Murine Muscle-derived Stem Cells Research Articles

Bone morphogenetic protein 4 rescues the bone regenerative potential of old muscle-derived stem cells via regulation of cell cycle inhibitors

BackgroundBone morphogenetic protein 4 (BMP4) promotes the osteogenic differentiation and the bone regenerative potential of muscle-derived stem cells (MDSCs). BMP4 also promotes the self-renewal of both embryonic and somatic stem cells; however, BMP4 signaling activity significantly decreases with age. Cyclin-dependent kinase inhibitors P16INK4A (P16) and P18INK4C (P18) induce early G1-phase cell cycle blockade by targeting cyclin-dependent kinase 4/6. It is still unclear if BMP4 affects the bone regenerative potential of old MDSCs through regulation of P16 and P18 expression.MethodsYoung and old MDSCs were isolated from 3 week (young) and 2-year-old (old) mice. In vitro cell proliferation and multipotent differentiation were performed for young and old MDSCs both before and after BMP4/GFP transduction. Cell cycle genes were analyzed using Q-PCR. The bone regenerative potential of young and old MDSCs transduced with BMP4/GFP were compared using Micro-CT and histological analysis. The bone regenerative potential of young and old MDSCs was also compared between single and double transduction (higher BMP4 levels expression). The cell proliferation, mitochondrial function and osteogenic differentiation was also compared in vitro between cells that have been transduced with BMP4GFP (single and double transduction). The correlation of bone regeneration capacity of young and old MDSCs with P16 and P18 expression was further evaluated at 10 days after cell transplantation using histology and western blot analysis.ResultsOld murine MDSCs (MDSCs) exhibit reduced proliferation and multi-lineage differentiation potential with or without BMP4 stimulation, when compared to young murine MDSCs. Old MDSCs express significantly higher P16 and lower P18, with more cells in the G0/1 phase and fewer cells in the G2/M phase, compared to young MDSCs. Old MDSCs retrovirally transduced to express BMP4 regenerated less bone in a critical size skull defect in CD-1 nude mice when compared to young retrovirally transduced MDSCs expressing similar BMP4 levels and contribute less to the new regenerated new bone. Importantly, both young and old MDSCs can regenerate more bone when BMP4 expression levels are increased by double-transduction with the retroviral-BMP4/GFP. However, the bone regeneration enhancement with elevated BMP4 was more profound in old MDSCs (400% at 2 weeks) compared to young MDSCs (200%). Accordingly, P18 is upregulated while P16 is downregulated after BMP4 transduction. Double transduction did not further increase cell proliferation nor mitochondrial function but did significantly increase Osx expression in both young and old MDSCs. Old MDSCs had even significant higher Osx levels as compared to young MDSCs following double transduction, while a similar Alp expression was observed between young and old MDSCs after double transduction. In addition, at 10 days after cell transplantation, old MDSCs having undergone double transduction regenerated bone more rapidly as showed by Alcian blue and Von Kossa staining. Western blot assays demonstrated that old MDSCs after retro-BMP4/GFP double transduction have significantly lower P18 expression levels when compared to young BMP4-transduced MDSCs. In addition, P18 expression was slightly increased in old MDSCs after double transduction when compared to single transduction. P16 expression was not detectable for both young and two old BMP4/GFP transduced MDSCs groups.ConclusionsIn summary, BMP4 can offset the adverse effect of aging on the osteogenic differentiation and the bone regenerative potential of old MDSCs via up-regulation of P18 and down-regulation P16 expression.

A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair

BackgroundOsteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study.MethodIn this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair.ResultsWe observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining.ConclusionsWe conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

Muscle-Derived Stem Cell-Enriched Scaffolds Are Capable of Enhanced Healing of a Murine Volumetric Muscle Loss Defect.

Volumetric muscle loss secondary to traumatic or surgical causes can lead to functional and aesthetic impairments. The authors hypothesize that an implantable muscle-derived stem cell-enriched collagen scaffold could significantly augment muscle regeneration in a murine model of volumetric muscle loss. Murine muscle-derived stem cells were isolated using a modified preplating technique and seeded onto type 1 collagen scaffolds to create the muscle-derived stem cell-enriched collagen scaffolds. Murine rectus femoris defects of 5 mm were created and randomized to one of three conditions (n = 6 per group): untreated controls, collagen scaffold only, and muscle-derived stem cell-enriched collagen scaffolds. In vivo muscle healing was quantified using micro-computed tomography. Muscle explants were analyzed using standard histology and whole-mount immunofluorescence at 8 weeks. In vivo experiments demonstrated significantly greater quadriceps cross-sectional area in the muscle-derived stem cell-enriched collagen scaffold group compared with controls on micro-computed tomography (0.74 ± 0.21 versus 0.55 ± 0.06 versus 0.49 ± 0.04 ratio of experimental to naive quadriceps cross-sectional area; p < 0.05). Muscle explants of the muscle-derived stem cell-enriched collagen scaffold group demonstrated significantly higher cellular density compared with controls (1185 ± 360 versus 359 ± 62 versus 197 ± 68 nuclei/high-power field; p < 0.01). Immunofluorescence for laminin and myosin heavy chain confirmed formation of organized muscle fibers within the defect of the muscle-derived stem cell-enriched collagen scaffold group only. However, appreciable confocal colocalization of myosin heavy chain with green fluorescent protein expression was low. The results of this study indicate that muscle-derived stem cell-enriched scaffolds significantly improved skeletal muscle regeneration in a murine muscle defect model. Based on the low fluorescent colocalization, host progenitor cells appear to contribute significantly to intradefect myogenesis, suggesting that deployment of a viable muscle-derived stem cell-enriched scaffold stimulates a regenerative mitogen response in native tissues.

Sustained Release of Bone Morphogenetic Protein 2 via Coacervate improves Muscle Derived Stem Cell Mediated Cartilage Regeneration in MIA-induced Osteoarthritis

Objectives:Individuals who participate in sports have an increased risk of osteoarthritis (OA), characterized by articular cartilage degeneration. Currently, there is no cure for OA with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. Previously, we have demonstrated that murine MDSCs retrovirally transduced to express chondrogenic proteins (BMPs) differentiate into chondrocytes and enhance cartilage repair in vivo. Direct injection of therapeutic proteins can promote cartilage healing; however, they have relatively short half-lives requiring muitiple injections of high dosages. This presents a challenge in terms of maintaining adequate local BMP levels and could negatively affect both injured and normal structures and lead to side effects such as osteophyte formation. Gene therapy is a promising approach that addresses this problem; however, its utilization in clinical applications is much further down the road. In order to circumvent viral transduction of cells for cartilage regeneration, we developed a unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) incorporated with BMP2 (BMP2 coacervate). In this study, we show that sustained delivery of BMP2 via a BMP2 coacervate can induce the differentiation of MDSCs to a chondrocyte lineage for in vivo cartilage regeneration and healing in a Monoiodoacetate (MIA)-induced osteoarthritis model.Methods:mMDSCs were isolated from muscle biopsies via a modified pre-plated technique. The BMP2 coacervates were prepared as previously described. The release profiles of BMP2 coacervate were tested by ELISA. The chondrogenic effects that delivery of BMP2 had on MDSCs were evaluated by RT-PCR. The efficacy of MDSC with BMP2 coacervate were evaluated in vivo in a MIA-induced OA model in C57B6 mice. Mice received an intraarticular injection of 20μl of MIA to induce osteoarthritic lesions. Two weeks after MIA injection, mice were injected with PBS (control), MDSC + free BMP2, or MDSC + BMP2 coacervate. Histologic evaluations of cartilage regeneration were conducted at week 4. Institutional Animal Care and Use Committee approval was obtained prior to all animal studies.Results:Release profiles of BMP2 showed sustained release for more than 28 days demonstrating sustained release. The chondrogenic differentiation of MDSCs following BMP2 delivery was assayed using cell culture. The mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (P<0.05). Multi-dosage free BMP2 demonstrated significantly higher Aggrecan expression compared to single dose free BMP2 (p<0.05). Col2A and aggrecan expression in the BMP coacervate group was superior to both single and mult-dose free BMP2 delivery (p<0.05) (Fig 1A). Histologic examination demonstrated superior cartilage repair and integration in the BMP2 coacervate group (Fig. 1B).Conclusion:This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle-derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Our results demonstrate an effective method for prolonged exposure to BMP2 via a non-gene therapy approach which is preferred and clinically translatable.

446. Gender Differences in the Osteogenic & Chondrogenic Potential of Human Muscle-Derived Stem Cells In Vitro

Introduction: It has been shown that murine muscle-derived stem cells (MDSCs) are capable of multipotent differentiation1xSee all References. These murine cells exhibit sexual dimorphism of osteogenic and chondrogenic potentials1xSee all References. Previous studies have shown that male mouse MDSCs (M-MDSCs) differentiate into chondrocytes more effectively and display better cartilage regeneration potentials than female MDSCs (F-MDSCs)2xSee all References. M-MDSCs have also been shown to be more osteogenic and demonstrate more rapid bone formation compared to F-MDSCs1. In vivo murine studies have also demonstrated that M-MDSCs form more bone than F-MDSCs1xSee all References, 3xSee all References. Adipose derived stem cells also display sexual dimorphism in their multipotent differentiation potential4xSee all References. This study further investigates this sexual dimorphism in human muscle-derived stem cells (hMDSCs). Methods: Two populations of human MDSCs, one male and one female, were used in these experiments. (1) Osteogenesis: M- and F-hMDSCs 3D pellets were cultured in osteogenic medium containing 100ng/ml bone morphogenic protein 2 (BMP-2) for 28d according to literature1xSee all References. Cell pellets were fixed in neutral buffered formalin and subjected to micro CT scanning. After scanning, cell pellets were embedded in NEG50 freezing medium for sectioning. Von Kossa staining was used to detect mineralization. Osteocalcin immunohistochemistry (IHC) was used to detect osteogenic differentiation. (2) Chondrogenesis: M- and F-MDSC 3D pellets were cultured for 24d in chondrogenic medium using Stem Pro complete chondrogenic medium. After culture, all pellets were fixed and sectioned. Alcian blue and Col2A1 were used as indicators of chondrogenesis, and the percentage of each pellet stained with Alcian blue staining was quantified. Results: Osteogenesis: MicroCT analysis indicated that M-hMDSCs had significantly more pellet mineralization compared to F-MDSCs (p<0.001) (Fig. 1A, 1BFig. 1A, 1B). Von Kossa staining showed stronger brown-black mineral deposition in male pellets than female pellets (Fig. 1CFig. 1C). Osteocalcin IHC indicated higher expression in male hMDSCs than female hMDSCs (Fig. 1DFig. 1D). Chondrogenesis: Alcian blue staining demonstrated that male cells have stronger blue cartilage matrices than female cells (Fig. 2AFig. 2A). Quantification of the blue matrix indicated significantly more matrix in male hMDSC pellets than female pellets (Fig. 2BFig. 2B). Col2A1 IHC showed stronger staining in male hMDSCs than female hMDSCs (Fig. 2CFig. 2C). Conclusions: Our results suggest that both gender hMDSCs can undergo osteogenic and chondrogenic differentiation, but male hMDSCs are more osteogenic and chondrogenic than female hMDSCs. Therefore, sex difference is an important factor to consider in the development and application of cell-based therapies for bone and articular cartilage repair.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

Role of donor and host cells in muscle‐derived stem cell‐mediated bone repair: differentiation vs. paracrine effects

Murine muscle-derived stem cells (MDSCs) have been shown capable of regenerating bone in a critical size calvarial defect model when transduced with BMP 2 or 4; however, the contribution of the donor cells and their interactions with the host cells during the bone healing process have not been fully elucidated. To address this question, C57/BL/6J mice were divided into MDSC/BMP4/GFP, MDSC/GFP, and scaffold groups. After transplanting MDSCs into the critical-size calvarial defects created in normal mice, we found that mice transplanted with BMP4GFP-transduced MDSCs healed the bone defect in 4 wk, while the control groups (MDSC-GFP and scaffold) demonstrated no bone healing. The newly formed trabecular bone displayed similar biomechanical properties as the native bone, and the donor cells directly participated in endochondral bone formation via their differentiation into chondrocytes, osteoblasts, and osteocytes via the BMP4-pSMAD5 and COX-2-PGE2 signaling pathways. In contrast to the scaffold group, the MDSC groups attracted more inflammatory cells initially and incurred faster inflammation resolution, enhanced angiogenesis, and suppressed initial immune responses in the host mice. MDSCs were shown to attract macrophages via the secretion of monocyte chemotactic protein 1 and promote endothelial cell proliferation by secreting multiple growth factors. Our findings indicated that BMP4GFP-transduced MDSCs not only regenerated bone by direct differentiation, but also positively influenced the host cells to coordinate and promote bone tissue repair through paracrine effects.

Human myogenic endothelial cells exhibit chondrogenic and osteogenic potentials at the clonal level

We have previously reported the high regenerative potential of murine muscle-derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC-like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration.

Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

BackgroundDespite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.Methodology/Principal FindingsSkeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs.ConclusionsThe methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.

Role of angiogenesis after muscle derived stem cell transplantation in injured medial collateral ligament

We performed this study to investigate the therapeutic role of vascular endothelial growth factor (VEGF) in medial collateral ligament (MCL) healing. Murine muscle derived stem cells (MDSCs) obtained via the preplate technique were retrovirally transduced to express: (1) VEGF and nLacZ (MDSC-VEGF), (2) soluble fms-like tyrosine kinase-1 (sFLT1, a VEGF-specific antagonist) and nLacZ (MDSC-sFLT1), and (3) nLacZ (MDSC-nLacZ). After transecting the MCL of immunodeficient rats, 5 × 10(5) cells of each of the transduction groups list above were transplanted into the MCL injury site. A control group was injected with phosphate-buffered saline (PBS) only. Immunohistochemical staining demonstrated that there were more Isolectin B4 and β-galactosidase double positive cells in the rats transplanted with MDSC-VEGF transduced cells than the other groups at week 1. Capillary density was significantly higher in the MDSC-VEGF group than the other groups at week 2; however, there were no significant differences in the biomechanical assessment between the MDSC-VEGF and MDSC-nLacZ groups. On the other hand, the MDSC-sFLT1 group revealed a lower capillary density than the other two groups and the functional ligament healing of the MDSC-sFLT1 group was significantly decreased compared to the other groups when assessed biomechanically. The findings of the present study suggest that angiogenesis plays a critical role in the healing process of injured MCL.

Isolation of murine muscle-derived stem cells with preplate technique combined with limited dilution technique

Isolation of murine muscle-derived stem cells with preplate technique combined with limited dilution technique

Regenerative medicine based on muscle stem cells

Members of the Stem Cell Research Center (SCRC) have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by means of the cells' adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Most of these cell populations have displayed characteristics similar to those of skeletal muscle satellite cells; however, we also have identified a unique population of muscle‐derived stem cells (MDSCs). The MDSCs exhibit long‐term proliferation and high self‐renewal abilities, increased resistance to stress, and multipotency with the ability to differentiate, in vitro and in vivo, into a variety of tissue types including: muscular (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages. In contrast to other myogenic cell types, MDSCs are very efficient at engrafting and regenerating a variety of musculoskeletal tissues due to their increased ability to survive post‐implantation because of their high expression of anti‐oxidants. MDSCs activate host cells and are influenced by environmental cues released within injured tissues, which have been shown to impact the MDSCs regenerative capacity in various tissues. Some of the results regarding the crosstalk between the donor cells and host cells/tissues will be presented. Potential strategies are being explored to increase angiogenesis and prevent scar tissue formation within injured tissues as a means to further improve the regenerative potential of these cells. Finally recent results will be presented that suggest that the blood vessel walls harbor several cell types, including myo‐endothelial cells and pericytes that are likely the place of origin of the murine MDSCs discussed above. These human blood vessels derived cells are being tested for their regenerative potential in various tissues. The results outlined above open new avenues by which researchers could use muscle stem cell–based gene therapy and tissue engineering to improve tissue regeneration.

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt-1 (sFlt-1), on the chondrogenesis of skeletal muscle-derived stem cells (MDSCs) in a rat model of osteoarthritis (OA). We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP-4) in combination with MDSCs expressing VEGF or sFlt-1. In vivo, a combination of sFlt-1- and BMP-4-transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt-1- and BMP-4-transduced MDSCs compared with a combination of VEGF- and BMP-4-transduced MDSCs or with BMP-4-transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP-4-transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes. Our results demonstrate that MDSC-based therapy involving sFlt-1 and BMP-4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

In vitro interaction between muscle-derived stem cells and nucleus pulposus cells

Background context Current therapies for intervertebral disc degeneration (IDD) are aimed at treating the clinical symptoms arising from IDD rather than directly treating the underlying problem. Pathophysiology of IDD is characterized by a progressive decrease in proteoglycan content and cell density in the nucleus pulposus (NP). A cell-based therapy is a promising concept that uses various cell types to repopulate the disc in an attempt to slow, stop, or reverse the progressive loss of proteoglycans. Stem cells appear to be an excellent candidate for this purpose, based on their ability to differentiate into various connective tissue lineages. The muscle tissue could serve as a good source of adult stem cells because of its vast abundance through out the human body. Purpose To examine the interaction between the nucleus pulposus cells (NPCs) and the muscle-derived stem cells (MDSCs) in vitro. Study design NPCs and MDSCs were cocultured and proteoglycan production and cell proliferation were evaluated. Methods Various ratios of human NPCs were cocultured for 2 weeks with murine MDSCs (transduced with retro/LacZ) in a monolayer culture. Each well contained an admixture of cells with NPC-to-MDSC ratios of 0:100, 25:75, 50:50, 75:25, 100:0. Glycosaminoglycan (GAG) content (1,9 dimethylmethylene blue [DMMB]), newly synthesized proteoglycan ( 35S incorporation), and DNA content were measured, and cultures were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-Gal) for cell counting. Results The NPC-to-MDSC ratio of 75:25 resulted in a significant increase in GAG content compared with NPCs alone. All coculture ratios showed increase in GAG content in comparison with MDSC culture alone. In addition, cocultures showed a significant increase in 35S incorporation normalized to DNA content in comparison with MDSC alone. Conclusions The data from this study shows a synergistic effect between MDSCs and NPCs resulting in an upregulated proteoglycan synthesis and NPCs proliferation in vitro.

245. Muscle-Derived Stem Cells Display an Extended, but Not Unlimited, Expansion Capability: Implication for Muscle Regeneration

Stem cells are frequently considered the optimal cell type for regenerative cell-based therapies; however they generally represent a small fraction of cells isolated from a biopsy or other cell source. Ex vivo cell expansion is a necessary step to obtain clinically relevant numbers of cells. In addition, stem cells are often theorized as cells with unlimited long-term expansion potential. The purpose of this study is to test the long-term expansion capability of a population of muscle-derived stem cells. We first examined the proliferation kinetics of murine muscle-derived stem cells (MDSCs) to determine if they obey Hayflick's limit. We determined that these cells can be expanded for more than 300 population doublings (PDs) with no indications of replicative senescence. Next we examined how the molecular and behavioral stem cell phenotype, including the regenerative capacity, changes over time. We find that the MDSC population continues to maintain a relatively low level of desmin expression ( 65%) throughout the expansion. We observe that up to 200 PDs the MDSCs readily differentiate to form multinucleated myotubes, however expansion beyond 200 PDs leads to a decline in the number of cells entering the post-mitotic differentiated state. Remarkably, MDSC are capable of regenerating dystrophin expressing muscle fibers upon implantation in mdx muscular dystrophy model even after 200 population doublings. However, expansion beyond 200 PDs resulted in a subsequent decline in regeneration efficiency. Observed phenotypic changes highlight the inevitable aging of cells that results from cell expansion. Several findings including loss of contact inhibition, ability to grow on soft agar and an increase in numerical chromosomal abnormalities suggests that the MDSC may have become transformed. While the MDSC demonstrate a highly extended functional lifetime for muscle regeneration, we find that this potential is not unlimited.