The Immunogenicity Of Neural Progenitor Cells Differentiated From Embryonic Stem Cells And The Effectiveness Of Dendritic Cells Derived From The Same Embryonic Stem Cells On Transplantation Tolerance Of Such Neural Progenitor Cells

The pluripotency of embryonic stem cells make them one of the best cell types for regenerative medicine. Animal models that transplant embryonic stems or derived cell types in cerebral ischemic treatment have proven that these cells can replace the loss neurons, recovery the network, rebuild synapse connection and enhance the recovery of neural function. Therefore, the ESC has become the most considerable cell type in CNS cell replacement therapy(CRT). However, it is important to evaluate the immunological mechanisms that may determine integration and acceptance, or the rejection of such grafts. The previously held view that the brain was an absolute"immunologically privileged site"allowing indefinite survival without rejection of grafts of cells has proven to be wrong. Most interestingly, ESCs in different species express low levels of major histocompatibility complex (MHC) class I but no class II molecules, making them obtain considerable immunology"privilege". Based on series of investigation, however, this opinion has also proven to be not the case, as grafts they could face the same challenge of donor's immunology system in brain as in periphery. Therefore,it is important to evaluate the immunogenicity of ESCs or their derived cells, the transplantation immunity mechanisms in the brain and the measures involving in immunodepression or immunotolerance in these cells transplant. This experiment investigated the immunogenicity of ES-D3 ESCs and neural progenitor cells (NPCs) differentiated from them and the transplantation immunity phenomena when NPCs were grafted in ischemic rat brain. We also introduced another cell type derived from the same ES-D3 ESCs, embryonic stem cells derived dendritic cells (esDCs), to induce the immunotolrence for NPCs grafted in ischemic rat brain.Part I Differentiation of ES-D3 Embryonic Stem Cells into Neural Progenitor Cells in vitroObjective To induce ES-D3 embryonic stem cells into neural progenitor cells by"five-steps"methods.Method Prior to differentiation, ESCs was passaged free of feeder cells on gelatinized flasks in complete medium, further supplemented with 1000U/ml of recombinant leukaemia inhibitory factor (rLIF) to prevent their differentiation. Single cell suspensions were plated onto bacteriological plastic in the absence of rLIF to encourage the formation of EBs. After 4 days in culture, EBs were plated onto tissue culture grade plastic in non-serum medium to enrich nestin positive cells. The cells were then amplied for 6 days by cultured in basic fibroblast factor (bFGF), followed by the differentiation of neural-like cells from them though removing bFGF from media combination with the use of micotinamide.Result By"five-steps"method we acquired about 80% nestin-postive cells from ESCs. These cells can be subcultured and maintain proliferation in medium supplemented with bFGF. They can also differentiated into neural-like cells expressing Tuj-1 or GFAP in bFGF-free media combination with the use of micotinamide, which further identified their neural progenitor cell nature.Conclusion We acquired NPCs by differentiating ESCs into nestin-postive cells. These cells may be useful for further neurobiology study as well as potential transplant therapy. Part II Immunogenicity of Neural Progenitor Cell and Its Transplantation Immunity Phenomena after Grafted in Ischemic Rat BrainObjective To study the immunogenicity of ESCs and NPCs in vitro and immunoreactions in vivo after NPCs were grafted in ischemic rat brain. Method1 To evaluate the expression of major major histocompatibility complex class (MHC) I and II on ESCs and NPCs by flow cytometry (FCM) and to investigate the inducible expression of MHC-I, II on NPCs by 20ng/ml interferon-γinduction.2 Six groups of male wistar rats were accepted MCAo or sham surgeries, which are as follows: I: MCAo+NPCs transplant II: MCAo+PBS (-) injection III: MCAo (no treatment) IV: sham+NPCs transplanted V: sham+PBS (-) injection VI: sham (no treatment)Two weeks after surgery these rats accepted lateral cerebral ventricle injection according to their groups design. A proliferation assay of lymphocytes dissociated from cervical lymph nodes and histological evaluation of CD4, CD8 and ED1 positive cells in brain were performed two weeks after transplant.ResultES-D3 ESCs express low level MHC-I but no MHC-II molecules. After differentiation into NPCs the MHC II molecules expression was no change but MHC I had a significant upregulation. However, when treatment with IFN-γthe NPCs further moderately increased MHC-I molecules and present a slight but measurable up-regulation of MHC-II. Proliferation assays of lymphocytes revealed significant difference between MCAo and sham rats (p<0.05). However, neither in MCAo nor in sham groups was there a significant difference among animals treated with or without NPCs.Immunological responses: a few of CD4, CD8 and ED1 positive cells emerged in sham animals or around the injection site. Compared with sham animals, MCAo rats significantly express CD4, CD8 and ED1 molecules around ischemic lesions. CD4-positive cells increased in ischemic cortex (P<0.01) and striatum (P<0.01). There is no significant difference in cortex in CD4 molecules expression among MCAo groups (P>0.05) but in striatum rats accepted NPCs transplant showed a higher CD4 expression compared with other MCAo groups (P<0.01). Compared with sham groups, MCAo rats show a significant increase CD8 molecules expression in cortex (P<0.05) and striatum (P<0.05). However, there is no significant difference among MCAo animals, whether compared among groups or between cortex and striatum. ED1 molecules expression was similar with CD4 and present a higher expression in striatum than cortex and this difference reaching statistical significance in I groups rats(P<0.05).interesting, there is an increase tendency in striatum in sham rat accepted NPCs graft(P<0.01)。Conclusion ESCs and derived NPCs express certain level of MHC-I molecules but do not constructively express MHC-II molecules, whereas in context of IFN-γthey can inducibly unregulated both molecules especially express detectable MHC-II molecules which indicated a possible immune-response directed to both MHC-I and II molecules on these cells in an inflammation context of ischemic brain. The modes of proliferation of lymphocytes and infiltration of immunoreactive cells suggested that ischemic lesion itself was the main cause for the immunoreactions in brain, whereas the distinguishing reaction of inflammation cells, for example the higher expression of CD4 and ED1 molecules in striatum in rats accepted NPCs graft, indicated there is also a possible graft rejection directed to NPCs including the active antigen processing and CD4+Th1 reaction in situ. Part III Differentiation of ES-D3 Embryonic Stem Cells into Dendritic Cells in vitroObjective To directly differentiate ES-D3 embryonic stem cells into tolerance dendritic cells.Method Prior to differentiation, ESCs was passaged free of feeder cells on gelatinized flasks in complete medium, further supplemented with 1000 U/ml of rLIF to prevent their differentiation. Single cell suspensions were plated onto bacteriological plastic in the absence of rLIF to encourage the formation of EBs. After 14 days in culture, EBs were plated onto tissue culture grade plastic in complete medium further supplemented with 25 ng/ml murine GM-CSF and 10ng /ml rmIL-3 and cultured for 10 days. By flow cytometry (FCM) the expression of MHC-I and II, CD11c, CD80, CD86 and CD83 on the differentiated cells were evaluated. Meanwhile, after inducd maturation by LPS, the morphology change and the expression of MHC-II and CD80 molecules of these cells were investigated and mixed leukocyte reactions (MLR) were performed to study the allogeneic immunostimulatory function of matured or immature esDCs.Result By cultured in IL-3 and GM-CSF, the early dendritic cells first appeared around days 4~5 of culture and rapidly expanded over the ensuing days. This population consistently regenerated following routine harvesting. Replating of esDCs onto fresh tissue culture plastic had no effect on their proliferative potential. These cells express moderate MHC-I and high CD11c but no CD80, CD86, CD83 or MHC-II molecules, which confirmed them the immature dendrite cells. However, when stimulated with LPS, these cells presented mature dendritic cell morphology and up-regulated MHC-II and CD80 molecules on cell surface. Allogeneic MLR suggested the mature esDCs had a significant immunostimulator activity (P<0.05) but the immature esDCs not (P>0.05). Conclusion We successfully acquired esDCs though directly differentiating ESCs into DCs by use of cytokine GM-CSF and IL-3. This is a novel method to obtain limitless imDCs.Part IV Effectiveness of esDCs on NPCs Transplantation Tolerance in Ischemic Rat BrainObjective To induce NPCs transplantation tolerance in brain by esDCsMethod1 To obtain NPCs makered by eGFP from pCX-eGFP ES-D3 ESCs.2 All of wistar rats were accepted MCAo surgery and divided in two groups based on pretreatment with or without esDCs. All of animals were transplant by pCX-eGFP NPCs two weeks after MCAo. A proliferation assay of lymphocytes dissociated from cervical lymph nodes, grading of the survival of the grafted cells , histological evaluation of CD4,CD8 and ED1 positive cells in brain and detection of mRNA level of IL-10 and IFN-γin ischemic lesions by reverse transcriptase-polymerase chain reaction(RT-PCR) were performed two weeks after graft.Result Pretreatment with esDCs decreased CD4 positive cells infiltration in striatum (P<0.05), but had no effect on other inflammation cells distribution (P>0.05). There was no difference in grafts survival, lymphocytes proliferation or expression level of IL-10 and IFN-γbetween two groups (P>0.05).Conclusion Pretreatment with esDCs may reduce CD4 positive cells reaction in ischemic striatum, which provides evidence for the esDCS inductivity to CD4+T cells allograft response. However, there was no evidence associated with cytokine-dependent Tregs effect on tolerance involved this mode, and in this time point, the esDCs did not significantly influence the graft survive, which indicate further investigation was needed.