Mesenchymal stem cells at the intersection of cell and gene therapy

Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secret bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases

Use of glycol chitosan modified by 5β-cholanic acid nanoparticles for the sustained release of proteins during murine embryonic limb skeletogenesis

Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-Agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol-chitosan–5β-cholanic-acid-conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission-electron-microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% of released after 48-hours. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age (E)13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC-nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I + MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I + MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I + MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I + MSC could be a novel therapeutic approach in patients who have inadequate fracture repair

Human bone marrow mesenchymal stem cells : a systematic reappraisal via the genostem experience

Genostem (acronym for “Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side”) has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendonWork supported by the European Community (Key action 1.2.4-3 Integrated Project Genostem, contract No 503161)

Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities

5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation

The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors

Defective proliferation and osteogenic potential with altered immunoregulatory phenotype of native bone marrow-multipotential stromal cells in atrophic fracture non-union

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing