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WO1998033515A1 - EFFETS STIMULANTS DE bFGF ET DE BMP-2 SUR UNE DIFFERENCIATION OSTEOGENIQUE DE CELLULES SOUCHES MESENCHYMATEUSES - Google Patents

EFFETS STIMULANTS DE bFGF ET DE BMP-2 SUR UNE DIFFERENCIATION OSTEOGENIQUE DE CELLULES SOUCHES MESENCHYMATEUSES Download PDF

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WO1998033515A1
WO1998033515A1 PCT/US1998/002143 US9802143W WO9833515A1 WO 1998033515 A1 WO1998033515 A1 WO 1998033515A1 US 9802143 W US9802143 W US 9802143W WO 9833515 A1 WO9833515 A1 WO 9833515A1
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bfgf
bmp
bone
cells
day
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PCT/US1998/002143
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James E. Dennis
Arnold I. Caplan
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Case Western Reserve University
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Priority to AU61446/98A priority Critical patent/AU6144698A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C12N2533/14—Ceramic
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • Bone marrow is a complex tissue composed of hematopoietic and mesenchymal elements.
  • the stroma of bone marrow is composed of a highly organized network of mesenchymal cells and extracellular matrix that provides structural and functional support for hematopoiesis.
  • mesenchymal progenitor cells exist which are capable of differentiating into several different mesenchymal tissues including bone and cartilage (1); we refer to these cells as mesenchymal stem cells (MSCs) (2, 3).
  • bFGF basic fibroblast growth factor
  • BMP-2 bone morphogenic protein-2
  • the study that resulted in the present invention examined the combined effects of bFGF and BMP-2 on the proliferation and osteogenic differentiation of rat bone marrow-derived MSCs in our above-referenced culture system. Combined treatment with the mitogenic factor bFGF and differentiation factor BMP-2 stimulated osteogenesis more than exposure to each factor alone. The results provide a rational basis for their clinical application.
  • the invention provides a method of enhancing osteogenic differentiation of culture-expanded mesenchymal stem cells.
  • the method comprises exposing such mesenchymal stem cells to both bFGF and a member of the TGF- ⁇ superfamily.
  • This method particularly comprises successive exposure of mesenchymal stem cells to BMP-2 in combination with dexamethasone followed by contact of the cells with bFGF.
  • the invention provides for concurrent contact of the cells with BMP-2 and bFGF.
  • the invention provides a method of accelerating osteogenic differentiation of culture-expanded mesenchymal stem cells.
  • the method comprises exposing such mesenchymal stem cells to both bFGF and a member of the TGF- 3 superfamily.
  • This method particularly comprises successive exposure of mesenchymal stem cells to BMP-2 in combination with dexamethasone followed by contact of the cells with bFGF.
  • the invention further provides a composition comprising the combination of human isolated, culture-expanded mesenchymal stem cells with BMP-2, an osteoinductive agent such as dexamethasone and bFGF.
  • Rat marrow MSCs were plated at a density of 5 x 10 3 cells/cm 2 in 24-well culture plates and treated for 6 days with no factors (control, C); BMP-2 (50 ng/ml) (B); bFGF (2.5 ng/ml) (F); and a combination of bFGF and BMP-2 (FB) in complete medium containing 10% FBS and 10 7 M Dex. Thereafter, the medium was replaced with "osteogenic" medium (complete medium plus 10 "7 M Dex, ascorbate (50 ⁇ g/ml) and 0-glycerophosphate (10 mM).
  • FIG. 3 Phase contrast micrographs of MSC cultures on day 11 (xlOO): control (a); BMP-2 (50 ng/ml) (b); bFGF (2.5 ng/ml) (c); and combined treatment with bFGF and BMP-2 (d).
  • the cultures were prepared as described in the above description for Fig. 2. Note the mineralizing early bone nodules (n) present in the cultures treated with bFGF and BMP-2. An uncalcified small cell colony (c) is also observed in the bFGF-treated culture.
  • FIG.3 Photographs of von Kossa-stained MSC cultures on day 18 (xl.3): control (a), BMP-2 (50 ng/ml) (b); bFGF (c) (2.5 ng/ml); and combined treatment with bFGF and BMP-2 (d).
  • control a
  • BMP-2 50 ng/ml
  • bFGF bFGF
  • c 2.5 ng/ml
  • BMP-2 bFGF
  • d combined treatment with bFGF and BMP-2
  • Figure 6 Effects of early (Days 1-4) and/or late (Days 4-7) addition of 2.5 ng/ml bFGF and 25 ng/ml BMP-2 on osteogenic differentiation of rat marrow MSCs.
  • Factors were administered on day 1 to Dex-treated MSC cultures and removed on day 4 or added on day 4 and removed on day 7 or both. The cultures were prepared as described in the legend for Fig. 2. Calcium content was measured on day 18. in parallel with this assay, DNA content of matching samples was also determined. All data are expressed per 0.1 ⁇ g of DNA. Each measurement is the mean of triplicate cultures. Standard deviation (SD) of the mean is shown by transverse bars.
  • SD Standard deviation
  • FIG. 7 Histologic features of a section of a ceramic cube loaded with MSCs exposed to both bFGF and BMP-2 (xlOO). Cubes were harvested from host rats 6 weeks postimplantation. After fixation and decacification, histologic sections were prepared and stained with Mallory-Heidenhain. The new bone (b) is formed along the walls of individual pores. Decalcified ceramic material (c) appears as acellular space stained lightly.
  • FBS fetal bovine serum
  • DMEM-LG Dulbecco's modified Eagle's medium containing low glucose
  • trypsin-EDTA antibiotic-antimycotic solution (penicillin, streptomycin and fungizon)
  • Superscript II reverse transcriptase dNTP mix, dithiothreitol, 5X first strand buffer, oligo(dt) 12 ., 8 , RNase H, 10X polymerase chain reaction (PCR) buffer, MgCl 2 Taq DNA polymerase, and Hae 111 restriction fragments of ⁇ XHA DNA were purchased from Gibco BRL (Gaithersburg, MD).
  • Tyrode's salts, dexamethasone (Dex), calf thymus DNA, 3, 5-diaminobenzoic acid dihydrochloride (DAB A), and calcium assay kit were obtained from Sigma Chemical Co. (St. Louis, MO).
  • Fibronectin was purchased from Collaborative Biomedical (Bedford, MA).
  • Calf serum was procured from Hyclone Laboratories (Logan, UT).
  • Total RNA isolation kit was purchased from Qiagen Inc. (Chatsworth, CA).
  • Recombinant bovine basic fibroblast growth factor (bFGF) was purchased from Boehringer Mannheim (Indianapolis, IN).
  • BMP-2 bone morphogenic protein-2
  • Falcon plasticware including 24- and 96-well culture plates, was purchased from Becton-Dickinson Labware (Franklin Lakes, IN). Porous calcium phosphate ceramic cubes were generously provided by Zimmer/Bristol Myers Squibb (Warsaw, IN). Fisher 344 rats were purchased from Charles River Laboratory (Wilmington, MA) and 10% neutral buffered formalin was from Fisher Scientific (Orangetown, NY).
  • MSC cultures were prepared from the bone marrow of femurs and tibias harvested from 2-month-old male F344 rats by a technique previously described (25). Briefly, the bones were cleaned of adherent soft tissue, the epiphyses removed with a rongeur, and the marrow harvested by inserting a syringe needle (18-gauge) into one end of the bone and flushing with complete medium (DMEM-LG supplemented with antibiotic-antimycotic solution and 10% FBS) into a 60-mm culture dish. A cell suspension was obtained by drawing the marrow into syringes sequentially three times through needles of decreasing size (gauge 18, 20, 22, respectively).
  • the cells were then centrifuged, counted, seeded at a density of 5 X 10 7 in 7 ml of complete medium per 100-mm culture dish, and cultured at 37°C in 95% humidified air and 5 % C0 2 .
  • non-adherent cells were removed by changing the medium; thereafter, the medium was changed every 3-4 days.
  • the cells were liberated by exposure to 0.25% trypsin/1 mM EDTA for 5 minutes at 37°C, followed by the addition of one-half volume of calf serum to stop the reaction.
  • the released cells were then centrifuged, resuspended in complete medium, and seeded at 5 X 10 3 cells/cm 2 in 24-well plates for biochemical and PCR analyses or in 100-mm culture dishes to generate cells for in vivo ceramic cube assays.
  • the attached cells were exposed to bFGF and/or BMP-2. Exposure of the marrow MSC cultures to bFGF and BMP-2
  • the MSC cultures were exposed continuously to 2.5 ng/ml recombinant bovine bFGF and/or recombinant human BMP-2 at concentrations up to 100 ng/ml in 0.5 ml of complete medium in the presence of 10 "7 M Dex for 6 days.
  • Optimal dose of bFGF determined by measuring DNA content and ALP activity at various time points, plateaued at a dose of 2.5 ng/ml.
  • Control cultures were maintained without added bFGF or BMP-2, but in the presence of 10 "7 M Dex. The medium was changed once on day 4 and replaced with medium containing fresh growth factors.
  • DNA content of the cultures was assayed with the techniques described by Gillery et al. (27). Briefly, the cells in each well of 24-well plates were rinsed with Tyrode's solution and then fixed with ethanol. Freshly prepared DABA solution (80 mg/ml, 0.2 ml) was added to each well. The standard curve was obtained by performing the DABA reaction in culture wells containing various concentrations of calf thymus DNA. The plates were then incubated for 45 minutes at 60°C.
  • the reaction between DABA and DNA was stabilized by adding 1.5 ml of 1 M HC1 to every well and the intensity of fluorescence was measured at 420 nm excitation and 490 nm emission in a spectrophotofluorometer (American Instrument Co., Silver Spring, MA). The DNA content was determined from a standard curve.
  • Total RNA was extracted with a commercial kit following the manufacturer's instructions. The purity and amount of isolated RNA were assessed by spectrophotometric measurement at 260 and 280 nm. Total RNA (1.5 ⁇ g) was reverse transcribed to cDNA at 42 °C for 50 minutes in a volume of 20 ⁇ l containing
  • IX first strand buffer 250 mM Tris, pH 8.3, 375 mM KC1 and 15 mM MgCl 2
  • Superscript II RNase H-free reverse transcriptase
  • DNA amplification included an initial denaturation at 94°C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C (osteocalcin primers) or 60 °C (actin primers) for 1 minute, and extension at 72 °C for 1 minute. The final cycle included 5 minutes for extension.
  • the cultures were fixed with neutral buffered formalin and stained by the method of von Kossa (26). Freshly prepared 2% silver nitrate was added to the plates (0.5 ml/well), which were incubated in the dark for 10 minutes. The plates were rinsed with distilled water and then exposed to bright light for 15 minutes. The reaction was terminated by rinsing thoroughly with distilled water. The strongly stained nodules were counted under a dissecting microscope.
  • the ceramic composite assay was performed to test the in vivo osteogenic potential of cultured cells (6-8,26). First passage cells exposed to test agents for 6 days were rinsed with Tyrode's solution, harvested by trypsinization, resuspended at 5 x 10 6 cells/ml in serum-free DMEM-LG medium, and placed in a 5-ml tube containing 3-mm porous calcium phosphate ceramic cubes precoated with fibronectin (7). After producing a slight vacuum to release airpockets from the ceramic cubes, the tubes were placed in a CO 2 incubator at 37°C for 2 hours to allow the cells to attach to the ceramic surface. The cubes were then implanted subcutaneously into syngeneic F344 male rats.
  • the ceramics were harvested 6 weeks postimplantation, fixed in 10% neutral buffered formalin, and processed for routine histology. The entire sample was serially sectioned and every 7th and 8th sections were stained with Mallory-Heidenhain. Each stained section was examined and scored for bone on a grading scale of 0 to 4 as previously described (26). The scores of all sections were combined and divided by the number of sections graded to determine the overall score of each ceramic cube.
  • results obtained were expressed as the mean ⁇ SD (standard deviation of the mean) of triplicate or quadruplicate cultures. Differences between experimental groups were determined with Student's t-test. For analyses of histologic score, Kruskall-Wallis One Way Rank test was used. Differences at P ⁇ 0.05 were considered significant. In pilot experiments, treatments with bFGF, BMP-2 and both factors did not induce bone nodule formation without Dex in the cultures. Therefore, the experiments reported here were performed with continuous exposure to Dex for the 17-day duration of the experiment.
  • osteocalcin mRNA expression was assessed as late markers of mature osteoblast functions.
  • Osteocalcin, mRNA expression RT-PCR analyses revealed that combined treatment with 2.5 ng/ml bFGF and 50 ng/ml BMP-2 induced early expression of osteocalcin MRNA on day 11, while treatment with bFGF alone produced a relatively weak expression (Fig. 2). On day 14, strong mRNA expression was detected in bFGF- and bFGF + BMP-2-treated cultures. In contrast, BMP-2-treated cultures had only low but detectable levels of osteocalcin MRNA even at this late time.
  • Bone nodule formation Consistent with early expression of osteocalcin mRNA, mineralizing bone-like nodules appeared in bFGF + BMP-2-treated cultures on day 11 (Fig. 3). In bFGF-treated cultures, uncalcified cell groupings were obser@,ed. On day 18, cultures treated with bFGF alone or in combination with 50 ng/ml BMP-2 developed a substantial number of calcified bone nodules (Fig. 4). In contrast, the number of bone nodules in BMP-2-treated cultures was much smaller than that in bFGF-treated cultures; the size of the nodules in BMP-2-treated cultures was similar to that in bFGF-treated cultures.
  • Figure 7 shows a representative section from the histologic analysis of a cube loaded with MSCs treated with bFGF + BMP-2.
  • the morphologic appearance of the bone which formed was histologically identical for all cubes seeded with the control cells or cells pre-treated with bFGF, BMP-2, or both factors.
  • bFGF is a mitogen for rat marrow MSCs and, in the presence of Dex, stimulates their osteogenic differentiation.
  • the MSCs were cultured with Dex-supplemented medium, since it was reported to be essential for induction of in vitro osteogenesis in various culture systems (9, 10).
  • Treatment with bFGF in the absence of Dex results in no osteogenesis.
  • an initial 3 day-exposure to bFGF is more effective at inducing bone formation than if bFGF is added later (Fig.6). This suggests that bFGF acts on early Dex-committed osteoprogenitor cells and/or uncommitted MSCs responsive to Dex at an earlier differentiation stage.
  • BMP-2 slightly induces bone nodule formation and calcium deposition compared with the control cultures.
  • a weak osteogenic potential of BMP-2-treated MSCs was further confirmed with the in vivo ceramic cube assay.
  • Rickard et al. (21) demonstrated that BMP-2 (50 ng/ml) acted synergistically with Dex to increase ALP activity and vitamin D-induced mRNA expression for type I collagen and osteocalcin in rat primary stromal cell cultures.
  • they did not assess actual in vitro bone formation represented by bone nodule formation and calcium deposition as presented here.
  • the methods of cell preparation were different from ours.
  • BMP-2 added BMP-2 to whole marrow primary cultures containing adherent and non-adherent cell populations for days 1-3 and then re-exposed the adherent cell fraction to BMP-2 after removing the non-adherent cells.
  • BMP-2 25-100 ng/ml greatly stimulated ALP activity, osteocalcin production, and bone nodule formation (31-33). Discrepancies between our results and those of others may be due to differences in the differentiation stage of the cells used, the age of the donor animals, and species differences.
  • BMP-2 was also reported to stimulate adipogenic and chondrongenic differentiation in cultures of a murine mesenchymal pluripotential line C3H10T1/2 (23). However, after a 6-day treatment of MSCs with 50 and 100 ng/ml BMP-2,
  • Rodan SB Wesolowski G, Thomas KA, Yoon K, Rodan GA 1989 Effects of acidic and basic fibroblast growth factors on osteoblastic cells. Conn Tissue Res 20:283-288.
  • Bone moiphogenetic protein 2 stimulates osteogenesis but does not affect chondrogenesis in osteochondrogenic differentiation of periosteum-derived cells. J Bone Miner Res 9: 1195-1204.

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Abstract

Le stroma de moelle osseuse contient des précurseurs mésenchymateux à potentiel multiple, qui peuvent se différencier en cellules ostéoblastiques; l'invention se réfère à ces cellules comme étant des cellules souches mésenchymateuses (MSC). Le facteur de croissance des fibroblastes de base (bFGF) et la protéine morphogénétique osseuse 2(BMP-2) ont été impliqués dans le processus régulatoire ostéogène en raison de leurs activités mitogène et de différenciation, respectivement. Cette étude examine et compare les effets de bFGF et de BMP-2 sur une différenciatio nostéogénique in vitro induite par dexaméthasone sur des MSC obtenues à partir de moelle de rat. Une exposition à bFGF pendant 6 jours a stimulé de façon marquante la croissance cellulaire, et a provoqué une différenciation ostéoblastique, comme le montre l'expression d'ARNm d'ostéocalcine (jour 14); une formation de nodule osseux (jour 18), et un dépôt de calcium (jour 18). Ces résultats montrent que bFGF permet d'améliorer l'activité mitogène et le développement ostéogène de MSC de moelle traitées à la dexaméthasone. Par contre, BMP-2 n'a pas provoqué une ostéogénèse de façon aussi forte que bFGF. L'exposition à BMP-2 a ainsi légèrement augmenté le nombre de nodules osseux et la teneur en calcium, par comparaison au témoin. L'exposition de MSC à BMP-2 et bFGF a provoqué l'expression d'ARNm d'ostéocalcine, et la minéralisation de nodules de type osseux dès le 11ème jour, et a résulté en une amélioration de formation osseuse de façon plus marquée que l'un des facteurs seul. En accord avec ces résultats, des cubes de céramique en phosphate de calcium poreux implantés in vitro, qui avaient été chargés en MSC pré-exposées à b-FGF et BMP-2, ont présenté des valeurs histologiques plus élevées pour ce qui est de la formation osseuse, que ceux comportant des MSC pré-exposées soit à bFGF, soit à BMP-2. Ces données montrent qu'un traitement combiné au bFGF ou à la BMP-2 permet d'améliorer de façon synergétique l'activité ostéogène de bFGF dans la culture de MSC de moelle de rat.
PCT/US1998/002143 1997-02-05 1998-02-04 EFFETS STIMULANTS DE bFGF ET DE BMP-2 SUR UNE DIFFERENCIATION OSTEOGENIQUE DE CELLULES SOUCHES MESENCHYMATEUSES WO1998033515A1 (fr)

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Cited By (13)

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WO1999047156A3 (fr) * 1998-03-14 1999-12-29 Creative Biomolecules Inc Compositions pour moduler la differentiation des cellules comprenant un lipide et un morphogene
WO2001006949A3 (fr) * 1999-07-28 2001-05-10 Interface Biotech As Reparation in vivo de defauts osseux et/ou du cartilage
WO2002067978A1 (fr) * 2001-02-23 2002-09-06 Wyeth Utilisation des protéines bmp pour potentialiser la chondrogenèse par les cellules cd105+ tirées de la moelle osseuse humaine
WO2005065704A1 (fr) * 2004-01-09 2005-07-21 Saitama Medical School Agent de prévention et de traitement pour maladies osseuses
AU2004200550B2 (en) * 1999-07-28 2006-07-20 Interface Biotech A/S Cell cultivation method for the preparation of chondroblasts/chondrocytes
EP1619242A4 (fr) * 2003-03-25 2006-09-06 Japan Science & Tech Agency Commande de l'induction de la differenciation de cellules souches et de la capacite de differenciation
US8337827B2 (en) 2006-02-16 2012-12-25 Universite Libre de Bruxelies Method for osteogenic differentiation of bone marrow stem cells (BMSC) and uses thereof
AU2009203682B2 (en) * 2008-01-11 2014-07-03 Bone Therapeutics S.A. Osteogenic differentiation of bone marrow stem cells and mesenchymal stem cells using a combination of growth factors
US9125906B2 (en) 2005-12-28 2015-09-08 DePuy Synthes Products, Inc. Treatment of peripheral vascular disease using umbilical cord tissue-derived cells
WO2016011438A1 (fr) * 2014-07-18 2016-01-21 Case Western Reserve University Dosage de péricytes pour la migration transendothéliale
US9498501B2 (en) 2003-06-27 2016-11-22 DePuy Synthes Products, Inc. Postpartum cells derived from umbilical cord tissue, and methods of making and using the same
US10179900B2 (en) 2008-12-19 2019-01-15 DePuy Synthes Products, Inc. Conditioned media and methods of making a conditioned media
CN110938669A (zh) * 2019-12-20 2020-03-31 南昌大学第二附属医院 无机磷酸盐刺激瓣膜间质细胞成骨分化的验证方法

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