| The shortage of platelet supply and the potential risk of transfusion-relatedinfectious diseases make the platelet transfusion facing serious challenges. To find asecure and adequate platelet substitute, many researchers devote themselves to inducingstem cells to megakaryocytes and platelets in vitro, which still faces a lot of technicalproblems. And the molecular regulation mechanisms under stem cell-megakaryocyteinduction demand fully understanding as well. In present study, we took the cord blood-derived hematopoietic stem/progenitor cells as initiating cells and induced them intomegakaryoblasts. We first optimized the megakaryocyte differentiation system and thenchose miR-125b, of which the endogenous expression change could match the stage ofmegakaryocyte differentiation, for further mechanism study. After that, we systematicallyidentified the role of miR-125b in the differentiation of megakaryocytes, and revealed itsmolecular regulation mechanism further.The study consists of three parts：Part1: Establish and optimize the system for inducing cord blood-derivedhematopoietic stem/progenitor cells into megakaryoblasts.Methods: We first established the megakaryocyte differentiation system withmesenchymal stem cells feeder layer. Mesenchymal stem cells were obtained from bonemarrow (BM-MSCs) or umbilical cord (UC-MSCs) and characterized with surfacemarkers and differentiation capacity. The expression profiles of hematopoietic cytokinesby these MSCs were identified and compared by semi-quantitative and quantitative RT-PCR. Mononuclear cells were obtained from cord blood by density gradientcentrifugation. The impact of MSC co-culture on megakaryocyte formation wascompared by using two culture systems： the direct contact system and non-contactsystem, mononuclear cells incubated only with induction media were set as control.Megakaryocyte development was judged by cell proliferation, surface-marker expressionand the detection of megakaryocytes specific genes. We also optimized the feeder-freesystem by comparing two kinds of basal medium, Xvivo-15and Stemspan.Results: BM-MSCs and UC-MSCs constitutively expressed hematopoietic cytokinesthat promote megakaryocyte proliferation and differentiation. MSC direct contact co-culture system promoted the proliferation and survival of HS/PCs, but the effect onmegakaryocyte differentiation is not remarkable；non-contact co-culture system couldpromote HS/PCs expansion and megakaryocytes differentiation, BM-MSCs appeared to be better than UC-MSCs for megakaryocyte induction；we checked the expression ofseveral miRNAs in the suspension cells cultured in five different co-culture systems andfound that only the expression of miR-125b was higher in mature megakaryocytes thanin the immature cells, so we speculated that miR-125b may play a pivotal role inmegakaryopoiesisPart2: MiR-125b promotes megakaryocyte differentiation of cord blood-derivedhematopoietic stem/progenitor cells.Methods: To detect the endogenous expression of miR-125b during megakaryocytedifferentiation, we first examined the total cellular expression of miR-125b by Q-PCRanalysis in differentiating magakaryoid cells collected every three day and in isolatedhuman megakaryoblasts at distinct developmental stages. In monocuclear cells, by miR-125b mimics or its inhibitor transfection, we evaluated the effect of miR-125b over-expression or down-regulation on megakaryocyte markers and genes important formegakaryocytes lineage. K562cells, a human leukemia cell line, can be induced into cellswith megakaryoblastic characteristics by phorbol myristate acetate(PMA) treatment. Tofurther evaluate the role of miR-125b in megakaryoid differentiation, stable miR-125bover expressing K562cell line was built and used to reveal the imapcts of miR-125b over-expression on megakaryocyte differentiation.Results: During megakaryocyte differentiation, the progressive increase of miR-125bstarted with relatively low expression in early stages and continual increased as terminaldifferentiation progresses. Of interest, the expression of miR-125b was markedly elevatedin late-stage megakaryocytes and platelets; on the other hand, miR-125b mimicstransfection increased miR-125b level in human umbilical cord blood mononuclear cellsand promoted megakaryocyte differentiation. And miR-125b inhibitor retardedmegakaryocyte differentiation. The stable miR-125b over-expressing K562cellsshowed similar results in megakaryocytic differentiation as miR-125b mimics transfectedmononuclear cells. Thus, we proposed miR-125b as a positive regulator ofmegakaryopoiesis.Part3: Identify and verify miR-125b target gene in megakaryocyte differentiation.Methods: By consulting literatures and performing integrative bioinformaticanalysis, we identified several possible target genes of miR-125b and finally focused onp19INK4D, a cell-cycle regulator. P19INK4Dis one of the putative target genes of miR-125b,consequently, it will be down-regulated when miR-125b is up-regulated. To figure outwhether p19INK4Dmediates miR-125b’s function in megakaryocyte differentiation, the endogenous expression of p19INK4D, its correlate kinetics with miR-125b were detectedwith Q-PCR during megakaryocyte differentiation and platelet production initiated fromCD34+hematopoietic cells. Next, we transfected two human leukemia cell lines, K562and UT-7, with siRNAs directed to p19INK4Dand confirmed the effect of RNAi-mediatedknockdown of p19INK4Din proliferation and megakaryocyte differentiation.Results: Using luciferase reporter assay, Q-PCR and western blot, we demonstratedthat p19INK4D, which conceiving complementary sites on the3’UTR to the seed sequenceof miR-125b, is a direct target of miR-125b. And the mRNA expression level of p19INK4Dwas inversely related to miR-125b. P19INK4DsiRNA mimics transfection decreasedp19INK4Dlevel in K562and UT-7cells and promoted megakaryocyte differentiation at themean time. Thus, we showed by integrative analysis of the transcriptome ofhematopoietic cells that miR-125b may exert its effect on Mk differentiation by repressionof p19INK4Dexpression.In conclusion, based on previous progresses in our laboratory, we compared severalkinds of megakaryocytes induction systems. And this part of work might laid thefoundation for further optimization of megakaryocyte differentiation system. The ex vivogenerated megakaryocytic progenitors will also be useful in clinical transfusion.Furthermore, we found the endogenous expression of miR-125b matched the stage ofmegakaryocyte differentiation, and the endogenous expression of miR-125b elevatedduring megakaryocyte differentiation. Then we evaluated the effect of miR-125b over-expression or down-regulation on the expression of MK markers and genes important forMK lineage. After that, we systematically identified the role of miR-125b in promotingthe differentiation of megakaryocytes, and revealed a cell cycle regulator, p19INK4D,served as its molecular mediator during this process.
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