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.     

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.     

Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian‐blue staining, qRT‐PCR, and quantification of alkaline phosphatase (ALP) activity. Pre‐differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)‐β was always required for chondrogenic differentiation and deposition of a collagen‐type‐II‐positive extracellular matrix, while bone morphogenetic protein (BMP)‐2, ‐4, ‐6, ‐7, aFGF, and IGF‐I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF‐β, however, without preventing collagen type X expression. bFGF or parathyroid hormone‐like peptide (PTHrP) inhibited the TGF‐β‐responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up‐regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor‐independent pathway. While TGF‐β was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II⁺/collagen type X^? cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes. J. Cell. Physiol. 223: 84–93, 2010. © 2009 Wiley‐Liss, Inc.     

Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway

Mesenchymal stem cells (MSCs) are multi-potent, and the chondrogenesis of MSCs is affected by mechanical stimulation. The aim of this study was to investigate, using a rotary cell culture system (RCCS) bioreactor, the effects of microgravity on the chondrogenic differentiation of human adipose-derived MSCs (ADSCs), which were cultured in pellets with or without the chondrogenic growth factor TGF-β1. In addition, we evaluated the role of the p38 MAPK pathway in this process. The real-time PCR and histological results show that microgravity has a synergistic effect on chondrogenesis with TGF-β1. The p38 MAPK pathway was activated by TGF-β1 alone and was further stimulated by microgravity. Inhibition of p38 activity with SB203580 suppressed chondrocyte-specific gene expression and matrix production. These findings suggest that the p38 MAPK signal acts as an essential mediator in the microgravity-induced chondrogenesis of ADSCs.     

BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.     

Soluble factors from ASCs effectively direct control of chondrogenic fate

Background and objectives: Adipose tissue‐derived stem cells (ASCs) have great potential for regenerative medicine. For molecular understanding of specific functional molecules present in ASCs, we analysed 756 proteins including specific chondrogenic functional factors, using high‐throughput nano reverse‐phase liquid chromatography–electrospray ionization–tandem mass spectrometry. Materials, methods and results: Of these proteins, 33 were identified as chondrogenic factors or proteins including type 2 collagen, biglycan, insulin‐like growth factor‐binding protein and transforming growth factor‐beta 1 (TGF‐β1). ASCs are a possible cell source for cartilage regeneration as they are able to secrete a number of functional cytokines including chondrogenesis‐inducing molecules such as TGF‐β1 and bone morphogenetic protein 4 (BMP4). The chondrogenic phenotype of cultured ASCs was effectively induced by ASC‐culture media (CM) containing BMP4 and TGF‐β1, and maintained after pre‐treatment for 14 days in vitro and subcutaneous implantation in vivo. Chondrogenic differentiation efficiency of cultured ASCs and cultured mouse skin‐derived progenitor cells (SPCs) depended absolutely on ASC CM‐fold concentration. Cell density was also a very important factor for chondrogenic behaviour development during differentiation of ASCs and SPCs. Conclusion: ASC CM‐derived TGF‐β1‐induced chondrogenic differentiation of ASCs resulted in significant reduction in chondrogenic activity after inhibition of the p38 pathway, revealing involvement of this MAPK pathway in TGF‐β1 signalling. On the other hand, TGF‐β1 signalling also led to SMAD activation that could directly increase chondrogenic activity of ASCs.     

In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells

There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs.     

Characteristics of stem cells derived from the degenerated human intervertebral disc cartilage endplate

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP). The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs) were compared with bone-marrow MSCs (BM-MSCs). Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD) pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering.     

Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases

Cartilage repair by mesenchymal stem cells (MSCs) often occurs in diseased joints in which the inflamed microenvironment impairs chondrogenic maturation and causes neocartilage degradation. In this environment, melatonin exerts an antioxidant effect by scavenging free radicals. This study aimed to investigate the anti-inflammatory and chondroprotective effects of melatonin on human MSCs in a proinflammatory cytokine-induced arthritic environment. MSCs were induced toward chondrogenesis in the presence of interleukin-1 β (IL-1β) or tumor necrosis factor α (TNF-α) with or without melatonin. Levels of intracellular reactive oxygen species (ROS), hydrogen peroxide, antioxidant enzymes, and cell viability were then assessed. Deposition of glycosaminoglycans and collagens was also determined by histological analysis. Gene expression of chondrogenic markers and matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. In addition, the involvement of the melatonin receptor and superoxide dismutase (SOD) in chondrogenesis was investigated using pharmacologic inhibitors. The results showed that melatonin significantly reduced ROS accumulation and increased SOD expression. Both IL-1β and TNF-α had an inhibitory effect on the chondrogenesis of MSCs, but melatonin successfully restored the low expression of cartilage matrix and chondrogenic genes. Melatonin prevented cartilage degradation by downregulating MMPs. The addition of luzindole and SOD inhibitors abrogated the protective effect of melatonin associated with increased levels of ROS and MMPs. These results demonstrated that proinflammatory cytokines impair the chondrogenesis of MSCs, which was rescued by melatonin treatment. This chondroprotective effect was potentially correlated to decreased ROS, preserved SOD, and suppressed levels of MMPs. Thus, melatonin provides a new strategy for promoting cell-based cartilage regeneration in diseased or injured joints.     

A comparison of the involvement of p38, ERK1/2 and PI3K in growth factor-induced chondrogenic differentiation of mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are under investigation as an alternative cell source for the engineering of cartilage tissue in three-dimensional (3D) scaffolds. However, little is known about the intracellular mechanisms involved in the chondrogenic differentiation of MSCs. This study investigated the signaling pathways evoked by TGF-β1 and IGF-1 that mediated chondrogenic differentiation in adult rat bone-marrow derived MSCs in (i) monolayer on plastic and (ii) a 3D collagen-GAG scaffold. The data demonstrated involvement of the p38 pathway, but not ERK1/2 or PI3K in TGF-β1-induced chondrogenic differentiation in monolayer. Similarly, when the MSCs were seeded onto a collagen-GAG scaffold and treated with TGF-β1, the chondrogenic differentiation was dependent upon p38. In contrast, IGF-1-induced chondrogenic differentiation in monolayer involved p38, ERK1/2, as well as PI3K. The phosphorylation of Akt occurred downstream of PI3K and phospho-Akt was found to accumulate in the nucleus of IGF-1-treated cells. When MSCs were seeded onto the collagen-GAG scaffold and exposed to IGF-1, PI3K was required for chondrogenesis. These findings highlight the respective and differential involvement of p38, ERK1/2 and PI3K in growth factor-induced chondrogenesis of MSCs and demonstrates that intracellular signaling pathways are similar when differentiation is stimulated in a 2D or 3D environment.     

Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair

Transplantation of bone-marrow derived mesenchymal stem cells (MSCs) has potential therapeutic effects on cardiac muscle repair. However, the underlying mechanism remains not completely clarified. Here we show that transplantation of MSCs significantly increased local recruitment of macrophages to facilitate cardiac muscle repair. MSCs-induced recovery of cardiac function and attenuation of fibrosis after injury were all abolished by either impaired macrophage infiltration, or by MSCs depletion after macrophage recruitment. However, angiogenesis seemed to be only affected by depletion of macrophages, but not by depletion of MSCs, suggesting that macrophages are responsible for the augmented angiogenesis after MSCs transplantation, while MSCs do not directly contribute to angiogenesis in the functional cardiac repair. Moreover, high level of transforming growth factor β 1 (TGFβ1) was detected in macrophages and high level of BMP7 was detected in MSCs, suggesting that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGFβ1 by macrophages. Our study thus sheds new insight on the interaction of MSCs and macrophages in a functional cardiac repair triggered by MSCs transplantation.     

Secretion of rat tracheal epithelial cells induces mesenchymal stem cells to differentiate into epithelial cells

Bone marrow MSCs (mesenchymal stem cells) can differentiate into various tissue cells, including epithelial cells. This presents interesting possibilities for cellular therapy, but the differentiation efficiency of MSCs is very low. We have explored specific inducing factors to improve the epithelial differentiation efficiency of MSCs. Under inducing conditions, MSCs differentiated into epithelial cells and expressed several airway epithelial markers using RTE (rat tracheal epithelial) cell secretions. Rat cytokine antibody array was used to detect cytokines of the RTE secretion components, in which 32 kinds of protein were found. Seven proteins [TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), VEGF (vascular endothelial growth factor), BDNF (brain-derived neurotrophic factor), TGFβ1 (transforming growth factor β1), MMP-2 (metalloproteinases-2), OPN (osteopontin) and activin A in RTE secretions] were assayed using ELISA kits. The four growth factors (VEGF, BDNF, TGFβ1 and activin A) were involved in regulating stem cell growth and differentiation. We speculated that these four play a vital role in the differentiation of MSCs into epithelial cells by triggering appropriate signalling pathways. To induce epithelial differentiation, MSCs were cultured using VEGF, BDNF, TGFβ1 and activin A. Differentiated MSCs were characterized both morphologically and functionally by their capacity to express specific markers for epithelial cells. The data demonstrated that MSCs can differentiate into epithelial cells induced by these growth factors.     

Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation,composition, and micromechanical properties of nucleus pulposus constructs

Introduction

Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs.

Methods

Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-β3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM).

Results

GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-β or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs.

Conclusions

The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies.     

Identity and ranking of colonic mesenchymal stromal cells

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.     

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10^?8 and 10^?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10^?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.     

Synovium-derived stem cell-based chondrogenesis

Synovium is considered a candidate source of cells for cartilage tissue engineering. Compared with mesenchymal stem cells (MSCs) from other sources, synovium-derived stem cells (SDSCs) have a higher capacity for chondrogenic differentiation. Our objective was to define cocktails of growth factors that support the growth and chondrogenic differentiation of SDSCs in chemically defined medium. We established a fast and highly selective technique of negative isolation of SDSC populations. The individual and combined effects of three growth factors-transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2)-were evaluated in serum-free pellet cultures of SDSCs for the chondrogenesis of SDSCs using histology, biochemical analysis, and real-time RT-PCR. In vitro studies identified TGF-beta1 as the key factor for both the growth and chondrogenesis of SDSCs. The highest rates of SDSC growth were observed with the synergistic interaction of all three factors. With respect to chondrogenic differentiation of SDSCs, the interaction of TGF-beta1 and IGF-I applied simultaneously was superior to the sequential application of these two factors or any other combination of growth factors studied. Based on these findings, we propose a two-step protocol for the derivation of chondrogenic SDSCs: a cocktail of TGF-beta1, IGF-I, and FGF-2 is applied first to induce cell growth followed by a cocktail of TGF-beta1 and IGF-I applied to induce chondrogenesis.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.