Zhengguo Wang

The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential

Adipose-derived stem cells (ADSCs) represent a valuable source of stem cells for regenerative medicine, but the loss of their stemness during in vitro expansion remains a major roadblock. We employed a microgravity bioreactor (MB) to develop a method for biomaterial-free-mediated spheroid formation to maintain the stemness properties of ADSCs. ADSCs spontaneously formed three-dimensional spheroids in the MB. Compared with monolayer culture, the expression levels of E-cadherin and pluripotent markers were significantly upregulated in ADSC spheroids. Spheroid-derived ADSCs exhibited increased proliferative ability and colony-forming efficiency. By culturing the spheroid-derived ADSCs in an appropriate induction medium, we found that the multipotency differentiation capacities of ADSCs were significantly improved by spheroid culture in the MB. Furthermore, when ADSCs were administered to mice with carbon tetrachloride-induced acute liver failure, spheroid-derived ADSCs showed more effective potentials to rescue liver failure than ADSCs derived from constant monolayer culture. Our results suggest that spheroid formation of ADSCs in an MB enhances their stemness properties and increases their therapeutic potential. Therefore, spheroid culture in an MB can be an efficient method to maintain stemness properties, without the involvement of any biomaterials for clinical applications of in vitro cultured ADSCs.

Allogeneic adipose-derived stem cells with low immunogenicity constructing tissue-engineered bone for repairing bone defects in pigs.

The ideal cells for tissue engineering should have the following characteristics: easy obtainment, safety, immune privilege, the capability of self-renewal, and multipotency. Adipose-derived stem cells (ADSCs) are a promising candidate. However, the immunogenicity of allogeneic mesenchymal stem cells limits their long-term benefits. In this study, we introduced human cytomegalovirus US2/US3 gene into the ADSCs to decrease the expression of MHC I protein of ADSCs and reduce the activation of T-cells of the recipient animals. Moreover, the biosafety and biological characteristics of ADSCs transfected with the US2/US3 genes (ADSCs-US2/US3) were similar to normal ADSCs. Then we took ADSCs-US2/US3 to construct a tissue-engineered bone for repairing bone defects in pigs and found that there were no great differences in repair effects or healing time between the allogeneic ADSCs-US2/US3 group and the autologous ADSC group. These results suggest that allogeneic ADSCs-US2/US3 have the advantages of biological safety, low immunogenicity, and effective osteogenesis. Such barely immunogenic ADSCs will be crucial for the success of future tissue-regenerative approaches.

Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects.

AIMS Bone defects induced by different causes are difficult to replace and repair. We sought to repair bone defects by transplantation of genetically modified adipose-derived stem cells (ADSC) and acellular bone matrix (ACBM). METHODS We constructed the biologic material of ACBM and evaluated its mechanical properties, general biocompatibility and biosafety. ADSC isolated from minipigs were cultured in vitro and then transfected by recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) plasmids, respectively. Subsequently, the compounds of ACBM/ADSC/rhBMP-2/rhVEGF were used to repair bone defects of the ulna in minipigs. X-ray examination, radionuclide bone imaging and single photon emission computerized tomography (SPECT) were employed to monitor the therapeutic effects 2, 4, 8 and 12 weeks after operation. Histologic experiments were carried out 12 weeks after operation. RESULTS ACBM had no or weak antigenicity and the natural mechanical properties of ACBM were preserved. In vitro, ADSC transfected by rhBMP-2 and rhVEGF, respectively, could release rhBMP-2 or rhVEGF for at least 4 weeks. The X-ray, radionuclide bone imaging and SPECT examinations indicated that the compound of ACBM/ADSC/rhBMP-2/rhVEGF had better treatment effects on bone defects compared with the controls. CONCLUSIONS Scaffolds, seed cells and bioactive factors are key points in tissue engineering. This research indicates that ACBM is a good biologic material for tissue repair, and ACBM/ADSC/rhBMP-2/rhVEGF can accelerate bone formation significantly.

Stromal Cell-Derived Factor-1 Receptor CXCR4-Overexpressing Bone Marrow Mesenchymal Stem Cells Accelerate Wound Healing by Migrating into Skin Injury Areas

Stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. This study investigated the hypothesis that bone marrow-derived mesenchymal stem cells (BMSCs) accelerate skin wound healing in the mouse model by overexpression of CXCR4 in BMSCs. We compared SDF-1 expression and skin wound healing times of BALB/c mice, severe combined immunodeficiency (SCID) mice, and immune system-deficient nude mice after (60)Co radiation-induced injury of their bone marrow. The occurrence of transplanted adenovirus-transfected CXCR4-overexpressing male BMSCs in the wound area was compared with the occurrence of untransfected male BALB/c BMSCs in (60)Co-irradiated female mice skin wound healing areas by Y chromosome marker analyses. The wound healing time of BALB/c mice was 14.00±1.41 days, whereas for the nude and SCID mice it was 17.16±1.17 days and 19.83±0.76 days, respectively. Male BMSCs could be detected in the surrounding areas of (60)Co-irradiated female BALB/c mice wounds, and CXCR4-overexpressing BMSCs accelerated the wound healing time. CXCR4-overexpressing BMSCs migrate in an enhanced manner to skin wounds in a SDF-1-expression-dependent manner, thereby reducing the skin wound healing time.

Adipose-derived stem cells induced dendritic cells undergo tolerance and inhibit Th1 polarization

Adipose tissue-derived stem cells (ADSC) have been shown to possess stem cell properties such as transdifferentiation, self-renewal and therapeutic potential. However, the property of ADSC to accommodate immune system is still unknown. In this study, ADSC were cocultured with allogenetic dendritic cells (DC), and then treated DC were mixed with allogenetic CD4+ T cells. The results demonstrated that ADSC could downregulate costimulatory molecules, including CD80, CD83, CD86, and cytokine secretion such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α, while upregulate indoleamine-2,3-dioxygenase (IDO) of allogenetic DC. In addition, treated DC could inhibit CD4+ T cell activation and naïve T cells toward Th1 polarization. The results suggest that ADSC could negatively modulate immunity and induce immune tolerance, which provide a promising strategy in transplantation or autoimmune disease.

Bifunctional polyethersulfone hollow fiber with a porous, single-layer skin for use as a bioartificial liver bioreactor

A bioartificial liver bioreactor requires a bifunctional hollow fiber that is hemocompatible on one side and cytocompatible on the other side. In this study, we developed a single-layer skin polyethersulfone (PES) hollow fiber with smooth inner surface and rough/porous outer surface for an artificial liver bioreactor. The hemocompatibility of the inner surface was evaluated by hemolysis, complement activation and clotting time. The cytocompatibility of the outer surface with HepG2 cells was examined by morphology, proliferation and liver-specific functions. The inner surface of the PES hollow fiber exhibited lower hemolysis and complement activation than cellulose acetate (CA) hollow fiber and a prolonged blood coagulation time. HepG2 cells readily adhered to the outer surfaces of the PES hollow fibers, and proliferated to form multicellular aggregates with time. Furthermore, HepG2 cells cultured on the outer surface of the PES hollow fiber exhibited higher proliferation ability and liver-specific functions than those grown on the CA hollow fiber. These results suggest that the single-layer skin PES hollow fiber is a bifunctional hollow fiber with good hemocompatibility on the inner side and cytocompatibility on the outer side. Thus, porous and single-layer skin PES hollow fibers may have potential as materials for an artificial liver bioreactor.

Three-dimensional culture in a microgravity bioreactor improves the engraftment efficiency of hepatic tissue constructs in mice

Tissue-engineered liver using primary hepatocytes has been considered a valuable new therapeutic modality as an alternative to whole organ liver transplantation for different liver diseases. The development of clinically feasible liver tissue engineering approaches, however, has been hampered by the poor engraftment efficiency of hepatocytes. We developed a three-dimensional (3D) culture system using a microgravity bioreactor (MB), biodegradable scaffolds and growth-factor-reduced Matrigel to construct a tissue-engineered liver for transplantation into the peritoneal cavity of non-obese diabetic severe combined immunodeficient mice. The number of viable cells in the hepatic tissue constructs was stably maintained in the 3D MB culture system. Hematoxylin–eosin staining and zonula occludens-1 expression revealed that neonatal mouse liver cells were reorganized to form tissue-like structures during MB culture. Significantly upregulated hepatic functions (albumin secretion, urea production and cytochrome P450 activity) were observed in the MB culture group. Post-transplantation analysis indicated that the engraftment efficiency of the hepatic tissue constructs prepared in MB cultures was higher than that of those prepared in the static cultures. Higher level of hepatic function in the implants was confirmed by the expression of albumin. These findings suggest that 3D MB culture systems may offer an improved method for creating tissue-engineered liver because of the higher engraftment efficiency and the reduction of the initial cell function loss.