Comparation Of The Effects Of Transdifferentiated And Untransdifferentiated Human Umbilical Mesenchymal Stem Cells On Functional Recovery After Rat Traumatic Brain Injury

Traumatic brain injury (TBI) is a common disease that severely affects quality of life. Most patients suffer from long-term disabilities in cognition, movement and sensation. Despite large efforts, there is no effective treatment for TBI, especially for cognitive deficits.There are numerous strategies being investigated in the treatment of TBI. There is accumulating evidence that suggest cellular transplantation is one of the most promising options. A number of different cell types are being investigated for seeding. In theory, embryonic stem cells (ESCs) are the best choice in this regard, but their use in clinical applications is hindered by moral and ethical concerns, as well as the scarcity of fetal tissue. Bone marrow stromal cells (BMSCs) are currently the most widely used seeding cells for both experimental and clinical studies. Unfortunately, the cell number and proliferation/differentiation capacity of BMSCs significantly decrease with age. Furthermore, the harvesting of BMSCs involves an invasive and painful procedure. Thus, the search for possible alternative MSCs is ongoing. In recent years, human umbilical mesenchymal stem cells (HUMSCs) were discovered and have attracted extensive attention as they have several advantages over ESCs or BMSCs:(a) no ethical or moral concerns;(b) rich sources and very rapid expansion in vitro; and (c) easily available with noninvasive and painless procedure. These advantages have excited great interest in the scientific community in the use of HUMSCs for cell-based therapies in central nervous system (CNS) injury.Recently, a number of groups reported that implanted untransdifferentiated HUMSCs secreted neurotrophins, which provided a therapeutic benefit to CNS diseases. Conversely, some groups showed that transdifferentiated HUMSCs differentiated into neural-like cells in vitro. Most recently, using a protocol described by Hermann et al., we successfully transdifferentiated HUMSCs into neurospheres, with the major characteristics of neural stem cells. We subsequently transplanted the HUMSC-derived neurospheres (HUMSC-NSs) into a complete transection model of spinal cord injury in rats. After10weeks, we found that some HUMSC-NSs differentiated into neural-like cells, with therapeutic benefits to locomotor functional recovery. Thus, we decided to test which would be better to transplant for CNS injury, transdifferentiated or untransdifferentiated HUMSCs. To our knowledge, no comparative study has been undertaken with the same conditions, even though there has always been an unresolved controversy about whether it is necessary to transdifferentiate mesenchymal stem cells (MSCs) prior to transplantation.Based on the above research background, we use HUMSCs as seeding cells in this study. Firstly, we established the protocol to isolate and culture HUMSCs and investigated the basic characteriscs of HUMSCs. Secondly, to establish the protocol to induce HUMSCs into HUMSC-NSs and then into neural-like cells. Finally, We conducted a comprehensive comparison of cognitive functional recovery, tissue structure, neural differentiation and neurotrophin secretion in a rat model of TBI. As the transplantation was a xenotransplantation, we also looked at immune rejection without immunosuppressants.This study includes three chapters:Chapter Ⅰ. Isolatiton, culture and identification of human umbilical mesenchymal stem cellsObjective:to establish the protocol to isolation, culture and identification of HUMSCs and investigate the basic characteriscs of HUMSCs.Methods:to obtain HUMSCs by using collagen digestion protocol, and also the morphological, proliferation ability and phenotypic charateristics were investigated.Results:we obtained HUMSCs successfully by using collagen digestion method. HUMSCs express the marker of MSCs and some adhesion molecules phenotype, but not express hematopoietic and endothelial phenotypes. The results are in accordance with the criteria of MSCs by The International Society for Cellular Therapy. Moreover, HUMSCs have strong proliferation ability, and the proliferation rates have no differences between passage1and25, and the doubling time of cells is26.74-27.68h.Conclusions:we have successfully established a protocol to isolate and culture HUMSCs. HUMSCs have the similar morphologies and phenotype with BMSCs, and also have very strong proliferation ability. It is a promising cell types for future studies and application.Chapter Ⅱ. Transdifferentiation of human umbilical mesenchymal stem cells into neuroectodermal-like cellsObjective:to establish the protocol to induce HUMSCs differente into HUMSC-NSs and then into neural-like cells.By the above protocol, we firstly want to obtain a new source of neural stem cells, and secondly to test the neuronal differentiation ability of HUMSCs. And also, to get some data of HUMSCs and HUMSC-NSs in neurotrophinc serection. It will provide new experimental data for the following research in vivo.Methods:The transdifferentiation protocols included two steps. Firstly, the HUMSCs were induced in neurospheres induction medium:neurobasal medium supplemented with20ng/ml epidermal growth factor (EGF),20ng/ml basic fibroblast growth factor (bFGF) and B27(1:50).3-4days later, small HUMSC-NSs could be observed. Secondly, to investigate the terminal differentiation of HUMSC-NSs, HUMSC-NSs were induced in neurobasal medium supplemented with0.5μmol/L all-trans-retinoic acid,1%FBS,5%horse serum,1%N2supplement. Cells were differentiated for two weeks. We added fresh N2every3days and change medium every6days. The results of transdifferentiation including the two steps were examined using immunofluorescence and western blot. And also, elisa was performed to quantify the protein levels of BDNF and NT-3in HUMSCs and HUMSC-NSs conditioned medium.Results:HUMSCs were induced into HUMSC-NSs successfully. More importantly, HUMSC-NSs displayed most of the chanracteristics of neural stem cells and could differentiate into neural-like cells in vitro. However the BDNF and NT-3levels of HUMSC-NSs conditioned medium was lower than that of HUMSCs conditioned medium.Conclusions:we successfully established the protocol to induce HUMSCs to differentiate into HUMSC-NSs, and then into neural-like cells, showing HUMSCs could be transdifferentiated into neuroectodermal-like cells in vitro. We find new source for neural stem cells. But in vitro transdifferentiattion procedures could weaken the secretion characteristics of cells. Therefore, who is more efficacious between transdifferentiated and untransdifferentiated HUMSCs is still need to evaluate in vivo. Chapter III. Comparison of the effects of transdifferentiated and untransdifferentiated human umbilical mesenchymal stem cells on cognitive functional recovery after rat traumatic brain injuryObjective:to determine the necessity of transdifferentiation of MSCs prior to transplantation and investigate the mechanisms for grafted cells promoting functional recovery in animals suffered TBI.Methods:TBI animal models were established in adult rat using a weight-drop device. For the different treatments, the rats were divided into four experimental groups:(a) Sham group,(b) Matrigel group,(c) HUMSCs group and (d) HUMSC-NSs group. Seven days after TBI, rats received cellular transplantation. For HUMSCs group,10μl Matrigel containing1×105HUMSCs were injected into the injured hippocampus; for HUMSC-NSs group,10μl Matrigel containing1×105HUMSC-NSs were injected; for Matrigel group, only10μl Matrigel was injected; Sham group received no injection. The overall animals did not receive any immunosuppressant. We compared cognitive functional recovery, tissue structure, neuronal differentiation, and neurotrophin secretion between groups using Morris water maze test, cavitation analysis, immunochemistry and Western blot, and enzyme-linked immunosorbent assay.Results:Firstly, HUMSCs significantly improved cognitive functional recovery compared with HUMSC-NSs. Secondly, the percentage cavitation of the brain in the HUMSCs group is significantly smaller than that of HUMSC-NSs group. Thirdly there was no obvious immune response in the HUMSCs or HUMSC-NSs groups. Fourthly, most of the grafted HUMSC-NSs remain undifferentiated like HUMSCs, with very few differentiating into neural-like cells. Finally, HUMSCs secreted higher levels of BDNF and NT-3than HUMSC-NSs in vivo.Conclusions:HUMSCs are more efficacious to cognitive functional recovery and tissue structural protection than HUMSC-NSs, following implantation in TBI in rats. Therefore in vitro neuronal transdifferentiation of HUMSCs may not be necessary prior to their transplantation. Cellular replacement is unlikely the mechanism for grafted cells promoting functional recovery in animals suffered TBI and neuroprotection by neurotrophins may be one of the reasons for grafted cells promoting functional recovery in animals suffered TBI.