MicroRNAs miR-26a, miR-26b, and miR-29b accelerate osteogenic differentiation of unrestricted somatic stem cells from human cord blood

Hans-Ingo Trompeter 0 2 Janine Dreesen 0 2 Eugenie Hermann 0 2 Katharina M Iwaniuk 0 2 Markus Hafner 1 Neil Renwick 1 Thomas Tuschl 1 Peter Wernet 0 2 0 University Dusseldorf, Medical Faculty, Institute for Transplantation Diagnostics and Cell Therapeutics (ITZ) , Dusseldorf D-40225 , Germany 1 Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University , New York, NY 10065 , USA 2 University Dusseldorf, Medical Faculty, Institute for Transplantation Diagnostics and Cell Therapeutics (ITZ) , Dusseldorf D-40225 , Germany Background: MicroRNAs are a population of short non-coding RNAs with widespread negative regulatory impact on mRNA translation. Unrestricted somatic stem cells (USSC) are a rare population in human cord blood that can be induced into cells representative of all three germinal layers. Here we analyzed the functional impact of miRNAs on the osteogenic differentiation in USSC. Results: Gene expression profiling identified 20 microRNAs that were consistently upregulated during osteogenic differentiation of two different USSC cell lines (SA5/73 and SA8/25). Bioinformatic target gene prediction indicated that among these microRNAs, miR-10a, -22, -26a, -26b, and -29b recognize transcripts that encode a set of proteins inhibiting osteogenesis. We subsequently verified osteo-inhibitory CDK6, CTNNBIP1, HDAC4, and TOB1 and osteopromoting SMAD1 as targets of these microRNAs. In Western blot analyses demonstrated that endogenous levels of CDK6 and HDAC4 were downregulated during osteogenic differentiation of USSC and reduced following ectopic expression of miR-26a/b and miR-29b. In contrast, endogenous expression of SMAD1, targeted by miR-26a/b, was unaltered during osteogenic differentiation of USSC or following ectopic expression of miR-26a/b. Functional overexpression analyses using microRNA mimics revealed that miR-26a/b, as well as miR-29b strongly accelerated osteogenic differentiation of USSC as assessed by Alizarin-Red staining and calcium-release assays. Conclusions: miR-26a/b and miR-29b are upregulated during osteogenic differentiation of USSC and share target genes inhibiting osteogenesis. Furthermore, these microRNAs accelerate osteogenic differentiation, likely mediated by osteo-inhibitory proteins such as CDK6 and HDAC4. - Background microRNA (miRNA)-mediated translational repression is an important regulatory mechanism in multiple cellular processes. miRNAs are a subpopulation of small RNAs, averaging 22 nucleotides in length, which inhibit translation through sequence-specific binding to target sites within the 30-UTRs of mRNAs. Following transcription, miRNAs are processed in a two-step mechanism involving the RNAses DROSHA and DICER [1,2] and subsequently integrated into the RNA-induced silencing complex [3,4], thereby unfolding their regulatory potential to regulate mRNAs [5-7]. Exhibiting stage- and tissue-specific expression patterns during development [8,9], miRNAs not only function as key regulatory molecules in multiple cellular processes including apoptosis [10,11], cancer [12], proliferation [13], development [14], and differentiation [15] but also control stemness and pluripotency of embryonic stem cells e.g. by repressing pluripotency factors OCT4, SOX2 and KLF4 [16]. Osteogenesis is a highly coordinated process involving transcription factors, such as RUNX2 and OSTERIX [17,18], BMP2, and other factors, that drive committed stem cells toward fully differentiated osteocytes (reviewed in [19-21]). Osteogenesis is promoted through several signalling pathways, including WNT/-catenin, BMP, JAK/STAT, and MAPK [22-27]. Several miRNAs modulate osteogenic differentiation: miR-125b negatively regulates osteoblastic differentiation through targeting VDR, ErbB2, and Osterix [28,29]; miR-133 (targeting RUNX2) and miR-135 (recognizing SMAD5) inhibit differentiation of mouse osteoprogenitors [30]; miR-26a and miR-29b facilitate osteogenic differentiation of human adipose tissue-derived stem cells (hADSCs), and positively modulate mouse osteoblast differentiation [31,32]. Multiple other miRNAs, including miR-9, -17, -27, -30, -96, -106, 138, -181, -182, -320, and 326, have been linked to osteogenesis [30]. Unrestricted somatic stem cells (USSC) are a rare CD45-negative population in human cord blood [33]. These cells can be discriminated from CB-MSC and BMMSC by their HOX expression pattern which resembles that of H9 embryonic stem cells [34]. Adherently-growing in vitro USSC can be induced to cells representative of all three germinal layers on a clonal level [35] and have been successfully reprogrammed to a pluripotent ES-like state [36]. Undergoing miRNA-supported cell cycle arrest, USSC can be differentiated into cells of neural lineage with miRNAs acting as network-like regulators [37-39]. USSC also differentiate into functional hepatic-like cells [40,41] as well as along osteogenic and chondrogenic lineages [33]. Upon induction with dexamethasone, ascorbic acid, and -glycerol phosphate (DAG), USSC differentiate into osteoblasts as evidenced by calcium phosphate deposition, bone-specific ALP-activity, increase in Ca2+-release, and expression of the osteogenic marker proteins osteocalcin, osteopontin, bone sialo-protein, and collagen type I [33]. Bony reconstitution was observed following implantation of USSC into nude rat femurs [33]. Beside their differentiation potential, USSC also fulfil regenerative functions in acute spinal cord trauma [42]. Here we analyzed the impact of miRNAs on osteogenic differentiation of USSC. We identified a set of miRNAs upregulated upon induction of osteogenesis, co-ordinately regulating a distinct set of genes known to inhibit osteogenesis. Among these inhibitors, CDK6, CTNNBIP1, HDAC4, TGFB3, and TOB1 were experimentally identified as targets of miR-26a, miR-26b, and miR-29b. These miRNAs were functionally identified as accelerators of osteogenic differentiation of USSC. Results Differential miRNA expression during osteogenic differentiation of USSC To assess the impact of miRNAs on osteogenic differentiation of USSC we studied two USSC lines (USSC SA5/73 and USSC SA8/25) that were induced to osteogenic differentiation using DAG as described [33]. As strong calcification of USSC during osteogenic differentiation impacts RNA isolation, we restricted our analyses to day 7 of differentiation. miRNA expression profiles of native and day 7 osteo-differentiated USSC were analyzed using the RT-PCR-based TaqMan Assay (Pool A) covering 377 miRNAs [43]. In SA5/73, 220 miRNAs were expressed and 124 miRNAs were upregulated by a factor 2 in differentiated cells. In SA8/25, 225 miRNAs were expressed and 196 miRNAs were upregulated during osteogenic differentiation. Interestingly, only 30 miRNAs were commonly upregulated in both USSC lines. In follow-up analyses we focused on 20 of these microRNAs (Figure 1), which were not only upregulated by a factor 2 but also present at high expres (...truncated)