Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression

Abstract

We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.hsa-mir199a ͉ hsa-mir346 ͉ stem cells ͉ plasticity H uman multipotent stromal cells (hMSCs) from bone marrow, also known as mesenchymal stem cells, are progenitor cells capable of differentiating into a variety of mature tissues (1). hMSCs can be isolated easily as single cell clones and differentiated into osteoblasts and adipocytes as well as other cellular phenotypes in culture. The cells hold great promise for therapy but the molecular mechanisms that govern the plasticity and differentiation remain unclear. Recently, the existence and function of a class of small, noncoding RNA molecules known as microRNAs (miRNAs) have gained attention as regulatory molecules. These genomically encoded RNAs undergo several modifications before being converted into mature 21-23 base pair transcripts capable of gene silencing. Biogenesis of functional miRNAs involves several enzymes, including Dicer and Drosha. Previous studies demonstrated that decreased expression of Dicer in Drosophila, zebrafish, and mice restricts the differentiation potential of stem cells (2-5). Studies have also demonstrated that specific miRNAs regulate gene expression during germ line development and cellular differentiation (2-6), including playing key roles in hematopoiesis as well as myogenic and neurogenic function (7-11). However, much of the work on miRNAs has focused on simpler organisms with very little data on human miRNAs. In this study we investigated the role of miRNAs in hMSCs, focusing first on the need for miRNA processing enzymes in hMSC differentiation. Then, we identified the key miRNAs that may regulate differentiation of hMSCs. Finally, we demonstrated that two of the miRNAs target leukemia inhibitory factor (LIF), the expression of which decreases as hMSCs differentiate. Results Generation of a Stable Knockdown of Dicer and Drosha in hMSCs.As a first step toward identifying the role of miRNAs in the differentiation of hMSCs, we analyzed the effect of decreasing the expression of key proteins...

Discovery of circulating microRNAs associated with human prostate cancer using a mouse model of disease

“…37,38 hsa-miR-346 may function in the differentiation of mesenchymal stem cells from bone marrow by targeting the leukaemia inhibitory factor. 39 Our data indicate that these miRNAs are also associated with prostate cancer and could be useful circulating markers of this disease. Using a large prostate cancer expression dataset from the MSKCC cohort, we showed that the levels of hsa-miR-141, hsa-miR-298 and hsa-miR-375 were increased in localized primary tumors and metastases.…”

A Differentiation-Based MicroRNA Signature Identifies Leiomyosarcoma as a Mesenchymal Stem Cell-Related Malignancy

“…It has previously been shown that miRNA expression and processing are required for other differentiation processes of the mesenchymal lineage, including osteocyte, adipocyte, 24 skeletal muscle (miR181a), 26 and cardiac muscle (miR-1 and miR-133). 27 This is the first report, however, of miRNA requirement for smooth muscle differentiation of mesenchymal stem cells.…”

“…Similarly, several let-7 members, including 7b, are also modulated during osteocytic and adipocytic differentiation. 24 Other miRNAs in the SM differentiation signature include miR-155, with a known role in B-cell differentiation, 29 and miR-18a, a member of the miR-17-92 family, which regulates stem cell renewal and displays pro-proliferative and oncogenic potential. 13 Our experiments show that at least some of these miRNAs (ie, miR-155) play an active role in SM differentiation and are not mere byproducts of the actual 'driver' alterations.…”

“…24 As a proof-of-principle of the functional requirement of miRNAs in SMC differentiation, we investigated the effect of knocking down Dicer in our in vitro model, using siRNA oligonucleotides. Dicer down-regulation impaired full SMC differentiation, as attested by morphological changes, reduced SM markers such as calponin, smooth muscle-22-alpha (SM22␣), and ASMA protein levels, and more importantly, lack of SM-MHC mRNA up-regulation, considered the hallmark of mature SMCs (Figure 2, A-D).…”

Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients

“…19 Very few studies have evaluated DICER1 or DROSHA activity in MSC and they focused on their role in differentiation of MSCs. 20,21 Thus, to gain insight into the biological effects of low DICER1 and DROSHA expression in MSC-MDS, we further analyzed mature microRNA expression profile, showing a trend towards global downexpression in MDS-MSC when compared with normal MSC. These results could be associated with the inherent biological diversity among MDS subtypes.…”