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.
Many biotechnology applications require the evolution of enhanced protein stability. Using polymerase chain reaction-based recovery of engineered clones during the screen enrichment phase, we describe a yeast display method capable of yielding engineered proteins having thermal stability that substantially exceeds the viability threshold of the yeast host. To this end, yeast-enhanced green fluorescent protein destabilized by dual-loop insertion was engineered to possess a substantially enhanced resistance to thermal denaturation at 70°C. Stabilized proteins were secreted, purified and found to have three- to six-fold increased resistance to thermal denaturation. The validated method enables yeast display-based screens in previously inaccessible regions of the fitness landscape.
Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture derived platelets (CDPs) from human peripheral blood CD34+ cells can be genetically altered and may eventually be used for transfusions. Using microfluidics, the time-dependent incorporation of CD41+CD42+ CDPs into clots was measured using only 54,000 CDP doped into 27 µL of human whole blood perfused over collagen at a wall shear rate of 100 s−1. Using fluorescently labeled human platelets (instead of CDPs) doped between 0.25 and 2 % of total platelets, incorporation was highly quantitative and allowed monitoring of anti-αIIbβ3 antagonism that occurred after collagen adhesion. CDPs were only 15 % as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized with αIIbβ3 inhibition. Transient transfection allowed the monitoring of GFP+ human CDP incorporation into clots. This assay quantifies genetically-altered CDP function under flow.
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