Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells

Abstract

Background: Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs.

Methodology/principal findings: In this study, we employed a library of miRNA inhibitors to evaluate the role of miRNAs in early osteogenic differentiation of human MS cells. We discovered that miR-148b, -27a and -489 are essential for the regulation of osteogenesis: miR-27a and miR-489 down-regulate while miR-148b up-regulates differentiation. Modulation of these miRNAs induced osteogenesis in the absence of other external differentiation cues and restored osteogenic potential in high passage number human MS cells.

Conclusions/significance: Overall, we have demonstrated the utility of the functional profiling strategy for unraveling complex miRNA pathways. Our findings indicate that miRNAs regulate early osteogenic differentiation in human MS cells: miR-148b, -27a, and -489 were found to play a critical role in osteogenesis.

Figure 1. Inhibition of miRNAs dramatically affect osteogenic differentiation of hMSC.

(A) Fifteen miRNA inhibitors were selected in the primary screen for their effect on AP activity in differentiated hMSC. Data are representative of a single screen performed in triplicate. (B) Seven inhibitors were confirmed as hits in separate experiments. miRNA mimics for four miRNAs demonstrated no effect on AP activity, -miR-189, -153, -133a and -486. For three other miRNAs, miR -148b, -27a and -489, mimics had an effect that was opposite to one of the corresponding inhibitors. Data are representative of three independent experiments performed in triplicate. (mean+/−SD). *, **: Student's ttest p value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.

Figure 2. Adjustment of the miRNAs activity alone induced osteogenic differentiation of hMSC in the absence of other differentiation stimuli.

Cells transfected with miRNA inhibitors or mimics alone or in combination, demonstrated drastic increase in total AP activity (A), and up-regulation of SPP1, another marker of early ostogenesis (B) . Data are representative of three independent experiments performed in triplicate. (mean+/−SD). *, **: Student's ttest p value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.

Figure 3. Alteration of the miRNAs activity rescues osteogenic potential in transfected hMSC with high passage number.

(A) Bone marrow hMSC with high passage number demonstrate a decrease in basal level of AP activity and poor response to differentiation media. hMSC were plated in 96-well plates and incubated either in propagation (P, grey bar) or osteogenic (O, black bar) media for 6 days. Data normalized to relative AP activity values obtained from passage 21 hMSC incubated in propagation media. (B and C) Transfection with miRNA inhibitors and mimics restore differentiation in over-propagated hMSC incubated in propagation (B) or differentiation (C) media. hMSC propagated for certain number of passages (16, white bar, 18, grey bar or 21 , black bar) plated in 96-well plates and transfected with inhibitors and mimics (as indicated, all at 25 nM). Data are representative of three independent experiments performed in triplicate. (mean+/−SD). *, **: Student's ttest p value between treated cells and corresponding control group, * - p<0.01, ** - p<0.05.

Figure 4. Negative effect of miRNAs, miR-489 and -27a, on osteogenesis in hMSC is, at least in part, mediated by repression of GCA gene expression.

(A–G) Transfection with inhibitors of miR-489 and -27a results in up-regulation of GCA protein expression. (A–F). hMSC were transfected with control molecule, ic1 (25 nM) or with combination of inhibitors, i489+i27a (12.5 nM each). GCA protein expression was determined by immunofluorescence as described in Materials and Methods. Cells were stained with Hoechst 33342 (A–C) and GCA specific primary antibody, followed by a secondary antibody (E,F). Control wells were treated with secondary antibody only and demonstrate no specific staining (D). (G) Quantitation of GCA immunohystochemistry. Data shown represents three independent transfections, four fields each. (mean+/−SD). ** - Student's ttest p value between treated cells and corresponding control group, p<0.05. (H) RT-PCR-based study of gene expression demonstrated that the expression of GCA mRNA is regulated by miR-489 and -27a. hMSC were transfected with Inhibitor Control molecule 1 (IC1, 25 nM) or with a combination of inhibitors for miR-27 and -489 (i27a+i489, 12.5 nM each). Transfected cells were incubated for 2 days in propagation media and then harvested for totat RNA. (I) siRNA-mediated knockdown of GCA (grancalcin) resulted in a significant decrease of AP activity in hMSC under differentiation conditions. hMSC were transfected with Control siRNA, RUNX2 and GCA siRNA (each at 50 nM). Cells were switched to osteogenic media 24 hr after transfection and harvested 6 days after the switch. Data shown represents two independent transfection experiments performed in duplicate (H) or triplicate (I) (mean+/−SD). **: Student's ttest p value between treated cells and corresponding control group, p<0.05.