Hailang Luo

MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis.

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis, are the most common causes of bone tissue destruction. Recently, human periodontal ligament tissue‐derived mesenchymal stem cells (PDLSCs), a population of multipotent stem cells, have been used to reconstruct tissues destroyed by chronic inflammation. However, the impact of the local inflammatory microenvironment on tissue‐specific stem cells and the mechanisms controlling the effects of the local inflammatory environment remain poorly understood. In this study, we found that the multidifferentiation potential of mesenchymal stem cells (MSCs) isolated from periodontitis‐affected periodontal ligament tissue (P‐PDLSCs) was significantly lower than that of MSCs isolated from healthy human periodontal ligament tissue (H‐PDLSCs). Inflammation in the microenvironment resulted in an inhibition of miR‐17 levels, and a perturbation in the expression of miR‐17 partly reversed the differentiation potential of PDLSCs in this microenvironment. Furthermore, inflammation in the microenvironment promoted the expression of Smad ubiquitin regulatory factor one (Smurf1), an important negative regulator of MSC osteogenic differentiation. Western blotting and 3′ untranslated regions (3′‐UTR) reporter assays confirmed that Smurf1 is a direct target of miR‐17 in PDLSCs. Our data demonstrate that excessive inflammatory cytokine levels, miR‐17, and Smurf1 were all involved in a coherent feed‐forward loop. In this circuit, inflammatory cytokines led to direct activation of Smurf1 and downregulation of miR‐17, thereby increasing degradation of Smurf1‐mediated osteoblast‐specific factors. The elucidation of the molecular mechanisms governing MSC osteogenic differentiation in a chronic inflammatory microenvironment could provide us with a better knowledge of chronic inflammatory disorder and improve stem cell‐mediated inflammatory bone disease therapy. STEM CELLS 2011;29:1804–1816

The protection of MSCs from apoptosis in nerve regeneration by TGFβ1 through reducing inflammation and promoting VEGF-dependent angiogenesis.

Our previous report demonstrated that autologous adipose-derived mesenchymal stem cells (ADSCs) combined with xenogeneic acellular nerve matrix (XANM) can support the regeneration of defective nerves. Although ADSCs had the potential to replace Schwann cells in engineered-tissue nerves, apoptosis easily obstructed the ability to treat serious nerve injury in the host, such as a >50-mm-long nerve defect. In the present study, we found that, in combination with transforming growth factor β1 (TGFβ1), an ADSCs-XANM graft was sufficient to support the regeneration of a 50-mm sciatic nerve defect, which was not achieved using an ADSCs-XANM graft alone. Based on this finding, we further investigated how TGFβ1 coordinated with ADSCs to enhance nerve regeneration. In vitro, cell culture experiments demonstrated that TGFβ1 did not have a direct effect on ADSC proliferation, apoptosis, the cell cycle, or neural differentiation. The expression of VEGF, however, was significantly increased in ADSCs cultured with TGFβ1. In vivo, fluorescence labeling experiments demonstrated that the survival of transplanted ADSCs inoculated with XANM-TGFβ1 was higher than with XANM. Further study showed that TGFβ1 was capable of impairing the host immune response that was trigged by transplanted XANM. Additionally, we discovered that XANM-ADSCs in immunodeficient mice had apoptosis rates similar to XANM-ADSCs-TGFβ1 over a short time course (7 days). Once we blocked VEGF with a neutralizing antibody, the protective effect of TGFβ1 was impaired over a long time course (28 days). These results suggested that TGFβ1 was capable of enhancing the regenerative capacity of an XANM-ADSCs graft, mainly by protecting transplanted ADSCs from apoptosis. This effect was achieved in part through decreasing inflammation and promoting VEGF-dependent angiogenesis.

Expansion and Delivery of Adipose-Derived Mesenchymal Stem Cells on Three Microcarriers for Soft Tissue Regeneration

Cell/microcarrier combinations can be injected to repair tissue defects, but whether currently available microcarriers can be utilized to repair different tissue defects remains unknown. Here, we compared the suitability of fabricated micronized acellular dermal matrix (MADM), micronized small intestinal submucosa (MSIS), and gelatin microspheres as expansion and delivery scaffolds for adipose-derived mesenchymal stem cells (ADSCs). The results of MTS assay, scanning electron microscopy (SEM), and flow cytometry suggested that the three microcarriers all have good biocompatibility. Quantitative polymerase chain reaction revealed enhanced epidermal growth factor, vascular endothelial growth factor, basal fibroblast growth factor, and transforming growth factor-β expression levels after ADSCs had been cultured on MADM or MSIS for 5 days. After culturing ADSCs on microcarriers in osteogenic medium for 7 days, the expression levels of bone formation-related genes were enhanced. ADSC/microcarrier treatment accelerated wound closure. The ADSC/MADM and ADSC/MSIS combinations retained more of the original implant volume at 1 month postimplantation than ADSC/gelatin microspheres combination in soft-tissue augmentation studies. All implants displayed fibroblast and capillary vessel infiltrations; but ectopic bone formation did not occur, and the calvarial defect repair results were unfavorable. Our study demonstrates the potential utility of these microcarriers not only as a cell-culture substrate but also as a cell-transplantation vehicle for skin regeneration and soft-tissue reconstruction.

Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects

Reconstruction of large area bone defect with mechanical integrity to the skeleton is important for patients rehabilitation. However with the limitation of scaffold material and suitable seed cell sources, the best treating strategy remains to be identified though various tissue engineering methods were reported. In this study, we investigated the feasibility of applying calcined bovine bone (CBB) which was coated by allograft bone marrow mesenchymal stem cells (BMSC)-sheet as a 3D scaffold material in bone repairing tissue engineering. The new scaffold material was implanted into osteoporosis rat cranial bone defects and repairing critical size bone defects (8 mm diameter). Data showed that CBB-BMSC-sheet combination had a stronger potential in osteogenic differentiation and mineralized formation both in vitro and in vivo than CBB-BMSC combination. In in vitro study BMSC-sheet had a more feasible characteristic upon bone repairing including richer ECM, larger mineralized area and stronger ALP activity in addition with a significant higher mRNA expression of osteogenic maker such as BMP-2, b-FGF, Col 1a1, OSX and Runx-2 than the control group. In in vivo study 3D reconstruction of micro CT, HE staining and bone strength results showed that newly formed bone in CBB-BMSC-sheet group was significant higher than that in CBB-BMSC group at 4, 8 and 12 weeks after transplantation in the aspect of area and volume. What was more, results indicated that allograft BMSC-sheet had survivaled in the scaffold material and participated in the newly formed bone which had the same thickness with surrounding autologous bone tissues after transplantation. Results of our study demonstrated that CBB-BMSC-sheet combination was a promising strategy in healing of large area bone defect in osteoporosis.

Construction of tissue-engineered cornea composed of amniotic epithelial cells and acellular porcine cornea for treating corneal alkali burn

Although acellular corneas have been reported to be a potential substitute for allogeneic cornea transplantation to treat corneal injury, severe corneal injury is hard to repair due to inflammation and neovascularization. The use of the amniotic membrane as a graft in ocular surface reconstruction has become widespread because of the anti-inflammatory and anti-angiogenic properties of amniotic epithelial cells (AECs). Our objective was to construct a tissue-engineered cornea (TEC) composed of an acellular porcine cornea (APC) and AECs to repair severe corneal injury. Corneal cells were completely removed from the prepared APC, and the microstructure, mechanical properties, and stability of a natural porcine cornea (NPC) was maintained. In vitro, MTT and flow cytometry analyses showed that the APC did not negatively affect cell viability and apoptosis. In vivo, corneal pocket and subcutaneous transplantation demonstrated that the APC was incapable of trigging accepted immune response. AECs isolated from the human amniotic membrane have proliferation potential and present healthy morphology before 6 passages. After 7 days of culture on the surface of the APC, the AECs were stratified into 5-6 layers. We found that the AECs reconstituted the basement membrane that had been disrupted by the decellularization process. ELISA results showed that after culturing the TEC, the culture medium contained anti-inflammatory and anti-angiogenic growth factors, such as MIF, IL6, Fas-L, and PDEF. Finally, the results of lamellar keratoplasty to treat an alkali burn showed that the transplanted TEC was transparent and completely inoculated into the host cornea. However, the transplanted APC was degraded due to host rejection. Therefore, we conclude that a TEC composed of AECs and an APC holds great potential for the repair of severe corneal injury.

GSK3β is a checkpoint for TNF-α-mediated impaired osteogenic differentiation of mesenchymal stem cells in inflammatory microenvironments.

BACKGROUND The fate and differentiation of mesenchymal stem cells (MSCs) depend on various microenvironmental cues. In chronic inflammatory bone disease, bone regeneration is inhibited. The present study therefore sought to identify the underlying molecule mechanisms. METHODS We isolated periodontal ligament stem cells (PDLSCs), a new population of MSCs, from the periodontal ligament tissues of periodontitis patients and healthy controls (p-PDLSCs and h-PDLSCs). The secretion of inflammatory cytokines, like TNF-α, IL-1β, IL-6 and IL-8, after LPS stimulation was measured by ELISA. The expressions of p-GSK3β and GSK3β in two types of PDLSCs were detected by Western blot. TOPFlash was used to assay the Tcf/Lef transcriptional activity. Knockdown of GSK3β by siRNA and over-expression of GSK3β by adenoviruses were performed to confirm the role of GSK3β in the impaired osteogenic differentiation of PDLSCs under inflammatory microenvironment. RESULTS We demonstrated that p-PDLSCs displayed impaired osteogenic capacity than h-PDLSCs. Upon inflammatory stimulation, monocytes, but not PDLSCs, released inflammatory cytokines among which TNF-α directly act on PDLSCs and suppressed their osteogenic differentiation. TNF-α induced the phosphorylation of GSK3β, the deactivated form of GSK3β, which increased nuclear β-catenin and Lef-1 accumulation, and eventually reduced the Runx2-associated osteogenesis in PDLSCs. Over-expression of GSK3β rescued osteogenesis in TNF-α-stimulated PDLSCs, whereas inactivation of GSK3β was sufficient to liberate the β-catenin/Lef-1/Runx2 pathway. CONCLUSION GSK3β plays an obligatory role in the TNF-α-mediated inhibition of osteogenesis in MSCs. GENERAL SIGNIFICANCE The strategy to target GSK3β may provide a potential approach to bone regeneration in inflammatory microenvironments.

Mesenchymal Stem Cells Prevent Hypertrophic Scar Formation via Inflammatory Regulation when Undergoing Apoptosis

The cutaneous wound-healing process can lead to hypertrophic scar formation, during which exaggerated inflammation has been demonstrated to have an important role. Therefore, an exploration of strategies designed to regulate this inflammatory process is warranted. Mesenchymal stem cells (MSCs) have recently been demonstrated to regulate inflammation in various diseases. In this regard, using a rabbit model, we locally injected human mesenchymal stem cells (hMSCs) derived from bone marrow to treat hypertrophic scar formation, and explored their underlying mechanisms. We found that hMSC therapy efficiently regulated inflammation and prevented scar formation. We attributed the therapeutic effects of hMSCs to their secretion of an anti-inflammatory protein, TNF-alpha-stimulated gene/protein 6 (TSG-6). Unexpectedly, after injection, the number of surviving hMSCs decreased markedly and the hMSCs underwent extensive apoptosis, which was demonstrated to promote their secretion of TSG-6, partially through the activation of caspase-3. Moreover, H2O2-induced apoptotic hMSCs showed higher inflammatory regulatory abilities. The inhibition of caspase-3 decreased the inflammatory regulatory abilities of hMSCs and attenuated their therapeutic effects. Our results demonstrate that hMSCs can efficiently prevent hypertrophic scar formation via inflammatory regulation. In addition, we found that apoptosis has an important role in the activation of the inflammatory regulatory abilities of hMSCs.

Licochalcone A up-regulates of FasL in mesenchymal stem cells to strengthen bone formation and increase bone mass

The role of bone marrow-derived mesenchymal stem cells(BMSCs)in the pathogenesis and therapy of osteoporosis has drawn increasing attention in recent years. In the development of osteoporosis, it has been demonstrated that many changes occurred in the behavior of BMSCs. For example, the biological system of FasL pathways mediated differentiation of ERK and GSK-3β-catenin pathway was damaged. Here we found that 0.35 mg/L Licochalcone A (L-A) had a strong effect in increasing the osteogenic differentiation and mineralization of BMSCs both in vivo and in vitro by up-regulating FasL and further playing a role in regulating the ERK and GSK-3β-catenin systems. It has also demonstrated that the administration of L-A could restore the biological function of the damaged BMSCs differentiation by recovering or protecting bone mass in a disease state through activating the endosteal bone formation and partially inhibiting bone resorption in acute estrogen deficiency model. Results of our study suggested that careful titration of MSC was response to L-A and up-regulated FasL pathways mediating differentiation of ERK and GSK-3β-catenin biological systems under disease state in vivo, restore the impaired function, is one of the ways of L-A relieve or treatment osteoporosis.

Tissue-engineered nerve constructs under a microgravity system for peripheral nerve regeneration.

Mesenchymal stem cells (MSCs) seeded in a 3D scaffold often present characteristics of low proliferation and migration, which affect the microstructure of tissue-engineered nerves (TENs) and impair the therapeutic effects of nerve defects. By promoting MSC differentiation and mass/nutrient transport, rotary cell culture systems (RCCSs) display potential for advancing the construction of MSC-based TENs. Thus, in this study, we attempted to construct a TEN composed of adipose-derived mesenchymal stem cells (ADSCs) and acellular nerve graft (ANG) utilizing an RCCS. Compared to TENs prepared in a static 3D approach, MTT and cell count results displayed an increased number of ADSCs for TENs in an RCCS. The similarity in cell cycle states and high rates of apoptosis in the static 3D culture demonstrated that the higher proliferation in the RCCS was not due to microgravity regulation but a result of preferential mass/nutrient transport. Quantitative PCR and ELISA indicated that the RCCS promoted the expression of ADSC neural differentiation-associated genes compared to the static 3D culture. Furthermore, this difference was eliminated by adding the Notch1 signaling pathway inhibitor DAPT to the 3D static culture. TEM, axon immunostaining, and retrograde labeling analysis after sciatic nerve transplantation indicated that the TENs prepared in the RCCS exhibited more regenerative characteristics for repairing peripheral nerves than those prepared in a static 3D approach. Therefore, these findings suggest that the RCCS can modulate the construction, morphology, and function of engineered nerves as a promising alternative for nerve regeneration.

Liver extracellular matrix promotes BM-MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway.

In cell‐based therapies for liver injuries, the clinical outcomes are closely related to the surrounding microenvironment of the transplanted bone marrow mesenchymal stem cells (BM‐MSCs). However, whether liver‐specific ECM (L‐ECM), as one of major microenvironment signals, could regulate the therapeutic effect of BM‐MSCs through changing their biological characteristics is unclear. This study aimed to investigate the hepatogenicity and underlying mechanism of L‐ECM as well as its potential regulative role in the MSC‐based liver recovery. L‐ECM was prepared by homogenization of decellularized whole porcine liver. After three‐dimensional culture with or without the presence of L‐ECM, BM‐MSCs expressed hepatocyte‐specific genes and proteins in an L‐ECM concentration‐dependent manner. Further analysis showed that L‐ECM could activate specific types of integrins (ITGs) as well as their downstream signalling pathways. When the cell/ECM interaction was enhanced by incorporating BM‐MSCs with Mn2+, ITGs were activated and the hepatogenic capacity of L‐ECM was improved. The regeneration of rat livers from either acute or chronic fibrosis could also be accelerated after transplantation of Mn2+‐treated BM‐MSCs. L‐ECM therefore promotes hepatic differentiation of BM‐MSCs via the ITG pathway and plays a therapeutically beneficial role for stem cell‐based liver regeneration. Copyright