Comparison of rat mesenchymal stem cells derived from bone marrow,synovium, periosteum,adipose tissue,and muscle

Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model. This study was supported in part by grants from the Japan Latest Osteoarthritis Society and from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University (to T.M.), and by the Japan Society for the Promotion of Science (grant no. 18591657 to I.S.). Recombinant human bone morphogenetic protein-2 was kindly provided by Astellas Pharma.     

BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration

Mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of lineages and to renew themselves without malignant changes, and thus hold potential for many clinical applications. However, it has not been well characterized how different the properties of MSCs are depending on the tissue source in which they resided. We previously reported a novel technique for the prospective MSC isolation from bone marrow, and revealed that a combination of cell surface markers (LNGFR and THY-1) allows the isolation of highly enriched MSC populations. In this study, we isolated LNGFR⁺ THY-1 ⁺ MSCs from synovium using flow cytometry. The results show that the synovium tissue contained a significantly larger percentage of LNGFR ⁺ THY-1 ⁺ MSCs. We examined the colony formation and differentiation abilities of bone marrow-derived MSCs (BM-MSCs) and synovium-derived MSCs (SYN-MSCs) isolated from the same patients. Both types of MSCs exhibited a marked propensity to differentiate into specific lineages. BM-MSCs were preferentially differentiated into bone, while in the SYN-MSC culture, enhanced adipogenic and chondrogenic differentiation was observed. These data suggest that the tissue from which MSCs are isolated should be tailored according to their intended clinical therapeutic application.     

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6

Recent interest has focused on mesenchymal stem cells (MSC) for tissue engineering and regenerative therapy of cartilage defects. MSC originating from adipose tissue (ATSC) are attractive as they are easily available and abundant. They have similar properties like bone marrow derived MSC (BMSC), except for a reduced chondrogenic potential under standard culture conditions driven by TGFbeta. Aim of this study was to search for possible differences explaining the reduced differentiation capacity of ATSC and to eliminate it by adaptation of induction protocols. Expanded MSC were analyzed for their growth factor and related receptor repertoire and ATSC spheroid cultures were supplemented with BMP-2,-4,-6,-7, TGFbeta, FGFa, FGFb, IGF-1, and PTHrP alone or in combination with TGFbeta. In contrast to BMSC, ATSC showed reduced expression of BMP-2, -4, and -6 mRNA and did not express TGFbeta-receptor-I protein. Consistent with this, increased concentrations of TGFbeta did not improve chondrogenesis of ATSC. BMP6 treatment induced TGFbeta-receptor-I expression and combined application of TGFbeta and BMP-6 eliminated the reduced chondrogenic potential of ATSC inducing a gene expression profile similar to differentiated BMSC. Like in BMSC, chondrogenesis of ATSC was associated with hypertrophy according to premature collagen Type X expression, upregulation of alkaline-phosphatase activity and in vivo calcification of spheroids after ectopic transplantation in SCID mice. In conclusion, a distinct BMP and TGFbeta-receptor repertoire may explain the reduced chondrogenic capacity of ATSC in vitro, which could be compensated by exogenous application of lacking factors. Further studies should now be directed to induce chondrogenesis in the absence of hypertrophy.     

In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel

Graft implantation is one of the more popular procedures for repairing cartilage defects; however, sacrifices of the donor site have been an issue. Mesenchymal stem cells (MSCs) are a fascinating source for regenerative medicine because they can be harvested in a less invasive manner and are easily isolated and expanded, with multipotentiality including chondrogenesis. MSCs can be isolated from various adult mesenchymal tissues including synovium. Here, we attempted to form cartilage from the composites of synovium-derived MSCs with collagen gel in vitro. After 21 days of culture, the composites had increased their cartilage matrix, as demonstrated by toluidine blue staining and immunohistochemistry for type II collagen. The composites consisting of 5×10⁷ and 10⁸ cells/ml in gel were richer in proteoglycans than those consisting of lower cell densities. After 1 day, MSCs/gel composites contracted and the diameter decreased by 30%; however, they were stable thereafter. Round cells with short processes producing collagen fibrils showing a similar morphology to that of chondrocytes were seen in the composites by transmission electron microscopy. During composite culture, chondroitin sulfate and mRNA expression for cartilage-related genes increased, demonstrating cartilage maturation. Using an optimized method, we obtained cartilage discs with a diameter of 7 mm and a thickness of 500 μm. Our procedure should thus make it possible to produce a large cartilage matrix in vitro. The tissue engineering of autologous cartilage from the composites of synovium-derived MSCs with collagen gel in vitro for transplantation may be a future alternative to graft implantation for patients with cartilage defects. This study is supported in part by grants from the Japanese Society for the Promotion of Science (16591478), the Japanese Orthopaedics and Traumatology Foundation, and the Nakatomi Foundation to I.S., and the Japanese Society for the Promotion of Science (16591477), the Japanese Sports Medicine Foundation, the Japanese Latest Osteoarthritis Society, and the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University to T.M.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full‐length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein‐2 (BMP‐2), transforming growth factor‐beta 1 (TGF‐β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full‐factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1550–1557, 2013     

Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit

We previously compared mesenchymal stem cells (MSCs) from a variety of mesenchymal tissues and demonstrated that synovium-MSCs had the best expansion and chondrogenic ability in vitro in humans and rats. In this study, we compared the in vivo chondrogenic potential of rabbit MSCs. We also examined other parameters to clarify suitable conditions for in vitro and in vivo cartilage formation. MSCs were isolated from bone marrow, synovium, adipose tissue, and muscle of adult rabbits. Proliferation potential and in vitro chondrogenic potential were compared. Toxicity of the tracer DiI for in vitro chondrogenesis was also examined. MSCs from each tissue were embedded in collagen gel and transplanted into full thickness cartilage defects of rabbits. Cartilage matrix production was compared histologically. The effects of cell density and periosteal patch on the in vivo chondrogenic potential of synovium-MSCs were also examined. Synovium- and muscle-MSCs had a higher proliferation potential than other cells. Pellets from synovium- and bone-marrow-MSCs showed abundant cartilage matrix. DiI had no significant influence on in vitro cartilage formation. After transplantation into cartilage defects, synovium- and bone-marrow-MSCs produced much more cartilage matrix than other cells. When synovium-MSCs were transplanted at a higher cell density and with a periosteal patch, more abundant cartilage matrix was observed. Thus, synovium- and bone-marrow-MSCs had greater in vivo chondrogenic potential than adipose- and muscle-MSCs, but synovium-MSCs had the advantage of a greater proliferation potential. Higher cell density and a periosteum patch were needed to obtain a high production of cartilage matrix by synovium-MSCs.     

Effects of various antimicrobial agents on multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells

Antimicrobial drugs of several classes play an important role in the treatment of bone and joint infections. In addition to fighting pathogenic microorganisms, the effects of drugs on local tissues and cells are also related to the course and prognosis of bone and joint infections. The multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells (MSCs) is essential for tissue repair after local injury, which is directly related to the recovery of bone, cartilage, and medullary adipose tissue. Our previous studies and the literature indicate that certain antimicrobial agents can regulate the differentiation potential of bone marrow-derived MSCs. Here, in order to systematically analyze the effects of various antimicrobial drugs on local tissue regeneration, we comprehensively review the studies on the effects of these drugs on MSC differentiation, and classify them according to the three differentiation directions (osteogenesis, chondrogenesis, and adipogenesis). Our review demonstrates the specific effects of different antimicrobial agents on bone marrow-derived MSCs and the range of concentrations at which they work, and provides a basis for drug selection at different sites of infection.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have strong potential in regeneration of musculoskeletal tissues including cartilage and bone. The microenvironment, comprising of scaffold and soluble factors, plays a pivotal role in determining the efficacy of cartilage tissue regeneration from MSCs. In this study, we investigated the effect of a three-dimensional synthetic-biological composite hydrogel scaffold comprised of poly (ethylene glycol) (PEG) and chondroitin sulfate (CS) on chondrogenesis of MSCs. The cells in CS-based bioactive hydrogels aggregated in a fashion which mimicked the mesenchymal condensation and produced cartilaginous tissues with characteristic morphology and basophilic extracellular matrix production. The aggregation of cells resulted in an enhancement of both chondrogenic gene expressions and cartilage specific matrix production compared to control PEG hydrogels containing no CS-moieties. Moreover, a significant down-regulation of type X collagen expression was observed in PEG/CS hydrogels, indicating that CS inhibits the further differentiation of MSCs into hypertrophic chondrocytes. Overall, this study demonstrates the morphogenetic role of bioactive scaffold-mediated microenvironment on temporal pattern of cartilage specific gene expressions and subsequent matrix production during MSC chondrogenesis.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

Introduction  

The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.     

Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO(2)) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO(2)). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO(2) proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO(2) also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO(2) was found to be a more potent promoter of chondrogenesis than expansion at 5% pO(2). Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO(2) compared to 5% pO(2). Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO(2) also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a chondrogenic phenotype for use in cartilage repair therapies or to promote hypertrophy of cartilaginous grafts for endochondral bone repair strategies.     

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells

We investigated chondrogenesis of cell-mediated sox9 gene therapy as a new treatment regimen for cartilage regeneration. pIRES2-EGFP vector containing a full-length mouse sox9 cDNA was transfected into bone marrow-derived mesenchymal stem cells (MSCs) by lipofection and chondrogenic differentiation of these cells was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich micromass aggregate with EGFP expressing MSCs was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice to analyze in vivo chondrogenesis. A massive tissue formation in about 2mm diameter was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained. These results indicated that cell-mediated sox9 gene therapy could be a novel strategy for hyaline cartilage damage.     

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.     

PTHrP isoforms have differing effect on chondrogenic differentiation and hypertrophy of mesenchymal stem cells

While several isoforms of parathyroid hormone-related peptide (PTHrP) have been commercially available, the difference in their effect has not been widely studied. The purpose of this study was to determine which isoform most effectively promoted chondrogenesis and suppressed hypertrophy from mesenchymal stem cells (MSCs). MSCs isolated from fresh bone marrow were cultured in pellet in chondrogenic medium containing 5 ng/ml of transforming growth factor (TGF)-β(3). From day 14 of culture, subsets of pellets were additionally treated with one of the four PTHrP isoforms (1-34, 1-86, 7-34, and 107-139) at 100 nM. After a further 2 weeks of in vitro culture, pellets were harvested for analysis. PTHrPs 1-34 and 1-86 significantly decreased the DNA level (p<0.05) while PTHrPs 7-34 and 107-139 significantly increased DNA level (p<0.05) compared with the control treated with TGF-β(3) only. Glycosaminoglycan per DNA significantly increased when treated with PTHrPs 1-34 and 1-86 (p<0.05) while it significantly decreased with PTHrPs 7-34 and 107-139 (p<0.05). PTHrP 1-34 significantly increased the gene and protein expression of the chondrogenic marker COL2A1, and decreased those of hypertrophic markers COL10A1 and alkaline phosphatase while other isoforms showed inconsistent effects. All of PTHrP isoforms significantly suppressed the gene and protein expression of indian hedgehog (p<0.05) while all isoforms except PTHrP 107-139 significantly reduced the gene and protein expression of patched 1 (p<0.05). In conclusion, of several PTHrP isoforms, PTHrP 1-34 most significantly enhanced chondrogenesis and suppressed hypertrophy in MSCs, supporting its use for cartilage tissue engineering.