| Background:Changing the cell status of human pluropotant stem cells,during the stages of stem cell culture and hematopoietic differentiation,can deeply change the process of hematopoiesis,which is also the important way to explore the related molecular mechanisms.We try to use external stimulus(based on the culture on nanomaterial surface)and the transcription profile changes(The up-or down-regulation of the key signaling pathways or genes)to interfere on the hematopoietic processes before or during hematopoiesis,to discover the regulation mechanisms influencing the hematopoietic differentiation efficiency,and look for a better way to improve it.The stem cell microenvironment plays a key role in the self-renewal and differentiation of stem cells.Nanomaterials can mimic the microenvironment of stem cells in vitro,and interact with stem cells to change the transcription profile of the stem cells,thereby regulating the proliferation and differentiation of stem cells.Previous studies have found that Petri dishes fabricated by different nanomaterials have different effects on cultured cells.We applied petri dishes coated with new types of nanomaterial on the culture stage of hESCs culture,and then induced hESCs to hematopoietic differentiation so as to explore their positive influence on the hematopoietic differentiation potentials of human embryonic stem cells,and try to elucidate the corresponding cellular/molecular mechanisms.The RUNX1 gene plays an important role in hematopoietic development.Earlier studies of our group had found that the overexpression of RUNX1b in the early stage can block the transition from mesoderm to hematopoiesis.The deep sequencing results of the co-culture samples overexpressing RUNX1b from the early stage demonstrated that along with overexpression of RUNX1b,P18 was significantly upregulated while HOXA9 was dramamtically downregulated,suggesting that the two genes might be involved in the inhibitory effects of RUNX1b overexpression on hematopoiesis.Therefore,we established inducible hESC lines for above genes respectively to explore their promotion effects on hematopoiesis and corresponding cellur/molecular mechanisms.Methods:the genes and signaling pathways that were involed in the effects of colloidal self-assembled nanomaterials on the hematopoietic differentiation potentials of hESCs,during the stages of stem cells culture,were investigated by using our unique in vitro hematopoiesis system and.inducible gene overexpression/silence system based on transposon vector piggyBac.hESCs were cultured with nanomaterials,and then co-cultured with AGM-S3 cells.The effeciency of hematopoietic differentiation was detected at different periods so as to find out the nanomaterials that can enhance the hematopoietic potentials of hESCs.Doxycycline(DOX)was added at different stages of hematopoiesis to induce the overexpression of target genes,and then their influence on hematopoiesis was detected by flow cytometry at different time points.Finally,molecular biological methods(including WB detection,qRT-PCR,RNA-seq,RNA interference,small molecule inhibitor etc.)and cytological methods(flow cytometry,hematopoietic colony formation assay,cell sorting,MGG staining,etc.)were combined to analyze their influence on efficiency of hematopoiesis and explore the corresponding cellular/molecular mechanisms.Results:Culturing hESCs with colloidal self-assembled nanomaterials during the stage of stem cells culture can promote its hematopoietic differentiation potentials.Inhibition of MAPK signaling pathway or overexpression of MT1E gene during hESCs culture can increase the production of hematopoietic cells in a comprehensive way,which is mostly consistent with the effects caused by cSAPs,and indicates that they probably participate in the corresponding molecular control mechanisms.Overexpressing P18 or HOXA9 during the late stage of hematopoietic differentiation can promote efficiency of hematopoiesis.P18 overexpression at the early stage(D0)strongly blockes the hematopoiesis,but hematopoiesis can be comprehensively promoted if P18 is overexpressd starting from D10 or later.Its promotion effects on hematopoiesis at the late stage is related to NF-κB signaling and the changes of cell cycle status.The HOXA9 overexpression starting from D4 or later can promote the production of myeloid progenitors while decrease the ones of erythroid progenitors,which is closely related to NF-κB signaling and the changes of cell cycle status. |
