Potential Of Bone Marrow-derived Mesenchymal Stem Cells Research Articles

Abstract 063: Evaluation of a Combination Therapy of Bone Marrow-Derived Stem Cells and Serelaxin Against Established Renal Fibrosis in Normotensive and Hypertensive Models of Disease

Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapies. To overcome this, we demonstrated that the anti-fibrotic drug, serelaxin (recombinant human relaxin; RLX), could prevent renal fibrosis progression in a unilateral ureteric obstruction (UUO)-induced murine model of interstitial renal disease and improve the ability of injected human bone marrow-derived mesenchymal stem cells (BM-MSCs) to attenuate renal injury. To advance the therapeutic potential of this combination therapy, we have now investigated its effects as an intervention therapy in the UUO model, and in a uninephrectomised/deoxycorticosterone acetate/drinking saline water (1K/DOCA/salt) model of hypertension. 7-8 week old male C57B6J mice were subjected to UUO or 1K/DOCA/salt (2.4mg/day, s.c; 21 days). Sub-groups of injured mice (n=6/group) were administered RLX (0.5mg/kg/day; s.c) alone, BM-MSCs (1x10 6 /mouse; i.v) alone or both combined 3-days post-UUO or 14 days post-1K/DOCA/salt-treatment. Untreated injured mice and sham-operated or 1K-mice were included as respective controls. Mice were killed 10 days post-UUO or 21 days post-1K/DOCA/salt treatment (7 days post-intervention) for tissue collection and analysis. UUO- and 1K/DOCA/salt-injured mice underwent significant renal damage, inflammation and (interstitial and total collagen-induced) fibrosis at the time-points studied, as determined by histological and biochemical analyses (all p<0.01 vs respective uninjured controls). All 1K/DOCA/salt-treated mice had systolic blood pressure levels of 140-150mmHg over the experimental period, compared to 1K-mice having 115-120mmHg (as measured by tail cuff plethysmography; p<0.01). Unlike BM-MSCs alone, the combined effects of BM-MSCs and RLX significantly reduced established UUO-induced renal damage and fibrosis, primarily by reducing myofibroblast-induced collagen deposition (all p<0.05 vs BM-MSCs alone). Treatment-induced effects of the hypertensive model are currently being evaluated and will be presented. These findings confirm that agents that can effectively reduce established fibrosis can enhance the therapeutic potential of BM-MSCs in treating kidney diseases, when added in combination.

Effect of Porous Chitosan Microspheres Loaded with Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stem Cells on Regeneration of Tibia Defect

Objective. Repair of bone defects represents a grave clinical challenge because of the tremendous difficulties in the recovery of bone function and regeneration of bone loss. Therefore, we investigated the effects of platelet-rich plasma-loaded (PRP) porous chitosan microspheres (PCMs) on the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and the proliferation and differentiation potential of BMSCs loaded by PCMs in vitro. We also established the model of bone defect repair in rat tibia to further explore the effects of PCMs loaded with PRP and BMSCs on bone regeneration. Methods. MTT assay was used to detect the proliferative ability of BMSCs after hypoxia/reoxygenation (H/R) treatment and the proliferative ability of BMSCs loaded by PCMs; polymerase chain reaction (PCR) was used to detect the expression of alkaline phosphatase (ALP), type I collagen (Col I), and type II collagen (Col II) in BMSCs after hypoxia and in BMSCs induced by PRP-loaded PCMs; PCR was used to detect the expression of Runt-associated transcription factor 2 (Runx2) and osteocalcin (OC) in the newly generated bone tissue; micro-CT scanning was applied to measure the bone mineral density and bone volume of the newly generated bone tissue in rats. Results. BMSCs still have the normal potential of proliferation and differentiation after H/R treatment. PCMs can provide a larger surface for the attachment of BMSCs, facilitating cell proliferation. Loaded by PCMs, PRP can be slowly released, effectively stimulating the differentiation of BMSCs. PCM/PRP/BMSC composites increased the expression levels of Runx2 and OC in the newly generated bone in rat tibia defect and the bone mineral density. Moreover, the composites improved the rate of regenerated bone volume. Conclusion. The application of PCM/PRP/BMSC composites is promising in the repair of tibia defects.

In vivo administration of G9a inhibitor A366 decreases osteogenic potential of bone marrow-derived mesenchymal stem cells.

Epigenetic mechanisms such as histone methylation are considered as one of the most important mediators that control stem cell behaviors such as proliferation, senescence and differentiation. G9a, a histone methyltransferase, has recently generated intense attention as potential target for controlling many diseases such as cancers. The aim of the present study was to evaluate the effect of in vivo administration of A366, a G9a inhibitor, on proliferative and differentiation potential of bone marrow-derived mesenchymal stem cells (BM-MSCs). We inhibited G9a using intraperitoneally administration of A366, and we evaluated BM-MSC proliferation and differentiation behaviors in vitro. Colony formation assay of BM-MSCs at primary culture showed that in vivo administration of A366 reduced the colony forming capacity of BM-MSCs. Moreover, PDT of BM-MSC isolated from A366-treated rats was higher than control, especially in the early passages. BM-MSC isolated from A366-treated rats showed higher adipogenic potential compared to the control at the early passages as determined by gene expression and Oil Red staining. Whereas, osteogenic potential of BM-MSC isolated from A366-treated rats was lower than control, especially at early passages. Our results suggest that the epigenetic modifier such as A366, which seems to be a therapeutic approach for controlling diseases such as cancer, might also influence the proliferation and differentiation capacity of MSCs both in vitro and in vivo. Moreover, epigenetic modifying chemicals seem to be a strategy to manipulate MSC expansion capacity and differentiation propensity, as well as to efficiently involvement of MSCs in tissue homeostasis, cell-based therapy and tissue engineering.

Antifibrotic potential of bone marrow‑derived mesenchymal stem cells in biliary atresia mice

Biliary atresia (BA) is a rare and severe disease that affects infants where a fibroinflammatory process destroys the bile ducts, leading to fibrosis and biliary cirrhosis, and mortality if untreated. Bone marrow‑derived mesenchymal stem cells (BMMSCs) have been considered as a promising therapy in fibrotic diseases. The aim of the present was to investigate the anti‑fibrotic roles of BMMSC transplantation in a BA mouse model. Mouse BA models were established by Rhesus rotavirus administration to neonatal mice. The results revealed that the liver enzyme and bilirubin metabolism levels, and the levels of the oxidative stress marker malondialdehyde (MDA) and the fibrosis marker were all increased in the BA model, while the liver tissue levels of superoxide dismutase and glutathione peroxidase were reduced. The hematoxylin and eosin and Masson's trichrome staining revealed severe liver fibrosis and collagen accumulation in BA livers. However, these indicators were all reversed once the BA mice were administered the BMMSC inoculation. In conclusion, the present study demonstrated the anti‑fibrotic potential of BMMSCs in BA mice, which may provide a novel approach to ameliorate the fibrotic response in BA patients.

Lentiviral delivery of biglycan promotes proliferation and increases osteogenic potential of bone marrow-derived mesenchymal stem cells in vitro

Mesenchymal stem cells (MSCs) are multipotent progenitor nonhematopoietic cells that have emerged as an attractive cell source for tissue engineering. Biglycan (BGN), an extracellular matrix component, has been reported to play a crucial role in bone formation. However, the role of BGN in MSCs remains unknown. Using lentiviral gene delivery, we sought to investigate the cellular effects of BGN on proliferation and osteogenic potential of bone marrow-derived MSCs in vitro. We found that MSCs with lentiviral transduction of BGN exhibited an increase in growth ability and the percentage of the S phase, together with significantly reduced mRNA levels of p21 and p27. Furthermore, lentiviral transduction of BGN in MSCs also increased alkaline phosphatase activity, stimulated the mineralization capacity, and enhanced expression of Runx2, Osteocalcin, collagen I, transforming growth factor (TGF)-β1, p-Smad2 and p-Smad3. Taken together, our data suggest that BGN overexpression positively regulates proliferation and osteogenic potential of MSCs, and TGF-β signaling pathway may be involved in BGN-induced osteogenic potential of MSCs.

Nanog Reverses the Effects of Organismal Aging on Mesenchymal Stem Cell Proliferation and Myogenic Differentiation Potential

Although the therapeutic potential of mesenchymal stem cells (MSCs) is widely accepted, loss of cell function due to donor aging or culture senescence are major limiting factors hampering their clinical application. Our laboratory recently showed that MSCs originating from older donors suffer from limited proliferative capacity and significantly reduced myogenic differentiation potential. This is a major concern, as the patients most likely to suffer from cardiovascular disease are elderly. Here we tested the hypothesis that a single pluripotency-associated transcription factor, namely Nanog, may reverse the proliferation and differentiation potential of bone marrow-derived MSC (BM-MSC) from adult donors. Microarray analysis showed that adult (a)BM-MSC expressing Nanog clustered close to Nanog-expressing neonatal cells. Nanog markedly upregulated genes involved in cell cycle, DNA replication, and DNA damage repair and enhanced the proliferation rate and clonogenic capacity of aBM-MSC. Notably, Nanog reversed the myogenic differentiation potential and restored the contractile function of aBM-MSC to a similar level as that of neonatal (n)BM-MSC. The effect of Nanog on contractility was mediated--at least in part--through activation of the TGF-β pathway by diffusible factors secreted in the conditioned medium of Nanog-expressing BM-MSC. Overall, our results suggest that Nanog may be used to overcome the effects of organismal aging on aBM-MSC, thereby increasing the potential of MSC from aged donors for cellular therapy and tissue regeneration.

Dynamic expression characteristics of Notch signal in bone marrow-derived mesenchymal stem cells during the process of differentiation into hepatocytes

Notch signaling is often involved in early development which helps to determine the differentiation state and fate of stem cells destined to form different tissues in the body. Its role in the differentiation of BM-MSCs (bone marrow-derived mesenchymal stem cells) is much less clear. As there is great interest in the potential of BM-MSCs as a source of cells for treating liver damage, it is important to understand if Notch signaling promotes or suppresses BM-MSCs differentiation into hepatocytes. In the present study, RT-PCR, Western blot results and morphologic changes demonstrated that BM-MSCs could successfully differentiate into hepatocytes in our special induction system including the tissue extract of damaged liver. On the 21st day when the differentiation direction was determined in BM-MSCs, the mRNA level of Jagged2, Delta1, Delta3, Notch1, Notch2, Notch3 and Presenilin1, was significantly lower than that on days 0, 7, and 11. In the further experiments, down-regulation of Notch signaling was shown to be critical for BM-MSCs to differentiate into hepatocytes, as increased Jagged1 resulted in up-regulated Notch activation leading to higher levels of expression of Hes1 and Hey1, which completely blocked Albumin expresion in BM-MSCs. These results in our study showed that Notch signaling in BM-MSCs was necessary to initiate differentiation into hepatocytes, but must be down-regulated for the differentiation to proceed continuously.