CTLA-4 mediates inhibitory function of mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult

Hypoxia promotes osteogenesis but suppresses adipogenesis of human mesenchymal stromal cells in a hypoxia-inducible factor-1 dependent manner.

BACKGROUND: Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages. METHODOLOGY/PRINCIPAL FINDINGS: Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O(2)) or hypoxic (less than 2% O(2)) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis. CONCLUSIONS/SIGNIFICANCE: Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches

Percentage of CD-positive cell populations after 2 week incubation under hypoxia.

<p>Percentage of CD-positive cell populations after 2 week incubation under hypoxia.</p

Percentage of CD-positive cell populations after 2 week incubation under normoxia.

<p>Percentage of CD-positive cell populations after 2 week incubation under normoxia.</p

Hypoxia suppresses adipogenic and promotes osteogenic differentiation of human MSCs.

<p>(<b>a</b>) Oil-Red-O stain for the analysis of adipogenesis and von-Kossa stain for the analysis of osteogenesis of MSCs incubated under normoxia (≈18% pO₂) or hypoxia (1% pO₂) for 4 weeks using either osteogenic or adipogenic differentiation medium (scale indicated on the figures; n = 6). (<b>b</b>) HIF1A, (<b>c</b>) VEGFA, (<b>d</b>) PPARG and (<b>e</b>) RUNX2 gene expression of MSCs incubated under normoxia (≈18% pO₂) or hypoxia (1% pO₂) for 2 weeks using either osteogenic or adipogenic differentiation medium as obtained by real-time PCR (n = 6; unpaired t-test; dotted-line indicates normalisation to gene expression of undifferentiated cells; * p<0.05). (<b>f</b>) Analysis of osteogenesis by von-Kossa stain of MSCs incubated under normoxia (≈18% pO₂) without treatment, or with either 250 µM DFX and 100 µM DMOG, respectively (scale indicated on the figures; n = 3).</p

Characterization of human MSCs by their surface marker expression and their ability to differentiate into mesenchymal lineages.

<p>(<b>a</b>) Bone marrow derived human MSCs express CD13, CD44, CD73, CD90, and CD105 but do not express CD45, CD34, CD14, and CD19; they differentiate into different mesenchymal lineages such as (<b>b</b>) adipogenic, (<b>c</b>) osteogenic and (<b>d</b>) chondrogenic lineages (scale indicated on the figures; n = 8).</p

Patient information and experiments conducted.

*<p>nonsteroidal anti-inflammatory drugs,</p>#<p>hip osteoarthritis,</p>§<p>Western-Blot.</p

Hypoxia induces HIF-1α and HIF-1-target-gene expression.

<p>(<b>a</b>) HIF1A gene expression of human MSCs obtained by real-time PCR (n = 6; unpaired t-test). (<b>b</b>) HIF-1alpha and beta-actin protein expression obtained by immunoblot. (<b>c</b>) Hypoxia-induced HIF-1-target-gene expression of VEGFA, LDHA, GLUT1, and PGK1 (n = 6; 2-weeks data; one sample t-test; dotted-line indicates normalization to gene expression of normoxic cells; *** p<0.001; ** p<0.01; * p<0.05).</p

Primersets used.

<p>Primersets used.</p