Upregulation of miR-135b Is Involved in the Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients

<div><p>Previous studies have demonstrated that mesenchymal stem cells from multiple myeloma (MM) patients (MM-hMSCs) display a distinctive gene expression profile, an enhanced production of cytokines and an impaired osteogenic differentiation ability compared to normal donors (ND-hMSCs). However, the underlying molecular mechanisms are unclear. In the present study, we observed that MM-hMSCs exhibited an abnormal upregulation of miR-135b, showing meanwhile an impaired osteogenic differentiation and a decrease of SMAD5 expression, which is the target of miR-135b involved in osteogenesis. By gain and loss of function studies we confirmed that miR-135b negatively regulated hMSCs osteogenesis. We also found that MM cell-produced factors stimulated ND-hMSCs to upregulate the expression of miR-135b. Importantly, treatment with a miR-135b inhibitor promoted osteogenic differentiation in MM-hMSCs. Finally, we observed that MM cell-derived soluble factors could induce an upregulation of miR-135b expression in ND-hMSCs in an indirect coculture system and the miR-135b expression turned to normal level after the removal of MM cells. Collectively, we provide evidence that miR-135b is involved in the impaired osteogenic differentiation of MSCs derived from MM patients and might therefore be a promising target for controlling bone disease. </p> </div

MM-hMSCs exhibit a different miR-135b expression during osteogenic differentiation compared to ND-hMSCs.

<p>(A) The osteogenic differentiation of ND-hMSCs and MM-hMSCs gradually progressed when exposed to osteogenic induction medium (OM) <i>in </i><i>vitro</i> as shown by qualitative ALP staining. However, the ALP increase in MM-hMSCs is lower compared to ND-hMSCs. (B) miR-135b relative expression, as detected by quantitative real time PCR, decreases during osteogenesis significantly in both ND-hMSCs and MM-hMSCs. There is a delay of miR-135b decrease in MM-hMSCs. n=5/group. * and # indicate <i>p</i><0.05, compared to day 0 for ND-hMSCs and MM-hMSCs, respectively. All values are normalized to day 0. (C) MM-hMSCs with impaired osteogenic differentiation show a less increasing level of SMAD5 during osteogenic differentiation compared to ND-hMSCs. One representative result of three is shown.</p

miR-135b expression is higher in MM BM-derived hMSCs showing impaired osteogenic differentiation potential.

<p>(A) MM-hMSCs show a reduced ALP activity, a marker for osteogenic differentiation. ND-hMSCs (n=7) and MM-hMSCs (n=12) are cultured in osteogenic medium (OM) for 72 hours and the ALP activity is measured quantitatively by the alkaline phosphatase yellow (pNPP) liquid substrate system for ELISA (Sigma-Aldrich, Bornem, Belgium). ** <i>p</i><0.01 (B) miR-135b expression is significantly upregulated in MM-hMSCs compared to ND-hMSCs by quantitative real time PCR. The miR-135b expression of all hMSCs samples is normalized to the miR-135b expression of U266 MM cells. * <i>p</i><0.05 (C) miR135b expression is inversely correlated with the ALP activity in MM-hMSCs. The relative expression of miR-135b in hMSCs derived from 12 MM patients was plotted versus their ALP activity.</p

miR-135b negatively regulates SMAD5 expression.

<p>(A) Transfection with miR-135b inhibitor or mimic leads to a decrease or an increase of miR-135b expression in hMSCs. One of three independent experiments is shown. (B) miR-135b is confirmed to target SMAD5 in hMSCs. Transfection with miR-135b inhibitor or mimic leads to an increase or a decrease of SMAD5 expression, respectively, by Western blot in hMSCs. One of three independent experiments is shown. (C) HEK 293 cells were cotransfected with the luciferase reporters carrying wild-type or mutated SMAD5 3’UTR, and 50 nM miR-135b mimic or negative control (miR-C) for 48 h. The luminescence of Renilla luciferase was normalized to that of firefly luciferase, and the relative luminescence units was plotted. Normalized data are shown as mean±SD. n=3. ***, p<0.001; NS, not significant. (D) MM-hMSCs with upregulated miR-135b expression have a lower SMAD5 expression as shown by Western blot analysis. Left panels show the blots; right panels show densitometric analysis using ImageJ software. *<i>p</i><0.05.</p

DataSheet_1_Multipotent mesenchymal stromal cells as treatment for poor graft function after allogeneic hematopoietic cell transplantation: A multicenter prospective analysis.pdf

IntroductionPoor graft function (PGF) is a rare but serious complication of allogeneic hematopoietic cell transplantation (alloHCT). Due to their hematopoietic supporting properties and immune regulatory effects, multipotent mesenchymal stromal cells (MSC) could be considered a good candidate to help to restore bone marrow (BM) niches homeostasis and facilitate hematopoiesis after alloHCT.MethodsWe prospectively assessed the efficacy and safety of ex-vivo expanded BM-derived MSC from third-party donor in a series of 30 patients with prolonged severe cytopenia and PGF after alloHCT. This multicenter trial was registered at www.clinicaltrials.gov (#NTC00603330).ResultsWithin 90 days post-MSC infusion, 53% (95% CI, 35 – 71%) of patients improved at least one cytopenia (overall response, OR) and 37% (95% CI, 19 - 54%) achieved a complete hematological response (CR: absolute neutrophil count, ANC >0.5 x 109/L, Hb > 80g/L and platelet count > 20 x 109/L with transfusion independence). Corresponding response rates increased to 67% (95% CI, 50 - 84%) OR and 53% (95% CI, 35 - 71%) CR within 180 days after MSC infusion. A significant decrease in red blood cells and platelets transfusion requirement was observed after MSC (median of 30-days transfusion requirement of 0.5 and 0 from d90-120 post-MSC versus 5 and 6.5 before MSC, respectively, p ≤0.001). An increase in ANC was also noted by day +90 and +180, with 3/5 patients with severe neutropenia having recovered an ANC > 1 x 109/L within the 90-120 days after MSC infusion. Overall survival at 1 year post-MSC was 70% (95% CI, 55.4 – 88.5), with all but one of the patients who achieved CR being alive. A single infusion of third-party MSC appeared to be safe, with the exception of one deep vein thrombotic event possibly related to the intervention.DiscussionIn conclusion, a single i.v. infusion of BM-derived MSC from third party donor seemed to improve hematological function after alloHCT, although spontaneous amelioration cannot be excluded. Comparative studies are warranted to confirm these encouraging results.</p