| In recent years,an exciting breakthrough in stem cell biology is that a limited number of genes can be activated to reprogram somatic cells into induced pluripotent stem cells(iPSCs).Some advantages of these cells provide a good source of cells for regenerative medicine,such as broad differentiation potential,higher safety and so on.Scientists have established a variety of animals’ iPSCs,including mice and human.However,on affiliation,rodents and humans apart far away,many studies which can produce functions and effects in mice do not necessarily obtain the same effects on human beings.Therefore,it’s necessary to establish nonhuman primates’ iPSCs and their derived cells,such as neural stem cells(NSCs),and explore the profound developmental problems and molecular mechanisms of these cells,which will lay the foundation for the basic research of human-related cells and the regenerative medicine treatment of human-related diseases in the future.This thesis consists of two parts,the first part is that establishing NSCs which can passage stably for a long time derived from cynomolgus monkeys induced pluripotent stem cells,and then assessing the ability of these cells to integrate and it’s safety in animals.The second part is to try to establish tree shrew induced pluripotent stem cells(tsiPSCs).Although primates’ neural stem cells can be obtained by various methods,but the problem is that these cells can’t be passaged stably for a long time in vitro.In the first part,we try to differentiate two kinds of cynomolgus monkeys induced pluripotent stem cells which were reprogrammed by retroviruses and Sendai virus into neural stem cells with small molecule compounds.This process is divided into two steps:first of all,we differentiate Cynomolgus monkeys iPSCs into Embryoid body(EB)by five small molecule compounds,containing bFGF,CHIR99021,SB431642,Compound E and LDN193189,followed by using four small molecules(bFGF,under CHIR99021,SB431642 and hLIF)we obtain NSCs which can passage stably,these cells expressed markers of NSCs,such as Sox1,Pax6 and Nestin and so on.More importantly,we obtained NSCs derived from a single cell,which may play an important role in basic research and regenerative medicine therapy of nervous system-related diseases in the future.Finally,we inject single-cell-derived NSCs which can express GFP into the brains of healthy adult mice to assess the ability of these cells to integrate and its security in the body.The second part is reprogramming the tree shrew embryo fibroblasts into iPSCs with retroviruses which can express four pluripotent-related macaque transcription factors(Oct4,Sox2,Klf and 4c-Myc).The iPSCs of mice,non-human primates and human iPSCs cell lines were established after their ESCs had been obtained,because culture systems of the two kinds of pluripotent stem cells(iPSCs and ESCs)are identical.As the tree shrews’ ESCs has not been established,so establishing a culture system which can allow the tsiPSCs to passage stably will be the key to obtain tsiPSCs.Based on the culture system of human pluripotent stem cells and mouse pluripotent stem cell,referring to other species of pluripotent stem cell culture system,we obtained cell clones which are similar to human pluripotent stem cells under the culture system of human pluripotent stem cells by add in recombinant human leukemia inhibitory factor(hLIF).However,these clones begin to differentiate after passaging for twice,not obtain long-term stably passaged tsiPSCs.In neural stem cells,we obtained two kinds of NSCs passaged stably which derived from two kinds of iPSCs.More importantly,we have also established a single-cell-derived NSCs.These cells will play an important role in drug screening,establishing relevant cell and animal models,as well as the study of brain development mechanism of primates in the future,which will promote the study of human brain development and its related diseases ultimately,providing support for the treatment of nervous system-related diseases in the future.This paper also attempted to establish tree shrews induced pluripotent stem cells which can passage stably for a long time,although we did not achieve the intended purpose,but we are also make it a good attempt,which can provide a beneficial reference for establishing long-term stably passaged tsiPSCs or iPSCs derived other species in the future. |
