Efficiency of adenoviral vector mediated CTLA4Ig gene delivery into mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy.</p><p><b>METHODS</b>We constructed the recombinant adenovirus containing CTLA4Ig gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4Ig expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model.</p><p><b>RESULTS</b>Recombinant adenovirus pAd-CTLA4Ig was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4Ig protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P < 0.05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4Ig expression was observed in the liver, intestine, kidney and spleen 30 days post-transplantation.</p><p><b>CONCLUSIONS</b>Our results indicate that adenoviral vectors could efficiently transfer CTLA4Ig gene into MSCs and sustain long-term stable expression in vitro and in vivo.</p>

Advances in studying the differentiation into neurons of mesenchymal stem cells and their application / 中国病理生理杂志

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into marrow and non-marrow cell types, such as adipocytes, chondrocytes, myocytes, and so on. In recent years, many researchers have studied whether MSCs are capable of differentiation into neurons in vivo and ex vivo. The result that MSCs-derived neurons express NSE and NF, but don't express GFAP suggests MSCs can differentiate into neurons, some researchers have achieved success in promoting functional recovery in Pakinsons and transactional spinal cord injury rat models by use of MSCs-derived neurons. Therefore, MSCs-derived neurons will play an important role in the therapy for a variety of diseases of the nervous system. [

Macaca irus bone mesenchymal stem cells differentiate into neuron-like cells induced by cryptotanshinone in vitro / 中国病理生理杂志

AIM: To determine the optimal protocol and condition in which macaca irus mesenchymal stem cells (MSCs) are induced to differentiate into neuron-like cells by cryptotanshinone in vitro. METHODS: MSCs from macaca irus bone marrow were generated in vitro and induced with cryptotanshinone. The morphological changes of MSCs were evaluated by microscope. The positive percentages of neurofilament (NF), neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression were measured by immunocytochemistry with ABC staining. RESULTS: The result showed that MSCs were positive for CD29, CD44, CD105, CD166, and negative for CD34, CD71, CD80 and CD86. After induced with cryptotanshinone, MSCs began to display neuronal morphologies, such as contracted multipolar cell body and formed extensive networks. The percentages of positive NSE, NF expression were 68.3%?3.5%, 70.3%?1.5%, respectively. CONCLUSION: Macaca irus MSCs could be induced to differentiate into neuron-like cells in vitro by cryptotanshinone and might be applied in cell transplantation and gene therapy in nervous system disorders.

Bone marrow mesenchymal stem cells modified by adenoviral vector containing CTLA4Ig inhibit immune response ex vivo / 中国病理生理杂志

AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibits the lymphocyte proliferation ex vivo.