WO2012054501A1 - Human multipotent embryonic stem cell-like progenitor cells - Google Patents
Human multipotent embryonic stem cell-like progenitor cells Download PDFInfo
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- WO2012054501A1 WO2012054501A1 PCT/US2011/056737 US2011056737W WO2012054501A1 WO 2012054501 A1 WO2012054501 A1 WO 2012054501A1 US 2011056737 W US2011056737 W US 2011056737W WO 2012054501 A1 WO2012054501 A1 WO 2012054501A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Definitions
- the present invention relates to a population of precursor/progenitor cells, particularly enriched with multipotent embryonic stem cell-like mesenchymal common progenitor cells (MCPCs), and method for enriching the same.
- MCPCs multipotent embryonic stem cell-like mesenchymal common progenitor cells
- hESCs Human embryonic stem cells
- hESCs Human embryonic stem cells
- MSCs Mesenchymal stem cells
- MSCs are multipotent stem cells that can differentiate into a variety of cell types. MSCs have been isolated from placenta, adipose tissue, lung, bone marrow, dental pulp, and blood. Cell types that MSCs have been shown to differentiate into in vitro or in vivo osteoblasts, chondrocytes, myocytes, adipocytes, and beta-pancreatic islets cells. MSCs were found to be rare in bone marrow, representing ⁇ 1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. Pittenger et al.
- mesenchymal (stem) cells were uniformly positive for SH2, SH3, CD29, CD44, CD71, CD90, CD 106, CD 120a, CD 124, and many other surface proteins, while the mesenchymal cells were negative for other markers of the hematopoietic lineage, including the lipopolysaccharide receptors CD14, CD34, and the leukocyte common antigen CD45.
- MSCs are identified by the expression of many molecules including CD44 and CD 105 and are negative for the hematopoietic markers CD34, CD45, and CD 14.
- amniotic mesenchymal stromal cells and human chorionic mesenchymal stromal cells could be isolated from placenta.
- the surface antigen expression of these cells is given in Table A below, showing that they cannot express CD45, CD34, CD14 and HLA-DR (Parolini et al, Stem Cells 26: 300-311, 2008).
- Table A Specific antigen expression at passages 2-4 for amniotic mesenchymal stromal cells and human chorionic mesenchymal stromal cells
- Caplice (US 7,790,453 B2) taught blood-derived, adult smooth muscle progenitor cells which were positive for CD34.
- the smooth muscle progenitor cells disclosed by Caplice are not characterized as mesenchymal stromal stem/progenitor cells and said progenitor cells have limited differentiation potential.
- Lucas et al. (US 7,259,01 1 B2) taught isolated human pluripotent adult stem cells (PPASCs) expressing CD13, CD34, CD56, and CD117.
- the PPASCs according to Lucas et al. did not express CD 10, CD 14, and stage specific embryonic antigen SSEA2.
- the PPASCs are not characterized as mesenchymal stromal stem/progenitor cells, either.
- Hariri (US 7,468,276 B2) taught isolated human placental stem cells that are OCT4 + and CD34 + .
- the human placental stem cells disclosed by Hariri were SSEA3 " and SSEA4 " .
- the human placental stem cells of Hariri were not characterized as mesenchymal stromal stem/progenitor cells.
- Edinger et al. discloses non-adherent, CD34 + CD45 " stem cells isolated from placenta.
- the placental stem cells according to Edinger et al. are non-adherent, and thus were not mesenchymal.
- tissue engineering an enriched population of multipotent stem cells that are juvenile and prolonged self-renewal are desired.
- the present invention provides a plurality of embryonic stem cell-like precursor cells, which is an enriched population of multipotent human mesenchymal common progenitor cells (MCPCs).
- the cells are isolated from a human somatic tissue by a systemic screening of human mesenchymal stromal stem/progenitor cells followed by a cell sorting by a cell antigen selected from the group consisting of CD34, CD1 17, CD133, CD201, GloboH and combination thereof, and cultured in a medium supplemented with at least one or more steroids, and one or more growth factors.
- MCPCs multipotent human mesenchymal common progenitor cells
- the invention provides an enriched population of multipotent human mesenchymal common progenitor cells (MCPCs), which are identified as mesenchymal stromal stem/progenitor cells having at least the following characteristics: CD14 + , CD34 + , CD117 + , CD133 + (AC133 + ), CD201 + , Nesting SSEA3 + , SSEA4 + , and GloboH + .
- MCPCs multipotent human mesenchymal common progenitor cells
- the invention provides a method for producing the enriched population of multipotent human MCPCs according to the invention, comprising isolating from a human somatic tissue by a systemic screening of human mesenchymal stromal stem/progenitor cells followed by a cell sorting by a cell antigen selected from the group consisting of CD34, CD1 17, CD133, CD201, GloboH and combination thereof, and culturing in a medium supplemented with at least one or more steroids selected from the group consisting of a corticosteroid and cholesterol and one or more growth factors selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), insulin, platelet-derived growth factor (PDGF), IL-6, and thrombopoietin (TPO).
- EGF epidermal growth factor
- FGF fibroblast growth factor
- IGF insulin-like growth factor
- PDGF platelet-derived growth factor
- IL-6 thrombop
- the invention provides a composition comprising the enriched population of multipotent human MCPCs according to the invention encapsulated in alginate.
- the invention provides a feeder cell layer for stem cell culture comprising the enriched population of multipotent human MCPCs of the invention.
- the invention provides a stem cell niche comprising the enriched population of multipotent human MCPCs according to the invention seeded on a scaffold.
- Fig. 1 shows the cell morphology of human placenta amnionic mesenchymal cells.
- Fig 1A is a phase contrast image of AM-MSCs-CD34 + cells.
- Fig IB is a phase contrast image of AM-MSCs-CD34 " cells.
- Fig. 2 shows the profiling of cell surface marker of AM-MSCs-CD34 + and AM-MSCs-CD34 " cells; wherein Fig. 2A shows the expression of CD29, CD44, CD73, CD90, CD105, CD31, CD56, EGFR, and PDGFR; and Fig. 2B shows the expression of CD34, CD1 17, CD133, SSEA1, SSEA3, SSEA4, GloboH, and CD201.
- FIG. 3 A provides a schematic illustration of neural and oligodendrocyte differentiation of CD34 sorted MSCs.
- Fig. 3B provides a schematic illustration of dopaminergic neuron differentiation of CD34 sorted MSCs.
- FIG. 4 shows TuJl, TH, and MAP2 expression of CD34 + or CD34 " AM-MSC after dopaminergic neuron induction
- Fig. 4A provides TuJl (top), GFAP (middle), and DAPI (bottom) immunofluorescence staining images of CD34 + AM-MSC induced neurons
- Fig. 4B provides TuJl (top), GFAP (middle), and DAPI (bottom) immunofluorescence staining images of CD34 " AM-MSC induced neurons
- Fig. 4C provides TH (top), MAP2 (middle), and DAPI (bottom) immunofluorescence staining images of CD34 + AM-MSC induced neurons
- Fig. 4D provides TH (top), MAP2 (middle), and DAPI (bottom) immunofluorescence staining images of CD34 " AM-MSC induced neurons.
- Fig. 5 shows the cell morphology of human endometrium mesenchymal cells; wherein Fig 5A is a phase contrast image of EnMSCs-CD34 + cells; and Fig. 5B is a phase contrast image of EnMSCs-CD34 " cells.
- Fig. 6 shows the cardiomyogenic differentiation potentials of EnMSCs-CD34 + ; wherein Fig 6A provides Troponin T (top), myosin heavy chain (MHC) (middle), and DAPI (bottom) immunofluorescence staining images of CD34 + EnMSCs after cardiomyogenic induction; and Fig 6B provides Troponin T (top), myosin heavy chain (MHC) (middle), and DAPI (bottom) immunofluorescence staining images of CD34 " EnMSCs after cardiomyogenic induction.
- Fig 6A provides Troponin T (top), myosin heavy chain (MHC) (middle), and DAPI (bottom) immunofluorescence staining images of CD34 " EnMSCs after cardiomyogenic induction.
- MHC myosin heavy chain
- DAPI bottom
- Fig. 7 shows the numbers of expanded hematopoietic stem cells (HSCs) with specific surface marker(s) when co-culturing with murine MS-5 feeder, MSCs-CD34 + feeder, or MSCs-CD34 " feeder.
- HSCs hematopoietic stem cells
- the article “a” or “an” means one or more than one (that is, at least one) of the grammatical object of the article, unless otherwise made clear in the specific use of the article in only a singular sense.
- a plurality of precursor cells are isolated from human somatic tissues, and identified as mesenchymal stromal stem/progenitor cells expressing at least CD14, CD34, CDl 17, CD133 (AC133), CD201, Nestin, SSEA3, SSEA4, and GloboH (called as "MCPCs").
- the MCPCs may be obtained by a method comprising the steps of isolating from a human somatic tissue by a systemic screening of human mesenchymal stromal stem/progenitor cells followed by a cell sorting by a cell antigen selected from the group consisting of CD34, CDl 17, CD133, CD201, GloboH, and combination thereof, and culturing in a medium supplemented with at least one or more steroids selected from the group consisting of a corticosteroid, cholesterol, and combination thereof, and one or more growth factors selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), insulin, platelet-derived growth factor (PDGF), IL-6, thrombopoietin (TPO), and combination thereof.
- the MCPCs of the invention adhere to a tissue culture surface, which is different from the CD34 + , CD45 " placental stem cells as disclosed in Edinger et al. (US 2008
- the MCPCs of the invention can be isolated from human somatic tissues including but not limited to neonatal placenta (e.g. amnion, chorion, and umbilical cord), endometrium, gingival, bone marrow, and adipose.
- the MCPCs are isolated from placenta, endometrium, and gingival. More preferably, the MCPCs are isolated from placenta amniotic tissue.
- the MCPCs isolated from placenta amniotic tissue are called as AM-MSCs-CD34 + cells; the MCPCs isolated from endometrium are called as EnMSCs-CD34 + cells; and the MCPCs isolated from gingival are called as GMSCs-CD34 + cells.
- AM-MSCs-CD34 + cells, EnMSCs-CD34 + cells, and GMSCs-CD34 + cells have consistent profiling of cell surface markers expression.
- the MCPCs exhibited sphere-like clonogenicty in early passages and expressed multipotent embryonic stem cell (ESCs) like characteristics in vitro.
- the MCPCs of the invention are shorter than CD34 " MSCs. Specifically, the MCPCs of the invention have a higher growth rate as compared to CD34 " MSCs or unsorted MSCs, indicating that MCPCs of the invention are more proliferative and younger.
- the MCPCs homogenously express embryonic (e.g. Oct-4, Nanog, Rex-1, Sox-2), sternness (e.g. CD1 17, CD34, CD44) surface antigens, in addition to present various lineage markers, including MSC (e.g. CD29, CD90, CD73, CD105, CD106), hem-angiogenic (e.g. AC133, CD34), myo-nurogenic (e.g. CD54, Nestin, NSE). Further, the MCPCs of the invention are prolonged self-renewal.
- the MCPCs e.g., AM-MSCs-CD34 + cells
- the MCPCs could retain their specific cell marker expression as CD34, CD54, CD1 17, and AC133 positive, and their MSC marker expression even after 20 passages.
- the invention provides an enriched population of multipotent human mesenchymal common progenitor cells (MCPCs), which are identified as mesenchymal stromal stem/progenitor cells which have at least the following characteristics: CD14 + , CD34 + , CD1 17 + , CD133 + (AC133 + ), CD201 + , Nesting SSEA3 + , SSEA4 + , and GloboH + .
- MCPCs multipotent human mesenchymal common progenitor cells
- the enriched population of multipotent human MCPCs further have at least one of the following characteristics: CD44 + , CD54 + , CD56 + , CD105 + , CD146 + , and PDGFR + .
- the enriched population of multipotent human MCPCs are identified as mesenchymal stromal stem/progenitor cells, having the following characteristics: CD14 + , CD34 + , CD1 17 + , CD133 + (AC133 + ), CD201 + , Nesting SSEA3 + , SSEA4 + , GloboH + , CD44 + , CD54 + , CD56 + , CD105 + , CD146 + and PDGFR + .
- the enriched population of multipotent human MCPCs have the potentials to differentiate into cells or tissues of ectodermal lineage, mesodermal lineage, and endodermal lineage.
- AM-MSCs-CD34 cells were examined and found that they were multipotent in differentiation of various types of somatic cells, including endoderm, mesoderm, or ectoderm cells.
- the enriched population of multipotent human MCPCs have the potentials of adipogenic differentiation, osteogenic differentiation, chondrogenic differentiation, neurogenic differentiation, cardiomyogenic differentiation, endothelial differentiation, and hepatic differentiation.
- the invention provides a method for producing an enriched population of multipotent human MCPCs, comprising isolating from a human somatic tissue by a systemic screening of human mesenchymal stromal stem/progenitor cells and a cell sorting by a cell antigen selected from the group consisting of CD34, Nestin, CD1 17, CD133 and combination thereof; and culturing in a medium supplemented with at least one or more steroids and one or more growth factors.
- the term "steroid” refers to a type of organic compound that contains a specific arrangement of four cycloalkane rings that are joined to each other.
- the steroid used in the medium according to the method may be one selected from the group consisting of a corticosteroid, cholesterol and combination thereof.
- corticosteroid refers to a class of steroid hormone.
- examples of corticosteroid include but are not limited to a group A corticosteroid (e.g. hydrocortisone, hydrocortisone acetate, and cortisone acetate), a group B corticosteroid (e.g. triamcinolone acetonide, triamcinolone alcohol, and mometasone), a group C corticosteroid (e.g. betamethasone, betamethasone sodium phosphate, and dexamethasone), and a group D corticosteroid (e.g. hydrocortisone- 17-valerate, aclometasone dipropionate, and hydrocortisone- 17-butyrate).
- group A corticosteroid e.g. hydrocortisone, hydrocortisone acetate, and cortisone acetate
- group B corticosteroid e.g. triamcinolone
- growth factor refers to a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation, which can regulate a variety of cellular processes, and typically act as signaling molecules between cells.
- the growth factor used in the medium according to the method may be one or more selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), insulin, platelet-derived growth factor (PDGF), IL-6, thrombopoietin (TPO), and combination thereof.
- the medium may be further supplemented with and one or more vitamins.
- vitamin refers to a nutrient in tiny amounts by an organism.
- examples of vitamins include but are limited to vitamin A, vitamin B-complex, vitamin E, biotin, p-aminobenzoic acid, menadione, and combination thereof.
- the medium may be also supplemented with one or more compounds selected from the group consisting of uracil, sodium acetate, ribose, Guanine HC1, deoxyribose, adenosine, adenine sulphate, ferric nitrate and combination thereof.
- the cell sorting is performed by using fluorescence-activated cell sorting (FACS) flow cytometry.
- FACS fluorescence-activated cell sorting
- the cell sorting is a CD34, CD117, CD133, CD201, or GloboH cell antigen FACS.
- the MCPCs may be encapsulated in alginate to form a composition.
- a feeder cell layer for stem cell culture is provided as well.
- the feeder cell layer comprises the enriched population of multipotent human MCPCs of the invention.
- the enriched population of multipotent human MCPCs may be seeded on the scaffold to form a stem cell niche.
- the invention provides a better source of ESC-like clonogenic stem cells that are derived from non-embryonic neonatal or adult tissue and multipotent in differentiation to various types of cells, than known sources, such as ESCs.
- the MCPCs of the invention were found to be of proliferative ability and differentiation potentials. They are of a great potential to be used for clinical regenerative therapies.
- the invention is further described in the following non-limiting examples.
- Example 1 Preparation and Characterization of AM-MSCs-CD34 + Cells [0046] Amnionic Mesenchymal Cell Isolation
- amnionic epithelial cells (Am-EpCs)
- washed amnion membrane was cut into 2-3 cm 2 fragments and incubated in 100 ml of 0. 1% Trypsin-EDTA (Sigma; St Louis, MO) with 1 * Hanks balanced salt solution (Gibco; CAT# 14185-052; Grand Island, NY) for an about 250 cm 2 membrane, with 4 times of each 15 min reactions in water bath, at 37°C.
- AM-MSCs Collection of clonogenic amnionic mesenchymal stromal cells
- AM-MSCs amnionic mesenchymal cells isolation
- the Am-EpCs depleted amnion membrane was subjected to wash with Hank's buffer one time and digested with collagenase 1A at 37°C for 45-60 minutes.
- An appropriate volume of Hank's buffer and a 40 ⁇ nylon cell strainer were used to collect the clongenic AM-MSCs.
- AM-MSCs were plated in CELL-BIND T75 flasks at a density of 5 ⁇ 10 4 cells per cm 2 and incubated in a 5% CO2, 37°C.
- the collected AM-MSCs were incubated in a medium containing Medium 199 (the Ml 99 conditioned medium) [(Lonza CAT# 12-1 18F; Switzerland), supplemented with fetal bovine serum (FBS), epidermal growth factor (EGF), and hydrocortisone.
- FBS fetal bovine serum
- EGF epidermal growth factor
- hydrocortisone Fresh medium were changed in every 3-4 days during the purification incubation, and cells expanded to 80% confluence in 7 days.
- the attached culturing cells were harvested with 0. 1% Trypsin-EDTA, and split into the new passage culture with a seeding density of l lO 5 cells per T75 flask.
- the collected CM-MSCs can be cultured in RPMI- 1640 (GIBCO; Grand Island, NY) supplemented with fetal bovine serum (FBS) (10%), sodium pyruvate (0.1 mM), basic fibroblast growth factor (bFGF), and EGF (10 ng/ml). Cells were split when they reached to 70-80% confluence, the culture medium was changed every 3-4 days.
- FBS fetal bovine serum
- bFGF basic fibroblast growth factor
- EGF EGF
- the expanded primary AM-MSCs cultured at passages 2-3 were used to label with CD34 antibody.
- Up to 3 ⁇ 10 6 cells were sorted by a FACS Aria flow cytometry (BD Bioscience, San Jose, CA) following the manufacture's instruction.
- CD34 positive (CD34 + ) and CD34 negative (CD34 ) cells were then analyzed, sorted, and collected.
- AM-MSCs-CD34 + and AM-MSCs-CD34 populations were re-analyzed for the positive fraction and expanded in the Ml 99 conditioned medium as described above or the RPMI conditioned medium.
- the cell morphology of AM-MSCs (passage 3) are given in Fig. 1, wherein the shape of AM-MSCs-CD34 + cells (Fig. 1A) is shorter than AM-MSCs-CD34 " cells (Fig. IB) and both showed stable in later passages.
- the initial doubling time of AM-MSCs-CD34 " cells is about 42 hours, which is longer than that of AM-MSCs-CD34 + at being 34 hours.
- the CD34 expression of AM-MSCs would be checked for each five passages in the following days. After CD34 + sorting 20 passages, CD34 + AM-MSC could still retain their specific cell marker expression as CD34, CD54, CD1 17, and CD133 (AC133) positive and their MSC marker expression.
- CD34 + AM-MSC could express more CD117, CD133, SSEAl, SSEA3, SSEA4, Globo H, and CD201 than CD34 " AM-MSC (Fig. 2B).
- AM-MSCs-CD34 + cells express "early genes" including Sox-2, Oct-4, Rex-1, and Nanog, ectodermal lineage genes, e.g. neurogenic differentiation markers including Nestin, NSE, NFM, NCAM, MAP2, mesodermal lineage genes, e.g. cardiomyogenic differentiation markers including MyoD, GATA-4, and MLC-2a, and endodermal lineage genes, e.g. hepatic differentiation makers including Albumin and HGF.
- ectodermal lineage genes e.g. neurogenic differentiation markers including Nestin, NSE, NFM, NCAM, MAP2, mesodermal lineage genes, e.g. cardiomyogenic differentiation markers including MyoD, GATA-4, and MLC-2a
- endodermal lineage genes e.g. hepatic differentiation makers including Albumin and HGF.
- Example 2 Induction of Differentiation
- the AM-MSCs-CD34 + cells at the number of 2 ⁇ 10 5 at passage 5 were used for the vasculogenic differentiation induction.
- Harvested cells were cultured in EGM-2 medium (Cambrex) for a 7 days induction.
- AM-MSCs-CD34 + cells at passages 4-6 were harvested for induction of cardiomyogenic differentiation.
- a cardiomyocytic differentiation agent 5-azacytidine (Sigma) was added into the medium to make a 5 ⁇ final concentration.
- CD34 + AM-MSCs trans differentiated easily into cardiomyocytes expressing MyoD, GATA-4, MLC-2a genes (data not shown).
- the AM-MSCs-CD34 cells obtained by the method as mentioned in Example 1 and expanded passages 5-6 were used for multi-lineage differentiation inductions.
- the adipogenic, osteogenic, chondrogenic, and neurogenic differentiation protocols were used by the methods given below.
- the AM-MSCs or AM-MSCs-CD34 pre-conditioning in Dulbecco's modified Eagle's medium (DMEM/LG, GIBCO) supplemented with 10% FBS (Hyclone) were used for the lineage differentiation cultures shown as following:
- Adipogenesis DMEM/LG medium supplemented with 10% FBS, 0.5 mM isobutyl-methylxanthine, 1 ⁇ dexamethasone, 10 ⁇ insulin, 200 ⁇ indomethacin.
- Osteogenesis DMEM/LG medium supplemented with 10% FBS, 0.1 ⁇ dexamethasone, 50 ⁇ ascorbate-2 -phosphate, 10 mM ⁇ -glycerolphosphate.
- CM Chondrogenesis
- Neurogenesis DMEM/LG medium supplemented with 5 ⁇ g/ml insulin, 200 ⁇ indomethacin, 0.5 mM isobutyl-methylxanthine. (Reagents used above for differentiation were all from Sigma; St.Louis, MO)
- the cell density was 3 x l0 4 cells/cm 2 .
- a higher cell density of l-2x l0 5 /10 ⁇ was used for chondrosphere formation.
- Medium was changed every three to four days for all differentiation assays, and cells were fixed for histochemical staining after 14 days of adipogenic, osteogenic, chondrogenic differentiation. After 14 days, intracellular oil droplets were formed under Oil Red O stain, and calcified extracellular matrix was present and positive for von Kossa staining (data not shown).
- AM-MSCs-CD34 + cells formed cartilage ball in 3 days.
- AM-MSCs-CD34 + cells cultured in neurogenic differentiation medium (Zuk's protocol, P4, Day 21) exhibited neural morphology and expressed neural markers including Nestin, NSE, NFM, NCAM, and MPA2, while AM-MSCs-CD34 " cells did not (data not shown).
- Stepl Neurosphere Formation: Cells were seeded at a density of 1000 per well with neurosphere medium (NS medium).
- NS Medium DMEM/HG/F12 (1 : 1) + l x B27 + 20 ng/ml EGF + 20 ng/ml FGF2 +2 ⁇ g/ml Heparin.
- Primary neurospheres (selecting mainly floating neurospheres) larger than 75 um were counted after 7 days in vitro.
- Step2 Neural Differentiation Assay: Dissociated neurospheres to single cells by trypsin-EDTA solution and culture the cells with: DMEM/F12 + 5% FBS for 24 hrs. These cells were treated with specific neural cell differentiation medium.
- Fig. 3A is a schematic illustration of neural and oligodendrocyte differentiation of CD34 sorted MSCs.
- Fig. 3B is a schematic illustration of dopaminergic neuron differentiation of CD34 sorted MSCs.
- CD34 + AM-MSC induced neurons were TuJl, TH, and MAP2 positive, while only a small population of CD34 " AM-MSC induced neurons expressed said markers (Fig. 4).
- CD34 + AM-MSCs expressed early genes and showed multipotent differentiation potential. Specific gene expression of CD34 + AM-MSCs is provided in Table 2 below.
- Example 3 EnMSCs-CD34 + cells, GMSCs-CD34 + cells, and CD34 + MSCs Enriched from Other Somatic Tissues
- AM-MSCs-CD34 + cells, EnMSCs-CD34 + cells, and GMSCs-CD34 + cells were identified as having consistent profiling of cell surface marker expression.
- the mesenchymal common progenitor cells (MCPCs) of the invention were CD14 + , CD34 + , Nesting CD117 + , CD133 + (AC133 + ), SSEA3 + , and SSEA4 + . Further, the MCPCs of the invention were also characterized as CD44 + , CD54 + , CD105 + ,
- CD146 + CD146 + , or PDGFR + .
- Table 5 Potentials of endothelial differentiation and chondrogenic differentiation.
- EnMSCs CD34+ CD31+, KDR+, CD34+: Bigger cartilage ball (CD34+/CD34-) more tube formation. (Diameter > 2x).
- CD34- CD31+, KDR+, less CD34-: Little cartilage ball tube formation. (Diameter ⁇ 100 ⁇ ).
- GMSCs CD34+ CD31+, KDR+, CD34+: Bigger cartilage ball
- CD34- CD31+, KDR+, less CD34-: Little cartilage ball tube formation. (Diameter ⁇ 100 ⁇ ).
- Table 7 Potentials of neurogenic differentiation and cardiomyogenic differentiation.
- CD34- Myosin Heavy Chain+
- CD34- Nestin+, Troponin T-.
- EnMSCs CD34+ Nestin+, TuJl+, CD34+: Myosin Heavy Chain+, (CD34+/CD34-) GFAP-, Troponin T+.
- CD34- Myosin Heavy Chain+
- CD34- Nestin+, TuJl+, Troponin T-.
- GMSC CD34+ Nestin+, TuJl+, CD34+: Myosin Heavy Chain+,
- CD34- Myosin Heavy Chain+
- CD34- Nestin+, TuJl+, Troponin T-.
- CD34+ MSCs can be enriched from many other somatic tissues according to the method of the invention. Normally, only 2-3% of MSCs are CD34 + . After isolated and enriched in culture, percentage of CD34 + MSCs ranging from 15% to 78% depending on which tissue they were isolated from and the donor (See Table 8 below). The culture-enriched CD34 + MSCs can be subject to FACS cell sorting for further enrichment to obtain a population of MSCs enriched with 99% or more CD34+ MSCs.
- Example 4 MCPCs as Feeder Cells
- MCPCs of the invention were used to prepare a stromal feeder for expansion of hematopoietic stem cells (HSCs).
- Stromal cells MS-5, or MSCs
- 2 ⁇ 4 ⁇ 10 4 CD34 + mononuclear cells (M Cs) were co-cultured with feeder in 1 ml HSC medium (X- VIVO 10 + 50 ng/ml SCF + 50 ng/ml Flt-3L + (20 ng/ml)10U/ml TPO + 10 ng/ml IL-6).
- suspension cells were counted and subjected to flow cytometry analysis (for CD34+CD38-, CD34+CD133+, CD34+CXCR4+, etc.).
Abstract
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KR1020137012722A KR20140001900A (en) | 2010-10-18 | 2011-10-18 | Human multipotent embryonic stem cell-like progenitor cells |
CN201180057856.7A CN104204190B (en) | 2010-10-18 | 2011-10-18 | Human multipotent embryonic stem cell-like progenitor cells |
CA2815097A CA2815097A1 (en) | 2010-10-18 | 2011-10-18 | Human multipotent embryonic stem cell-like progenitor cells |
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EP3613847A1 (en) * | 2013-03-13 | 2020-02-26 | The University of Queensland | A method of isolating cells for therapy and prophylaxis |
EP3004328A4 (en) * | 2013-05-30 | 2016-12-07 | Cells For Cells | Chorion-derived mscs cells and conditioned media as inducer for angiogenesis application for the treatment of cardiac degeneration. |
BR102013033827B1 (en) | 2013-12-27 | 2021-09-08 | Regenera - Medicina Veterinária Avançada Ltda. | MULTIPOTENT AND IMMUNOCOMPATIBLE CONCENTRATE OF STEM CELLS AND BIOPHARMACEUTICALS AND DOSAGE FORM THAT INCLUDE IT |
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US11103537B2 (en) | 2016-06-23 | 2021-08-31 | Tithon Biotech Inc. | Cells expressing parathyroid hormone 1 receptor and uses thereof |
CN108728408B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Canine fetal membrane mesenchymal stem cell, preparation method and culture medium used by same |
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