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 four-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 sixfold 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.

Keywords: Embryonic stem cells; Hematopoiesis; Megakaryocyte; miRNA.

Figure 1. MicroRNA screen in differentiating human embryonic stem cells

(A) A miRNA mimic library was screened in hematopoietic progenitors derived from the H9 human embryonic stem cell line. hESCs (SSEA-3⁺SSEA-4⁺) were plated on matrigel on Day 0 and were subjected to a 8-day serum free differentiation protocol in a 6-well plate. CMPs (CD41⁺CD235⁺CD43⁺) emerge from the adherent layer into suspension as single cells on Days 7, 8 and 9. Day 8 CMPs were transfected via electroporation with miRNA mimics in 96-well plates and then cultured for 4 to 5 days after which erythroid (CD235⁺CD41⁻), megakaryocyte (CD41⁺CD42⁺) and myeloid (CD18⁺CD235⁻) populations were assessed by 96-well flow cytometry. (B) Representative flow cytometry plots are shown.

Figure 2. microRNA screen results for regulators of megakaryocyte populations

466 miRNA mimics were screened in hESC derived Day 8 CMPs in six 96-well plates. (A) The deviation for each miRNA mimic from the mean plate value of %megakaryocyte (CD41⁺CD42⁺) population is shown. The top miRNA enhancers and inhibitors are labeled. Dotted lines represent 2 standard deviations. (B) The top hits were re-tested in replicates. The fold change in % megakaryocyte population as a result of miRNA mimic transfection is shown. Data are representative of 2 to 5 independent transfections.

Figure 3. miR-105 and miR-150 enhance megakaryocyte population from H9 hESC derived hematopoietic progenitors

Day 8 CMPs were transfected with 2 μM miR-105 mimic, miR-150 mimic, or a non-specific negative control. Cell populations were analyzed by flow cytometry on Day 12. (A) Representative flow cytometry plots for megakaryocyte (CD41⁺CD42⁺) population are shown. (B) The fold change in % megakaryocyte population as a result of miR mimic transfection is shown. Data are representative of 3 to 4 independent transfections. (C) Representative flow cytometry plots for erythroid (CD235⁺CD41⁻) population are shown. (D) The fold change in % erythroid population as a result of miR mimic transfection is shown. Data are representative of 3 to 4 independent transfections.

Figure 4. miR-105 enhances megakaryocyte production in vitro from adult CD34⁺ cells

CD34⁺ cells isolated from the peripheral blood of mobilized adult donors were transfected with 2 μM miR-105 mimic, miR-150 mimic or a negative control. Cells were cultured for 14 days post transfection in a megakaryocyte lineage promoting medium containing TPO, SCF, IL-6 and IL-9. Cells were assayed for colony forming units 4 days post transfection. (A) Representative flow cytometry plots for megakaryocyte (CD41⁺CD42⁺) population on Day 7 are shown. (B) The fold change in % megakaryocyte population as a result of miR mimic transfection is shown. Data are representative of 5 independent transfections. (C) The fold change in megakaryocyte yield as a result of miR mimic transfection is shown. Megakayocyte yield is defined as the number of CD41⁺CD42⁺ in culture on Day 7 per CD34⁺ cell seeded within 24 hrs post transfection. Data are representative of 5 independent transfections. (D) Megakaryocyte colony forming units per 2500 transfected cells for 3 separate donors are shown. Data are representative of 3-4 plates of colony assay. (E) Representative megakaryocyte colony image of miR-105 transfected cells is shown.

Figure 5. miR-105 is expressed endogenously in differentiating CD34⁺ cells

CD34⁺ cells isolated from the peripheral blood of mobilized adult donors were cultured in megakaryocyte conditions. Endogenous miRNA expression was assayed by quantitative RT-PCR on Day 0 and Day 7 for 3 different donors. Expression levels were measured relative to RNU6B endogenous control.

Figure 6. miR-105 regulates c-myb expression

(A) CD34⁺ cells isolated from the peripheral blood of mobilized adult donors and CMPs derived from hESCs were cultured in megakaryocyte conditions. C-Myb expression was assessed by quantitative RT-PCR. 18s rRNA was used as the endogenous control (B) Day 8 hESC derived CMPs were transfected with 2 μM c-Myb siRNA or a negative control siRNA. Cells were analyzed by flow cytometry 72 hours post transfection. Representative flow cytometry plots for megakaryocyte (CD41⁺CD42⁺) population are shown (C) Mature sequences of miR-105 and miR-105 with its seed region scrambled are shown (D) HEK-293 cells were cotransfected with 0.075 μg/ml reporter plasmid and 6.7 nM miR mimic or non-specific control in 96 well plates. Culture medium was assayed 48 hr post transfection. The dual reporter vector construct is shown. The relative gaussia luciferase to secreted alkaline phosphotase signal is shown. The signal for luciferase/alkaline phosphotase for the control mimic was set to a value of 1. Data are representative of 4 separate transfections in 96-well plate assay. (E) K-562 cells were transfected with control miRNA mimic, miR-150, miR-105 and siRNA against c-Myb. MYB and GAPDH protein levels were detected with western blot analysis.