cord (UC), bone marrow-derived stem cell (BM-MSC) and human dermal fibroblast (NHDF) cell lines used in this study. All cultures were grown and propagated using the same seeding and passage densities. Circles indicate which cultures were used for RNA isolation. Late cultures were harvested just prior to senescence. Two UC cultures were harvested at 38 doublings (UC-38) to compare the effect of trypsinization on gene expression. All other cultures were lysed directly in their culture flasks prior to RNA isolation.
  • AC amnion/chorion
  • UC umbilical cord
  • BM-MSC bone marrow-derived stem cell
  • NHDF human dermal fibroblast
  • FIG. 11 Line plot of relative expression levels of 8215 genes in amnion/chorion (AC), umbilical cord (UC), bone marrow-derived stem cell (BM-MSC) and human dermal fibroblast (DF) cells.
  • the number associated with each cell line designation on the X-axis indicates the number of days the cell line was cultured prior to evaluation of gene expression levels.
  • the chart was generated from RNA expression data analyzed by GeneSpring software. AC-03 was used as the selected condition.
  • FIG. 12 Subset of the all genes list showing genes over-expressed ⁇ 6-fold in AC-03 for amnion/chorion (AC), umbilical cord (UC), bone marrow-derived stem cell (BM-MSC) and human dermal fibroblast (DF) cells.
  • AC amnion/chorion
  • UC umbilical cord
  • BM-MSC bone marrow-derived stem cell
  • DF human dermal fibroblast
  • FIG. 13 Placental stem cell-specific or umbilical cord stem cell-specific genes found by fold change filtering for amnion/chorion (AC), umbilical cord (UC), bone marrow-derived stem cell (BM-MSC) and human dermal fibroblast (DF) cells.
  • AC amnion/chorion
  • UC umbilical cord
  • BM-MSC bone marrow-derived stem cell
  • DF human dermal fibroblast
  • Placental stem cells are stem cells, obtainable from a placenta or part thereof, that adhere to a tissue culture substrate and have the capacity to differentiate into non-placental cell types.
  • Placental stem cells can be either fetal or maternal in origin (that is, can have the genotype of either the fetus or mother, respectively).
  • the placental stem cells and placental stem cell populations of the invention are fetal in origin.
  • Populations of placental stem cells, or populations of cells comprising placental stem cells can comprise placental stem cells that are solely fetal or maternal in origin, or can comprise a mixed population of placental stem cells of both fetal and maternal origin.
  • the placental stem cells, and populations of cells comprising the placental stem cells can be identified and selected by the morphological, marker, and culture characteristic discussed below.
  • placental stem cells of the present invention when cultured in primary cultures or in cell culture, adhere to the tissue culture substrate, e.g., tissue culture container surface (e.g., tissue culture plastic).
  • tissue culture substrate e.g., tissue culture container surface
  • Placental stem cells in culture assume a generally fibroblastoid, stellate appearance, with a number of cytoplasmic processes extending from the central cell body.
  • the placental stem cells are, however, morphologically differentiable from fibroblasts cultured under the same conditions, as the placental stem cells exhibit a greater number of such processes than do fibroblasts. Morphologically, placental stem cells are also differentiable from hematopoietic stem cells, which generally assume a more rounded, or cobblestone, morphology in culture.
  • Placental stem cells of the present invention and populations of placental stem cells, express a plurality of markers that can be used to identify and/or isolate the stem cells, or populations of cells that comprise the stem cells.
  • the placental stem cells, and stem cell populations of the invention include stem cells and stem cell-containing cell populations obtained directly from the placenta, or any part thereof (e.g., amnion, chorion, placental cotyledons, and the like).
  • Placental stem cell populations also includes populations of (that is, two or more) placental stem cells in culture, and a population in a container, e.g., a bag. Placental stem cells are not, however, 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 other stem cell types. Because the placental stem cells can express CD73 and CD105, they can have mesenchymal stem cell-like characteristics.
  • the placental stem cells can express CD200 and HLA-G, a fetal-specific marker, they can be distinguished from mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells, which express neither CD200 nor HLA-G.
  • mesenchymal stem cells e.g., bone marrow-derived mesenchymal stem cells, which express neither CD200 nor HLA-G.
  • the lack of expression of CD34, CD38 and/or CD45 identifies the placental stem cells as non-hematopoietic stem cells.
  • the invention provides an isolated stem cell that is CD200 + or HLA-G + .
  • said stem cell is a placental stem cell.
  • the stem cell is CD200 + and HLA-G + .
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + and CD105 + .
  • said CD200 + or HLA-G + stem cell facilitates the formation of embryoid-like bodies in a population of placental cells comprising the stem cells, under conditions that allow the formation of embryoid-like bodies.
  • said placental stem cell is isolated away from placental cells that are not stem cells.
  • said placental stem cell is isolated away from placental stem cells that do not display these markers.
  • the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a CD200 + or HLA-G+placental cell, whereby said cell is a placental stem cell.
  • said selecting comprises selecting a placental cell that is both CD200 + and HLA-G + .
  • said selecting comprises selecting a placental cell that is also CD73 + and CD105 + .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + and CD105 + .
  • said selecting comprises selecting a placental cell that also facilitates the formation of embryoid-like bodies in a population of placental cells comprising the stem cells, under conditions that allow the formation of embryoid-like bodies.
  • the invention provides an isolated population of cells comprising, e.g., that is enriched for, CD200 + , HLA-G + stem cells.
  • said population is a population of placental cells.
  • 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 CD200 + , HLA-G + stem cells.
  • 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.
  • said stem cells are also CD73 + and CD105 + .
  • said stem cells are also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + and CD105 + .
  • said isolated population produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • said population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these markers.
  • the invention also provides a method of selecting a placental stem cell population from a plurality of placental cells, comprising selecting a population of placental cells wherein 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 cells are CD200 + , HLA-G + stem cells.
  • said selecting comprises selecting stem cells that are also CD73 + and CD105 + .
  • said selecting comprises selecting stem cells that are also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said selecting comprises selecting stem cells that are also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + and CD105 + .
  • said selecting also comprises selecting a population of placental stem cells that forms one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • the invention provides an isolated stem cell that is CD73 + , CD105 + , and CD200 + .
  • said isolated stem cell is an isolated placental stem cell.
  • said stem cell is HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + .
  • the isolated CD73 + , CD105 + , and CD200 + stem cell facilitates 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.
  • said placental stem cell is isolated away from placental cells that are not stem cells.
  • said placental stem cell is isolated away from placental stem cells that do not display these markers.
  • the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a CD73 + , CD105 + , and CD200 + placental cell, whereby said cell is a placental stem cell.
  • said selecting comprises selecting a placental cell that is also HLA-G + .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + .
  • said selecting additionally comprises selecting a CD73 + , CD105 + , and CD200 + stem cell that facilitates 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 facilitate formation of embryoid-like bodies.
  • the invention provides an isolated population of cells comprising, e.g., that is enriched for, CD73 + , CD105 + , CD200 + stem cells.
  • said stem cells are placental stem cells.
  • 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 CD73 + , CD105 + , CD200 + stem cells.
  • at least about 70% of said cells in said population of cells are CD73 + , CD105 + , CD200 + stem cells.
  • at least about 90%, 95% or 99% of said cells in said population of cells are CD73 + , CD105 + , CD200 + stem cells.
  • said stem cells are HLA-G + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + .
  • said population of cells produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • said population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention also provides a method of selecting a placental stem cell population from a plurality of placental cells, comprising selecting a population of placental cells wherein 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 cells are CD73 + , CD105 + , CD200 + stem cells.
  • said selecting comprises selecting stem cells that are also HLA-G + .
  • said selecting comprises selecting stem cells that are also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said selecting comprises selecting stem cells that are also CD34 ⁇ , CD38 ⁇ and CD45 ⁇ . In another specific embodiment, said selecting comprises selecting stem cells that are also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , and HLA-G + . In another specific embodiment, said selecting additionally 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 that is CD200 + and OCT-4 + .
  • the stem cell is CD73 + and CD105 + .
  • the stem cell is a placental stem cell.
  • said stem cell is HLA-G + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + and HLA-G + .
  • the stem cell facilitates the production of one or more embryoid-like bodies by a population of placental cells that comprises the stem cell, when the population is cultured under conditions that allow the formation of embryoid-like bodies.
  • said placental stem cell is isolated away from placental cells that are not stem cells.
  • said placental stem cell is isolated away from placental stem cells that do not display these markers.
  • the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a CD200 + and OCT-4 + placental cell, whereby said cell is a placental stem cell.
  • said selecting comprises selecting a placental cell that is also HLA-G + .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + and HLA-G + .
  • said selecting comprises selecting a placental stem cell that also facilitates the production of one or more embryoid-like bodies by a population of placental cells that comprises the stem cell, when the population is cultured under conditions that allow the formation of embryoid-like bodies.
  • the invention also provides an isolated population of cells comprising, e.g., that is enriched for, CD200 + , OCT-4 + stem cells.
  • CD200 + , OCT-4 + stem cells 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 CD200 + , OCT-4 + stem cells.
  • at least about 70% of said cells are said CD200 + , OCT-4 + stem cells.
  • at least about 90%, 95%, or 99% of said cells are said CD200 + , OCT-4 + stem cells.
  • said stem cells are CD73 + and CD105 + .
  • said stem cells are HLA-G + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + and HLA-G + .
  • the population produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • said population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention also provides a method of selecting a placental stem cell population from a plurality of placental cells, comprising selecting a population of placental cells wherein 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 cells are CD200 + , OCT-4 + stem cells.
  • said selecting comprises selecting stem cells that are also CD73 + and CD105 + .
  • said selecting comprises selecting stem cells that are also HLA-G + .
  • said selecting comprises selecting stem cells that are also CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , CD73 + , CD105 + and HLA-G + .
  • the invention further provides an isolated stem cell that is CD73 + , CD105+ and HLA-G + .
  • the stem cell is a placental stem cell.
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is OCT-4 + .
  • said stem cell is CD200 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , OCT-4 + and CD200 + .
  • said stem cell facilitates the formation of embryoid-like bodies in a population of placental cells comprising said stem cell, when the population is cultured under conditions that allow the formation of embryoid-like bodies.
  • said placental stem cell is isolated away from placental cells that are not stem cells.
  • said placental stem cell is isolated away from placental stem cells that do not display these characteristics.
  • the invention also provides a method of selecting a placental stem cell from a plurality of placental cells, comprising selecting a CD73 + , CD105 + and HLA-G+placental cell, whereby said cell is a placental stem cell.
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said selecting comprises selecting a placental cell that is also OCT-4 + .
  • said selecting comprises selecting a placental cell that is also CD200 + .
  • said selecting comprises selecting a placental cell that is also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , OCT-4 + and CD200 + .
  • said selecting comprises selecting a placental cell that also facilitates the formation of one or more embryoid-like bodies in a population of placental cells that comprises said stem cell, when said population is culture under conditions that allow the formation of embryoid-like bodies.
  • the invention also provides an isolated population of cells comprising, e.g., that is enriched for, CD73 + , CD105 + and HLA-G + stem cells.
  • said stem cells are placental stem cells.
  • 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 CD73 + , CD105 + and HLA-G + stem cells.
  • at least about 70% of said cells are CD73 + , CD105 + and HLA-G + .
  • at least about 90%, 95% or 99% of said cells are CD73 + , CD105 + and HLA-G + stem cells.
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are OCT-4 + .
  • said stem cells are CD200 + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , OCT-4 + and CD200 + .
  • said population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention also provides a method of selecting a placental stem cell population from a plurality of placental cells, comprising selecting a population of placental cells wherein a majority of said cells are CD73 + , CD105 + and HLA-G + .
  • said majority of cells are also CD34 ⁇ , CD38 ⁇ and/or CD45 ⁇ .
  • said majority of cells are also CD200 + .
  • said majority of cells are also CD34 ⁇ , CD38 ⁇ , CD45 ⁇ , OCT-4 + and CD200 + .
  • the invention provides an isolated stem cell that 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 formation of embryoid-like bodies.
  • said stem cell is CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cell is CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cell is OCT4 + .
  • said stem cell is OCT4 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said placental stem cell is isolated away from placental cells that are not stem cells.
  • said placental stem cell is isolated away from placental stem cells that do not display these characteristics.
  • the invention further provides a population of isolated placental cells comprising, e.g., that is enriched for, CD73 + , CD105 + stem cells, wherein said population forms one or more embryoid-like bodies under conditions that allow formation of embryoid-like bodies.
  • 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.
  • said stem cells are CD34 ⁇ , CD38 ⁇ or CD45 ⁇ .
  • said stem cells are CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said stem cells are OCT-4 + .
  • said stem cells are OCT-4 + , CD34 ⁇ , CD38 ⁇ and CD45 ⁇ .
  • said population has been expanded, for example, has been passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
  • said population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention further provides an isolated stem cell that is OCT-4 + and which facilitates 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 formation of embryoid-like bodies.
  • said stem cell is CD73 + and CD105 + .
  • said stem cell is CD34 ⁇ , CD38 ⁇ , or CD45 ⁇ .
  • said stem cell is CD200 + .
  • said stem cell is CD73 + , CD105 + , CD200 + , CD34 ⁇ , CD38 ⁇ , and CD45 ⁇ .
  • said placental stem cell is isolated away from placental cells that are not stem cells.
  • said placental stem cell is isolated away from placental stem cells that do not display these characteristics.
  • the invention also provides a population of isolated cells comprising, e.g., that is enriched for, OCT-4 + stem cells, wherein said population forms one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
  • 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 OCT4 + stem cells.
  • said stem cells are CD73 + and CD105 + .
  • said stem cells are CD34 ⁇ , CD38 ⁇ , or CD45 ⁇ .
  • said stem cells are CD200 + .
  • said stem cells are CD73 + , CD105 + , CD200 + , CD34 ⁇ , CD38 ⁇ , and CD45 ⁇ .
  • said population has been expanded, for example, passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
  • said population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • 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 + .
  • said stem cells are additionally CD90 + and CD45 ⁇ .
  • said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said stem cell or population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • said isolated placental stem cell is non-maternal in origin.
  • 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.
  • 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 ⁇ .
  • said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • 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 ⁇ comprising isolating said cell from placental perfusate.
  • 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 ⁇ .
  • said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said isolated placental stem cell is non-maternal in origin.
  • 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.
  • said stem cell or population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention provides a method of obtaining a placental stem cell that is CD10 + , CD13 + , CD33 + , CD45 ⁇ , CD117 ⁇ and CD133 ⁇ comprising isolating said cell from placental perfusate.
  • 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 ⁇ .
  • said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said isolated placental stem cell is non-maternal in origin.
  • At least about 90%, at least about 95%, or at least 99% of said cells in said isolated population of placental stem cells are non-maternal in origin.
  • said stem cell or population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention provides a method of obtaining a placental stem cell that is CD10 ⁇ , CD33 ⁇ , CD44 + ; CD45 ⁇ , CD117 ⁇ comprising isolating said cell from placental perfusate.
  • 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 ⁇ .
  • said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.
  • said isolated placental stem cell is non-maternal in origin.
  • At least about 90%, at least about 95%, or at least 99% of said cells in said isolated population of placental stem cells are non-maternal in origin.
  • said stem cell or population of placental stem cells is isolated away from placental stem cells that do not display these characteristics.
  • the invention provides a method of obtaining a placental stem cell that is CD10 ⁇ , CD13 ⁇ , CD33 ⁇ , CD45 ⁇ , and CD117 + comprising isolating said cell from placental perfusate.
  • the invention provides an isolated placental stem cell that 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 about 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.
  • said stem cell or population of placental stem cells is isolated away from placental cells that are not stem cells.
  • 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 display these characteristics.
  • 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 ⁇ , comprising isolating said cell from placental perfusate.
  • the invention provides a placental stem cell that is CD200 + and CD10 + , as determined by antibody binding, and CD117 ⁇ , as determined by both antibody binding and RT-PCR.
  • the invention provides a placental stem cell that is CD10 + , CD29 ⁇ , CD54 + , CD200 + , HLA-G + , HLA class I ⁇ and ⁇ -2-microglobulin ⁇ .
  • the invention provides placental stem cells, wherein the expression of at least one marker is at least two-fold higher than for a mesenchymal stem cell (e.g., a bone marrow-derived mesenchymal stem cell).
  • 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 99%, of said cells in said isolated population of placental stem cells, are non-maternal in origin.
  • 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 assessed by an aldehyde dehydrogenase activity assay.
  • ALDH aldehyde dehydrogenase
  • said ALDH assay uses ALDEFLUOR® (Aldagen, Inc., Ashland, Oreg.) as a marker of aldehyde dehydrogenase activity.
  • said plurality is between about 3% and about 25% of cells in said population of cells.
  • 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 assessed by an aldehyde dehydrogenase activity assay that uses ALDEFLUOR® as an indicator of aldehyde dehydrogenase activity.
  • said plurality is between about 3% and about 25% of cells in said population of cells.
  • said population of placental stem cells or umbilical cord stem cells shows at least three-fold, or at least five-fold, higher ALDH activity than a population of bone marrow-derived mesenchymal stem cells having the same number of cells and cultured under the same conditions.
  • the invention provides any of the above placental stem cells, or populations of placental stem cells, wherein the stem cell or population of placental stem cells has been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more, or expanded for 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.
  • the karyotype of the cells, or at least about 95% or about 99% of the cells in said population is normal.
  • the cells, or cells in the population of cells are non-maternal in origin.
  • Isolated placental stem cells, or isolated populations of placental stem cells, bearing 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 two or more of the above placental stem cell populations to form a placental stem cell population.
  • 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 about 99:1.
  • any three, four, five or more of the above-described placental stem cells or placental stem cell populations can be combined.
  • the invention further provides placental stem cells that 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).
  • the invention provides an isolated population of placental stem cells that is produced according to a method comprising perfusing a mammalian placenta that has been drained of cord blood and perfused to remove residual blood; perfusing said placenta with a perfusion solution; and collecting said perfusion solution, wherein said perfusion solution after perfusion comprises a population of placental cells that comprises placental stem cells; and isolating a plurality of said placental stem cells from said population of cells.
  • the perfusion solution is passed through both the umbilical vein and umbilical arteries and collected after it exudes from the placenta.
  • Populations of placental stem cells produced by this method typically comprise a mixture of fetal and maternal cells.
  • 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.
  • Populations of placental stem cells produced by this method typically are substantially exclusively fetal in origin; that is, e.g., greater than 90%, 95%, 99%, or 99.5% of the placental stem cells in the population are fetal in origin.
  • the 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 at least 99.5% of said population of placental cells.
  • the placental stem cells collected by perfusion comprise fetal and maternal cells.
  • the placental stem cells collected by perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% fetal cells.
  • the invention provides a composition comprising a population of isolated placental stem cells collected 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 the placental stem cells described herein that is produced according to a method comprising digesting placental tissue with a tissue-disrupting enzyme to obtain a population of placental cells comprising placental stem cells, and isolating a plurality of placental stem cells from the remainder of said placental cells.
  • the whole, or any part of, the placenta can be digested to obtain placental stem cells.
  • said placental tissue is a whole placenta, an amniotic membrane, chorion, a combination of amnion and chorion, or a combination of any of the foregoing.
  • the tissue-disrupting enzyme is trypsin or collagenase.
  • 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 at least 99.5% of said population of placental cells.
  • Gene profiling confirms that isolated placental stem cells, and populations of isolated placental stem cells, are distinguishable from other cells, e.g., mesenchymal stem cells, e.g., bone marrow-derived stem cells.
  • mesenchymal stem cells e.g., bone marrow-derived stem cells.
  • the placental stem cells described herein can be distinguished from mesenchymal 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 in comparison to bone marrow-derived mesenchymal stem cells.
  • placental stem cells can be distinguished from mesenchymal stem cells on the basis of the expression of one or more gene, the expression of which is significantly higher (that is, at least twofold higher) in placental stem cells than in mesenchymal stem cells, wherein the one or more gene is(are) ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2 ⁇ l, 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, T
  • 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 placental stem cells, the identity of which is confirmed can be clonal, e.g., a population of placental stem cells expanded form a single placental stem cell, or a mixed population of stem cells, e.g., a population of cells comprising solely placental stem cells that are expanded 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 placental stem cells. For example, a population of cells, e.g., clonally-expanded cells, is selected if the expression of one or more of these genes is significantly higher in a sample from the population of cells than in an equivalent population of mesenchymal stem cells. Such selecting can be of a population from a plurality of placental stem cells populations, from a plurality of cell populations, the identity of which is not known, etc.
  • Placental stem cells can be selected on the basis of the level of expression of one or more such genes as compared to the level of expression in said one or more genes in a mesenchymal stem cell control.
  • the level of expression of said one or more genes in a sample comprising an equivalent number of mesenchymal stem cells is used as a control.
  • the control, for placental stem cells tested under certain conditions is a numeric value representing the level of expression of said one or more genes in mesenchymal stem cells under said conditions.
  • the placental stem cells of the invention display 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% DMEM-LG (Gibco), 40% MCDB-201(Sigma), 2% fetal calf serum (FCS) (Hyclone Laboratories), 1 ⁇ insulin-transferrin-selenium (ITS), 1 ⁇ lenolenic-acid-bovine-serum-albumin (LA-BSA), 10 ⁇ 9 M dexamethasone (Sigma), 10 ⁇ 4 M ascorbic acid 2-phosphate (Sigma), epidermal growth factor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor (PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 U streptomycin.
  • FCS fetal calf serum
  • ITS insulin-transferrin-selenium
  • LA-BSA 1 ⁇
  • the isolated populations of placental stem cells described above, and populations of placental stem cells generally, can comprise about, at least, or no more than, 1 ⁇ 10 5 , 5 ⁇ 10 5 , 1 ⁇ 10 6 , 5 ⁇ 10 6 , 1 ⁇ 10 7 , 5 ⁇ 10 7 , 1 ⁇ 10 8 , 5 ⁇ 10 8 , 1 ⁇ 10 9 , 5 ⁇ 10 9 , 1 ⁇ 10 10 , 5 ⁇ 10 10 , 1 ⁇ 10 11 or more placental stem cells.
  • placental stem cells The growth of the placental stem cells described herein, as for any mammalian cell, depends in part upon the particular medium selected for growth. Under optimum conditions, placental stem cells typically double in number in 3-5 days.
  • the placental stem cells of the invention adhere to a substrate in culture, e.g. the surface of a tissue culture container (e.g., tissue culture dish plastic, fibronectin-coated plastic, and the like) and form a monolayer.
  • tissue culture container e.g., tissue culture dish plastic, fibronectin-coated plastic, and the like
  • embryoid-like bodies that is, three-dimensional clusters of cells grow atop the adherent stem cell layer.
  • Cells within the embryoid-like bodies express markers associated with very early stem cells, e.g., OCT-4, Nanog, SSEA3 and SSEA4.
  • Cells within the embryoid-like bodies are typically not adherent to the culture substrate, as are the placental stem cells described herein, but remain attached to the adherent cells during culture.
  • Embryoid-like body cells are dependent upon the adherent placental stem cells for viability, as embryoid-like bodies do not form in the absence of the adherent stem cells.
  • the adherent placental stem cells thus facilitate the growth of one or more embryoid-like bodies in a population of placental cells that comprise the adherent placental stem cells.
  • the cells of the embryoid-like bodies are thought to grow on the adherent placental stem cells much as embryonic stem cells grow on a feeder layer of cells.
  • Mesenchymal stem cells e.g., bone marrow-derived mesenchymal stem cells, do not develop embryoid-like bodies in culture.
  • the present invention further provides methods of collecting and isolating placental stem cells.
  • stem cells are obtained from a mammalian placenta using a physiologically-acceptable solution, e.g., a stem cell collection composition.
  • a stem cell collection composition is described in detail in related U.S. Provisional Application No. 60/754,969, entitled “Improved Medium for Collecting Placental Stem Cells and Preserving Organs,” filed on Dec. 29, 2005.
  • the stem cell collection composition can comprise any physiologically-acceptable solution suitable for the collection and/or culture of stem cells, for example, a saline solution (e.g., phosphate-buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.
  • a saline solution e.g., phosphate-buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl. etc.
  • a culture medium e.g., DMEM, H.DMEM, etc.
  • the stem cell collection composition can comprise one or more components that tend to preserve placental stem cells, that is, prevent the placental stem cells from dying, 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 culturing.
  • Such components can be, e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF- ⁇ inhibitor; and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromid
  • the stem cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, an RNase, or a DNase, or the like.
  • tissue-degrading enzymes include, but are not limited to, collagenases (e.g., collagenase I, II, III or IV, a collagenase from Clostridium histolyticum , etc.); dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.
  • the stem cell collection composition can comprise a bacteriocidally or bacteriostatically effective amount of an antibiotic.
  • 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 (e.g., ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc.
  • the antibiotic is active against Gram(+) and/or Gram( ⁇ ) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus , and the like.
  • the stem cell collection composition can also comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM 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 cellular viability (e.g., a synthetic or naturally occurring colloid, a polysaccharide such as dextran or a polyethylene glycol present at about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 ⁇ M to about 100 ⁇ M); a reducing agent (e.g., N-acetylcysteine present at about 0.1
  • a human placenta is recovered shortly after its expulsion after birth.
  • the placenta is recovered from a patient after informed consent and after a complete medical history of the patient is taken and is associated with the placenta.
  • the medical history continues after delivery.
  • Such a medical history can be used to coordinate subsequent use of the placenta or the stem cells harvested therefrom.
  • human placental stem cells can be used, in light of the medical history, for personalized medicine for the infant associated with the placenta, or for parents, siblings or other relatives of the infant.
  • the umbilical cord blood and placental blood Prior to recovery of placental stem cells, the umbilical cord blood and placental blood are removed. In certain embodiments, after delivery, the cord blood in the placenta is recovered.
  • the placenta can be subjected to a conventional cord blood recovery process.
  • a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665).
  • the needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to aid in draining cord blood from the placenta.
  • cord blood recovery may be performed commercially, e.g., LifeBank USA, Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell.
  • the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.
  • a placenta is transported from the delivery or birthing room to another location, e.g., a laboratory, for recovery of cord blood and collection of stem cells by, e.g., perfusion or tissue dissociation.
  • the placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28° C.), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed in an insulated container.
  • the placenta is transported in a cord blood collection kit substantially as described in pending U.S. Pat. No. 7,147,626.
  • the placenta is delivered to the laboratory four to twenty-four hours following delivery.
  • the proximal umbilical cord is clamped, preferably within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery.
  • the proximal umbilical cord is clamped after cord blood recovery but prior to further processing of the placenta.
  • the placenta prior to stem cell collection, can be stored under sterile conditions and at either room temperature or at a temperature of 5 to 25° C. (centigrade).
  • the placenta may be stored for a period of for a period of four to twenty-four hours, up to forty-eight hours, or longer than forty eight hours, prior to perfusing the placenta to remove any residual cord blood.
  • the placenta is harvested from between about zero hours to about two hours post-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 solution of heparin or warfarin sodium can be used.
  • the anticoagulant solution comprises a solution of heparin (e.g., 1% w/w in 1:1000 solution).
  • the exsanguinated placenta is preferably stored for no more than 36 hours before placental stem cells are collected.
  • the mammalian placenta or a part thereof, once collected and prepared generally as above, can be treated in any art-known manner, e.g., can be perfused or disrupted, e.g., digested with one or more tissue-disrupting enzymes, to obtain stem cells.
  • stem cells are collected from a mammalian placenta by physical disruption of part of all of the organ.
  • the placenta, or a portion thereof may be, e.g., crushed, sheared, minced, diced, chopped, macerated or the like.
  • the tissue can then be cultured to obtain a population of stem cells.
  • the placental tissue is disrupted using, e.g., in, a stem cell collection composition (see Section 5.2.1 and below).
  • Placenta can be dissected into components prior to physical disruption and/or enzymatic digestion and stem cell recovery.
  • Placental stem cells can be obtained from all or a portion of the amniotic membrane, chorion, umbilical cord, placental cotyledons, or any combination thereof, including from a whole placenta.
  • placental stem cells are obtained from placental tissue comprising amnion and chorion.
  • placental stem cells can be obtained by disruption of a small block of placental tissue, e.g., a block of placental tissue that is about 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, provided that the method of disruption 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 organ viable, as determined by, e.g., trypan blue exclusion.
  • Stem cells can generally be collected from a placenta, or portion thereof, at any time within about the first three days post-expulsion, but preferably between about 8 hours and about 18 hours post-expulsion.
  • the disrupted tissue is cultured in tissue culture medium suitable for the proliferation of placental stem cells (see, e.g., Section 5.3, below, describing the culture of placental stem cells).
  • stem cells are collected by physical disruption of placental tissue, wherein the physical disruption includes enzymatic digestion, which can be accomplished by use of one or more tissue-digesting enzymes.
  • the placenta, or a portion thereof, may also be physically disrupted and digested with one or more enzymes, and the resulting material then immersed in, or mixed into, a stem cell collection composition.
  • a preferred stem cell collection composition comprises one or more tissue-disruptive enzyme(s).
  • Enzymatic digestion preferably uses a combination of enzymes, e.g., a combination of a matrix metalloprotease and a neutral protease, for example, a combination of collagenase and dispase.
  • 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, dispase, and hyaluronidase or a combination of LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase.
  • enzymes that can be used to disrupt placenta tissue include papain, deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, or elastase.
  • Serine proteases may be inhibited by alpha 2 microglobulin in serum and therefore the medium used for digestion is usually serum-free.
  • EDTA and DNase are commonly used in enzyme digestion procedures to increase the efficiency of cell recovery.
  • the digestate is preferably diluted so as to avoid trapping stem cells within the viscous digest.
  • tissue digestion enzymes can be used. Typical concentrations for tissue digestion enzymes include, e.g., 50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100 U/mL for elastase.
  • Proteases can be used in combination, that is, two or more proteases in the same digestion reaction, or can be used sequentially in order to liberate placental stem cells.
  • a placenta is digested first with an appropriate amount of collagenase I at about 1 to about 2 mg/ml for, e.g., 30 minutes, followed by digestion with trypsin, at a concentration of about 0.25%, for, e.g., 10 minutes, at 37° C.
  • Serine proteases are preferably used consecutively following use of other enzymes.
  • the tissue can further be disrupted by the addition of a chelator, e.g., ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA) to the stem cell collection composition comprising the stem cells, or to a solution in which the tissue is disrupted and/or digested prior to isolation of the stem cells with the stem cell collection composition.
  • a chelator e.g., ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA)
  • a digestion can proceed as follows. Approximately a gram of placental tissue is obtained and minced. The tissue is digested in 10 mL of a solution comprising about 1 mg/mL collagenase 1A and about 0.25% trypsin at 37° C. in a shaker at about 100 RPM. The digestate is washed three times with culture medium, and the washed cells are seeded into 2 T-75 flasks. The cells are then isolated by differential adherence, and characterized for, e.g., viability, cell surface markers, differentiation, and the like.
  • the placental stem cells collected will comprise a mix of placental stem cells derived from both fetal and maternal sources.
  • the placental stem cells collected will comprise almost exclusively fetal placental stem cells.
  • Stem cells can be isolated from disrupted tissue by differential trypsinization (see Section 5.2.5, below) followed by culture in one or more new culture containers in fresh proliferation medium, optionally followed by a second differential trypsinization step.
  • Placental stem cells can also be obtained by perfusion of the mammalian placenta.
  • Methods of perfusing mammalian placenta to obtain stem cells are disclosed, e.g., in Hariri, U.S. Application Publication No. 2002/0123141, and in related U.S. Provisional Application No. 60/754,969, entitled “Improved Medium for Collecting Placental Stem Cells and Preserving Organs,” filed on Dec. 29, 2005.
  • Placental stem cells can be collected by perfusion, e.g., through the placental vasculature, using, e.g., a stem cell collection composition as a perfusion solution.
  • a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein.
  • the flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta.
  • the perfusion solution is forced through the placenta using a pump, e.g., a peristaltic pump.
  • the umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected to a sterile connection apparatus, such as sterile tubing.
  • a sterile connection apparatus such as sterile tubing.
  • the sterile connection apparatus is connected to a perfusion manifold.
  • the placenta In preparation for perfusion, the placenta is preferably oriented (e.g., suspended) in such a manner that the umbilical artery and umbilical vein are located at the highest point of the placenta.
  • the placenta can be perfused by passage of a perfusion fluid through the placental vasculature and surrounding tissue.
  • the placenta can also be perfused by passage of a perfusion fluid into the umbilical vein and collection from the umbilical arteries, or passage of a perfusion fluid into the umbilical arteries and collection from the umbilical vein.
  • the umbilical artery and the umbilical vein are connected simultaneously, e.g., to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution.
  • the perfusion solution is passed into the umbilical vein and artery.
  • the perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.
  • the perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall.
  • Placental cells that are collected by this method which can be referred to as a “pan” method, are typically a mixture of fetal and maternal cells.
  • the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is 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 circuit” method, are typically almost exclusively fetal.
  • perfusion using the pan method that is, whereby perfusate is collected after it has exuded from the maternal side of the placenta, results in a mix of fetal and maternal cells.
  • the cells collected by this method comprise a mixed population of placental stem cells of both fetal and maternal origin.
  • perfusion solely through the placental vasculature in the closed circuit method whereby perfusion fluid is passed through one or two placental vessels and is collected solely through the remaining vessel(s), results in the collection of a population of placental stem cells almost exclusively of fetal origin.
  • the closed circuit perfusion method can, in one embodiment, be performed as follows.
  • a post-partum placenta is obtained within about 48 hours after birth.
  • the umbilical cord is clamped and cut above the clamp.
  • the umbilical cord can be discarded, or can processed to recover, e.g., umbilical cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial.
  • the amniotic membrane can be retained during perfusion, or can be separated from the chorion, e.g., using blunt dissection with the fingers.
  • amniotic membrane is separated from the chorion prior to perfusion, it can be, e.g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g., the biomaterial described in U.S. Application Publication No. 2004/0048796.
  • an amniotic membrane biomaterial e.g., the biomaterial described in U.S. Application Publication No. 2004/0048796.
  • the umbilical cord vessels are exposed, e.g., by partially cutting the umbilical cord membrane to expose a cross-section of the cord.
  • the vessels are identified, and opened, e.g., by advancing a closed alligator clamp through the cut end of each vessel.
  • the apparatus e.g., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries.
  • the pump can be any pump suitable for the purpose, e.g., a peristaltic pump.
  • Plastic tubing, connected to a sterile collection reservoir, e.g., a blood bag such as a 250 mL collection bag, is then inserted into the placental vein.
  • the tubing connected to the pump is inserted into the placental vein, and tubes to a collection reservoir(s) are inserted into one or both of the placental arteries.
  • the placenta is then perfused with a volume of perfusion solution, e.g., about 750 ml of perfusion solution. Cells in the perfusate are then collected, e.g., by centrifugation.
  • the proximal umbilical cord is clamped during perfusion, and more preferably, is clamped within 4-5 cm (centimeter) of the cord's insertion into the placental disc.
  • the first collection of perfusion fluid from a mammalian placenta during the exsanguination process is generally colored with residual red blood cells of the cord blood and/or placental blood.
  • the perfusion fluid becomes more colorless as perfusion proceeds and the residual cord blood cells are washed out of the placenta.
  • 30 to 100 ml (milliliter) of perfusion fluid is adequate to initially exsanguinate the placenta, but more or less perfusion fluid may be used depending on the observed results.
  • the volume of perfusion liquid used to collect placental stem cells may vary depending upon the number of stem cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc.
  • the volume of perfusion liquid may 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.
  • the placenta is perfused with 700-800 mL of perfusion liquid following exsanguination.
  • the placenta can be perfused a plurality of times over the course of several hours or several days. Where the placenta is to be perfused a plurality of times, it may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and perfused with the stem cell collection 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, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., ⁇ -mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 ⁇ g/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 ⁇ g/ml).
  • a standard perfusion solution e.g., a
  • an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta is maintained or cultured for 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 collection of perfusate.
  • the perfused placenta can be maintained for one or more additional time(s), e.g., 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 perfused a second time with, e.g., 700-800 mL perfusion fluid.
  • the placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours.
  • perfusion of the placenta and collection of perfusion solution e.g., stem cell collection composition
  • perfusion of the placenta and collection of perfusion solution is repeated until the number of recovered nucleated cells falls below 100 cells/ml.
  • the perfusates at different time points can be further processed individually to recover time-dependent populations of cells, e.g., stem cells. Perfusates from different time points can also be pooled.
  • stem cells are collected at a time or times between about 8 hours and about 18 hours post-expulsion.
  • placental stem cells are believed to migrate into the exsanguinated and perfused microcirculation of the placenta where, according to the methods of the invention, they are collected, preferably by washing into a collecting vessel by perfusion.
  • Perfusing the isolated placenta not only serves to remove residual cord blood but also provide the placenta with the appropriate nutrients, including oxygen.
  • the placenta may be cultivated and perfused with a similar solution which was used to remove the residual cord blood cells, 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 perfused with said solution, and not otherwise treated to obtain stem cells (e.g., by tissue disruption, e.g., enzymatic digestion).
  • stem cells e.g., by tissue disruption, e.g., enzymatic digestion.
  • “significantly more” means at least 10% more.
  • Perfusion according to the methods of the invention yields significantly more placental stem cells than, e.g., the number of placental stem cells obtainable from culture medium in which a placenta, or portion thereof, has been cultured.
  • Stem cells can be isolated from placenta by perfusion with a solution comprising one or more proteases or other tissue-disruptive enzymes.
  • a placenta or portion thereof e.g., amniotic membrane, amnion and chorion, placental lobule or cotyledon, umbilical cord, or combination of any of the foregoing
  • tissue-disruptive enzymes in 200 mL of a culture medium for 30-minutes.
  • Stem cells from mammalian placenta can initially be purified from (i.e., be isolated from) other cells by Ficoll gradient centrifugation. Such centrifugation can follow any standard protocol for centrifugation speed, etc. In one embodiment, for example, cells collected from the placenta are recovered from perfusate by centrifugation at 5000 ⁇ g for 15 minutes at room temperature, which separates cells from, e.g., contaminating debris and platelets.
  • placental perfusate is concentrated to about 200 ml, gently layered over Ficoll, and centrifuged at about 1100 ⁇ g for 20 minutes at 22° C., and the low-density interface layer of cells is collected for further processing.
  • Cell pellets can be resuspended in fresh stem cell collection composition, or a medium suitable for stem cell maintenance, e.g., IMDM serum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY).
  • IMDM serum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY).
  • the total mononuclear cell fraction can be isolated, e.g., using Lymphoprep (Nycomed Pharma, Oslo, Norway) according to the manufacturer's recommended procedure.
  • isolated placental stem cells means to remove 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 from an organ is “isolated” when it is present in a population of cells that comprises fewer than 50% of the cells with which the stem cell is normally associated in the intact organ.
  • Placental cells obtained by perfusion or digestion can, for example, be further, or initially, isolated by differential trypsinization using, e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.). Differential trypsinization is possible because placental stem cells typically detach from plastic surfaces within about five minutes whereas other adherent populations typically require more than 20-30 minutes incubation.
  • the detached placental stem cells can be harvested following trypsinization and trypsin neutralization, using, e.g., Trypsin Neutralizing Solution (TNS, Cambrex).
  • aliquots of, for example, about 5-10 ⁇ 10 6 cells are placed in each of several T-75 flasks, preferably fibronectin-coated T75 flasks.
  • 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% CO 2 ). After 10 to 15 days, non-adherent cells are removed from the flasks by washing with PBS. The PBS is then replaced by MSCGM. Flasks are preferably examined daily for the presence of various adherent cell types and in particular, for identification and expansion of clusters of fibroblastoid cells.
  • MSCGM Mesenchymal Stem Cell Growth Medium
  • the number and type of cells collected from a mammalian placenta can be monitored, for example, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue specific or cell-marker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling. These techniques can be used, too, to identify cells that are positive for one or more particular markers.
  • standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue specific or cell-marker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in
  • a cell comprises a detectable amount of CD34; if so, the cell is CD34 + .
  • the cell is OCT-4 +
  • Antibodies to cell surface markers e.g., CD markers such as CD34
  • sequence of stem cell-specific genes such as OCT-4
  • Placental cells may be sorted using a fluorescence activated cell sorter (FACS).
  • Fluorescence activated cell sorting 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). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture.
  • cell surface marker-specific antibodies or ligands are labeled with distinct fluorescent labels. Cells are processed through the cell sorter, allowing separation of cells based on their ability to bind to the antibodies used.
  • FACS sorted particles may be directly deposited into individual wells of 96-well or 384-well plates to facilitate separation and cloning.
  • stem cells from placenta are sorted on the basis of expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4 and/or HLA-G. Ths can be accomplished in connection with procedures to select stem cells on the basis of their adherence properties in culture. For example, an adherence selection stem can be accomplished before or after sorting on the basis of marker expression. In one embodiment, for example, cells are sorted first on the basis of their expression of CD34; CD34 ⁇ cells are retained, and cells that are CD200 + HLA-G + , are separated from all other CD34 ⁇ cells.
  • cells from placenta are based on their expression of markers CD200 and/or HLA-G; for example, cells displaying either of these markers are isolated for further use.
  • Cells that express, e.g., CD200 and/or HLA-G can, in a specific embodiment, be further sorted 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.
  • placental cells are sorted 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 placental cells for further use.
  • any antibody specific for a particular marker, can be used, in combination with any fluorophore or other label suitable for the detection and sorting of cells (e.g., fluorescence-activated cell sorting).
  • Antibody/fluorophore combinations to specific markers include, but are not limited to, fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies against HLA-G (available from Serotec, Raleigh, N.C.), CD10 (available from BD Immunocytometry Systems, San Jose, Calif.), CD44 (available from BD Biosciences Pharmingen, San Jose, Calif.), and CD105 (available from R&D Systems Inc., Minneapolis, Minn.); phycoerythrin (PE) conjugated monoclonal antibodies against CD44, CD200, CD117, and CD 13 (BD Biosciences Pharmingen); phycoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibodies against CD33 and CD10 (BD Biosciences Pharmingen); allophycocyanin (APC) conjugated streptavidin and monoclonal antibodies against CD38 (BD Biosciences Pharmingen); and Biotinylated CD90 (BD Biosciences Pharmingen).
  • antibodies that can be used include, but are not limited to, CD133-APC (Miltenyi), KDR-Biotin (CD309, Abcam), CytokeratinK-Fitc (Sigma or Dako), HLA ABC-Fitc (BD), HLA DRDQDP-PE (BD), ⁇ -2-microglobulin-PE (BD), CD80-PE (BD) and CD86-APC (BD).
  • CD133-APC Miltenyi
  • KDR-Biotin CD309, Abcam
  • CytokeratinK-Fitc Sigma or Dako
  • HLA ABC-Fitc BD
  • HLA DRDQDP-PE HLA DRDQDP-PE
  • BD ⁇ -2-microglobulin-PE
  • CD80-PE CD80-PE
  • CD86-APC CD86-APC
  • antibody/label 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 the like.
  • Placental stem cells can be assayed for CD117 or CD133 using, for example, phycoerythrin-Cy5 (PE Cy5) conjugated streptavidin and biotin conjugated monoclonal antibodies against CD117 or CD133; however, using this system, the cells can appear to be positive for CD117 or CD133, respectively, because of a relatively high background.
  • phycoerythrin-Cy5 PE Cy5 conjugated streptavidin
  • biotin conjugated monoclonal antibodies against CD117 or CD133 the cells can appear to be positive for CD117 or CD133, respectively, because of a relatively high background.
  • Placental stem cells can be labeled with an antibody to a single marker and detected and/sorted. Placental stem cells can also be simultaneously labeled with multiple antibodies to different markers.
  • magnetic beads can be used to separate cells.
  • the cells may be sorted using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (0.5-100 ⁇ m diameter).
  • MCS magnetic activated cell sorting
  • a variety of useful modifications can be performed on 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. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation.
  • Placental stem cells can also be characterized and/or sorted based on cell morphology and growth characteristics. For example, placental stem cells can be characterized as having, and/or selected on the basis of, e.g., a fibroblastoid appearance in culture. Placental stem cells can also be characterized as having, and/or be selected, on the basis of their ability to form embryoid-like bodies. In one embodiment, for example, placental cells that are fibroblastoid 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, OCT-4 + placental cells that produce one or more embryoid-like bodies in culture are isolated from other placental cells.
  • 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 CULTTM medium (Stem Cell Technologies, Inc., Vancouver British Columbia)
  • Placental stem cells can be assessed for viability, proliferation potential, and longevity using standard techniques known in the art, such as trypan blue exclusion assay, fluorescein diacetate uptake assay, propidium iodide uptake assay (to assess viability); and thymidine uptake assay, MTT cell proliferation assay (to assess proliferation). Longevity may be determined by methods well known in the art, such as by determining the maximum number of population doubling in an extended culture.
  • Placental stem cells can also be separated from other placental cells using other techniques known in the art, e.g., selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; filtration; conventional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; electrophoresis; and the like.
  • other techniques known in the art e.g., selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; filtration; conventional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; electrophoresis; and the like.
  • Isolated placental stem cells, or placental stem cell population, or cells or placental tissue from which placental stem cells grow out can be used to initiate, or seed, cell cultures.
  • Cells are generally transferred to sterile tissue culture vessels either uncoated or coated with extracellular matrix or ligands such as laminin, collagen (e.g., native or denatured), gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane protein (e.g., MATRIGEL® (BD Discovery Labware, Bedford, Mass.)).
  • extracellular matrix or ligands such as laminin, collagen (e.g., 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 the culture of stem cells.
  • 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 (chick fibroblast basal medium) containing ITS (insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serum albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1, and penicillin/streptomycin; DMEM-HG (high glucose) comprising 10% fetal bovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM (Iscove's modified Dulbecco's medium) comprising 10% FBS, 10% horse serum, and hydrocortisone; M199 comprising 10% FBS, EGF, and heparin; ⁇ -MEM (
  • DMEM high or low glucose
  • Eagle's basal medium Eagle's basal medium
  • Ham's F10 medium F10
  • Ham's F-12 medium F12
  • Iscove's modified Dulbecco's medium Mesenchymal Stem Cell Growth Medium (MSCGM)
  • MSCGM Mesenchymal Stem Cell Growth Medium
  • Liebovitz's L-15 medium MCDB
  • DMEM/F 12 DMEM/F 12
  • RPMI 1640 advanced DMEM
  • DMEM/MCDB201 Sigma
  • CELL-GRO FREE CELL-GRO FREE
  • the culture medium can be supplemented with one or more components including, for example, serum (e.g., fetal bovine serum (FBS), preferably about 2-15% (v/v); equine (horse) serum (ES); human serum (HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one or more growth factors, for example, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (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 antimycotic agents to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination.
  • serum e
  • Placental stem cells can be cultured in standard tissue culture conditions, e.g., in tissue culture dishes or multiwell plates. Placental stem cells can also be cultured using a hanging drop method. In this method, placental stem cells are suspended at about 1 ⁇ 10 4 cells per mL in about 5 mL of medium, and one or more drops of the medium are placed on the inside of the lid of a tissue culture container, e.g., a 100 mL Petri dish. The drops can be, e.g., single drops, or multiple drops from, e.g., a multichannel pipetter. The lid is carefully inverted and placed on top of the bottom of the dish, which contains a volume of liquid, e.g., sterile PBS sufficient to maintain the moisture content in the dish atmosphere, and the stem cells are cultured.
  • a volume of liquid e.g., sterile PBS
  • the placental stem cells are cultured in the presence of a compound that acts to maintain an undifferentiated phenotype in the placental stem cell.
  • the compound is a substituted 3,4-dihydropyridimol[4,5-d]pyrimidine.
  • the compound is a compound having the following chemical structure: The compound can be contacted with a placental stem cell, or population of placental stem cells, at a concentration of, for example, between about 1 ⁇ M to about 10 ⁇ M.
  • an isolated placental stem cell, or isolated population of stem cells e.g., a stem cell or population of stem cells separated from at least 50% of the placental cells with which the stem cell or population of stem cells is normally associated in vivo
  • the stem cell or population of stem cells can be proliferated and expanded in vitro.
  • a population of placental stem cells can be cultured in tissue culture containers, e.g., dishes, flasks, multiwell plates, or the like, for a sufficient time for the stem cells to proliferate to 70-90% confluence, that is, until the stem cells and their progeny occupy 70-90% of the culturing surface area of the tissue culture container.
  • Placental stem cells can be seeded in culture vessels at a density that allows cell growth.
  • the cells may be seeded at low density (e.g., about 1,000 to about 5,000 cells/cm 2 ) to high density (e.g., about 50,000 or more cells/cm 2 ).
  • the cells are cultured at about 0 to about 5 percent by volume CO 2 in air.
  • the cells are cultured at about 2 to about 25 percent O 2 in air, preferably about 5 to about 20 percent O 2 in air.
  • the cells preferably are 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 agitated, for example, using a bioreactor.
  • Placental stem cells preferably are grown under low oxidative stress (e.g., with addition of glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, or the like).
  • the cells may be passaged.
  • the cells can be enzymatically treated, e.g., trypsinized, using techniques well-known in the art, to separate them from the tissue culture surface.
  • about 20,000-100,000 stem cells preferably about 50,000 stem cells, are passaged to a new culture container containing fresh culture medium.
  • the new medium is the same type of medium from which the stem cells were removed.
  • the invention encompasses populations of placental stem cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more.
  • Placental stem cell population can be isolated directly from one or more placentas; that is, the placental stem cell population can be a population of placental cells comprising placental stem cells obtained from, or contained within, perfusate, or obtained from, or contained within, disrupted placental tissue, e.g., placental tissue digestate (that is, the collection of cells obtained by enzymatic digestion of a placenta or part thereof).
  • Isolated placental stem cells of the invention can also be cultured and expanded to produce placental stem cell populations.
  • Populations of placental cells comprising placental stem cells can also be cultured and expanded to produce placental stem cell populations.
  • Placental stem cell populations of the invention comprise placental stem cells, for example, placental stem cells as described herein. In various embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in an isolated placental stem cell population are placental stem cells. That is, a placental stem cell population can comprise, e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% non-stem cells.
  • the invention provides methods of producing isolated placental stem cell population by, e.g., selecting placental stem cells, whether derived from enzymatic digestion or perfusion, that express particular markers and/or particular culture or morphological characteristics.
  • the invention provides a method of producing a cell population comprising selecting placental cells that (a) adhere to a substrate, and (b) express CD200 and HLA-G; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population comprising identifying placental cells that express CD200 and HLA-G, and isolating said cells from other cells to form a cell population.
  • the method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, and (b) express CD73, CD105, and CD200; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population comprising identifying placental cells that express CD73, CD105, and CD200, and isolating said cells from other cells to form a cell population.
  • the method of producing a cell population comprises selecting placental 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.
  • the invention provides a method of producing a cell population comprising identifying placental cells that express CD200 and OCT-4, and isolating said cells from other cells to form a cell population.
  • 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 for the formation of an embryoid-like body; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population comprising identifying placental cells that express 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 for the formation of an embryoid-like body, and isolating said cells from other cells to form a cell population.
  • the method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, and (b) express CD73, CD105 and HLA-G; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population comprising identifying placental cells that express CD73, CD105 and HLA-G, and isolating said cells from other cells to form a cell population.
  • the method of producing a cell population comprises selecting placental cells that (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 for the formation of an embryoid-like body; and isolating said cells from other cells to form a cell population.
  • the invention provides a method of producing a cell population comprising identifying placental cells that express OCT-4, 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 for the formation of an embryoid-like body, and isolating said cells from other cells to form a cell population.
  • Such cell populations can be used to treat any of the diseases or conditions listed hereinbelow. Such cell populations can also be used to assess populations of placental stem cells, e.g., as part of a quality control method.
  • the method can additionally comprise selecting placental cells that express ABC-p (a placenta-specific ABC transporter protein; see, e.g., Allikmets et al., Cancer Res. 58(23):5337-9 (1998)).
  • the method can also comprise selecting cells exhibiting at least one characteristic specific to, e.g., a mesenchymal stem cell, for example, expression of CD29, expression of CD44, expression of CD90, or expression of a combination of the foregoing.
  • the substrate can be any surface on which culture and/or selection of cells, e.g., placental stem cells, can be accomplished.
  • the substrate is plastic, e.g., tissue culture dish or multiwell plate plastic.
  • Tissue culture plastic can be coated with a biomolecule, e.g., laminin or fibronectin.
  • Cells e.g., placental stem cells
  • cells can be selected using an antibody or antibodies to one or more cell surface markers, for example, in flow cytometry or FACS. Selection can be accomplished using antibodies in conjunction with magnetic beads.
  • Antibodies that are specific for certain stem cell-related markers are known in the art. For example, antibodies to OCT-4 (Abcam, Cambridge, Mass.), CD200 (Abcam), HLA-G (Abcam), CD73 (BD Biosciences Pharmingen, San Diego, Calif.), CD105 (Abcam; BioDesign International, Saco, Me.), etc.
  • Antibodies to other markers are also available commercially, e.g., CD34, CD38 and CD45 are available from, e.g., StemCell Technologies or BioDesign International.
  • the isolated placental stem cell population can comprise placental cells that are not stem cells, or cells that are not placental cells.
  • Isolated placental stem cell populations can be combined with one or more populations of non-stem cells or non-placental cells.
  • an isolated population of placental stem cells can be combined with blood (e.g., placental blood or umbilical cord blood), blood-derived stem cells (e.g., stem cells derived from placental blood or umbilical cord blood), umbilical cord stem cells, populations of blood-derived nucleated cells, bone marrow-derived mesenchymal cells, bone-derived stem cell populations, crude bone marrow, adult (somatic) stem cells, populations of stem cells contained within tissue, cultured stem cells, populations of fully-differentiated cells (e.g., chondrocytes, fibroblasts, amniotic cells, osteoblasts, muscle cells, cardiac cells, etc.) and the like.
  • blood e.g., placental blood or umbilical cord blood
  • blood-derived stem cells e.g., stem cells derived from placental blood or umbilical cord blood
  • the invention provides a population of stem cells comprising placental stem cells and umbilical cord stem cells.
  • Cells in an isolated placental stem cell population can be combined with a plurality of cells of another type in ratios of about 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 about 1:100,000,000, comparing numbers of total nucleated cells in each population.
  • Cells in an isolated placental stem cell population can be
  • an isolated population of placental stem cells is combined with a plurality of hematopoietic stem cells.
  • hematopoietic stem cells can be, for example, contained within unprocessed placental, umbilical cord blood 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 unprocessed bone marrow; in total nucleated cells from bone marrow; in an isolated population of CD34 + cells from bone marrow, or the like.
  • Stem cells from postpartum placentas can be cultured in a number of different ways to produce a set of lots, e.g., a set of individually-administrable doses, of placental stem cells.
  • Such lots can, for example, be obtained from stem cells from placental perfusate or from enzyme-digested placental tissue.
  • Sets of lots of placental stem cells, obtained from a plurality of placentas can be arranged in a bank of placental stem cells for, e.g., long-term storage.
  • adherent stem cells are obtained from an initial culture of placental material to form a seed culture, which is expanded under controlled conditions to form populations of cells from approximately equivalent numbers of doublings.
  • Lots are preferably derived from the tissue of a single placenta, but can be derived from the tissue of a plurality of placentas.
  • stem cell lots are obtained as follows. Placental tissue is first disrupted, e.g., by mincing, digested with a suitable enzyme, e.g., collagenase (see Section 5.2.3, above).
  • the placental tissue preferably comprises, e.g., the entire amnion, entire chorion, or both, from a single placenta, but can comprise only a part of either the amnion or chorion.
  • the digested tissue is cultured, e.g., for about 1-3 weeks, preferably about 2 weeks. After removal of non-adherent cells, high-density colonies that form are collected, e.g., by trypsinization. These cells are collected and resuspended in a convenient volume of culture medium, and defined as Passage 0 cells.
  • Expansion cultures can be any arrangement of separate cell culture apparatuses, e.g., a Cell Factory by NUNCTM.
  • Cells in the Passage 0 culture can be subdivided to any degree so as to seed expansion cultures with, e.g., 1 ⁇ 10 3 , 2 ⁇ 10 3 , 3 ⁇ 10 3 , 4 ⁇ 10 3 , 5 ⁇ 10 3 , 6 ⁇ 10 3 , 7 ⁇ 10 3 , 8 ⁇ 10 3 , 9 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 4 , 2 ⁇ 10 4 , 3 ⁇ 10 4 , 4 ⁇ 10 4 , 5 ⁇ 10 4 , 6 ⁇ 10 4 , 7 ⁇ 10 4 , 8 ⁇ 10 4 , 9 ⁇ 10 4 , or 10 ⁇ 10 4 stem cells.
  • Passage 0 cells Preferably, from about 2 ⁇ 10 4 to about 3 ⁇ 10 4 Passage 0 cells are used to seed each expansion culture.
  • the number of expansion cultures can depend upon the number of Passage 0 cells, and may be greater or fewer in number depending upon 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, e.g., about 1 ⁇ 10 5 cells/cm 2 .
  • Cells can either be collected and cryopreserved at this point, or passaged into new expansion cultures as described above. Cells can be passaged, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times prior to use.
  • a record of the cumulative number of population doublings is preferably maintained during expansion culture(s).
  • the cells from a Passage 0 culture can be expanded for 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 doublings, or up to 60 doublings.
  • the number of population doublings, prior to dividing the population of cells into individual doses is between about 15 and about 30, preferably about 20 doublings.
  • the cells can be culture continuously throughout the expansion process, or can be frozen at one or more points during expansion.
  • Cells to be used for individual doses can be frozen, e.g., cryopreserved for later use.
  • Individual doses can comprise, e.g., about 1 million to about 100 million cells per ml, and can comprise between about 10 6 and about 10 9 cells in total.
  • Passage 0 cells are cultured for a first number of doublings, e.g., approximately 4 doublings, then frozen in a first cell bank.
  • Cells from the first cell bank are frozen and used to seed a second cell bank, the cells of which are expanded for a second number of doublings, e.g., about another eight doublings.
  • Cells at this stage are collected and frozen and used to seed new expansion cultures that are allowed to proceed for a third number of doublings, e.g., about eight additional doublings, bringing the cumulative number of cell doublings to about 20.
  • Cells at the intermediate points in passaging can be frozen in units of about 100,000 to about 10 million cells per ml, preferably about 1 million cells per ml for use in subsequent expansion culture. Cells at about 20 doublings 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 stem cell-containing composition.
  • the invention provides a method of making a placental stem cell bank, comprising: expanding primary culture placental stem cells from a human post-partum placenta for a first plurality of population doublings; cryopreserving said placental stem cells to form a Master Cell Bank; expanding a plurality of placental stem cells from the Master Cell Bank for a second plurality of population doublings; cryopreserving said placental stem cells to form a Working Cell Bank; expanding a plurality of placental stem cells from the Working Cell Bank for a third plurality of population doublings; and cryopreserving said placental stem cells in individual doses, wherein said individual doses collectively compose a placental stem cell bank.
  • the total number of population doublings is about 20.
  • said first plurality of population doublings is about four population doublings; said second plurality of population doublings is about eight population doublings; and said third plurality of population doublings is about eight population doublings.
  • said primary culture placental stem cells comprise placental stem cells from placental perfusate.
  • said primary culture placental stem cells comprise placental stem cells from digested placental tissue.
  • said primary culture placental stem cells comprise placental stem cells from placental perfusate and from digested placental tissue.
  • all of said placental stem cells in said placental stem cell primary culture are from the same placenta.
  • the method further comprises the step of selecting CD200 + or HLA-G+placental stem cells from said plurality of said placental stem cells from said Working Cell Bank to form individual doses.
  • said individual doses comprise from about 10 4 to about 10 5 placental stem cells.
  • said individual doses comprise from about 10 5 to about 10 6 placental stem cells.
  • said individual doses comprise from about 10 6 to about 10 7 placental stem cells.
  • said individual doses comprise from about 10 7 to about 10 8 placental stem cells.
  • the donor from which the placenta is obtained (e.g., the mother) is tested for at least one pathogen. If the mother tests positive for a tested pathogen, the entire lot from the placenta is discarded. Such testing can be performed at any time during production of placental stem cell lots, including before or after establishment of Passage 0 cells, or during expansion culture.
  • Pathogens for which the presence is tested can include, without limitation, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human immunodeficiency virus (types I and II), cytomegalovirus, herpesvirus, and the like.
  • Neuronal differentiation of placental stem cells can be accomplished, for example, by placing placental stem cells in cell culture conditions that induce differentiation into neurons.
  • a neurogenic medium comprises DMEM/20% FBS and 1 mM beta-mercaptoethanol; such medium can be replaced after culture for about 24 hours with medium consisting of DMEM and 1-10 mM betamercaptoethanol.
  • the cells are contacted with DMEM/2% DMSO/200 ⁇ M butylated hydroxyanisole.
  • the differentiation medium comprises serum-free DMEMIF-12, butylated hydroxyanisole, potassium chloride, insulin, forskolin, valproic acid, and hydrocortisone.
  • neuronal differentiation is accomplished by plating placental stem cells on laminin-coated plates in Neurobasal-A medium (Invitrogen, Carlsbad Calif.) containing B27 supplement and L-glutamine, optionally supplemented with bFGF and/or EGF. Placental stem cells can also be induced to neural differentiation by co-culture with neural cells, or culture in neuron-conditioned medium.
  • Neurobasal-A medium Invitrogen, Carlsbad Calif.
  • Placental stem cells can also be induced to neural differentiation by co-culture with neural cells, or culture in neuron-conditioned medium.
  • Neuronal differentiation can be assessed, e.g., by detection of neuron-like morphology (e.g., bipolar cells comprising extended processes) detection of the expression of e.g., nerve growth factor receptor and neurofilament heavy chain genes by RT-PCR; or detection of electrical activity, e.g., by patch-clamp.
  • a placental stem cell is considered to have differentiated into a neuronal cell when the cell displays one or more of these characteristics.
  • Adipogenic differentiation of placental stem cells can be accomplished, for example, by placing placental stem cells in cell culture conditions that induce differentiation into adipocytes.
  • a preferred adipogenic medium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum.
  • placental stem cells are fed Adipogenesis Induction Medium (Cambrex) and cultured for 3 days (at 37° C., 5% CO 2 ), followed by 1-3 days of culture in Adipogenesis Maintenance Medium (Cambrex). After 3-complete cycles of induction/maintenance, the cells are cultured for an additional 7 days in adipogenesis maintenance medium, replacing the medium every 2-3 days.
  • placental stem cells are cultured in medium comprising 1 ⁇ M dexamethasone, 0.2 mM indomethacin, 0.01 mg/ml insulin, 0.5 mM IBMX, DMEM-high glucose, FBS, and antibiotics.
  • Placental stem cells can also be induced towards adipogenesis by culture in medium comprising one or more glucocorticoids (e.g., dexamethasone, indomethasone, hydrocortisone, cortisone), insulin, a compound which elevates intracellular levels of cAMP (e.g., dibutyryl-cAMP; 8-CPT-cAMP (8-(4)chlorophenylthio)-adenosine, 3′,5′ cyclic monophosphate); 8-bromo-cAMP; dioctanoyl-cAMP; forskolin) and/or a compound which inhibits degradation of cAMP (e.g., a phosphodiesterase inhibitor such as isobutylmethylxanthine (IBMX), methyl isobutylxanthine, theophylline, caffeine, indomethacin).
  • glucocorticoids e.g., dexamethasone, indometha
  • a hallmark of adipogenesis is the development of multiple intracytoplasmic lipid vesicles that can be easily observed using the lipophilic stain oil red 0.
  • Expression of lipase and/or fatty acid binding protein genes is confirmed by RT/PCR in placental stem cells that have begun to differentiate into adipocytes.
  • a placental stem cell is considered to have differentiated into an adipocytic cell when the cell displays one or more of these characteristics.
  • Chondrogenic differentiation of placental stem cells can be accomplished, for example, by placing placental stem cells in cell culture conditions that induce differentiation into chondrocytes.
  • a preferred chondrocytic medium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum.
  • placental stem cells are aliquoted into a sterile polypropylene tube, centrifuged (e.g., at 150 ⁇ g for 5 minutes), and washed twice in Incomplete Chondrogenesis Medium (Cambrex). The cells are resuspended in Complete Chondrogenesis Medium (Cambrex) containing 0.01 ⁇ g/ml TGF-beta-3 at a concentration of about 1-20 ⁇ 10 5 cells/ml.
  • placental stem cells are contacted with exogenous growth factors, e.g., GDF-5 or transforming growth factor beta3 (TGF-beta3), with or without ascorbate.
  • exogenous growth factors e.g., GDF-5 or transforming growth factor beta3 (TGF-beta3)
  • Chondrogenic medium can be supplemented with amino acids including proline and glutamine, sodium pyruvate, dexamethasone, ascorbic acid, and insulin/transferrin/selenium. Chondrogenic medium can be supplemented with sodium hydroxide and/or collagen.
  • the placental stem cells may be cultured at high or low density. Cells are preferably cultured in the absence of serum.
  • Chondrogenesis can be assessed by e.g., observation of production of esoinophilic ground substance, safranin-O staining for glycosaminoglycan expression; hematoxylin/eosin staining, assessing cell morphology, and/or RT/PCR confirmation of collagen 2 and collagen 9 gene expression. Chondrogenesis can also be observed by growing the stem cells in a pellet, formed, e.g., by gently centrifuging stem cells in suspension (e.g., at about 800 g for about 5 minutes). After about 1-28 days, the pellet of stem cells begins to form a tough matrix and demonstrates a structural integrity not found in non-induced, or non-chondrogenic, cell lines, pellets of which tend to fall apart when challenged.
  • Chondrogenesis can also be demonstrated, e.g., in such cell pellets, by staining with a stain that stains collage, e.g., Sirius Red, and/or a stain that stains glycosaminoglycans (GAGs), such as, e.g., Alcian Blue.
  • a stain that stains collage e.g., Sirius Red
  • GAGs glycosaminoglycans
  • a placental stem cell is considered to have differentiated into a chondrocytic cell when the cell displays one or more of these characteristics.
  • Osteogenic differentiation of placental stem cells can be accomplished, for example, by placing placental stem cells in cell culture conditions that induce differentiation into osteocytes.
  • a preferred osteocytic medium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum, followed by Osteogenic Induction Medium (Cambrex) containing 0.1 ⁇ M dexamethasone, 0.05 mM ascorbic acid-2-phosphate, 10 mM beta glycerophosphate.
  • placental stem cells are cultured in medium (e.g., DMEM-low glucose) containing about 10 ⁇ 7 to about 10 ⁇ 9 M dexamethasone, about 10-50 ⁇ M ascorbate phosphate salt (e.g., ascorbate-2-phosphate) and about 10 nM to about 10 mM ⁇ -glycerophosphate.
  • Osteogenic medium can also include serum, one or more antibiotic/antimycotic agents, transforming growth factor-beta (e.g., TGF- ⁇ 1) and/or bone morphogenic protein (e.g., BMP-2, BMP-4, or a combination thereof).
  • Differentiation can be assayed using a calcium-specific stain, e.g., von Kossa staining, and RT/PCR detection of, e.g., alkaline phosphatase, osteocalcin, bone sialoprotein and/or osteopontin gene expression.
  • a placental stem cell is considered to have differentiated into an osteocytic cell when the cell displays one or more of these characteristics.
  • Differentiation of placental stem cells into insulin-producing pancreatic cells can be accomplished, for example, by placing placental stem cells in cell culture conditions that induce differentiation into pancreatic cells.
  • An example pancreagenic medium comprises DMEM/20% 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 media from nestin-positive neuronal cell cultures at 50/50 v/v. KnockOut Serum Replacement can be used in lieu of CBS. Cells are cultured for 14-28 days, refeeding every 3-4 days.
  • a placental stem cell is considered to have differentiated into a pancreatic cell when the cell displays one or more of these characteristics.
  • Myogenic (cardiogenic) differentiation of placental stem cells can be accomplished, for example, by placing placental stem cells in cell culture conditions that induce differentiation into cardiomyocytes.
  • a preferred cardiomyocytic medium comprises DMEM/20% CBS supplemented with retinoic acid, 1 ⁇ M; 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, Calif.) may be used in lieu of CBS.
  • placental stem cells are cultured in DMEM/20% CBS supplemented with 50 ng/ml Cardiotropin-1 for 24 hours.
  • placental stem cells can be cultured 10-14 days in protein-free medium for 5-7 days, then stimulated with human myocardium extract, e.g., produced by homogenizing human myocardium in 1% HEPES buffer supplemented with 1% cord blood serum.
  • human myocardium extract e.g., produced by homogenizing human myocardium in 1% HEPES buffer supplemented with 1% cord blood serum.
  • a placental stem cell is considered to have differentiated into a cardiac cell when the cell displays one or more of these characteristics.
  • Placental stem cells can be preserved, that is, placed under conditions that allow for long-term storage, or conditions that inhibit cell death by, e.g., apoptosis or necrosis.
  • Placental stem cells can be preserved using, e.g., a composition comprising an apoptosis inhibitor, necrosis inhibitor and/or an oxygen-carrying perfluorocarbon, as described in related U.S. Provisional Application No. 60/754,969, entitled “Improved Medium for Collecting Placental Stem Cells and Preserving Organs,” filed on Dec. 25, 2005.
  • the invention provides a method of preserving a population of stem cells comprising contacting said population of stem cells with a stem cell collection composition comprising an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon, wherein said inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of stem cells, as compared to a population of stem cells not contacted with the inhibitor of apoptosis.
  • said inhibitor of apoptosis is a caspase inhibitor.
  • said inhibitor of apoptosis is a JNK inhibitor.
  • said JNK inhibitor does not modulate differentiation or proliferation of said stem cells.
  • said stem cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in separate phases.
  • said stem cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in an emulsion.
  • the stem cell collection composition additionally comprises an emulsifier, e.g., lecithin.
  • said apoptosis inhibitor and said perfluorocarbon are between about 0° C. and about 25° C. at the time of contacting the stem cells.
  • said apoptosis inhibitor and said perfluorocarbon are between about 2° C. and 10° C., or between about 2° C. and about 5° C., at the time of contacting the stem cells.
  • said contacting is performed during transport of said population of stem cells.
  • said contacting is performed during freezing and thawing of said population of stem cells.
  • the invention provides a method of preserving a population of placental stem cells comprising contacting said population of stem cells with an inhibitor of apoptosis and an organ-preserving compound, wherein said inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis, in the population of stem cells, as compared to a population of stem cells not contacted with the inhibitor of apoptosis.
  • the organ-preserving compound is UW solution (described in U.S. Pat. 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., U.S.
  • said organ-preserving compound is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof.
  • the stem cell collection composition additionally comprises an oxygen-carrying perfluorocarbon, either in two phases or as an emulsion.
  • placental stem cells are contacted with a stem cell collection composition comprising an apoptosis inhibitor and oxygen-carrying perfluorocarbon, organ-preserving compound, or combination thereof, during perfusion.
  • said stem cells are contacted during a process of tissue disruption, e.g., enzymatic digestion.
  • placental stem cells are contacted with said stem cell collection compound after collection by perfusion, or after collection by tissue disruption, e.g., enzymatic digestion.
  • 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 said preservation, wherein a hypoxic condition is a concentration of oxygen that is less than normal blood oxygen concentration.
  • a hypoxic condition is a concentration of oxygen that is less than normal blood oxygen concentration.
  • said population of stem cells is exposed to said hypoxic condition for less than two hours during said preservation.
  • 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.
  • said population of stem cells is not exposed to shear stress during collection, enrichment or isolation.
  • the placental stem cells of the invention can be cryopreserved, e.g., in cryopreservation medium in small containers, e.g., ampoules.
  • Suitable cryopreservation medium includes, but is not limited to, culture medium including, e.g., growth medium, or cell freezing medium, for example commercially available cell freezing medium, e.g., C2695, C2639 or C6039 (Sigma).
  • Cryopreservation medium preferably comprises DMSO (dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).
  • Cryopreservation medium may comprise additional agents, for example, methylcellulose and/or glycerol.
  • Placental stem cells are preferably cooled at about 1° C./min during cryopreservation.
  • a preferred cryopreservation temperature is about ⁇ 80° C. to about ⁇ 180° C., preferably about ⁇ 125° C. to about ⁇ 140° C.
  • Cryopreserved cells can be transferred to liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoules have reached about ⁇ 90° C., they are transferred to a liquid nitrogen storage area. Cryopreservation can also be done using a controlled-rate freezer.
  • Cryopreserved cells preferably are thawed at a temperature of about 25° C. to about 40° C., preferably to a temperature of about 37° C.
  • Placental stem cell populations can be used to treat any disease, disorder or condition that is amenable to treatment by administration of a population of stem cells.
  • “treat” encompasses the cure of, remediation of, improvement of, lessening of the severity of, or reduction in the time course of, a disease, disorder or condition, or any parameter or symptom thereof.
  • Placental stem cells, and populations of placental stem cells 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 cells differentiated from stem cells.
  • placental stem cells can be injected into a damaged organ, and for organ neogenesis and repair of injury in vivo.
  • Such injury may be due to such conditions and disorders including, but not limited to, myocardial infarction, seizure disorder, multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia, inflammation, thyroiditis, age-related loss of cognitive function, radiation damage, cerebral palsy, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Leigh disease, AIDS dementia, memory loss, amyotrophic lateral sclerosis, muscular dystrophy, ischemic renal disease, brain or spinal cord trauma, heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma.
  • 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 can be used, in specific embodiments, 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's, Gaucher's disease (e.g., glucocerbrosidase deficiency), Hunter's, and Hurler's syndromes, Maroteaux-Lamy syndrome, fucosidosis (flicosidase deficiency), Batten disease (CLN3), as well as other gangliosidoses, mucopolysaccharidoses, and glycogenoses.
  • lysosomal storage diseases such as Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's disease (e.g., glucocerbrosidase deficiency), Hunter's, and Hurler's syndromes, Maroteaux-Lamy syndrome, fucosidosis
  • Isolated populations of placental stem cells may be used alone, or as autologous or heterologous transgene carriers in gene therapy, to correct inborn errors of metabolism, cystic fibrosis, adrenoleukodystrophy (e.g., co-A ligase deficiency), metachromatic leukodystrophy (arylsulfatase A deficiency) (e.g., symptomatic, or presymptomatic late infantile or juvenile forms), globoid cell leukodystrophy (Krabbe's disease; galactocerebrosidase deficiency), acid lipase deficiency (Wolman disease), glycogen storage disease, hypothyroidism, anemia (e.g., aplastic anemia, sickle cell anemia, etc.), Pearson syndrome, Pompe's disease, phenylketonuria (PKU), porphyrias, maple syrup urine disease, homoc
  • adrenoleukodystrophy
  • isolated populations of placental stem cells may be used in autologous or heterologous tissue regeneration or replacement therapies or protocols, including, but not limited to treatment of corneal epithelial defects, treatment of osteogenesis imperfecta, cartilage repair, facial dermabrasion, mucosal membranes, tympanic membranes, intestinal linings, neurological structures (e.g., retina, auditory neurons in basilar membrane, olfactory neurons in olfactory epithelium), burn and wound repair for traumatic injuries of the skin, or for reconstruction of other damaged or diseased organs or tissues.
  • therapies or protocols including, but not limited to treatment of corneal epithelial defects, treatment of osteogenesis imperfecta, cartilage repair, facial dermabrasion, mucosal membranes, tympanic membranes, intestinal linings, neurological structures (e.g., retina, auditory neurons in basilar membrane, olfactory neurons in olfactory epithelium), burn and wound repair for traumatic injuries of the skin, or for reconstruction of other damaged
  • an isolated population of placental stem cells is used in hematopoietic reconstitution in an individual that has suffered a partial or total loss of hematopoietic stem cells, e.g., individuals exposed to lethal or sub-lethal doses of radiation (whether industrial, medical or military); individuals that have undergone myeloablation as part of, e.g., cancer therapy, and the like, in the treatment of, e.g., a hematologic malignancy.
  • Placental stem cells can be used in hematopoietic reconstitution in individuals having anemia (e.g., aplastic anemia, sickle cell anemia, etc.).
  • the placental stem cells are administered to such individuals with a population of hematopoietic stem cells.
  • Isolated populations of placental-derived stem cells can be used in place of, or to supplement, bone marrow or populations of stem cells derived from bone marrow.
  • approximately 1 ⁇ 10 8 to 2 ⁇ 10 8 bone marrow mononuclear cells per kilogram of patient weight are infused for engraftment in a bone marrow transplantation (i.e., about 70 ml of marrow for a 70 kg donor).
  • 70 ml requires an intensive donation and significant loss of donor blood in the donation process.
  • An isolated population of placental stem cells for hematopoietic reconstitution can comprise, in various embodiments, about, at least, or no more than 1 ⁇ 10 5 , 5 ⁇ 10 5 , 1 ⁇ 10 6 , 5 ⁇ 10 6 , 1 ⁇ 10 7 , 5 ⁇ 10 7 , 1 ⁇ 10 8 , 5 ⁇ 10 8 , 1 ⁇ 10 9 , 5 ⁇ 10 9 , 1 ⁇ 10 10 , 5 ⁇ 10 10 , 1 ⁇ 10 11 or more placental stem cells.
  • placental stem cells can be used to treat patients having a 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.
  • a 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.
  • the disease, disorder or condition is chronic granulomatous disease.
  • the present invention further encompasses the treatment of an individual with a stem cell combination of the invention, wherein the individual has an anemia or disorder of the blood hemoglobin.
  • the anemia or disorder may be natural (e.g., caused by genetics or disease), or may be artificially-induced (e.g., by accidental or deliberate poisoning, chemotherapy, and the like).
  • the disease or disorder is a marrow failure syndrome (e.g., aplastic anemia, Kostmann syndrome, Diamond-Blackfan anemia, amegakaryocytic thrombocytopenia, and the like), a bone marrow disorder or a hematopoietic disease or disorder.
  • Placental stem cells can also be used to treat severe combined immunodeficiency disease, including, but not limited to, combined immunodeficiency disease (e.g., Wiskott-Aldrich syndrome, severe DiGeorge syndrome, and the like).
  • combined immunodeficiency disease e.g., Wiskott-Aldrich syndrome, severe DiGeorge syndrome, and the like.
  • the placental stem cells of the invention 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 to be cultured under the appropriate conditions to allow the cells to differentiate and populate 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.
  • placental stem cells and placental stem cell populations may be used for autologous and allogenic transplants, including matched and mismatched HLA type hematopoietic transplants.
  • the host is treated to reduce immunological rejection of the donor cells, or to create immunotolerance (see, e.g., U.S. Pat. Nos. 5,800,539 and 5,806,529).
  • the host is not treated to reduce immunological rejection or to create immunotolerance.
  • Placental stem cells can be used in therapeutic transplantation protocols, e.g., to augment or replace stem or progenitor cells of the liver, pancreas, kidney, lung, nervous system, muscular system, bone, bone marrow, thymus, spleen, mucosal tissue, gonads, or hair. Additionally, placental stem cells may be used instead of specific classes of progenitor cells (e.g., chondrocytes, hepatocytes, hematopoietic cells, pancreatic parenchymal cells, neuroblasts, muscle progenitor cells, etc.) in therapeutic or research protocols in which progenitor cells would typically be used.
  • progenitor cells e.g., chondrocytes, hepatocytes, hematopoietic cells, pancreatic parenchymal cells, neuroblasts, muscle progenitor cells, etc.
  • the invention provides for the use of placental stem cells, particularly CD200 + placental stem cells, as an adjunct to hair replacement therapy.
  • placental stem cells e.g., CD200 + placental stem cells
  • the number of stem cells injected can be, e.g., between about 100 and about 10,000 per injection, in a volume of about 0.1 to about 1.0 ⁇ L, though more ore fewer cells in a greater or lesser volume can also be used.
  • Administration of placental stem cells to facilitate hair regrowth can comprise a single injection or multiple injections in, e.g., a regular or a random pattern in an area in which hair regrowth is desired.
  • Known hair regrowth therapies can be used in conjunction with the placental stem cells, e.g., topical minoxidil. Hair loss that can be treated using placental stem cells can be naturally-occurring (e.g., male pattern baldness) or induced (e.g., resulting from toxic chemical exposure).
  • Placental stem cells and placental stem cell populations of the invention can be used for augmentation, repair or replacement of cartilage, tendon, or ligaments.
  • prostheses e.g., hip prostheses
  • joints e.g., knee
  • Cartilage tissue constructs can also be employed in major reconstructive surgery for different types of joints (see, e.g., Resnick & Niwayama, eds., 1988, Diagnosis of Bone and Joint Disorders, 2d ed., W. B. Saunders Co.).
  • the placental stem cells of the invention can be used to repair damage to tissues and organs resulting from, e.g., trauma, metabolic disorders, or disease.
  • the trauma can be, e.g., trauma from surgery, e.g., cosmetic surgery.
  • a patient can be administered placental stem cells, alone or combined with other stem or progenitor cell populations, to regenerate or restore tissues or organs which have been damaged as a consequence of disease.
  • the present invention provides compositions comprising placental stem cells, or biomolecules therefrom.
  • the placental stem cells of the present invention can be combined with any physiologically-acceptable or medically-acceptable compound, composition or device for use in, e.g., research or therapeutics.
  • Placental stem cell populations of the invention can be preserved, for example, cryopreserved for later use.
  • Methods for cryopreservation of cells, such as stem cells are well known in the art.
  • Placental stem cell populations can be prepared in a form that is easily administrable to an individual.
  • the invention provides a placental stem cell population that is contained within a container that is suitable for medical use.
  • a container can be, for example, a sterile plastic bag, flask, jar, or other container from which the placental stem cell population can be easily dispensed.
  • the container can be a blood bag or other plastic, medically-acceptable bag suitable for the intravenous administration of a liquid to a recipient.
  • the container is preferably one that allows for cryopreservation of the combined stem cell population.
  • the cryopreserved placental stem cell population can comprise placental stem cells derived from a single donor, or from multiple donors.
  • the placental stem cell population can be completely HLA-matched to an intended recipient, or partially or completely HLA-mismatched.
  • the invention provides a composition comprising a placental stem cell population in a container.
  • the stem cell population is cryopreserved.
  • the container is a bag, flask, or jar.
  • said bag is a sterile plastic bag.
  • said bag is suitable for, allows or facilitates intravenous administration of said placental stem cell population.
  • the bag can comprise multiple lumens or compartments that are interconnected to allow mixing of the placental stem cells and one or more other solutions, e.g., a drug, prior to, or during, administration.
  • the composition comprises one or more compounds that facilitate cryopreservation of the combined stem cell population.
  • said placental stem cell population is contained within a physiologically-acceptable aqueous solution.
  • said physiologically-acceptable aqueous solution is a 0.9% NaCl solution.
  • said placental stem cell population comprises placental cells that are HLA-matched to a recipient of said stem cell population.
  • said combined stem cell population comprises placental cells that are at least partially HLA-mismatched to a recipient of said stem cell population.
  • said placental stem cells are derived from a plurality of donors.
  • compositions for use in vivo can be formulated into pharmaceutical compositions for use in vivo.
  • Such pharmaceutical compositions comprise a population of placental stem cells, or a population of cells comprising placental stem cells, in a pharmaceutically-acceptable carrier, e.g., a saline solution or other accepted physiologically-acceptable solution for in vivo administration.
  • Pharmaceutical compositions of the invention can comprise any of the placental stem cell populations, or placental stem cell types, described elsewhere herein.
  • the pharmaceutical compositions can comprise fetal, maternal, or both fetal and maternal placental stem cells.
  • the pharmaceutical compositions of the invention can further comprise placental stem cells obtained from a single individual or placenta, or from a plurality of individuals or placentae.
  • compositions of the invention can comprise any number of placental stem cells.
  • a single unit dose of placental stem cells can comprise, in various embodiments, about, at least, or no more than 1 ⁇ 10 5 , 5 ⁇ 10 5 , 1 ⁇ 10 6 , 5 ⁇ 10 6 , 1 ⁇ 10 7 , 5 ⁇ 10 7 , 1 ⁇ 10 8 , 5 ⁇ 10 8 , 1 ⁇ 10 9 , 5 ⁇ 10 9 , 1 ⁇ 10 10 , 5 ⁇ 10 10 , 1 ⁇ 10 11 or more placental stem cells.
  • compositions of the invention comprise populations of cells that comprise 50% viable cells or more (that is, 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.
  • compositions of the invention can comprise one or more compounds that, e.g., facilitate engraftment (e.g., anti-T-cell receptor antibodies, an immunosuppressant, or the like); stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.
  • facilitate engraftment e.g., anti-T-cell receptor antibodies, an immunosuppressant, or the like
  • stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.
  • the pharmaceutical composition of the invention comprises about 1.25% HSA and about 2.5% dextran.
  • Other injectable formulations, suitable for the administration of cellular products, may be used.
  • the composition of the invention comprises placental stem cells that are substantially, or completely, non-maternal in origin.
  • the invention provides in one embodiment a composition comprising a population of placental stem cells that 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 cell when said population of placental cells is cultured under conditions that allow the formation of an embryoid-like 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 cell when said population of placental cells is cultured under conditions that allow the formation of an embryoid-like body; or a combination of the foregoing, wherein at least 70%,
  • the placental stem cells of the invention can be used to produce conditioned medium, that is, medium comprising one or more biomolecules secreted or excreted by the stem cells.
  • the conditioned medium comprises medium in which placental stem cells have grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.
  • the conditioned medium comprises medium in which placental stem cells have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100% confluence.
  • Such conditioned medium can be used to support the culture of a separate population of placental stem cells, or stem cells of another kind.
  • the conditioned medium comprises medium in which placental stem cells have been differentiated into an adult cell type.
  • the conditioned medium of the invention comprises medium in which placental stem cells and non-placental stem cells have been cultured.
  • the invention further comprises matrices, hydrogels, scaffolds, and the like that comprise a placental stem cell, or a population of placental stem cells.
  • Placental stem cells of the invention can be seeded onto a natural matrix, e.g., a placental biomaterial such as an amniotic membrane material.
  • a placental biomaterial such as an amniotic membrane material.
  • an amniotic membrane material can be, e.g., amniotic membrane dissected directly from a mammalian placenta; fixed or heat-treated amniotic membrane, substantially dry (i.e., ⁇ 20% H 2 O) amniotic membrane, chorionic membrane, substantially dry chorionic membrane, substantially dry amniotic and chorionic membrane, and the like.
  • Preferred placental biomaterials on which placental stem cells can be seeded are described in Hariri, U.S. Application Publication No. 2004/0048796.
  • Placental stem cells of the invention can be suspended in a hydrogel solution suitable for, e.g., injection.
  • Suitable hydrogels for such compositions include self-assembling peptides, such as RAD16.
  • a hydrogel solution comprising the cells can be allowed to harden, for instance in a mold, to form a matrix having cells dispersed therein for implantation. Placental stem cells in such a matrix can also be cultured so that the cells are mitotically expanded prior to implantation.
  • the hydrogel is, e.g., an organic polymer (natural or synthetic) that is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel.
  • Hydrogel-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block polymers such as polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively.
  • the hydrogel or matrix of the invention is biodegradable.
  • the formulation comprises an in situ polymerizable gel (see, e.g., U.S. Patent Application Publication 2002/0022676; Anseth et al., J. Control Release, 78(1-3):199-209 (2002); Wang et al., Biomaterials, 24(22):3969-80 (2003).
  • the polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
  • aqueous solutions such as water, buffered salt solutions, or aqueous alcohol solutions
  • polymers having acidic 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 reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.
  • acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.
  • the placental stem cells of the invention or co-cultures thereof can be seeded onto a three-dimensional framework or scaffold and implanted in vivo.
  • a three-dimensional framework or scaffold 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.
  • Nonwoven mats can be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.).
  • Foams composed of, e.g., poly( ⁇ -caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by processes such as freeze-drying, or lyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be used as scaffolds.
  • Placental stem cells of the invention can also be seeded onto, or contacted with, a physiologically-acceptable ceramic material including, but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BIOGLASS®, and mixtures thereof.
  • a physiologically-acceptable ceramic material including, but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BIOGLASS®, and mixtures thereof.
  • Porous biocompatible ceramic materials currently commercially available include SURGIBONE® (CanMedica Corp., Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® (Mathys, AG, Bettlach, Switzerland), and mineralized collagen bone grafting products such as HEALOSTM (DePuy, Inc., Raynham, Mass.) and VITOSS®, RHAKOSSTM, and CORTOSS® (Orthovita, Malvern, Pa.).
  • the framework can be a mixture, blend or composite of natural and/or synthetic materials.
  • placental stem cells can be seeded onto, or contacted with, a felt, which can be, e.g., composed of a multifilament yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.
  • a felt which can be, e.g., composed of a multifilament yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.
  • the placental stem cells of the invention can, in another embodiment, be seeded onto foam scaffolds that may be composite structures.
  • foam scaffolds 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 augmented.
  • the framework is treated, e.g., with 0.1M acetic acid followed by incubation in polylysine, PBS, and/or collagen, prior to inoculation of the cells of the invention in order to enhance cell attachment.
  • External surfaces of a matrix may be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma-coating the matrix, or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, and the like.
  • proteins e.g., collagens, elastic fibers, reticular fibers
  • glycoproteins e.g., glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sul
  • the scaffold comprises, or is treated with, materials that render it non-thrombogenic. These treatments and materials may 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 basement membrane proteins such as laminin and Type IV collagen, synthetic materials such as EPTFE, and segmented polyurethaneurea silicones, such as PURSPANTM (The Polymer Technology Group, Inc., Berkeley, Calif.).
  • the scaffold can also comprise anti-thrombotic agents such as heparin; the scaffolds can also be treated to alter the surface charge (e.g., coating with plasma) prior to seeding with placental stem cells.
  • Mammalian placental cells can be conditionally immortalized by transfection with any suitable vector containing a growth-promoting gene, that is, a gene encoding a protein that, under appropriate conditions, promotes growth of the transfected cell, such that the production and/or activity of the growth-promoting protein is regulatable by an external factor.
  • a growth-promoting gene is an oncogene such as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1a adenovirus or E7 protein of human papillomavirus.
  • External regulation of the growth-promoting protein can be achieved by placing the growth-promoting gene under the control of an externally-regulatable promoter, e.g., a promoter the activity of which can be controlled by, for example, modifying the temperature of the transfected cells or the composition of the medium in contact with the cells.
  • an externally-regulatable promoter e.g., a promoter the activity of which can be controlled by, for example, modifying the temperature of the transfected cells or the composition of the medium in contact with the cells.
  • tet tetracycline
  • tTA tet-controlled transactivator
  • tTA is a fusion protein of the repressor (tetR) of the transposon-10-derived tet resistance operon of Escherichia coli and the acidic domain of VP16 of herpes simplex virus.
  • the vector further contains a gene encoding a selectable marker, e.g., a protein that confers drug resistance.
  • a selectable marker e.g., a protein that confers drug resistance.
  • the bacterial neomycin resistance gene (neo R ) is one such marker that may be employed within the present invention.
  • Cells carrying neo R may be selected by means known to those of ordinary skill in the art, such as the addition of, e.g., 100-200 ⁇ g/mL G418 to the growth medium.
  • Transfection can be achieved by any of a variety of means known to those of ordinary skill in the art including, but not limited to, retroviral infection.
  • a cell culture may 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.
  • a placental cell culture prepared as described above may be infected after, e.g., five days in vitro by incubation for about 20 hours in one volume of conditioned medium and two volumes of DMEM/F12 containing N2 supplements.
  • Transfected cells carrying a selectable marker may then be selected as described above.
  • the substrate is a polyomithine/laminin substrate, consisting of tissue culture plastic coated with polyomithine (10 ⁇ g/mL) and/or laminin (10 ⁇ g/mL), a polylysine/laminin substrate or a surface treated with fibronectin.
  • Cultures are then fed every 3-4 days with growth medium, which may or may not be supplemented with one or more proliferation-enhancing factors. Proliferation-enhancing factors may be added to the growth medium when cultures are less than 50% confluent.
  • conditionally-immortalized placental stem cell lines can be passaged using standard techniques, such as by trypsinization, when 80-95% confluent. Up to approximately the twentieth passage, it is, in some embodiments, beneficial to maintain selection (by, for example, the addition of G418 for cells containing a neomycin resistance gene). Cells may also be frozen in liquid nitrogen for long-term storage.
  • Clonal cell lines can be isolated from a conditionally-immortalized human placental stem cell line prepared as described above. In general, such clonal cell lines may be isolated using standard techniques, such as by limit dilution or using cloning rings, and expanded. Clonal cell lines may generally be fed and passaged as described above.
  • Conditionally-immortalized human placental stem cell lines which may, but need not, be clonal, may generally be induced to differentiate by suppressing the production and/or activity of the growth-promoting protein under culture conditions that facilitate differentiation.
  • the conditions e.g., temperature or composition of medium
  • differentiation can be achieved by the addition of tetracycline to suppress transcription of the growth-promoting gene. In general, 1 ⁇ g/mL tetracycline for 4-5 days is sufficient to initiate differentiation.
  • additional agents may be included in the growth medium.
  • 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 on stem cell proliferation, expansion, and/or differentiation, compared to placental stem cells not exposed to such conditions.
  • environmental factors e.g., biomolecules, small inorganic molecules. etc.
  • molecules e.g., biomolecules, small inorganic molecules. etc.
  • the placental stem cells of the present invention are assayed for changes in proliferation, expansion or differentiation upon contact with a molecule.
  • the invention provides a method of identifying a compound that modulates the proliferation of a plurality of placental stem cells, comprising contacting said plurality of stem cells with said compound under conditions that allow proliferation, wherein if said compound causes a detectable change in proliferation 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 proliferation of placental stem cells.
  • said compound is identified as an inhibitor of proliferation.
  • said compound is identified as an enhancer of proliferation.
  • the invention provides a method of identifying a compound that modulates the expansion of a plurality of placental stem cells, comprising contacting said plurality of stem cells with said compound under conditions that allow expansion, wherein if 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 expansion of placental stem cells.
  • said compound is identified as an inhibitor of expansion.
  • said compound is identified as an enhancer of expansion.
  • the invention provides a method of identifying a compound that modulates the differentiation of a placental stem cell, comprising contacting said stem cells with said compound under conditions that allow differentiation, wherein if said compound causes a detectable change in differentiation of said stem cells compared to a stem cell not contacted with said compound, said compound is identified as a compound that modulates proliferation of placental stem cells.
  • said compound is identified as an inhibitor of differentiation.
  • said compound is identified as an enhancer of differentiation.
  • Placental stem cells are obtained from a post-partum mammalian placenta either by perfusion or by physical disruption, e.g., enzymatic digestion.
  • the cells are cultured in a culture medium comprising 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma), 2% fetal calf serum (FCS) (Hyclone Laboratories), 1 ⁇ insulin-transferrin-selenium (ITS), 1 ⁇ lenolenic-acid-bovine-serum-albumin (LA-BSA), 10 ⁇ 9 M dexamethasone (Sigma), 10 ⁇ 4 M ascorbic acid 2-phosphate (Sigma), epidermal growth factor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor (PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 U streptomycin.
  • DMEM-LG Gibco
  • MCDB-201 Sigma
  • the culture flask in which the cells are cultured is prepared as follows. T75 flasks are coated with fibronectin (FN), by adding 5 ml PBS containing 5 ng/ml human FN (Sigma F0895) to the flask. The flasks with FN solution are left at 37° C. for 30 min. The FN solution is then removed prior to cell culture. There is no need to dry the flasks following treatment. Alternatively, the flasks are left in contact with the FN solution at 4° C. overnight or longer; prior to culture, the flasks are warmed and the FN solution is removed.
  • FN fibronectin
  • Cultures of placental stem cells from placental perfusate are established as follows. Cells from a Ficoll gradient are seeded in FN-coated T75 flasks, prepared as above, at 50-100 ⁇ 10 6 cells/flask in 15 ml culture medium. Typically, 5 to 10 flasks are seeded. The flasks are incubated at 37° C. for 12-18 hrs to allow the attachment of adherent cells. 10 ml of warm PBS is added to each flask to remove cells in suspension, and mixed gently. 15 mL of the medium is then removed and replaced with 15 ml fresh culture medium. All medium is changed 3-4 days after the start of culture. Subsequent culture medium changes are performed, during which 50% or 7.5 ml of the medium is removed.
  • the culture is checked under a microscope to examine the growth of the adherent cell colonies.
  • adherent cells are harvested by trypsin digestion. Cells harvested from these primary cultures are designated passage 0 (zero).
  • Placental stem cell cultures are established from digested placental tissue as follows.
  • the perfused placenta is placed on a sterile paper sheet with the maternal side up.
  • Approximately 0.5 cm of the surface layer on maternal side of placenta is scraped off with a blade, and the blade is used to remove a placental tissue block measuring approximately 1 ⁇ 2 ⁇ 1 cm.
  • This placenta tissue is then minced into approximately 1 mm3 pieces. These pieces are collected into a 50 ml Falcon tube and digested with collagenase IA (2 mg/ml, Sigma) for 30 minutes, followed by trypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at 37° C. in water bath.
  • the resulting solution is centrifuged at 400 g for 10 minutes at room temperature, and the digestion solution is removed.
  • the pellet is resuspended to approximately 10 volumes with PBS (for example, a 5 ml pellet is resuspended with 45 ml PBS), and the tubes are centrifuged at 400 g for 10 minutes at room temperature.
  • the tissue/cell pellet is resuspended in 130 mL culture medium, and the cells are seeded at 13 ml per fibronectin-coated T-75 flask. Cells are incubated at 37° C. with a humidified atmosphere with 5% CO 2 . Placental Stem Cells are optionally cryopreserved at this stage.
  • Cryopreserved cells are quickly thawed in a 37° C. water bath. Placental stem cells are immediately removed from the cryovial with 10 ml warm medium and transferred to a 15 ml sterile tube. The cells are centrifuged at 400 g for 10 minutes at room temperature. The cells are gently resuspended in 10 ml of warm culture medium by pipetting, and viable cell counts are determined by Trypan blue exclusion. Cells are then seeded at about 6000-7000 cells per cm 2 onto FN-coated flasks, prepared as above (approximately 5 ⁇ 10 5 cells per T-75 flask). The cells are incubated at 37° C., 5% CO 2 and 90% humidity.
  • the cells After counting the cells and determining viability as above, the cells are centrifuged at 1000 RPM for 5 minutes at room temperature. Cells are passaged by gently resuspending the cell pellet from one T-75 flask with culture medium, and evenly plating the cells onto two FN-coated T-75 flasks.
  • exemplary populations of adherent placental stem cells are identified that express markers CD105, CD33, CD73, CD29, CD44, CD10, and CD90. These populations of cells typically does not express CD34, CD45, CD117 or CD133. Some, but not all cultures of these placental stem cells expressed HLA-ABC and/or HLA-DR.
  • Placental stem cells were obtained from the following sources: (1) placental perfusate (from perfusion of the placental vasculature); and enzymatic digestions of (2) amnion, (3) chorion, (4) amnion-chorion plate, and (5) umbilical cord.
  • placental perfusate from perfusion of the placental vasculature
  • the various placental tissues were cleaned in sterile PBS (Gibco-Invitrogen Corporation, Carlsbad, Calif.) and placed on separate sterile Petri dishes.
  • the various tissues were minced using a sterile surgical scalpel and placed into 50 mL Falcon Conical tubes.
  • the minced tissues were digested with 1 ⁇ Collagenase (Sigma-Aldrich, St. Louis, Mo.) for 20 minutes in a 37° C. water bath, centrifuged, and then digested with 0.25% Trypsin-EDTA (Gibco-Invitrogen Corp) for 10 minutes in a 37° C. water bath.
  • 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 ⁇ m cell strainers and once with 30 ⁇ m separation filters, to remove any residual extracellular matrix or cellular debris.
  • the manual trypan blue exclusion method was employed post digestion to calculate cell counts and assess cellular viability.
  • Cells were mixed with Trypan Blue Dye (Sigma-Aldrich) at a ratio of 1:1, and the cells were read on hemacytometer.
  • Cells that were HLA ABC ⁇ /CD45 ⁇ /CD34 ⁇ /CD133 + were selected for characterization. Cells having this phenotype were identified, quantified, and characterized by two of Becton-Dickinson flow cytometers, the FACSCalibur and the FACS Aria (Becton-Dickinson, San Jose, Calif., USA). The various placental cells were stained, at a ratio of about 10 ⁇ L of antibody per 1 million cells, for 30 minutes at room temperature on a shaker.
  • FITC Fluorescein Isothiocyanate
  • CD10 BD Immunocytometry Systems, San Jose, Calif.
  • CD44 BD Biosciences Pharmingen, San Jose, Calif.
  • CD105 R&D Systems Inc., Minneapolis, Minn.
  • PE Phycoerythrin
  • PE conjugated monoclonal antibodies against CD44, CD200, CD 117, and CD13
  • Phycoerythrin-Cy5 PE Cy5 conjugated Streptavidin and monoclonal antibodies against CD117
  • Phycoerythrin-Cy7 PE Cy7 conjugated monoclonal antibodies against CD33 and CD10
  • Allophycocyanin (APC) conjugated streptavidin and monoclonal antibodies against CD38 BD Biosciences Pharmingen
  • the cells were rinsed once to remove unbound antibodies and were fixed overnight with 4% paraformaldehyde (USB, Cleveland, Ohio) at 4° C. The following day, the cells were rinsed twice, filtered through a 30 ⁇ m separation filter, and were run on the flow cytometer(s).
  • Samples that were stained with anti-mouse IgG antibodies were used as negative controls and were used to adjust the Photo Multiplier Tubes (PMTs). Samples that were single stained with anti-human antibodies were used as positive controls and were used to adjust spectral overlaps/compensations.
  • placental cells from perfusate, amnion, or chorion, prior to any culture, was stained with 7-Amino-Actinomycin D (7AAD; BD Biosciences Pharmingen) and monoclonal antibodies specific for the phenotype of interest.
  • the cells were stained at a ratio of 10 ⁇ L of antibody per 1 million cells, and were incubated for 30 minutes at room temperature on a shaker. These cells were then positively sorted for live cells expressing the phenotype of interest on the BD FACS Aria and plated into culture. Sorted (population of interest) and “All” (non-sorted) placental cell populations were plated for comparisons.
  • the cells were plated onto a fibronectin (Sigma-Aldrich) coated 96 well plate at the cell densities listed in Table 1 (cells/cm 2 ). The cell density, and whether 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 96 Well Plate Culture Density of Plated Cells Conditions All Max.
  • Complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201 (Sigma); 2% fetal calf serum (Hyclone Labs.); 1 ⁇ insulin-transferrin-selenium (ITS); 1 ⁇ linoleic acid-bovine serum albumin (LA-BSA); 10 ⁇ 9 M dexamethasone (Sigma); 10 ⁇ 4 M ascorbic acid 2-phosphate (Sigma); epidermal growth factor 10 ng/mL (R&D Systems); and platelet-derived growth factor (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% CO 2 /37° C. incubator. On day 7, 100 ⁇ L of complete medium was added to each of the wells. The 96 well plate was monitored for about two weeks and a final assessment of the culture was completed on day 12. This is very early in the placental stem cell culture, and represents passage 0 cells.
  • FACSCalibur data was analyzed in FlowJo (Tree star, Inc) using standard gating techniques.
  • the BD FACS Aria data was analyzed using the FACSDiva software (Becton-Dickinson).
  • the FACS Aria data was analyzed using doublet discrimination gating to minimize doublets, as well as, standard gating techniques. All results were compiled in Microsoft Excel and all values, herein, are represented as average standard deviation (number, standard error of mean).
  • Post-digestion viability was assessed using the manual trypan blue exclusion method ( FIG. 1 ).
  • the average viability of cells obtained from the majority of the digested tissue was around 70%.
  • the BD FACS Aria data also identified amnion, perfusate, and chorion as providing higher numbers of HLA ABC ⁇ /CD45 ⁇ /CD34 ⁇ /CD133 + cells than the remaining sources.
  • HLA ABC ⁇ /CD45 ⁇ /CD34 ⁇ /CD133 + cells were identified and quantified from each cell source, its cells were further analyzed and characterized for their expression of cell surface markers HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200, and CD105.
  • Perfusate-derived cells were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 ( FIG. 4 ).
  • Amnion-derived cells were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 ( FIG. 5 ).
  • Umbilical cord-derived cells were consistently positive for HLA-G, CD33, CD90, CD38, CD105, and CD13, while the expression of CD117, CD10, CD44, and CD200 varied ( FIG. 8 ).
  • FIG. 9 A summary of all marker expression averages is shown in FIG. 9 .
  • Sorted perfusate-derived cells plated at a cell density of 40,600/cm 2 , resulted in small, round, non-adherent cells.
  • Two out of the three sets of non-sorted perfusate-derived cells, each plated at a cell density of 40,600/cm 2 resulted in mostly small, round, non-adherent cells with several adherent cells located around the periphery of well.
  • Non-sorted perfusate-derived cells, plated at a cell density of 93,800/cm 2 resulted in mostly small, round, non-adherent cells with several adherent cells located around the well peripheries.
  • Sorted amnion-derived cells plated at a cell density of 6,300/cm 2 , resulted in small, round, non-adherent cells.
  • Non-sorted amnion-derived cells plated at a cell density of 6,300/cm 2 , resulted in small, round, non-adherent cells.
  • Non-sorted amnion-derived cells plated at a cell density of 62,500/cm 2 resulted in small, round, non-adherent cells.
  • Sorted chorion-derived cells plated at a cell density of 6,300/cm 2 , resulted in small, round, non-adherent cells.
  • Non-sorted chorion-derived cells plated at a cell density of 6,300/cm 2 , resulted in small, round, non-adherent cells.
  • Non-sorted chorion-derived cells plated at a cell density of 62,500/cm 2 resulted in small, round, non-adherent cells.
  • This Example demonstrates an exemplary cell surface marker profile of placental stem cells.
  • Placental stem cells or umbilical cord stem cells, obtained by enzymatic digestion, in culture medium were washed once by adding 2 mL 2% FBS-PBS and centrifuging at 400 g for 5 minutes. The supernatant was decanted, and the pellet was resuspended in 100-200 ⁇ L 2% FBS-PBS.
  • 4 tubes were prepared with BDTM CompBeads (Cat# 552843) by adding 100 ⁇ l of 2% FBS-PBS to each tube, adding 1 full drop (approximately 60 ⁇ l) of the BDTM CompBeads Negative Control and 1 drop of the BDTM CompBeads Anti-Mouse beads to each tube, and vortexing.
  • Control tubes were prepared as follows: Volume Tube# Antibody Cat# Clone ⁇ L 1 Unstained — — — 2 IgG FITC/IgG PE// 555787, 555786, G18-145 10 ea IgG APC 550931
  • control and sample tubes were incubated in the dark at room temperature for 30 minutes. After incubation, the tubes were washed by adding 2 mL 2% FBS-PBS and centrifuging at 400 g for 5 minutes. The supernatant was decanted, and the pellet was resuspended in 100-200 ⁇ L 2% FBS-PBS and acquire on flow cytometer. All other antibodies were used following this procedure.
  • Matched placental stem cells from amniotic membrane and umbilical cord stem cells were analyzed using fluorescently-labeled antibodies and flow cytometry to identify cell surface markers that were present or absent. Markers analyzed included CD105 (proliferation related endothelial specific marker); CD200 (marker associated with regulatory function); CD34 (expressed on endothelial cells and on hematopoietic stem cells); CD10 (stem cell/precursor cell marker); cytokeratin K (epithelial marker); CD44 (cell migration, lymphocyte homing, hematopoeisis); CD45 (lineage marker); CD133 (marker for hematopoietic progenitor cells); CD117 (stem cell factor (c-Kit)); CD90 (expressed on primitive hematopoietic stem cells in normal bone marrow, cord blood and fetal liver cells); HLA ABC (pan MHC I, antigen presentation, immunogenicity); ⁇ -2-microglobulin (associates with MHC I
  • CD200 + , CD105 + , CD 10 + , CD34 ⁇ appears to be consistent over numerous such analyses.
  • This phenotype is additionally positive for CD90, CD44, HLA ABC (weak), P-2-microglobulin (weak), and cytokeratin K, and negative for HLA DR, DQ, DP, CD117, CD133, and CD45.
  • ALDEFLUOR® Assay Kit from Stem Cell Technologies, Inc. Typically, more primitive, undifferentiated stem cells demonstrate less ALDH activity than more differentiated stem cells.
  • the assay uses ALDEFLUOR®, a fluorescent ALDH substrate (Aldagen, Inc., Durham, N.C.). The manufacturer's protocol was followed.
  • the dry ALDEFLUOR® reagent is provided in a stable, inactive form.
  • the ALDEFLUOR® was activated by dissolving the dry compound in dimethylsulfoxide (DMSO) and adding 2N HCl, and was added immediately to the cells.
  • DMSO dimethylsulfoxide
  • a control tube was also established by combing the cells with ALDEFLUOR® plus DEAB, a specific inhibitor of ALDH.
  • BM-MSC bone marrow-derived mesenchymal stem cell line
  • ADSC adipose-derived stem cell line
  • HVT human villous trophoblast cell line
  • CD34 + stem cells purified from cord blood included four umbilical cord stem cell lines and three placental stem cell lines from amnion-chorion plate, a bone marrow-derived mesenchymal stem cell line (BM-MSC), an adipose-derived stem cell line (ADSC), a human villous trophoblast cell line (HVT), and CD34 + stem cells purified from cord blood.
  • the assay proceeded as follows. Sample concentration was adjusted to 1 ⁇ 10 6 cells/ml with Assay buffer provided with the ALDEFLUOR® Assay Kit. 1 mL of adjusted cell suspension into experimental and control tube for each of the cell lines tested, and 5 ⁇ l of DEAB was additionally added to the control tube labeled as control.
  • ALDEFLUOR® substrate was activated by adding 25 ⁇ l of DMSO to the dry ALDEFLUOR® Reagent, and let stand for 1 minute at RT. 25 ⁇ l of 2N HCL was added and mixed well. This mixture was incubated for 15 min at RT. 360 ⁇ l of ALDEFLUOR® Assay Buffer was added to the vial and mixed. The resulting mixture was stored at 2-8° C. during use.
  • Placental stem cell lines demonstrated ALDH activity of from about 3% to about 25% (3.53%, 8.76% and 25.26%).
  • Umbilical cord stem cell lines demonstrated ALDH activity of from about 16% to about 20% (16.59%, 17.01%, 18.44% and 19.83%).
  • BM-MSC and HVT were negative and 1.5% respectively for ALDH, but the adipose derived MSC is close to 30% ALDH + .
  • the positive control CD34 + cells purified from umbilical cord blood were, as expected, highly positive (75%) for ALDH.
  • This Example demonstrates one method of collecting placental stem cells by perfusion.
  • a postpartum placenta is obtained within 24 hours after birth.
  • the umbilical cord is clamped with an umbilical cord clamp approximately 3 to 4 inches about the placental disk, and the cord is cut above the clamp.
  • the umbilical cord is either discarded, or processed to recover, e.g., umbilical cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial.
  • Excess amniotic membrane and chorion is cut from the placenta, leaving approximately 1 ⁇ 4 inch around the edge of the placenta. The trimmed material is discarded.
  • the amniotic membrane is separated from the chorion using blunt dissection with the fingers.
  • the amniotic membrane is cut around the base of the umbilical cord with scissors, and detached from the placental disk.
  • the amniotic membrane can be discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial.
  • the fetal side of the remaining placental material is cleaned of all visible blood clots and residual blood using sterile gauze, and is then sterilized by wiping with an iodine swab than with an alcohol swab.
  • the umbilical cord is then clamped crosswise with a sterile hemostat beneath the umbilical cord clamp, and the hemostat is rotated away, pulling the cord over the clamp to create a fold.
  • the cord is then partially cut below the hemostat to expose a cross-section of the cord supported by the clamp.
  • the cord is clamped with a sterile hemostat.
  • the cord is then placed on sterile gauze and held with the hemostat to provide tension.
  • the cord is then cut straight across directly below the hemostat, and the edge of the cord near the vessel is re-clamped.
  • the vessels exposed as described above usually a vein and two arteries, are identified, and opened as follows.
  • a closed alligator clamp is advanced through the cut end of each vessel, taking care not to puncture the clamp through the vessel wall. Insertion is halted when the tip of the clamp is slightly above the base of the umbilical cord. The clamp is then slightly opened, and slowly withdrawn from the vessel to dilate the vessel.
  • Plastic tubing connected to a perfusion device or peristaltic pump, is inserted into each of the placental arteries.
  • Plastic tubing connected to a 250 mL collection bag, is inserted into the placental vein. The tubing is taped into place.
  • each bag is placed inside a plasma extractor without disturbing the pellet 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.
  • a sterile 1 mL syringe about 300-500 ⁇ L of cells is withdrawn from the collection bag, via a sampling 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 immediately cultured to isolate placental stem cells, or are cryopreserved for later use.
  • Neuronal differentiation of placental stem cells can also be accomplished as follows:
  • PPAR- ⁇ 2 Differentiation was also assessed by quantitative real-time PCR to examine the expression of specific genes associated with adipogenesis, i.e., PPAR- ⁇ 2, aP-2, lipoprotein lipase, and osteopontin.
  • PPAR- ⁇ 2 Two cultures of placental stem cells showed an increase of 6.5-fold 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 induction of adipogenesis.
  • placental stem cells obtained from perfusate were cultured in DMEM/MCDB-201 (Chick fibroblast basal 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 Mesenchymal Stem Cells (StemCell Technologies) supplemented with Mesenchymal Stem Cell Adipogenic Supplements (StemCell Technologies).
  • AIM2 comprised DMEM/MCDB-201 with 2% FCS and LA-BSA (1%).
  • Adipogenic differentiation of placental stem cells can also be accomplished as follows:
  • Osteogenic medium was prepared from 185 mL Cambrex Differentiation Basal Medium—Osteogenic and SingleQuots (one each of dexamethasone, 1-glutamine, ascorbate, pen/strep, MCGS, and ⁇ -glycerophosphate). Placental stem cells from perfusate were plated, at about 3 ⁇ 10 3 cells per cm 2 of tissue culture surface area in 0.2-0.3 mL MSCGM per cm 2 tissue culture area. Typically, all cells adhered to the culture surface for 4-24 hours in MSCGM at 37° C. in 5% CO 2 . Osteogenic differentiation was induced by replacing the medium with Osteogenic Differentiation medium. Cell morphology began to change from the typical spindle-shaped appearance of the adherent placental stem cells, to a cuboidal appearance, accompanied by mineralization. Some cells delaminated from the tissue culture surface during differentiation.
  • Osteogenic differentiation can also be accomplished as follows:
  • Pancreatic differentiation is accomplished as follows:
  • Myogenic (cardiogenic) differentiation is accomplished as follows:
  • Chondrogenic differentiation of placental stem cells is generally accomplished as follows:
  • the Example demonstrates the differentiation of placental stem cells into chondrogenic cells and the development of cartilage-like tissue from such cells.
  • Cartilage is an avascular, alymphatic tissue that lacks a nerve supply. Cartilage has a low chondrocyte density ( ⁇ 5%), however these cells are surprisingly efficient at maintaining the extracellular matrix around them.
  • articular cartilage which facilitates joint lubrication in joints
  • fibrocartilage which provides shock absorption in, e.g., meniscus and intervertebral disc
  • elastic cartilage which provides anatomical structure in, e.g., nose and ears. All three types of cartilage are similar in biochemical structure.
  • Joint pain is a major cause of disability and provides an unmet need of relief in the area of orthopedics.
  • Primary osteoarthritis (which can cause joint degeneration), and trauma are two common causes of pain.
  • Approximately 9% of the U.S. population has osteoarthritis of hip or knee, and more than 2 million knee surgeries are performed yearly.
  • Current treatments are more geared towards treatment of symptoms rather than repairing the cartilage.
  • Natural repair occurs when fibroblast-like cells invade the area and fill it with fibrous tissue which is neither as resilient or elastic as the normal tissue, hence causing more damage.
  • Treatment options historically included tissue grafts, subchondral drilling, or total joint replacement.
  • CARTICEL® an autologous chondrocyte injection
  • SYNVISC® and ORTHOVISC® which are hyaluronic acid injections for temporary pain relief
  • CHONDROGENTM an injection of adult mesenchymal stem cells for meniscus repair.
  • Placental and umbilical cord stem cells were isolated and purified from full term human placenta by enzymatic digestion.
  • Human MSC cells and HDF cells were purchased from Cambrex, and MC3T3 cells were purchased from American Type Culture Collection. All cell lines used were centrifuged into pellets in polypropylene centrifuge tubes at 800 RPM for 5 minutes and grown in both chondrogenic induction media (Cambrex) and non-inducing basal MSC media (Cambrex). Pellets were harvested and histologically analyzed at 7, 14, 21 and 28 days by staining for glycosaminoglycans (GAGs) with Alcian Blue, and/or for collagens with Sirius Red. Collagen type was further assessed with immunostaining. RNA analysis for cartilage-specific genes was performed at 7 and 14 days.
  • one placental stem cell line was cultured in chondrogenic induction medium in the form of cell pellets, either with or without bone morphogenic protein (BMP) at a final concentration of 500 ng/mL. Pellets were assessed for evidence of chondrogenic induction every week for 4 weeks. Results indicated that the pellets do increase in size over time. However, no visual differences were noted between the BMP + and BMP ⁇ samples. Pellets were also histologically analyzed for GAG's, an indicator of cartilage tissue, by staining with Alcian Blue. BMP + cells generally appeared more metabolically active with pale vacuoles whereas BMP ⁇ cells were smaller with dense-stained nuclei and less cytoplasm (reflects low metabolic activity).
  • BMP bone morphogenic protein
  • BMP + cells had stained heavily blue, while BMP ⁇ had stained only faintly.
  • BMP + and BMP ⁇ cells were roughly equivalently stained with Alcian Blue.
  • cell density decreased over time, and matrix overtook the pellet.
  • the MC3T3 negative cell line did not demonstrate any presence of GAG when stained with Alcian Blue.
  • Experiment 2 Based on the results of Experiment 1, a more detailed study was designed to assess the chondrogenic differentiation potential of two placental stem cell and two umbilical cord stem cell lines. In addition to the Alcian Blue histology, cells were also stained with Sirius Red, which is specific for type II collagen. Multiple pellets were made for each cell line, with and without induction media.
  • pellets from placental stem cells or umbilical cord stem cells were much larger than the MSC controls. Control pellets in non-induction media started to fall apart by Day 11, and were much smaller at 28 days than pellets developed by cells cultured in chondrogenic induction medium. Visually, there were no differences between pellets formed by placental stem cells or umbilical cord.
  • the UC67249 stem cell line which was initiated in dexamethasone-free media, formed larger pellets. Negative control MC3T3 cells did not form pellets; however, HDFs did form pellets.
  • pellets from all test groups were then subjected to histological analysis for GAG's and collagen.
  • pellets formed by the stem cells under inducing conditions were much larger and stayed intact better than pellets formed under non-inducing conditions.
  • Pellets formed under inducing conditions showed production of GAGs and increasing collagen content over time, and as early as seven days, while pellets formed under non-inducing conditions showed little to no collagen production, as evidenced by weak Alcian Blue staining.
  • the placental stem cells and umbilical cord stem cells appeared, by visual inspection, to produce tougher, larger pellets, and appeared to be producing more collagen over time, than the hMSCs.
  • Non-induced placental stem cells produced much less type II collagen, if any, compared to the induced stem cells. Over the 28-day period, cell density decreased as matrix production increased, a characteristic of cartilage tissue.
  • Placental adherent stem cells in culture are trypsinized at 37° C. for about 5 minutes, and loosened from the culture dish by tapping. 10% FBS is added to the culture to stop trypsinization.
  • the cells are diluted to about 1 ⁇ 10 4 cells per mL in about 5 mL of medium.
  • Drops (either a single drop or drops from a multi-channel micropipette are placed on the inside of the lid of a 100 mL Petri dish. The lid is carefully inverted and placed on top of the bottom of the dish, which contains about 25 ml of sterile PBS to maintain the moisture content in the dish atmosphere. Cells are grown for 6-7 days.
  • This Example demonstrates a scaled up isolation of placental stem cells by enzymatic digestion.
  • 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. Cells liberated by the digestion are washed 3 ⁇ with culture medium, distributed into four T-225 flasks and cultured as described in Example 1. Placental stem cell yield is between about 4 ⁇ 10 8 and 5 ⁇ 10 8 cells per 10 g starting material. Cells, characterized at passage 3, are predominantly CD10 + , CD90 + , CD105 + , CD200 + , CD34 ⁇ and CD45 ⁇ .
  • This Example demonstrates the isolation of placental stem cell and the production of a frozen stem cell-based product.
  • Placental tissue is dissected and digested, followed by primary and expansion cultures to achieve an expanded cell product that produces many cell doses.
  • Cells are stored in a two-tiered cell bank and are distributed as a frozen cell product. All cell doses derived from a single donor placenta are defined as a lot, and one placenta lot is processed at a time using sterile technique in a dedicated room and Class 100 laminar flow hood.
  • the cell product is defined as being CD105 + , CD200 + , CD10 + , and CD34 ⁇ , having a normal karyotype and no or substantially no maternal cell content.
  • Tissue Dissection and Digestion A placenta is obtained less than 24 hours after expulsion. Placental tissue is obtained from amnion, a combination of amnion and chorion, or chorion. The tissue is minced into small pieces, about 1 mm in size. Minced tissue is digested in 1 mg/ml Collagenase 1A for 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 primary culture is to establish cells from digested placental tissue.
  • the digested tissue is suspended in culture medium and placed into Corning T-flasks, which are incubated in a humidified chamber maintained at 37° C. with 5% CO 2 .
  • Half of the medium is replenished after 5 days of culture.
  • High-density colonies of cells form by 2 weeks of culture. Colonies are harvested with Trypsin-EDTA, which is then quenched with 2% FBS in PBS. Cells are centrifuged and resuspended in culture medium for seeding expansion cultures. These cells are defined as Passage 0 cells having doubled 0 times.
  • Expansion Culture Cells harvested from primary culture, harvested from expansion culture, or thawed from the cell bank are used to seed expansion cultures.
  • Cell Factories NUNCTM
  • NUNCTM Cell Factories
  • Cell seeds are counted on a hemacytometer with trypan blue, and cell number, viability, passage number, and the cumulative number of doublings are recorded.
  • Cells are suspended in culture medium to about 2.3 ⁇ 10 4 cells/ml and 110 ml/tray are seeded in the Cell Factories.
  • culture medium is removed and replaced with fresh medium, followed by another treatment with 5% CO 2 in air.
  • cells reach approximately 10 5 cells/cm 2
  • cells are harvested with Trypsin-EDTA, followed by quenching with 2% FBS in PBS. Cell are then centrifuged and resuspended in culture medium.
  • Cryopreservation Cells to be frozen down are harvested from culture with Trypsin-EDTA, quenched with 2% FBS in PBS, and counted on a hemacytometer. After centrifugation, cells are resuspended with 10% DMSO in FBS to 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 freezing container, which is placed in an isopropyl alcohol bath in a ⁇ 80° C. freezer. The following day, cells are transferred to liquid nitrogen.
  • a “lot” is defined as all cell doses derived from a single donor placenta. Cells maintained normal growth, karyotype, and cell surface maker phenotype for over 8 passages and 30 doublings during expansion culture. Given this limitation, doses comprise cells from 5 passages and about 20 doublings. To generate a supply of equivalent cells, a single lot is expanded in culture and is stored in a two-tiered cell bank and frozen doses. In particular, cells harvested from the primary culture, which are defined as Passage 0 cells having undergone 0 doublings, are used to initiate an expansion culture. After the first passage, approximately 4 doublings occur, and cells are frozen in a Master Cell Bank (MCB). Vials from the MCB are used to seed additional expansion cultures.
  • MCB Master Cell Bank
  • WCB Working Cell Bank
  • Frozen containers of cells are placed into a sealed plastic bag and immersed in a 37° C. water bath. Containers are gently swirled until all of the contents are melted except for a small piece of ice. Containers are removed from the sealed plastic bag and a 10 ⁇ volume of culture medium is slowly added to the cells with gentle mixing. A sample is counted on the hemacytometer and seeded into expansion cultures.
  • Frozen containers of cells are transferred to the administration site in a dry nitrogen shipper. Prior to administration, containers are placed into a sealed plastic bag and immersed in a 37° C. water bath. Containers are gently swirled until all of the contents are melted except for a small piece of ice. Containers are removed from the sealed plastic bag and an equal volume of 2.5% HSA/5% Dextran is added. Cells are injected with no further washing.
  • a maternal blood sample accompanies all donor placentas.
  • the sample is screened 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 prior to the receipt of test results, but continues only for placentas associated with maternal blood samples testing negative for all viruses. A lot is rejected if the donor tests positive for any pathogen.
  • the tests described in Table 3 are performed on the MCB, the WCB, and a sample of the cell dose material derived from a vial of the WCB. A lot is released only when all specifications are met.
  • Cells are placed in 1% paraformaldehyde (PFA) in PBS for 20 minutes and stored in a refrigerator until stained (up to a week). Cells are washed with 2% FBS, 0.05% sodium azide in PBS (Staining Buffer) and then resuspended in staining buffer. Cells are stained with the following antibody conjugates: CD105-FITC, CD200-PE, CD34-PECy7, CD10-APC. Cells are also stained with isotype controls. After 30 minute incubation, the cells are washed and resuspended with Staining Buffer, followed by analysis on a flow cytometer. Cells having an increased fluorescence compared to isotype controls are counted as positive for a marker.
  • PFA paraformaldehyde
  • Gene expression patterns from placental stem cells from amnion-chorion (AC) and umbilical cord (UC) were compared to gene expression patterns of multipotent bone marrow-derived mesenchymal stem cells (BM) and dermal fibroblasts (DF), the latter of which is considered to be terminally differentiated.
  • BM bone marrow-derived mesenchymal stem cells
  • DF dermal fibroblasts
  • Cells were grown for a single passage, an intermediate number of passages, and large number of passages (including until senescence). Results indicate that the number of population doublings has a major impact on gene expression.
  • a set of genes was identified that are up-regulated in AC and UC, and either down-regulated or absent in BM and DF, and that are expressed independent of passage number.
  • Placental stem cell- or umbilical cord stem cell-specific genes encodes a number of cytoskeleton and cell-to-cell adhesion proteins associated with epithelial cells and an immunoglobulin-like surface protein, CD200, implicated in maternal-fetal immune tolerance.
  • Placental stem cells and umbilical cord stem cells will be referred to collectively hereinafter in this Example as AC/UC stem cells.
  • BM (Cat# PT-2501) and DF (Cat# CC-2511) were purchased from Cambrex.
  • AC and UC originated from passage 0 tissue culture flasks. AC and UC in the flasks were obtained by digestion from a donor placenta designated 2063919. T-75 culture flasks were seeded at 6000 cells/cm 2 and cells were passaged when they became confluent. Population doublings were estimated from trypan blue cell counts. Cultures were assayed for gene expression after 3, 11-14, and 24-38 population doublings.
  • AC(6) and UC(6) were cultured in parallel with BM-MSC and DF.
  • BM-MSC and DF To maximize identifying a gene expression profile attributable to cellular origin and minimize exogenous influences all cells were grown in the same medium, seeded, and sub-cultured using the same criteria. Cells were harvested after 3 population doublings, 11-14 doublings, or 35 doublings or senescence, whichever came first. Genes whose expression in AC/UC stem cells are unchanged by time-in-culture and are up-regulated relative to BM and DF are candidates for AC/UC stem cell-specific genes.
  • FIG. 10 shows growth profiles for the four cell lines in the study; 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 were regarded as being in culture for a “short” period of time. A short-term DF sample was not collected. Intermediate length cultures, 11 to 14 doublings, were collected for all cell types. Long-term cultures were collected from all cell lines at about 35 population doublings or just prior to senescence, whichever came first. Senescence occurred before 15 doublings for BM and at 25 doublings for DF. The purchased BM and DF cells were expanded many times prior to gene analysis, and cannot be considered early-stage.
  • BM grown for three doublings are deemed a short-term culture.
  • BM-11 is operationally referred to as an intermediate length culture, but because senescence occurred at 14 doublings, BM-11 is most likely a long-term culture biologically.
  • Microarray analysis identifies patterns of gene expression, and hierarchical clustering (HC) attempts 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 over 22,000 probe sets (referred to as the “all genes list”), but many of these sets interrogate genes that are not expressed in any condition. To reduce the all genes list, genes not expressed or expressed at low levels (raw values below 250) in all samples were eliminated to yield a list of 8,215 genes.
  • Gene expression patterns of the 8215 genes were displayed using the line graph view in GeneSpring ( FIG. 11 ).
  • the x-axis shows the twelve experimental conditions and the y-axis shows the normalized probe set expression values on a log scale.
  • the y-axis covers a 10,000-fold range, 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 set, some genes have multiple probe sets) and runs across all twelve conditions as a single color. Colors depict relative expression levels, as described for the heatmaps, but the coloring pattern is determined by selecting one condition.
  • AC-03 is the selected condition in FIG. 11 .
  • Genes up-regulated relative to the normalized value are displayed by the software as red, and those that are down-regulated, are displayed as blue.
  • the obvious upward and downward pointing spikes in AC-03 through UC-11 indicate that many genes are differentially expressed across these conditions.
  • the striking similarity in the color patterns between AC-03 and UC-03 show that many of the same genes are up or down-regulated in these two samples.
  • Horizontal line segments indicate that a gene's expression level is unchanged across a number of conditions. This is most notable by comparing UC-36, UC-38, and UC-38-T. There are no obvious spikes, but there is a subtle trend in that a number of red lines between UC-36 and UC-38-T are below the normalized value of 1.
  • FIG. 12 shows genes differentially over-expressed, by six-fold or more relative to the baseline, in AC-03. The majority of genes up-regulated in AC-03 are also up-regulated in UC-03, and more divergent in BM and DF.
  • DMD dystrophin muscle contraction, cell shape and cell size dystrophy, Duchenne and control, muscle development Becker types
  • DSC3 desmocollin 3 homophilic cell-cell adhesion localized to desmosomes
  • DSG2 desmoglein 2 homophilic cell-cell adhesion localized to desmosomes
  • ELOVL2 elongation of very long chain fatty acid biosynthesis lipid biosynthesis fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 F2RL1 coagulation factor II (thrombin)
  • the experimental design compared cells cultured for short, medium, and long periods of time in culture.
  • each culture period has a characteristic set of differentially expressed genes.
  • AC-03 and UC-03 two hundred up-regulated genes regress to the mean after eight population doublings.
  • 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 are not actively dividing, they are metabolizing nutrients, signaling between themselves, and securing their location by remodeling the extracellular surroundings.
  • Gene expression by the intermediate length cultures is defined by rapid cell division and genes differentially expressed at this time are quite different from those differentially expressed during the early phase.
  • Many of the genes up-regulated in AC-11 and UC-11, along with BM-03 and DF-14, are involved in chromosome replication and cell division.
  • BM-03 appears biologically to be a mid-term culture. In this middle stage cell type-specific gene expression is overshadowed by cellular proliferation.
  • almost every gene over expressed in the short-term AC or UC cultures is down-regulated in the middle and later stage conditions. 143 genes were up-regulated ⁇ five-fold during this highly proliferative phase, constituting approximately 1.7% of the expressed genes.
  • the long-term cultures represent the final or senescent phase.
  • cells have exhausted their ability to divide, and, especially for AC and UC, the absolute number of differentially expressed genes is noticeably reduced. This may be the result of cells being fully adapted to their culture environment and a consequently reduced burden to biosynthesize.
  • late BM and DF cultures do not display this 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 the long-term cultures.
  • placental stem cell-specific gene list described here is diverse. COL4A1 and COL4A2 are coordinately regulated, 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. Tight cell-to-cell contact is characteristic of epithelial and endothelial cells and not typically associated with fibroblasts. Table 3 lists 16 genes, of the 46 total, characteristic to epithelial cells. Placental stem cells are generally described as fibroblast-like small spindle-shaped cells.
  • CD200 This morphology is typically distinct from BM and DF, especially at lower cell densities. Also of note is the expression pattern of CD200, which is present in AC/UC stem cell and absent in all BM and DF samples. Moreover, CD200 has been shown to be associated with immune tolerance in the placenta during fetal development (see, e.g., Clark et al., Am. J. Reprod. Immunol. 50(3):187-195 (2003)).
  • This subset of genes of 46 genes constitutes a set of molecular biomarkers that distinguishes AC/UC stem cells from bone marrow-derived mesenchymal stem cells or fibroblasts.
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