| ObjectivesMesenchymal stem cells (MSCs),which have many important functions such asregeneration repair, immunoregulation and hematopoietic support, have becomehotspot in the field of stem cell research and application in recent years. MSCs aremainly used in the treatment of immune diseases, degenerative diseases, damage andother refractory diseases. Umbilical cord-derived MSCs (UC-MSCs) have strongability of proliferation with low risk of infection of pathogenic microorganisms suchas the virus, and UC-MSCs are very suitable for large scale expansion andfreeze-stored in vitro and clinical application. Moreover, umbilical cords as the"waste" after birth, can be gained conveniently without any risks to puerperas andnewborns and without ethics problems. Therefore, human umbilical cord-derivedmesenchymal stem cells (hUC-MSCs) can be large-scalely produced as a kind of"stem cell medicine".Of course, for cell therapy, safety is the primary concern and needs to beresearched. Especially, non-human primates (NHPs) are the closest to human ingenetic relationship, tissue organization, pathophysiology, immunoregulation andmetabolism, and so the research value of NHPs is much better than other species ofexperiment animals. The present study was aimed to evaluate toxicity andimmunological effects of UC-MSCs administration in macaca fascicularis, and toestablish methods to isolate, culture and identify macaca fascicularis umbilicalcord-derived mesenchymal stem cells (mUC-MSCs), and to provide NHPs testsupport for the clinical application of allogeneic UC-MSCs.Contents and Methods1. Toxicity study and immunological effects of hUC-MSCs repeatedly intravenousadministrations in macaca fascicularis for4weeks.24monkeys were randomlydivided into4groups: the negative control group, the solvent control group, the hUC-MSCs low dose group (2x106cells/kg), and the hUC-MSCs high dose group(2x10~7cells/kg). The monkeys were administrated once a week for5times. During thetest, clinical observations were performed and body weights, body temperatures,electrocardiograms, blood cell counts, coagulation parameter, biochemistry, Tlymphocyte subsets(CD3, CD4, CD8and CD4/CD8), CD4+CD25+regulatory Tcells, Th1/Th2cytokines(IFN-γ, IL2, IL-4, IL-6, IL-5, TNF), T lymphocyteproliferation test, IgG and antibody of hUC-MSCs were monitored.2. To isolate and culture mUC-MSCs and to analyze their biological characteristics.We obtained the umbilical cord of macaca fascicularis by caesarean section, and thenumbilical cord was cut into pieces with scissors and was digested with collagenase. SomUC-MSCs were isolated from umbilical cord and subcultured. They were analyzedon morphology, growth curve, immunophenotype, cell cycle and multi-directionaldifferentiation potential.3. Toxicity study and immunological effects of mUC-MSCs (2x106cells/kg) singleadministration in macaca fascicularis. After3monkeys were administrated once,clinical observations were performed and body weights, body temperatures,electrocardiograms, blood cell counts, coagulation parameter, biochemistry, Tlymphocyte subsets, regulatory T cells, Th1/Th2cytokines, T lymphocyteproliferation test and IgG were monitored.Results1.Repeatedly intravenous administrations of hUC-MSCs had no toxicologicallysignificant and irreversible effects on the general physical activity, body weights,body temperatures, electrocardiograms, blood cell counts, coagulation parameter andbiochemistry in monkeys, and there were no apparent changes in injection local. Thepossible toxic reactions were reduction of PLT, serum TP, and P(P≤0.05). Afteradministrations, there were no statistically significant differences in T lymphocytesubsets, regulatory T cells, T lymphocyte proliferation test between experimentalgroups and control ones. Only at individual time points,there were statisticallysignificant differences in IFN-γ, IL-4and IL-6. Antibodies to hUC-MSCs were detected only in3monkeys.2. The shape of mUC-MSCs was fusiform. After the third passage, immunophenotypeof mUC-MSCs was CD29, CD73, CD90, CD105, CD166positive and CD14, CD31,CD34, CD45, CD40, CD80, CD86, HLA-DR negative. In special inductionconditions, mUC-MSCs could differentiate into osteocytes, adipocytes andchondrocytes in vitro. The cell cycle analyzed by flow cytometry showed that morethan90percent cells were in G0/G1phase.3.Single administration of mUC-MSCs had no toxicologically significant effects onthe general physical activity, body weights, body temperatures, electrocardiogramsand coagulation parameter in monkeys. After administration, RBC, HGB and HCTdeclined, and Retic increased(P≤0.05). And we found that Glb declined and A/Gincreased after administration (P≤0.05). Moreover, after administration, thepercentage of CD4+T cells and the ratio of CD4+/CD8+increased significantly(P≤0.05). On the fourth day after administration, CD4+CD25+Treg increased(P≤0.05). Before and after administration, T lymphocyte proliferation test and IgGhad no significant differences. Only at individual time points,there were statisticallysignificant changes in TNF, IL-4and IL-6.Conclusions1.This study successfully set up the methods to isolate, culture and identifymUC-MSCs, and showed that mUC-MSCs were similar to hUC-MSCs inmorphology, immunophenotype and multi-directional differentiation potential, andprovided NHPs test support for the clinical application of allogeneic UC-MSCs.2.This study proved the safety of intravenous administration of UC-MSCs based onxenogeneic and allogeneic patterns. In the present experimental condition, the safedosage of hUC-MSC of repeatedly intravenous administrations was2.0x10~7cells/kg,which was20times as much as the dose of clinical therapy.3.The study confirmed intravenous administrations of hUC-MSCs had little effects onT lymphocyte subsets in peripheral blood, T lymphocyte proliferation ability,CD4+CD25+regulatory T cells, IgG, Th1/Th2cytokines of healthy monkeys. But administration of mUC-MSC could effect T lymphocyte subsets and regulatory Tcells, which showed that there were maybe differences between xenogeneic infusionand allogeneic infusion of MSCs. |
PDF Full Text Request