Role of microRNAs 99b, 181a, and 181b in the differentiation of human embryonic stem cells to vascular endothelial cells

Abstract

MicroRNAs (miRNAs) are short noncoding RNAs, which post-transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development, and disease. We have recently developed a feeder- and serum-free protocol for direct derivation of endothelial cells (ECs) from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis-associated miRNAs (miR-126 and -210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular EC remains to be fully interrogated. Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES-EC differentiation. A miRNA microarray was performed to quantify hES-EC miRNA profiles during defined stages of endothelial differentiation. miR-99b, -181a, and -181b were identified as increasing in a time- and differentiation-dependent manner to peak in mature hESC-ECs and adult ECs. Augmentation of miR-99b, -181a, and -181b levels by lentiviral-mediated transfer potentiated the mRNA and protein expression of EC-specific markers, Pecam1 and VE Cadherin, increased nitric oxide production, and improved hES-EC-induced therapeutic neovascularization in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR-99b, -181a, and -181b comprise a component of an endothelial-miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs.

Figure 1

Lentiviral (LV)-mediated knockdown of Dicer in human embryonic stem-endothelial cells (hES-ECs). (A): Dicer mRNA expression during SA461 hES-EC differentiation. *Denotes significance, when compared with pluripotent D0 sample. ⁺Denotes significance, when compared with D4 and D10 hES-EC sample. (B): Dicer mRNA expression after LV-mediated knockdown. Pluripotent SA 461 hESCs (2 × 10⁴) were transduced with a multiplicity of infection of 25 prior to directed EC differentiation. Pluripotent samples (black bar), LV-shRNA SCR sequence (gray bar), and LV-shDicer sequence (white bars). Data are given as the mean ± SEM. *, p <.05; **, p <.01; ***, p <.001 versus the time-matched uninfected sample. (C): Western blot analysis of Dicer protein expression in SA461 hESCs. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SCR, scramble; shRNA, short hairpin RNA; UI, uninfected.

Figure 2

Dicer knockdown is correlated with lack of acquisition of endothelial cell (EC) phenotype. (A): Morphology of SA461 human embryonic stem cells subjected to LV-mediated Dicer knockdown or SCR sequence control (multiplicity of infection 25). Control cells (UI and SCR sequence samples) display morphological acquisition of a cobblestone-like appearance, typical of EC differentiation. Dicer knockdown prevents the appearance of morphological changes indicative of EC differentiation. Scale bar = 100 μm. (B): mRNA expression of VE Cadherin and Pecam1 in SA461 cells subjected to LV-mediated Dicer knockdown or UI and SCR sequence controls. D0 = white bars, D4 = black bars, D10 = dark gray bars, and D14 = light gray bars. Data are given as the mean ± SEM. *, p <.05; ***, p <.001 versus the time-matched uninfected sample. (C): Fluorescence-activated cell sorting analysis of increasing positive expression of VE Cadherin (FL1) and Pecam1 (FL2) observed in a time- and differentiation-dependent manner for more than 14 days of directed differentiation. LV-mediated Dicer knockdown reduced the population expressing one or both markers. Abbreviations: LV, lentiviral; SCR, scramble; UI, uninfected; VE, vascular endothelial.

Figure 3

Dicer knockdown prevents the acquisition of vascular bed marker genes and reduces nitric oxide (NO) production. (A): mRNA expression of vascular bed marker genes in H1 human embryonic stem cells (hESCs). Expression of all vascular bed markers are induced with differentiation in UI and SCR sequence controls but reduced with Dicer knockdown. D0 = white bars, D4 = black bars, D10 = dark gray bars, and D14 = light gray bars. Data are given as the mean ± SEM. *, p <.05; **, p <.01; ***, p <.001 versus the time-matched uninfected sample. (B): NO production in D0 and D14 H1 hES-endothelial cells subjected to lentiviral-mediated Dicer knockdown or UI and SCR sequence controls. Dicer knockdown suppresses NO production. Data are given as the mean ± SEM. **, p <.01; ***, p <.001 versus the time-matched uninfected sample or ⁺⁺, p <.01 versus pluripotent sample. Abbreviations: SCR, scramble; UI, uninfected.

Figure 4

MicroRNA (miRNA) microarray validation of miRNA transcriptome at early time points of endothelial lineage specification. (A): TaqMan and Northern blot validation of microarray expression. Expression of mature miR-99b, -181a, and -181b is induced in an endothelial differentiation-specific manner, when compared with time-matched pluripotent samples in SA461 hESC line. Data are given as the mean ± SEM. *, p <.05; **, p <.01; ***, p <.001 versus the time-matched pluripotent sample. (B): Expression of miR-99b, -181a, and -181b in H1 pluripotent, hES-EC, hES-HPC, and hES-neural cell. Data are given as the mean ± SEM. *, p <.05; **, p <.01; ***, p <.001 versus pluripotent sample. ⁺Versus the time-matched differentiated sample. Abbreviations: EC, endothelial cell; hES, human embryonic stem; HPC, hematopoietic progenitor cell; RQ: relative quantification.

Figure 5

Overexpression of miRNAs potentiates endothelial cell (EC) differentiation. (A): mRNA expression of VE Cadherin and Pecam1 in H1 human embryonic stem cells (hESCs) subjected to lentiviral-mediated overexpression of miR-99b, −181a, −181b, all together (miR ×3), or UI and SCR sequence controls. (B): Fluorescence-activated cell sorting analysis of positive expression of VE Cadherin (FL1) and Pecam1 (FL2) in hES-EC subjected to 14 days of directed differentiation. (C): Nitric oxide (NO) production in hES-ECs. miR-99b, −181a, −181b, all together (miR ×3), or overexpression potentiates NO production. Data are given as the mean ± SEM. **, p <.01; ***, p <.001 versus the time-matched uninfected sample or ⁺⁺, p <.01 versus pluripotent sample. D0 = white bars, D4 = black bars, D10 = dark gray bars, and D14 = light gray bars. Abbreviations: SCR, scramble; UI, uninfected; VE, vascular endothelial.

Figure 6

Expression of vascular bed markers after microRNA overexpression modulation. Fold expression of vascular bed marker genes in H1 human embryonic stem cells relative to time-matched lentiviral (LV)-shRNA scramble sequence controls. Data are given as the mean ± SEM. *, p <.05; **, p <.01 versus the time-matched LV-shRNA scramble sequence sample. Gray hatched line denotes scramble sequence reference value. Abbreviation: SCR, scramble; shRNA, short hairpin RNA.

Figure 7

Overexpression of miRNAs improves therapeutic angiogenesis. (A): Ischemic to contralateral blood flow ratio at 7, 14, and 21 days after injection of cells in the ischemic adductor muscle. (B): Photographs show typical laser Doppler images of blood flow captured from human embryonic stem cell-endothelial cell (GFP, miR-99b, -181a, and -181b overexpression groups)-injected mice at 21 days after induction of ischemia. The squares highlight the areas of interest (feet), where blood flow was calculated to determine the ischemic/contralateral ratio. Color scale from blue to red indicates progressive increases in blood flow. (C): Capillary density of ischemic adductor muscle (21 days postischemia). Capillary density is expressed as the number of capillaries to myofiber ratio. Data are mean ± SEM; *, p <.05 versus control GFP expressing virus. Abbreviation: GFP, green fluorescent protein.