Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules.

Human mesenchymal stem cells (hMSC) are easily isolated from the bone marrow by adherence to plastic surfaces. These cells show self-renewal capacity and multipotency. A unique feature of hMSC is their capacity to survive without serum. Under this condition hMSC neither proliferate nor differentiate but maintain their biological properties unaffected. Therefore, this should be a perfect platform to study the biological effects of defined molecules on these human stem cells. We show that hMSC treated for five days with retinoic acid (RA) in the absence of serum undergo several transcriptional changes causing an inhibition of ERK related pathways. We found that RA induces the loss of hMSC properties such as differentiation potential to either osteoblasts or adipocytes. We also found that RA inhibits cell cycle progression in the presence of proliferating signals such as epidermal growth factor (EGF) combined with basic fibroblast growth factor (bFGF). In the same manner, RA showed to cause a reduction in cell adhesion and cell migration. In contrast to these results, the addition of EGF+bFGF to serum free cultures was enough to upregulate ERK activity and induce hMSC proliferation and cell migration. Furthermore, the addition of these factors to differentiation specific media instead of serum was enough to induce either osteogenesis or adipogenesis. Altogether, our results show that hMSC's ability to survive without serum enables the identification of signaling factors and pathways that are involved in their stem cell biological characteristics without possible serum interferences

RA and EGF+bFGF effects on Erk phosphorylation and on cell cycle progression.

<p>A: Western blot analysis of hMSC that were cultured for 5 days in either DMEM alone (DMEM) or in the presence of 0.5 µM RA (RA) or in the presence of 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+bFGF), with or without the addition of 0.5 µM RA (EGF+bFGF+RA). As control, cells were cultured with 10%FBS (FBS), or with 0.5 µM RA in 10%FBS (FBS+RA). Whole-cell protein extracts from these differently treated cells were fractionated on a denaturating 12% polyacrylamide gel, transferred to nitrocellulose and detected with anti phosphorylated Erk antibody (pErk1/2). The membrane was stripped twice, one for detection with anti total Erk antibody (Erk1) and the second for β-actin antibody detection used as loading control. B: Densitometry analysis of A. The bars represent relative expression normalized to β-actin expression and referred to this ratio in DMEM. C–J: Cell cycle progression by FACS of hMSC that were cultured for 5 days with DMEM (C), 0.5 µM RA in DMEM (D), 20 ng/ml EGF (E), 5 ng/ml bFGF (F), 5 ng/ml bFGF +20 ng/ml EGF (G), or 5 ng/ml bFGF +20 ng/ml EGF +0.5 µM RA (H). In addition, hMSC were cultured for 2 days in DMEM (I) or in the presence of 0.5 µM RA (J) before replacement of the medium with 20 ng/ml EGF +5 ng/ml bFGF in DMEM for further 2 days. At the end of the experiment the cells were harvested by trypsinization, permeabilized and stained with propidium iodide to measure the DNA content by FACS.</p

Antibodies used for Western blot in this study.

<p>Antibodies used for Western blot in this study.</p

Periostin can rescue the effect of RA on hMSC osteogenesis.

<p>A: hMSC were treated for 5 days with either DMEM, 0.5 µM RA in DMEM (RA), 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+FGF), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (EGF+FGF+RA), 10%FBS (FBS), or 0.5 µM RA in 10%FBS (FBS+RA). The RNAs were purified and the cDNAs were synthesized using reverse transcriptase. Periostin transcript was quantified in each sample. The error bars represent relative expression normalized to RNF10 expression and referred to the relative expression on DMEM as mean ± s.e.m. All the treatments were performed in triplicates in two independent experiments with cDNA from different donors and the significance of the results was assessed using one way ANOVA and Tukey's multiple comparison test. B–G: hMSC were cultured in osteoblasts differentiation medium supplemented with 0.5 µM RA (B–D), or in 10%FBS +0.5 µM RA (negative control) (E–G), for 21 days over uncoated plastic (B and E), 2.5 µg laminin/well coated plastic (C and F), or 2.5 µg periostin/well coated plastic (D and G). Osteoblasts were stained with alizarin red. Size bars = 100 µm.</p

hMSC osteogenesis is inhibited by RA and enhanced by EGF+bFGF.

<p>hMSC were cultured for 21 days in osteoblasts differentiation medium without serum supplemented with 10% FBS (A), 10% FBS +0.5 µM RA (C), nothing (D), 0.5 µM RA (E), 20 ng/ml EGF +5 ng/ml bFGF (F), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (G), or were cultured in DMEM supplemented with 10% FBS (B) or with 20 ng/ml EGF +5 ng/ml bFGF (H) (negative controls). To visualize differentiation osteoblasts were stained with alizarin red after fixation in 70% ethanol. Size bars = 100 µm.</p

Primers used for QRT-PCR in this study.

<p>Primers used for QRT-PCR in this study.</p

Microarray bioinformatical analysis of the RA effects on hMSC cultured for 5 days without serum.

<p>A: Venn diagram showing the number of differentially expressed transcripts in DMEM and RA treated hMSC when compared to 10%FBS treated cells. B: The genes regulated specifically by RA (i.e. blue or yellow in the diagram depicted in A) were clustered by Ingenuity software. This software contains a database with different transcripts arranged in networks according to their known biological interactions. According to the number of transcripts regulated in each of these networks, the program scores them. The best scored network is depicted here. Direct interactions are represented by continuous arrows and direct by dotted ones. Increase in expression is represented by red color and decrease by green. The number under each transcript name is the logarithmic change in comparison to 10%FBS control cells RNA. The type of interaction is indicated by a label and the type of molecule is indicated by the shape of the box, being both detailed at the right of the figure.</p

hMSC adipogenesis is inhibited by RA and enhanced by EGF+bFGF.

<p>hMSC were cultured for 21 days in either adipocytes maintenance medium (days 7–8 and 15–16) or adipocytes induction medium (the rest of the days) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012689#s4" target="_blank">Materials and Methods</a> for seeing the media composition), without serum, supplemented with 10% FBS (A), 10% FBS +0.5 µM RA (C), nothing (D), 0.5 µM RA (E), 20 ng/ml EGF +5 ng/ml bFGF (F), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (G). In addition, hMSC were cultured in DMEM supplemented with 10% FBS (B) or with 20 ng/ml EGF +5 ng/ml bFGF (H) (negative controls). To visualize differentiation, adipocytes were stained with Oil red O after fixation with 4% paraformaldehyde. Size bars = 100 µm.</p