AU2001257436A1 - Isolation of mesenchymal stem cells and use thereof - Google Patents
Isolation of mesenchymal stem cells and use thereofInfo
- Publication number
- AU2001257436A1 AU2001257436A1 AU2001257436A AU2001257436A AU2001257436A1 AU 2001257436 A1 AU2001257436 A1 AU 2001257436A1 AU 2001257436 A AU2001257436 A AU 2001257436A AU 2001257436 A AU2001257436 A AU 2001257436A AU 2001257436 A1 AU2001257436 A1 AU 2001257436A1
- Authority
- AU
- Australia
- Prior art keywords
- collagenase
- cell
- cells
- specimen
- mesenchymal stem
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Description
ISOLATION OF MESENCHYMAL STEM CELLS AND USE THEREOF
FIELD OF THE INVENTION
[0001] The present invention is generally directed to isolation of mesenchymal stem cells and a method of differentiating these cells into various lineages.
BACKGROUND OF THE INVENTION
[0002] One of the major challenges facing tissue engineering is having a reliable and easily reproducible cell source for harvesting and expansion. Embryonic tissues " , bone marrow, and skeletal muscle4"6 have been identified as sources for the derivation of mesenchymal stem cells with defined pluripotential properties. Mesenchymal stem cells can replicate as undifferentiated cells and have the remarkable ability to differentiate into specific kinds of mesenchymal or connective tissues including osseous (bone), cartilagenous, adipose (fat), muscular, elasltic, and fibrous connective tissues. Although these cells are normally present at very low frequencies in bone marrow, a process for isolating, purifying and mitotically expanding the population of these cells in tissue culture has been described in U.S. Patent Nos. 5,486,359; 5,197,985; and 5,226,914.
[0003] Adult meenchymal stem cells go through a sequence of proliferation, commitment, differentiation and maturation. In a healthy individual, these cells take care of the replacement and regenration of human connective tissues.
[0004] The ability to grow mesenchymal stem cells from a reliable cell source is crucial to production of complex tissues and organs for the restoration of damaged or diseased tissue.
[0005] Skin tissue is composed of mesoderm and ectoderm. The mesodermal layer contains many different cell types, and most have a stromal phenotype. Although fibroblasts are ubiquitous and posess the same phenotypic properties in mammalian tissues and organs, their biological properties may differ, depending on their natural environment. While it is possible that most fibroblasts are "mature", incapable of transformation, there is evidence that there are "immature" fibroblasts (pluripotent mesenchymal stem cells) that are capable of transformation.
[0006] The molecular and cellular basis of directing an "immature" connective tissue phenotype to form bone, fat and cartilage is a remarkable biological phenomenon with enormous implications for disorders related to ostegenisis (e.g. the control of bone regenration and fracture
healing), adipogenisis (obesity and obesity-induced diabetes), and chondrogenisis (e.g., tissue egineering for numerous cartilage-regenration therapies including treatment of urinary incontinence).
[0007] For the foregoing reasons and deficiencies of the current state of the art, there exists a need for mesenchymal stem cells and especially mesenchymal stem cells derived from a reliable source and capable of being differentiated into different lineages of these cells, such as osteogenic, adipogenic and chondrogenic lieneages.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to isolation of mesenchymal stem cells, their isolation form a reliable source, and their differentiation into osteogenic, adipogenic and chondrogenic lineages. In accordance with one aspect of the invention, mesenchymal stem cells are isolated from a dermal layer of human skin, and more particularly from a postnatal human foreskin. In another aspect of the present invention, the isolated mesenchymal stem cells are differentiated into osteogenic, adipogenic, and chondrogenic lineages.
[0009] As aforesaid, the first aspect of the present invention provides a method for isolating human mesenchymal stem cells. The method comprises providing a specimen of human connective tissue; holding the specimen in a sterile environment for at least 2 days (preferably for 2 to 10 days and most preferably for 3 to 6 days) at about 4C; isolating a dermal layer from the specimen; contacting the dermal layer with collagenase, preferably collagenase I, to provide a collagenase cell suspension; collecting the supernatant from the cell suspension; centrifuging the supernatant to obtain a cell product, and resuspending the resulting cell product in the culture medium to promote cell growth. Preferably, the human connective tissue is obtained from a human skin, more preferably, from human postnatal foreskin. Preferably, the cells are subcultured using trypsin and EDTA for 5 minutes at 37C.
[0010] In another aspect of the invention, a human mesenchymal stem cell produced by the method described supra is disclosed.
[0011] In a third aspect of the invention, a method for obtaining osteoblasts is provided. The method comprises: providing a specimen of human connective tissue; holding the specimen in a sterile environment for 2-10 days at about 4C; isolating a dermal layer from the tissue; contacting the dermal layer with collagenase to provide a collagenase cell suspension; collecting the supernatant from the cell suspension; centrifuging the supernatant to obtain a cell product, and resuspending the resulting cell product in the culture medium with dexamethasone, beta-
glycerophosphate and ascorbic acid-2-phosphate in amounts sufficient to induce human mesenchymal stem cells to undergo osteogenic differentiation.
[0012] In a fourth aspect of the invention, a method for obtaining adipocytes is provided. The method comprises: providing a specimen of human connective tissue; holding the specimen in a sterile environment for 2-10 days at about 4C; isolating a dermal layer from the tissue; contacting the dermal layer with collagenase to provide a collagenase cell suspension; collecting the supernatant from the cell suspension; centrifuging the supernatant to obtain a cell product, and resuspending the resulting cell product in the culture medium with dexamethasone, 3- isobutyl-1-methylxanthine, insulin, and indomethacin in an amount sufficient to induce human mesenchymal stem cells to undergo adipogenic differentiation.
[0013] In a fifth aspect of the invention, a method for obtaining chondrocytes is provided. The method comprises: providing a specimen of human connective tissue; holding the specimen in a sterile environment for 2-10 days at about 4C; isolating a dermal layer from the tissue; contacting the dermal layer with collagenase to provide a collagenase cell suspension; collecting the supernatant from the cell suspension; centrifuging the supernatant to obtain a cell product, and resuspending the resulting cell product in the culture medium with L-glutamine, MEM nonessential amino acids, 2-mercaptoethanol, dexamethasone, ascorbic acid-2 -phosphate, and transforming growth factor 3 in an amount sufficient to induce human mesenchymal stem cells to undergo chondrogenic differentiation.
[0014] Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE FIGURES
[0015] For a more complete understanding of the invention, reference should be made to the figures, in which:
[0016] Figure 1 depicts the effect of adipogenic medium on mesenchymal stem cells in light microscopy and Oil-O-Red staining. Cells were seeded at 3x10 cells/cm in 2 well plates in control medium for 4 days. Subsequently, plates were cultured either in adipogenic medium or in control medium for 16 days. Before air-drying, the chamber slides for Oil-O-Red staining, pictures from cultures were taken in light microscopy.
[0017] Figure 2A depicts the effect of osteogenic medium on undifferentiated fibroblast cultures in alkaline phosphatase (AP) staining. Cells were grown in either control medium or in osteogenic medium for 4, 8, and 16 days.
[0018] Figure 2B shows the average of AP production of cells from 2 patients, treated either with osteogenic medium or with control medium after 4, 8, and 16 days of culture. Star marked values at Day 16 time point represent AP production of mesenchymal stem cells from bone marrow. Values were measured in quadruplicate values for each patient. P-Nitrophenol production is equivalent to alkaline phosphatase production.
[0019] Figure 3 A shows the effect on undifferentiated fibroblast cultures of osteogenic medium in von Kossa staining. Undifferentiated cells were seeded at 3x10 cells/cm in 35 mm dishes. Cells were grown either in control medium or in osteogenic medium for 4, 8, and 16 days.
[0020] Figure 3B shows the average of two patients for calcium deposition of cells with Osteogenic Medium (solid line) and cells with Control Medium (broken line) after 4, 8, and 16 days of treatment. Values were measured in quadruplicate values for each patient. Results were normalized to cell counts.
[0021] Figure 4A shows a RT-PCR upregulation of cbfal in osteogenic cells.
[0022] Figure 4B is a RT-PCR performed for ppar -2 showing upregualtion of transcription in cells treated with adipogenic medium. Lipoprotein lipase expression is strongly induced in adipogenic cells.
DETAILED DESCRIPTION OF THE INVENTION
[0023] In the present invention, the postnatal human dermal tissue was used as a source of pluripotential cells. Particularly, the present invention provides a method for isolating mesenchymal stem cells from postnatal human tissue, and a method of differentiate the mesenchymal stem cells into osteogenic, adipogenic, and chondrogenic lineages.
[0024] Osteogenic induction of mesenchymal stem cells has been reported for bone marrow, skeletal muscle, and embryonic stem cells. Osteogenic differentiation can be determined from a change in phenotype, the production of alkaline phosphatase, mineralization, accumulation of calcium, and the expression of genes specific for osteogenic induction.5'8'1 A transcription factor that is important for osteoblastic differentiation is cbfal .
Cbfal is also highly expressed in chondrocytes when becoming hypertrophic for bone formation.18 In one aspect of the present invention, we disclose, infra, how mesenchymal stem cells can be derived from postnatal dermal tissue (preferably, postnatal human foreskin tissue), and can be made to differentiate into an osteogenic lineage.
[0025] Adipogenic differentiation has been reported for postnatal mesenchymal stem cells and for C3/T10 cells, derived from embryonic tissue.14 Adipogenic differentiation can be determined with oil-O-red staining, which stains intracellular fat-filled vacuoles, expression of lipoprotein lipase in adipogenic cells, and over-expression or induction of expression of pparγ-2, which is the most specific marker for adipogenic induction.5'14'15'19 In another aspect of the present invention, we disclose infra how mesenchymal cells, derived from human skin (preferably postnatal human foreskin) can be made to differentiate into an adipogenic lineage. Although Pittenger et al. used the same markers in an attempt to demonstrate the potential of bone marrow mesenchymal stem cells to differentiate into an adipogenic lineage, they were unsuccessful in that they were able to produce adipocytes only, they were not able to induce fibroblasts from skin to become osteogenic and adipogenic.5
[0026] In yet another aspect of the invention, we disclose infra a method of differentiating mesenchymal stem cells derived from human skin (preferably, postnatal human foreskin) into chondrocytes.
[0027] Although markers for differentiated cells are widely used and recognized, stem cell markers are more difficult to define, and marker expression may depend on the pluripotential ability of the cells. For example, mesenchymal stem cells derived from bone marrow express endoglin (CD-105) and Thy-1 (CD-90), but do not express CD-34 and CD-133, which are expressed in hematopoetic mesenchymal stem cells5. The mesenchymal cells of the present invention show positive expression for CD-90 and CD-105, which is consistent with the expression seen with bone marrow derived stem cells.
[0028] It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
EXAMPLES
Cell Preparation and Culture Methods:
[0029] Human foreskin specimens are obtained during routine circumcisions from children aged 6 months to 18 years. The foreskin tissues are kept in sterile containers at 4°C for 3-6 days and are rinsed in betadine for 5 minutes prior to processing. The dermal layers are
isolated, sectioned into small pieces (1 mm3) and digested in 15 ml of a sterile phosphate buffered solution (PBS) containing 1.25 mg/ml Collagenase I (Worthington Biochemicals) for 2 hours at 37°C. After 2 hours. Subsequently, the supernatant is collected and centriftiged at 1000 RPM for 5 minutes to obtain a cell product. The resulting cell product is then resuspended in culture medium, plated on 100mm bacterial dishes (VWR scientific products, Willard, OH) and grown to confluency for 2 to 3 weeks at 95% humidity and 37°C.
[0030] The culture medium preferably consists of DMEM high glucose (GIBCO/BRL, Grand Island, NY) with 20% embryonic stem cell-certified fetal bovine serum (ES- FBS, GIBCO/BRL, Grand Island, NY), 1% antibiotics (GIBCO/BRL, Grand Island, NY), L- glutamine, (Sigma- Aldrich, St.Louis, MO), lOOmM MEM nonessential amino acids (Sigma- Aldrich, St.Louis, MO), 0.55mM 2-mercaptoethanol (Sigma-Aldrich, St.Louis, MO), and 2000U/ml leukemia inhibitory factor (LIF, R&D Systems). The cells can then be subcultured using 0.25% trypsin containing ImM EDTA for 5 minutes at 37°C.
[0031] For cell characterization, mesenchymal cells are seeded in 8 well chamber slides (nunc.Nalge) at a seeding density of 2500 cells per well. After three days, immunocytochemistry on cell surface markers for mesenchymal stem cells [CD 105 (Pharmingen International), CD90 (Santa Cruz Biotechnology, Inc.)] and hematopoetic stem cells [CD34 (Pharmingen International), CD 133] is performed.
[0032] Mesenchymal stem cells, as controls, can be purchased from Clonetics and cultured as recommended by the manufacturer's instructions.
Osteogenic Differentiation:
[0033] For the induction of osteogenic differentiation, the mesenchymal cells are seeded at 3000 cells/cm2 in 24 well plates (Falcon), 35 mm and 60 mm plates and cultured in DMEM low glucose medium (GIBCO/BRL, Grand Island, NY) with 10% fetal bovine serum (FBS, GIBCO/BRL, Grand Island, NY), 1% antibiotics (GIBCO/BRL, Grand Island, NY), and osteogenic supplements [lOOnM dexamethasone (Sigma-Aldrich, St.Louis, MO), lOmM beta- glycerophosphate (Sigma-Aldrich, St.Louis, MO), and 0.05mM ascorbic acid-2 -phosphate (Wako Chemicals)]. Control medium consists of DMEM high glucose with 15% ES-FBS, 1% antibiotics, L-glutamine, lOOmM MEM nonessential amino acids, and 0.55mM 2- mercaptoethanol. Medium changes are preferably performed every 3 days.
Adipogenic Differentiation:
[0034] For the induction of adipogenic differentiation, the cells are seeded at 3000 cells/cm in two-well chamber slides (Nunc/Nalge) and 60mm plates and cultured in DMEM low glucose medium with 10% FBS, 1% antibiotics, and adipogenic supplements [lμM dexamethasone, ImM 3-isobutyl-l-methylxanthine (Sigma-Aldrich, St.Louis, MO), lOμg/ml insulin (Sigma-Aldrich, St.Louis, MO), and 60μM indomethacin (Sigma-Aldrich, St.Louis, MO)]. All cells are preferably kept in control medium for four days, before adipogenic induction is started.
[0035] Control medium consists of DMEM high glucose with 15% ES-FBS, 1% antibiotics, L-glutamine, lOOmM MEM nonessential amino acids, and 0.55mM 2- mercaptoethanol. The medium was changed every 3 days.
Chondrogenic Differentiation:
[0036] For chondrogenic differentiation undifferentiated cells (e.g., in the amount of 0.25x106 cells) are spun down in 15 ml polystyrene conical tubes (Falcon) and grown as a pelleted micromass with 0.5ml of a defined chondrogenic differentiation medium. The medium preferably consists of DMEM high glucose with 15% ES-FBS, 1% antibiotics, L-glutamine, lOOmM MEM nonessential amino acids, 0.55mM 2-mercaptoethanol, lOOnM dexamethasone, 50μg/ml ascorbic acid-2-phosphate, and 2.5ng/ml transforming growth factor 3 (TGF- 3 , R&D Systems). After 21 days, with medium changes every 48 hours, pellets are harvested and assayed as described below.
Alkaline Phosphatase Assay:
[0037] Alkaline phosphatase enzyme cell activity is measured in quadruplicate cultures. After rinsing twice with warm PBS, the cells are incubated with 2-amino-2-methyl-l-propanol buffer, pH 10.3 (Sigma-Aldrich, St.Louis, MO, #221) with 40mg p-nitrophenyl phosphate (Sigma-Aldrich, St.Louis, MO, #104/40) added, at 37°C for 3 to 35 min. AP activity was calculated after measuring the absorbance of p-nitrophenol product formed at 405nm on a micro plate reader (Molecular Devices, Spectra Max Plus). As a standard, p-nitrophenol standard solution (Sigma-Aldrich, St.Louis, MO, #104-1) diluted in 2-amino-2-methyl-l-propanol buffer in concentrations from 0 to lOOnMol p-nitrophenol, is used. Enzyme activity is expressed as nMol p-Nitrophenol/min/106 cells.
Histochemical Analyses:
[0038] Alkaline phosphatase activity is determined histologically, according to the manufacturer's instructions (Sigma-Aldrich Kit #85). Briefly, cells are fixed in a citrate-acetone solution. An alkaline-dye mixture (fast blue RR solution with naphthol AS-MX phosphate alkaline solution) is added to the cells in the 35 mm culture dishes. The cell cultures should be protected from direct light. Prior to viewing the cell cultures are rinsed with deionized water and air-dried.
[0039] The presence of mineralization in cell culture is determined by von Kossa staining. The cell culture plates are fixed with 10% formaldehyde for 1 hour, incubated with 2% silver nitrate solution for 10 min in the dark, washed thoroughly with deionized water, and then exposed to UV-light for 15 min.
[0040] The presence of adipose elements in cell culture is determined with Oil-O-Red staining. The 2-well chamber slides are washed in deionized water and air-dried. The cells are incubated with oil red O staining solution for 15 minutes, rinsed with 50% ethanol 3 times, rinsed with distilled water, counterstained with Gill's hematoxilin for 30 sec to 1 min, and rinsed in deionized water 3 to 4 times. Chamber slides are mounted with water-based mounting media.
Cell Proliferation Assays:
[0041] Cells are seeded at 3000 cells/cm in 24 well plates. Cell numbers are determined after 4, 8, and 16 days in quadruplicate values. For the first time point (4 days), the medium is removed from the 24 well plates. The cells are rinsed once with PBS/EDTA, and incubated with 0.2 ml trypsin/EDTA for 10 minutes at 37°C. The cells are re-suspended with trypsin/EDTA solution several times to avoid cell clusters, before being transferred to 9.8 ml of Isoton fluid (Coulter Counter). Cells are counted as recommended by the manufacturer's instructions (Coulter Counter).
[0042] An MTT assay is performed after 4, 8, and 16 days. lOOμl of MTT reagent (Sigma-Aldrich, St. Louis, MO) is added to 1ml of medium for 3 hours. The cells are lysed and color is extracted with isopropanolol containing 0.1M HC1. Extinction is read in a biorad reader at 570nm against 655nm. Results are expressed as a cell count.
RT-PCR:
[0043] RNA is isolated from cultured cells and cell pellets with RNAzol reagent (Tel- Test Inc., Friendswood, TX) according to the manufacturer's protocol. RNA (2μg) is processed for c-DNA synthesis with Superscript II reverse transcriptase with random hexamers (Life Technologies, Rockville, MD). c-DNA is used for each PCR reaction, in a final volume of 30μl with 200nM dNTP, lOpM of each primer, 0.3U Taq-DNA-polymerase, reaction buffer, and MgCl2 (Life Technologies, Rockville, MD), in a PTC-100 cycler (MJ-Research Inc., Watertown, MA). The cycling conditions consist of 94°C for 2 minutes, annealing at 63°C for 40 seconds, and elongation at 72°C for 1 minute. Cycle numbers preferably vary between 22 and 37 cycles. All primers can obtained from Life Technologies. PCR products are quantified with NIH Image 1.62.
Immunocytochemistry:
[0044] Cells, can be grown on 8-well chamber slides (Nunc/Nalge) for 3 days were fixed in 2% formaldehyde for 5 minutes, in 4% formaldehyde for 5 minutes, and in ice-cold methanol. Cell layers are washed 3 times with PBS.
[0045] Cells are stained for CD34 (Pharmingen International), CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD 105 (Pharmingen International), and CD90 (Santa Cruz Biotechnology, Inc.).
Cultivation and Characterization of Human Mesenchymal Stem Cell Cultures:
[0046] Cells are digested in collagenase type I, transferred into bacterial plates, and grown in a defined medium. After digestion only a few of the cells would attach to the plate surfaces. Under phase contrast microscopy, the cells may appear as short slightly spindle shaped cells. After 2 to 3 weeks in culture, cells can reach 70-80% confluence. Immunocytochemically, the cells express the mesenchymal stem cell markers CD-90 (Thy-1) and CD-105 (endoglin), and do not express either CD-34 or CD 1'33 (Figure 2).
Induction of Human Mesenchymal Stem Cells into an Adipogenic Phenotype:
[0047] For adipogenic differentiation, the mesenchymal stem cells are treated with a defined medium with dexamethasone, insulin, indomethacin, and 3-isobutyl-l-methylxanthine. The cells tend to change their microscopic morphology from elongated to round. Within 8 days of exposure to the adipogenic medium, some cells may begin to form small intracellular
translucent vacuoles that gradually fill the cytoplasm, along the cell membrane. After 16 days in culture, approximately 20-30% of the cells may be completely filled with these vacuoles. Oil-O- Red stains these areas red, consistent with the presence of adipose (Figure 1). Control cells, grown in regular medium, tend not to show any signs of adipogenic differentiation and tend not to stain with Oil-O-Red at 8 or 16 days (Figure 1).
[0048] The adipogenic differentiation of the cells can be confirmed with RT-PCR for lipoprotein lipase (lpl), an adipocyte specific marker, and pparγ-2, a transcription factor highly expressed in adipocytes. To demonstrate equal amounts of RNA and c-DNA, β2-microglobulin as a housekeeping-gene can be used. After 16 days of treatment with adipogenic medium the cells tend to show strong expression of lpl and pparγ-2. In contrast, cells in the control medium do not express lpl and only minimal pparγ-2 expression can be detected on day 16 (Figures 4A- B). These results suggest that undifferentiated cells from dermal tissue can be induced into an adipogenic lineage.
Induction of Human Mesenchymal Stem Cells into an Osteogenic Phenotype:
[0049] Induction of human mesenchymal stem cells into an osteogenic phenotype is performed for 16 days. Phase contrast microscopy demonstrates that the mesenchymal cells, within 4 days of treatment, have a decreased spindle morphology and develop a round appearance with fingerlike excavations into the cell membrane.
[0050] Chemical staining for alkaline phosphatase shows a significant increase in the osteogenic medium treated cells by day 8 and 16 (Figure 2 A). The cells in the control medium do not show any significant difference in alkaline phosphatase staining at 8 or 16 days.
[0051 ] To confirm these findings, alkaline phosphatase activity in cells undergoing osteogenic induction is measured using a chemical assay (Figure 2B). There is approximately a six-fold increase in alkaline phosphatase production in cells treated with osteogenic medium at 4 days compared to cells treated with control medium (30.6nM p-Nitrophenol/min/106 cells and 8nM p-Nitrophenol/min/106 cells, respectively). The osteogenic induced cells produce an average of 66.5nM p-Nitrophenol/min/106 cells by day 8 and 109. InM p-Nitrophenol/min/106 cells by day 16. In contrast, the control cells produce an average of only 12.6nM p- Nitrophenol/min/106 cells by day 16.
[0052] As an additional control, commercially available bone-marrow-derived mesenchymal stem cells, which have been reported to be strongly responsive for induction of
alkaline phosphatase, are tested under the same conditions. Bone marrow cells can produce 120,2nM p-Nitrophenol/min/106 cells at Day 16, similar to the mesenchymal stem cells derived from dermal tissue.
[0053] A major feature of osteogenic induction is the presence of cell-associated mineralization. In previous studies cell associated mineralization has been shown by using von Kossa staining and assays measuring the calcium content of cell cultures. Von Kossa staining of cells in the osteogenic media is strongly positive by day 16 (Figure 3 A). In contrast, cells in the control medium do not stain with silver nitrate after days 8 or 16.
[0054] To further test for mineralization, calcium deposition by the cells is measured (Figure 3B). Cells undergoing osteogenic induction show a significant increase in calcium precipitation after 16 days (28.5mg/dl). In contrast, cells grown in control medium do not show any increase in calcium accumulation (0.32 mg/dl) on day 16.
[0055] Undifferentiated mesenchymal cells are additionally cultured in 6-well dishes for 6 months. Osteogenic medium is added to half of the wells and control medium is added to the rest. The osteogenic medium cells form bone-like tissue in vitro, which stains positively with von Kossa, confirming calcification.
[0056] Expression of cbfal, a transcription factor highly expressed in osteoblasts and hypertrophic chondrocytes, is analyzed in cells using RT-PCR. To demonstrate equal amounts of RNA and c-DNA, β2-microglobulin, as a housekeeping-gene, can be used. Cbfal expression is highest in osteogenic differentiating cells after 8 days and decreased slightly at 16 days. There is nearly no expression of cbfal in the controls after 8 and 16 days (Figures 4A-B).
[0057] The references cited herein, as set forth above and below, are incorporated herein by reference in their entirety.
References:
1. Pliard, A., Nifuji, A., Lamblin, D., Plee, E., Forest, C, and Kellermann, O. Controlled Conversion of an Immortilized Mesodermal Progenitor Cell Towards Osteogenic, Chondrogenic, or Adipogenic Pathways. J Cell Biol 130(6): 1461-72 (1995)
2. Atkinson, B.L., Fantle, K.S., Benedict, J.J., Huffer, W.E., and Gutierrez-Hartmann, A. Combination of Osteoinductive Bone Proteins Differentiates Mesenchymal C3H/10T1/2 Cells Specifically to the Cartilage Lineage. J Cell Biochem 65:325-339 (1997)
3. Pinney, D.F., Emerson, Jr., C.P. 10T1/2 Cells: An In vitro Mode for Molecular Genetic Analysis of Mesodermal Determination and Differentiation. Environ Health Perspect 80:221-227 (1989)
4. Mackay, A.M., Beck, S.C., Murphy, J.M., Barry, F.P., Chichester, CO., and Pitteneger, M.F. Chondrogenic Differentiation of Cultured Human Mesenchymal Stem Cells from Marrow. Tissue Engineering 4(4):415-428 (1998)
5. Pittenger, M.F., Mackay, A.M., Beck, S.C, Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 284:143-147, (1999)
6. Angele, P., Kujat, R., Nerlich, M., Yoo., J.,Goldberg, V., and Johnstone, B. Engineering of Osteochondral Tissue with Bone Marrow mesenchymal Progenitor Cells in a Derivatized Hyaluronan-Gelatin Composite Sponge. Tissue Engineering 5(6):545-553 (1999)
7. Young, H.E., Mancini, M ., Wright, R.P., Smith, J.C, Black, Jr., A.C, Reagan, C.R., and Lucas, P. A. Mesenchymal Stem Cells Reside Within the Connective Tissue of Many Organs Dev Dyn 202:137-144, (1995)
8. Lee, J.Y., Qu-Petersen, Z, Cao, B., Kimura, S., Jankowski, R., Cummins, J., Usas, A., Gates, C, Robbins, P., Wernig, G, Huard, J. Clonal Isolation of Muscle-derived Cells Capable of Enhancing Muscle Regeneration and Bone Healing. J Cell Biol 2000 Sep 4; 150(5): 1085-100
9. Bosch, P., Musgrave, D.S., Lee, J.Y., Cummins, J., Shuler, T., Ghivizzani, T.C, Evans, T., Robbins, T.D., Huard, J. Osteoprogenitor Cells within Skeletal Muscle. J Orthop Res 2000 Nov;18(6):933-44
10. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D. Molecular Biology of the Cell (Second Edition), Chapter 17, pp. 986-988 (1989)
11. Lee, S, Lumelsky, N., Studer, L., Auerbach, J.M., McKay, R.D. Efficient Generation of Midbrain and Hindbrain Neurons from Mouse Embryonic Stem Cells. Nature Biotech 2000 Jun Vol 18:675-79
12. Reubinoff, B.E., Pera, M.F., Fong, C, Trounson, A., Bongso, A. Embryonic Stem Cell Lines from Human Blastocytes: Somatic Differeniation In Vitro. Nature Biotech 2000 Apr Vol 18:399-404
13. Jaiswal, N., Haynesworth, S.E., Caplan, A.I., Bruder, S.P. Osteogenic Differentiation of Purified Culture-Expanded Human Mesenchymal Stem Cells In Vitro. J Cell Biochem 64:295- 312 (1997)
14. Lehmann, J.M., Lenhard, J.M., Oliver, B.B., Ringold, G.M., Kliewer, S.A. Peroxisome Proliferator-activated Receptors α and γ Are Activated by Indomethacin and Other Non-steroidal Ant-inflammatory Drugs. J Biol Chem 1997 Feb;272(6):3406-10
15. Rosen, E.D., Sarraf, P., Troy, A.E., Bradwin, G., Moore, K., Milstone, D.S., Spiegelman, B.M., Mortensen, R.M. PPAR gamma Is Required for the Differentiation of Adipose Tissue In Vitro and In Vivo. Mol Cell 1999 Oct;4(4):611-7
16. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R.T., Gao, Y.H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S., Kishimoto, T. Targeted Disruption of Cbfal Results in a Complete Lack of Bone Formation Owing to Maturational Arrest of Osteoblasts. Cell 1997 May 30;89(5):677-80
17. Karsenty, G. Role of Cbfal in Osteoblast Differentiation and Function. Semin Cell Dev Biol 2000 Oct;l l(5):343-6
18. Enomoto, H., Enomoto-Iwamoto, M., Iwamoto, M., Nomura, S., Himeno, M., Kitamura, Y., Kishimoto, T., Komori, T. Cbfal Is a Positive Regulatory Factor in Chondrocyte Maturation. J Biol Chem 2000 Mar; 275(12):8695-8702
19. Jaiswal, R.K., Jaiswal, N., Bruder, S.P., Mbalaviele, G., Marshak, D.R., Pittenger, M.F. Adult Human Mesenchymal Stem Cell Differentiation to the Osteogenic or Adipogenic Lineage Is Regulated by Mitogen-activated Protein Kinase. J Biol Chem 2000 Mar; 275(13):9645-9652
Claims (9)
1. A method for obtaining human mesenchymal stem cells, the method comprising:
(a) providing a specimen of human connective tissue;
(b) holding the specimen in a sterile environment for at least 2 days at about 4C;
(c) isolating a dermal layer from the specimen;
(d) contacting the dermal layer with collagenase to provide a collagenase cell suspension;
(e) collecting supernatant from the collagenase cell suspension;
(f) centrifuging the supernatant to obtain a cell product;
(g) culturing the cell product under conditions to promote cell growth; and (h) isolating mesenchymal stem cells from the culture.
2. The method of Claim 1, wherein the specimen is held in a sterile environment for 3-6 days.
3. The method of Claim 1, wherein the collagenase is collagenase I.
4. The method of Claim 1, wherein the human connective tissue is obtained from a human skin.
5. The method of Claim 4, wherein the human skin is foreskin.
6. A human mesenchymal stem cell produced by the method of claim 1.
7. A method for obtaining osteoblasts, the method comprising:
(a) providing a specimen of human connective tissue;
(b) holding the specimen in a sterile environment for at least 2 days at about 4C;
(c) isolating a dermal layer from the tissue;
(d) contacting the dermal layer with collagenase to provide a collagenase cell suspension;
(e) collecting supernatant from the collagenase cell suspension; (f) centrifuging the supernatant to obtain a cell product; and
(g) culturing the cell product under conditions to promote cell growth in contact with dexamethasone, beta-glycerophosphate and ascorbic acid-2-phosphate in amounts sufficient to induce human mesenchymal stem cells to undergo osteogenic differentiation.
8. A method for obtaining adipocytes, the method comprising:
(a) providing a specimen of human connective tissue;
(b) holding the specimen in a sterile environment for at least 2 days at about 4C;
(c) isolating a dermal layer from the tissue;
(d) contacting the dermal layer with collagenase to provide a collagenase cell suspension;
(e) collecting supernatant from the collagenase cell suspension;
(f) centrifuging the supernatant to obtain a cell product; and
(g) culturing the cell product under conditions to promote cell growth in contact with dexamethasone, 3-isobutyl-l-methylxanthine, insulin, and indomethacin in an amount sufficient to induce human mesenchymal stem cells to undergo adipogenic differentiation.
9. A method for obtaining chondrocytes, the method comprising:
(a) providing a specimen of human connective tissue;
(b) holding the specimen in a sterile environment for at least 2 days at about 4C;
(c) isolating a dermal layer from the tissue;
(d) contacting the dermal layer with collagenase to provide a collagenase cell suspension;
(e) collecting supernatant from the collagenase cell suspension;
(f) centrifuging the supernatant to obtain a cell product; and
(g) culturing the cell product under conditions to promote cell growth in contact with in contact with L-glutamine, MEM nonessential amino acids, 2- mercaptoethanol, dexamethasone, ascorbic acid-2 -phosphate, and transforming growth factor 3 in an amount sufficient to induce human mesenchymal stem cells to undergo chondrogenic differentiation.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US20042700P | 2000-04-28 | 2000-04-28 | |
| US60/200,427 | 2000-04-28 | ||
| PCT/US2001/013891 WO2001083709A1 (en) | 2000-04-28 | 2001-04-30 | Isolation of mesenchymal stem cells and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2001257436A1 true AU2001257436A1 (en) | 2002-01-31 |
| AU2001257436B2 AU2001257436B2 (en) | 2005-07-14 |
Family
ID=39321809
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2001257436A Ceased AU2001257436B2 (en) | 2000-04-28 | 2001-04-30 | Isolation of mesenchymal stem cells and use thereof |
| AU5743601A Pending AU5743601A (en) | 2000-04-28 | 2001-04-30 | Isolation of mesenchymal stem cells and use thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU5743601A Pending AU5743601A (en) | 2000-04-28 | 2001-04-30 | Isolation of mesenchymal stem cells and use thereof |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US20030211602A1 (en) |
| EP (1) | EP1285057B1 (en) |
| JP (1) | JP2003531604A (en) |
| AT (1) | ATE328067T1 (en) |
| AU (2) | AU2001257436B2 (en) |
| CA (1) | CA2407696A1 (en) |
| DE (1) | DE60120151D1 (en) |
| WO (1) | WO2001083709A1 (en) |
Families Citing this family (60)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030082152A1 (en) * | 1999-03-10 | 2003-05-01 | Hedrick Marc H. | Adipose-derived stem cells and lattices |
| US20050076396A1 (en) * | 1999-03-10 | 2005-04-07 | Katz Adam J. | Adipose-derived stem cells and lattices |
| US6777231B1 (en) * | 1999-03-10 | 2004-08-17 | The Regents Of The University Of California | Adipose-derived stem cells and lattices |
| US20050153442A1 (en) * | 1999-03-10 | 2005-07-14 | Adam Katz | Adipose-derived stem cells and lattices |
| US8105580B2 (en) | 2001-12-07 | 2012-01-31 | Cytori Therapeutics, Inc. | Methods of using adipose derived stem cells to promote wound healing |
| US7651684B2 (en) | 2001-12-07 | 2010-01-26 | Cytori Therapeutics, Inc. | Methods of using adipose tissue-derived cells in augmenting autologous fat transfer |
| US7595043B2 (en) * | 2001-12-07 | 2009-09-29 | Cytori Therapeutics, Inc. | Method for processing and using adipose-derived stem cells |
| US9597395B2 (en) | 2001-12-07 | 2017-03-21 | Cytori Therapeutics, Inc. | Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions |
| US20050008626A1 (en) * | 2001-12-07 | 2005-01-13 | Fraser John K. | Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions |
| US20060204556A1 (en) * | 2001-12-07 | 2006-09-14 | Cytori Therapeutics, Inc. | Cell-loaded prostheses for regenerative intraluminal applications |
| KR100930139B1 (en) | 2001-12-07 | 2009-12-07 | 사이토리 테라퓨틱스, 인크. | Systems and Methods for treating Patients with Processed Lipoaspirate Cells |
| US7771716B2 (en) | 2001-12-07 | 2010-08-10 | Cytori Therapeutics, Inc. | Methods of using regenerative cells in the treatment of musculoskeletal disorders |
| US20050095228A1 (en) * | 2001-12-07 | 2005-05-05 | Fraser John K. | Methods of using regenerative cells in the treatment of peripheral vascular disease and related disorders |
| US7514075B2 (en) * | 2001-12-07 | 2009-04-07 | Cytori Therapeutics, Inc. | Systems and methods for separating and concentrating adipose derived stem cells from tissue |
| US7585670B2 (en) * | 2001-12-07 | 2009-09-08 | Cytori Therapeutics, Inc. | Automated methods for isolating and using clinically safe adipose derived regenerative cells |
| US8404229B2 (en) * | 2001-12-07 | 2013-03-26 | Cytori Therapeutics, Inc. | Methods of using adipose derived stem cells to treat acute tubular necrosis |
| WO2004069172A2 (en) | 2003-01-30 | 2004-08-19 | The Government of the United States of America as represented by the Department of Veterans Affairs | Multilineage-inducible cells and uses thereof |
| AU2004219820B2 (en) * | 2003-03-10 | 2008-06-12 | Japan Science And Technology Agency | Marker for detecting mesenchymal stem cell and method of distinguishing mesenchymal stem cell using the marker |
| WO2004097720A1 (en) * | 2003-04-24 | 2004-11-11 | Koninklijke Philips Electronics N.V. | Non-invasive left ventricular volume determination |
| EP1711598A4 (en) * | 2004-01-30 | 2009-04-08 | Lifecord Inc | Method for isolating and culturing multipotent progenitor/stem cells from umbilical cord blood and method for inducing differentiation thereof |
| DE602005020120D1 (en) | 2004-03-03 | 2010-05-06 | Japan Science & Tech Agency | METHOD FOR DIFFERENTIATING MESENCHYMAL STEM CELLS TO STEROID-PRODUCING CELLS |
| JP5226928B2 (en) * | 2004-04-16 | 2013-07-03 | 株式会社カネカ | Cell transdifferentiation method |
| EP1844434A4 (en) * | 2005-01-10 | 2013-11-13 | Cytori Therapeutics Inc | Devices and methods for monitoring, managing, and servicing medical devices |
| US7759120B2 (en) * | 2005-03-02 | 2010-07-20 | Kps Bay Medical, Inc. | Seeding implantable medical devices with cells |
| US20060199265A1 (en) * | 2005-03-02 | 2006-09-07 | Wolf Michael F | Seeding implantable medical devices with cells |
| JP4921767B2 (en) * | 2005-10-14 | 2012-04-25 | 株式会社カネカ | Cell differentiation induction method |
| US9057052B2 (en) | 2005-10-21 | 2015-06-16 | Kaneka Corporation | Stem cell separating material and method of separation |
| US9694034B2 (en) * | 2005-10-31 | 2017-07-04 | Bio Solution Co., Ltd. | Artificial cartilage containing chondrocytes obtained from costal cartilage and preparation process thereof |
| GB0600972D0 (en) * | 2006-01-18 | 2006-03-01 | Univ Leeds | Enrichment of cells |
| JPWO2008156221A1 (en) * | 2007-06-20 | 2010-08-26 | Hoya株式会社 | Repair and treatment of bone defects by the factor and scaffold produced by chondrocytes capable of hypertrophy |
| JPWO2008156220A1 (en) | 2007-06-20 | 2010-09-09 | Hoya株式会社 | Repair and treatment of bone defects by cells and scaffolds induced by factors produced by chondrocytes capable of hypertrophy |
| WO2010021993A1 (en) | 2008-08-19 | 2010-02-25 | Cytori Therapeutics, Inc. | Methods of using adipose tissue-derived cells in the treatment of the lymphatic system and malignant disease |
| AU2010242780B2 (en) | 2009-05-01 | 2016-04-21 | Puregraft Llc | Systems, methods and compositions for optimizing tissue and cell enriched grafts |
| JP2011010581A (en) * | 2009-06-30 | 2011-01-20 | Kaneka Corp | Stem cell separator, separation filter for separating stem cell, method of separating stem cell using separator or separation filter, and method of recovering stem cell |
| JP5678360B2 (en) | 2009-09-28 | 2015-03-04 | 株式会社ジーシー | Method for culturing mesenchymal stem cells |
| SG10201407102QA (en) * | 2009-10-30 | 2014-11-27 | Univ North Carolina | Multipotent stem cells from the extrahepatic billary tree and methods of isolating same |
| WO2013106655A1 (en) | 2012-01-11 | 2013-07-18 | The Gid Group, Inc. | Method for processing adipose tissue and processing apparatus |
| US9206387B2 (en) | 2010-07-09 | 2015-12-08 | The Gid Group, Inc. | Method and apparatus for processing adipose tissue |
| US9260697B2 (en) | 2010-07-09 | 2016-02-16 | The Gid Group, Inc. | Apparatus and methods relating to collecting and processing human biological material containing adipose |
| US9296984B2 (en) | 2010-07-09 | 2016-03-29 | The Gid Group, Inc. | Tissue processing apparatus and method for processing adipose tissue |
| CN103200937B (en) | 2010-09-01 | 2020-07-03 | 托马斯杰弗逊大学 | Compositions and methods for muscle repair and regeneration |
| EP2625577B1 (en) | 2010-10-08 | 2019-06-26 | Terumo BCT, Inc. | Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system |
| KR101751355B1 (en) * | 2011-08-24 | 2017-07-04 | (주)아모레퍼시픽 | Adult stem cells derived human skin dermis |
| BR112015004003B1 (en) | 2012-09-06 | 2020-05-19 | The Gid Group Inc | apparatus for processing human biological material containing fibrous tissue and method for processing adipose tissue |
| EP3038629B1 (en) | 2013-09-05 | 2020-11-18 | GID BIO, Inc. | Method for processing adipose tissue |
| JP6633522B2 (en) | 2013-11-16 | 2020-01-22 | テルモ ビーシーティー、インコーポレーテッド | Cell growth in bioreactors |
| WO2015148704A1 (en) | 2014-03-25 | 2015-10-01 | Terumo Bct, Inc. | Passive replacement of media |
| WO2016049421A1 (en) | 2014-09-26 | 2016-03-31 | Terumo Bct, Inc. | Scheduled feed |
| WO2016210256A1 (en) * | 2015-06-25 | 2016-12-29 | The University Of Florida Research Foundation, Inc. | Conductive nonwoven mat and method of using the conductive nonwoven mat |
| WO2017004592A1 (en) | 2015-07-02 | 2017-01-05 | Terumo Bct, Inc. | Cell growth with mechanical stimuli |
| US11965175B2 (en) | 2016-05-25 | 2024-04-23 | Terumo Bct, Inc. | Cell expansion |
| US11685883B2 (en) | 2016-06-07 | 2023-06-27 | Terumo Bct, Inc. | Methods and systems for coating a cell growth surface |
| US11104874B2 (en) | 2016-06-07 | 2021-08-31 | Terumo Bct, Inc. | Coating a bioreactor |
| CA3035193A1 (en) | 2016-08-30 | 2018-03-08 | Lifecell Corporation | Systems and methods for medical device control |
| USD851777S1 (en) | 2017-01-30 | 2019-06-18 | Lifecell Corporation | Canister-type device for tissue processing |
| EP3656841A1 (en) | 2017-03-31 | 2020-05-27 | Terumo BCT, Inc. | Cell expansion |
| US11624046B2 (en) | 2017-03-31 | 2023-04-11 | Terumo Bct, Inc. | Cell expansion |
| EP3655518A4 (en) | 2017-07-18 | 2021-07-14 | GID Bio, Inc. | Adipose tissue digestion system and tissue processing method |
| US11285177B2 (en) | 2018-01-03 | 2022-03-29 | Globus Medical, Inc. | Allografts containing viable cells and methods thereof |
| US12043823B2 (en) | 2021-03-23 | 2024-07-23 | Terumo Bct, Inc. | Cell capture and expansion |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5486359A (en) * | 1990-11-16 | 1996-01-23 | Osiris Therapeutics, Inc. | Human mesenchymal stem cells |
| US5736396A (en) * | 1995-01-24 | 1998-04-07 | Case Western Reserve University | Lineage-directed induction of human mesenchymal stem cell differentiation |
| JP3543869B2 (en) * | 1995-03-07 | 2004-07-21 | 株式会社メニコン | Cultured skin and method for producing the same |
| CA2307743A1 (en) * | 1997-10-30 | 1999-05-14 | The General Hospital Corporation | Bonding of cartilaginous matrices using isolated chondrocytes |
-
2001
- 2001-04-30 AT AT01930948T patent/ATE328067T1/en not_active IP Right Cessation
- 2001-04-30 EP EP01930948A patent/EP1285057B1/en not_active Expired - Lifetime
- 2001-04-30 US US10/258,076 patent/US20030211602A1/en not_active Abandoned
- 2001-04-30 DE DE60120151T patent/DE60120151D1/en not_active Expired - Lifetime
- 2001-04-30 CA CA002407696A patent/CA2407696A1/en not_active Abandoned
- 2001-04-30 WO PCT/US2001/013891 patent/WO2001083709A1/en active IP Right Grant
- 2001-04-30 JP JP2001580318A patent/JP2003531604A/en not_active Withdrawn
- 2001-04-30 AU AU2001257436A patent/AU2001257436B2/en not_active Ceased
- 2001-04-30 AU AU5743601A patent/AU5743601A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1285057B1 (en) | Isolation of mesenchymal stem cells and use thereof | |
| AU2001257436A1 (en) | Isolation of mesenchymal stem cells and use thereof | |
| Doucet et al. | Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell‐based therapy applications | |
| Lee et al. | Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue | |
| Sottile et al. | Stem cell characteristics of human trabecular bone-derived cells | |
| Karaöz et al. | Isolation and in vitro characterisation of dental pulp stem cells from natal teeth | |
| Harkness et al. | Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential | |
| AU2012346645B2 (en) | Culture-medium composition for rejuvenating stem cells | |
| Hsu et al. | Isolation of the multipotent MSC subpopulation from human gingival fibroblasts by culturing on chitosan membranes | |
| Singhatanadgit et al. | Isolation and characterization of stem cell clones from adult human ligament | |
| AU2016227607B2 (en) | Cell culture method for mesenchymal stem cells | |
| CN107828722B (en) | Stem cell specifically expressing PD-1, and identification and separation method and application thereof | |
| AU2008202757A1 (en) | Adipose-derived stem cells and lattices | |
| WO2005113780A1 (en) | Adipose-derived stem cells and lattices | |
| WO2006017320A2 (en) | Compositions and methods for myogenesis of fat-derived stem cells expressing telomerase and myocardin | |
| Ahmed et al. | CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells | |
| Taihi et al. | Efficient isolation of human gingival stem cells in a new serum-free medium supplemented with platelet lysate and growth hormone for osteogenic differentiation enhancement | |
| RU2418855C1 (en) | Medium for cultivation of multipotent stromal cells from human adipose tissue and method of cultivation of said cells with its application | |
| WO2020013719A1 (en) | Stem cell culture method using plant secretion | |
| KR102533120B1 (en) | Composition for Facilitating the Osteogenic Differentiation of Stem Cell Spheroids Comprising Bone Morphogenetic Protein-4 as an Effective Ingredient | |
| WO2021227573A1 (en) | Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same | |
| KR101618380B1 (en) | Human bone-forming cells in the treatment of inflammatory rheumatic diseases | |
| MESSAT et al. | Transdifferentiation of Human Dental Pulp Mesenchymal Stem Cells into Spiral Ganglion-like Neurons | |
| KR20230138786A (en) | Composition for Facilitating the Differentiation of Stem Cell Organoids Comprising Transforming Growth Factor-β1 as an Effective Ingredient | |
| Joshi | Master of Veterinary |