The effects of thiazolidinediones on human bone marrow stromal cell differentiation in vitro and in thiazolidinedione-treated patients with type 2 diabetes

Abstract

Thiazolidinedione (TZD) therapy has been associated with an increased risk of bone fractures. Studies in rodents have led to a model in which decreased bone quality in response to TZDs is due to a competition of lineage commitment between osteoblasts (OBs) and adipocytes (ADs) for a common precursor cell, resulting in decreased OB numbers. Our goal was to investigate the effects of TZD exposure on OB-AD lineage determination from primary human bone marrow stromal cells (hBMSCs) both in vitro and in vivo from nondiabetic subjects and patients with type 2 diabetics. Our experimental design included 2 phases. Phase 1 was an in vitro study of TZD effects on the differentiation of hBMSCs into OBs and ADs in nondiabetic subjects. Phase 2 was a randomized, placebo-controlled trial to determine the effects of 6-month pioglitazone treatment in vivo on hBMSC differentiation using AD/OB colony forming unit assays in patients with type 2 diabetes. In vitro, TZDs (pioglitazone and rosiglitazone) enhanced the adipogenesis of hBMSCs, whereas neither altered OB differentiation or function as measured by alkaline phosphatase activity, gene expression, and mineralization. The ability of TZDs to enhance adipogenesis occurred at a specific time/stage of the differentiation process, and pretreating with TZDs did not further enhance adipogenesis. In vivo, 6-month TZD treatment decreased OB precursors, increased AD precursors, and increased total colony number in patients with type 2 diabetes. Our results indicate that TZD exposure in vitro potently stimulates adipogenesis but does not directly alter OB differentiation/mineralization or lineage commitment from hBMSCs. However, TZD treatment in type 2 diabetic patients results in decreased osteoblastogenesis from hBMSCs compared with placebo, indicating an indirect negative effect on OBs and suggesting an alternative model by which TZDs might negatively regulate bone quality.

Trial registration: ClinicalTrials.gov NCT00927355.

Conflict of interest statement

Disclosures: All authors have read the journal’s policy on disclosure of potential conflicts of interest. The authors confirm that there are no conflicts of interest.

Copyright © 2013 Mosby, Inc. All rights reserved.

Figures

Fig. 1. TZDs do not negatively alter osteoblastogenesis from hBMSCs

hBMSCs were cultured in growth or osteoblast differentiation medium (OB Diff) in the presence of varying concentrations of PIO- or ROSI-glitazone for 21–24 days. (A) hBMSCs were stained for mineralization with Alizarin Red S (ARS) top panel or alkaline phosphatase (ALP) bottom panel. (B) Alkaline phosphatase enzyme activity was quantified using PNPP as a substrate. (Rep. of 3 patients) (C) Gene expression was measured by real-time qRT-PCR (mean of 5 patients). (D) A cell viability assay was performed and data presented as fold change from control from 3–6 patients. Results are presented as mean +/−SEM. * p < 0.05. ns:not significant

Fig 2. TZDs do not negatively alter osteoblast differentiation from hBMSCs

(A) hBMSCs were pretreated with TZDs for 5 days or post-treated with TZDs for 5 days relative to initiation of OB differentiation as indicated and stained with alizarin Red S. (Rep. of 3 patients). Similar results were obtained with 14 days of pretreatment. (B) Cells were pretreated with TZDs for 5 days followed by 5 days in OB differentiation medium as indicated and ALP enzyme activity measured. (Rep. of 3 patients). (C) hBMSCs were treated with OB differentiation medium for 21 days and treated with Pio (1μM) or the phosphate transport inhibitor Foscarnet (Fos-1mM) as indicated. The resulting cells were stained for alizarin Red S. Foscarnet was used as a positive control for inhibition (Rep. of 3 patients). * p < 0.05. ns:not significant.

Fig. 3. TZDs stimulate adipogenesis from hBMSCs

hBMSCs were differentiated to adipocytes in the presence of growth (−) or adipocyte differentiation medium (AD Diff) with the indicated concentrations of PIO- or ROSI-glitazone for 14–24 days. (A) Lipid accumulation was fluorometrically quantified with Adipored and results are expressed as arbitrary units read at (485–538) (Rep. of 10 patients) (B) Lipid accumulation was measured in the presence or absence of differentiation medium and PIO and calculated as fold change (mean of 4 patients) (C) hBMSCs were treated as indicated and RNA analyzed for PPARγ2 and aP2 expression by qRT-PCR. Results are expressed as fold increase relative to untreated and representative of 7 patients. (D) hBMSCs were pretreated with TZDs for 5 days prior to addition of adipocyte differentiation medium (pretreat-gray bars) or TZDs added simultaneously (baseline-black bars) and the lipid from all samples fluorometrically quantified after 14 to 21 days. Results are expressed as fold change and representative of 7 patients. (E) RNA was harvested from cultures treated as in (D) and analyzed for the expression of PPARγ2 and aP2. (mean of 5 patients). (F) hBMCS were simultaneously treated with adipocyte differentiation medium (baseline-black bars) or treated 5 days after differentiation initiation (post-treat-gray bars). Lipid accumulation was fluorometrically quantified from all samples after 14 to 21 days and is expressed as fold change from untreated. (Rep. of 3 patients). Columns with different characters differ at p < 0.05. Results are presented as mean +/−SEM. ns:not significant

Fig. 4. Pioglitazone alters adipocyte CFU but has no effect on osteoblast CFU from human bone marrow

Whole bone marrow was diluted 1:20 in DMEM and colonies allowed to expand for twenty-one days. Cells were then treated with AD or OB differentiation medium and +/− PIO (1μM) as indicated. Colonies were treated with adipocyte (CFU-AD) or osteoblast (CFU-OB) differentiation medium and stained for adipocyte positive colonies with Oil-red-O or osteoblast positive colonies with either Alizarin Red S or NBT/BCIP (alkaline phosphatase activity). (A) Representative wells from a 6-well plate demonstrate both positive and negative colonies for the different stains. (B) The CFU-AD assay was performed in the presence or absence of 1μM PIO and positive colonies counted and expressed as a ratio relative to the total number of colonies. (mean of 7 patients). (C) Colonies were pretreated for 5–14 days with 1μM PIO prior to the initiation of adipogenesis and the percent of positive colonies relative counted and expressed as a relative ratio to the total number of colonies. (mean of 7 patients). (D) The CFU-OB assay was performed in the presence or absence of 1μM PIO and positive colonies counted and expressed as a ratio relative to the total number of colonies. (mean of 7 patients). Parallel plates were harvested for RNA analyses and analyzed for adipocyte marker genes, adiponectin shown (E), and osteoblast marker genes, osteocalcin shown (F) by qRT-PCR (Rep. of 7 patients). * p < 0.05. Results are presented as mean+/−SEM. ns:not significant

Fig. 5. Changes in bone marrow CFU-AD and OB from study subjects treated with Pioglitazone or placebo

Type-2 diabetic volunteers had baseline bone marrow aspirations and again following 26 weeks of treatment with placebo or PIO. (A) CFU-OB, both Alizarin Red S (ARS) and alkaline phosphatase (ALP) positive colonies were calculated as percent change from baseline for each patient and the mean calculated for each treatment group. (B) Changes in RNA levels from baseline to final visit of parallel CFU-OB assays were calculated for Runx2 and osteocalcin (Osc) from the PIO treated group. (C) The percent of CFU-AD positive colonies, based on Oil-red-O staining, was calculated as change from baseline for each patient and the mean calculated for each treatment group. (D) Changes in RNA levels from baseline to final visit of parallel CFU-AD assays were calculated for PPARγ2 and Adiponectin (AdipoQ) from the PIO treated group. (E) The total number of colonies was calculated from both the CFU-OB and CFU-AD assays, representing 12 wells per patient, for each patient group at baseline (B) and final (F) visit. Results are expressed as the mean of placebo (N=3) and 5 PIO (N=5) treated patients as indicated +/−SEM. *p<0.05 (paired student T-Test).