MicroRNA Screen of Human Embryonic Stem Cell Differentiation Reveals miR‐105 as an Enhancer of Megakaryopoiesis from Adult CD34+ Cells

Abstract

MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by 4-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235+CD41−), megakaryocyte (CD41+CD42+) and myeloid (CD18+CD235−) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a 6-fold enhancement in megakaryocyte production. MiR-513a, miR-571 and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34+ cells derived from peripheral blood. In addition, adult CD34+ cells express endogenous miR-105 during megakaryocyte differentiation. SiRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3’UTR construct and western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis.

Evaluation of microRNA Expression in Patients with Herpes Zoster

“…It could be inferred that a host may increase the miR-1276 expression to resistance the VZV infection, though the exact mechanism remains to be elucidated. MiR-571 [27] and miR-943 [28] have also been reported to play a putative role in different cell compartments involved in the fibrogenesis and inflammation processes during liver cirrhosis pathogenesis. This finding could explain the liver injury resulting from HZ infection.…”

Combinatorial effect of miR-486-5p & miR-22-3p mimics thrombopoietin’s impact on hematopoietic stem & progenitor cells

“…Based on published literature, miR concentrations from 1 nM to 3.6 µM have been used to transfect HSPCs. [33][34][35] In the following studies, since the seven MkMP miRs we examined are highly abundant in MkMPs, and due to the fact that multiple MkMPs are taken up by a single recipient CD34 + HSPC, 16 we hypothesized that higher concentrations of these seven miRs are likely delivered to CD34 + HSPC via MkMPs. Therefore, we chose 8 µM of miR mimics for the following experiments.…”

“…Several miRs have been previously reported as regulators of megakaryopoiesis. 33,[41][42][43][44] These and all in vitro studies of miRs in megakaryopoiesis were carried out in the presence of TPO. To our knowledge, there have been no reports of miRs promoting megakaryocytic differentiation of CD34 + HSPCs in the absence of TPO.…”

MicroRNA-mediated regulation of differentiation and trans-differentiation in stem cells

“…This method has been reported in several different studies for delivering shRNA in neural progenitor cells with knockdown efficiencies of ~60–90% [155, 156]. A recent study that involved human ESCs-derived myeloid progenitor cells reported on the use of electroporation to deliver 466 human miRNA mimics for screening purposes, demonstrating the feasibility of this method with an efficiency of ~50–80% [157]. …”