Dongyan Shi

Proliferation and osteogenesis of immortalized bone marrow-derived mesenchymal stem cells in porous polylactic glycolic acid scaffolds under perfusion culture

Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. However, the life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction could prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. Therefore, hMSCs transduced with hTERT (hTERT-hMSCs) could be used as a cell model for in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties. A perfusion culture system for proliferation and osteogenesis of hTERT-hMSCs or primary hMSCs in porous polylactic glycolic acid (PLGA) scaffolds is described here. A cell suspension of hTERT-hMSCs or primary hMSCs (5 x 10(5) cells/250 microL) was seeded and then cultured for 12 days in porous PLGA scaffolds (10 mm in diameter, 3 mm in height) under both static and perfusion culture systems. The seeding efficiency, proliferation, distribution and viability, and osteogenesis of cells in scaffolds were evaluated. The perfusion method generated higher scaffold cellularity and proliferation of cells in scaffolds, and hTERT-hMSCs showed the higher proliferation potential than primary hMSCs. Results from fluorescein diacetate (FDA) staining and scanning electron microscopy (SEM) demonstrated homogeneous seeding, proliferation, and viability of hTERT-hMSCs throughout the scaffolds in the perfusion culture system. On the contrary, the static culture yielded polarized proliferation favoring the outer and upper scaffold surfaces, and resulted in decreasing of cells in the central section of the scaffolds. A flow rate of 0.5 mL/min had an effect on osteogenic differentiation of cells in scaffolds. However, the osteogenic medium promoted the osteogenic efficiency of cells. Scaffolds with hTERT-hMSCs had the higher osteogenesis than scaffolds with primary hMSCs. Thus, these results suggest that the flow condition not only allow a better seeding efficiency and homogeneity but also facilitate uniform proliferation and osteogenic differentiation of hTERT-hMSCs in scaffolds. hTERT-hMSCs could be used as stem cell candidates for bone tissue engineering experiments.

Human mesenchymal stem cells are sensitive to abnormal gravity and exhibit classic apoptotic features

The aim of the present study was to investigate the effects of abnormal gravity on human mesenchymal stem cells (hMSCs). Strong magnetic field and magnetic field gradient generate a magnetic force that can add to or subtract from the gravitational force. In this study, this is defined as a high-magneto-gravitational environment (HMGE). The HMGE provides three apparent gravity levels, i.e. hypogravity (μg), hypergravity (2g) and normal gravity with strong magnetic field (1g) conditions. After hMSCs were subject to HMGE for 12 h, the proliferation, morphology, structure and apoptosis were investigated. Results showed that the proliferation of hMSCs was inhibited under μg condition. The abnormal gravity induced morphologic characteristics of apoptosis cells, such as cell shrinkage, membrane blebbing, nuclear chromatin condensation and margination, decreased cell viability, and increased caspase-3/7 activity. The rate of apoptosis under μg condition is up to 56.95%. The F-actin stress fibers and microtubules were disrupted under abnormal gravity condition. Under μg-condition, the expression of p53 at mRNA and protein levels was up-regulated more than 9- and 6 folds, respectively. The Pifithrin-α, an specific inhibitor of p53, inhibited the apoptosis and prevented the disruption of cytoskeleton induced by abnormal gravity. These results implied that hMSCs were sensitive to abnormal gravity and exhibited classic apoptotic features, which might be associated with p53 signaling.

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii

The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-γ and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.

Biological, immunological and regenerative characteristics of placenta-derived mesenchymal stem cell isolated using a time-gradient attachment method.

It has been verified that placenta contains multi-lineage mesenchymal stem cells (MSCs). We have used a time-gradient attachment method to isolate placenta-derived MSCs (PMSCs). The morphology, differentiation potential, immunogenicity and xenogenic reconstruction potential of these PMSCs were examined. The results showed that PMSCs isolated using the time-gradient attachment method showed higher potential of in vitro proliferation and multi-lineage differentiation. PMSCs isolated using the time-gradient attachment method showed a low immunogenicity. HLA-A gene fragment and no HLA-DR gene fragment were detected in PMSCs isolated using the time-gradient attachment method, and the mixed lymphocyte reaction (MLR) assay identified that these cells inhibited the proliferation of the allogeneic T-lymphocytes induced by PHA. The transplantation in calvaria of rats showed that PMSCs had the higher xenogenic reconstruction potential. Finally, the significance of PMSCs isolated using the time-gradient attachment method in experimental and clinical applications is discussed.

Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood

AIM To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product. METHODS Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential. RESULTS After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34(+) cells increased 43-fold. The granulocyte-macrophage colony- forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3- and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells. CONCLUSION HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.

Hepatogenic engineering from human bone marrow mesenchymal stem cells in porous polylactic glycolic acid scaffolds under perfusion culture

Bone marrow mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. We used mesenchymal stem cells from human bone marrow (hMSCs) as the seeding cells to investigate the potential of hepatocytic differentiation of hMSCs in porous polylactic glycolic acid (PLGA) scaffolds under perfusion induction. hMSCs were seeded and proliferated in PLGA scaffolds, and then induced into hepatocyte‐like cells with hepatogenic medium in perfusion and static cultures. The results showed that hMSCs could be induced into hepatocyte‐like cells in PLGA scaffolds with hepatogenic medium in both static and perfusion induction systems. However, perfusion induction was more effective for cellularity in PLGA scaffolds than in static induction. Cells in the scaffold induced by the hepatogenic medium expressed hepatocyte‐specific genes cytokeratin 19 (CK19), α‐fetoprotein (αFP), cytokeratin 18 (CK18), albumin and cytochrome P4503A4 (CYP3A4) in a time‐dependent manner. Induced cells stained positive for αFP and albumin. Induced cells also acquired the functional characteristics of hepatocytes, i.e. secretion of urea and albumin. In a comparison of survival and hepatogenic differentiation of hMSCs between perfusion and static induction, perfusion induction increased the survival and the uniform distribution of induced cells in scaffolds, which resulted in a higher efficiency of hepatogenesis in the PLGA construct with hMSCs. The oscillatory perfusion induction system combined with the hepatogenic medium should be a valuable and convenient tool for in vitro hepatic tissue engineering using hMSCs. Copyright