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A Reduction in CD90 (THY-1) Expression Results in Increased Differentiation of Mesenchymal Stromal Cells

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A Reduction in CD90 (THY-1) Expression Results in Increased Differentiation of Mesenchymal Stromal Cells

Daniela A Moraes et al. Stem Cell Res Ther.

Abstract

Background: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs.

Methods: We investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors.

Results: The present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression.

Conclusion: Our study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro.

Keywords: CD90; Differentiation; Fibroblast; Mesenchymal stem cells; Mesenchymal stromal cells; Thy-1.

Figures

Fig. 1
Fig. 1
Reduction of CD90 in MSCs. a Non-transduced mesenchymal stromal cells (MSC) and MSCs transduced with lentiviral particles expressing short hairpin (sh)RNA against CD90 (shRNA CD90 MSC) were analysed by flow cytometry. An accentuated decrease in CD90 expression is observed in shRNA CD90 MSC (thick line), whereas non-transduced MSCs (slim line) expressed high levels of CD90. The shaded histogram indicates staining with isotype control antibody. Representative histograms from dental pulp MSCs are shown. b Significant decrease of CD90 median fluorescence intensity (MFI) on shRNA CD90 MSCs when compared to non-transduced MSCs. (MFI = MFI marker – MFI isotype). Bar graphs represent the average mean fluorescence intensity as the median ± SD of CD90-FITC on cell lines used in this work; n = 7; ****p < 0.001. c Relative mRNA expression levels of CD90 in MSC, shRNA control MSC, and shRNA CD90 MSC had consistently low expression of CD90. Data are presented as mean ± SD of experiments performed in triplicate; *p < 0.05
Fig. 2
Fig. 2
CD90-negative selection of shRNA CD90 MSCs by magnetic-activated cell sorting. Mesenchymal stromal cells (MSCs) expressing short hairpin (sh)RNA CD90 (shRNA CD90 MSC) were magnetically labelled with CD90 microbeads, and MSC populations negatively selected for CD90 (CD90-negative MSCs). a Histogram superposition: isotype control (shaded histogram grey), non-transduced MSCs (black slim line), shRNA control MSCs (grey line), shRNA CD90 MSCs (blue line), and CD90-negative MSCs (black thick line).Representative histograms from a dental pulp MSC group are shown. b Graph showing mean fluorescence intensity (MFI) of CD90 marker (MFI = MFI marker – MFI isotype) on cells; n = 7, independent experiments with MSCs derived from 7 tissue donors; ****p < 0.001
Fig. 3
Fig. 3
Reduction of CD90 expression does not affect mesenchymal stromal cell (MSC) morphology and proliferation rate. a Representative phase contrast microscopy images of MSCs derived from dental pulp. All MSCs displayed a spindle-like morphology exhibiting relatively thin processes extending from the cell bodies. The photographs shown here are representative of all samples analysed (n = 7). b Proliferation curves of non-transduced MSCs, short hairpin (sh)RNA control MSCs, shRNA CD90 MSCs, and CD90-negative MSCs. Data shown represent the mean ± SD of two independent experiments performed in triplicate with dental pulp MSCs from two tissue donors; *p < 0.05
Fig. 4
Fig. 4
T-cell proliferation assays. Assays were performed using carboxyfluorescein succinimidyl ester (CFSE)-labelled human peripheral blood mononuclear cells (PBMC) activated with phytohaemagglutinin and co-cultured with or without human dental pulp mesenchymal stromal cells (MSC) for 5 days. a Representative histograms from dental pulp MSC cytometry analysis are shown (n = 3). b Histograms showing number of CFSE-labelled activated cells. c Histograms showing percentage proliferation of CFSE-labelled CD8+ T cells. n = 3; *p < 0.05. sh short hairpin
Fig. 5
Fig. 5
Reduction of CD90 expression leads to a reduction in the expression of CD44 and CD166 in mesenchymal stromal cells (MSCs). a MSCs (slim line) and shRNA CD90 MSCs (thick line) were analysed by flow cytometry to evaluate their expression of CD44 and CD166. The results showed a significant reduction of CD44 and CD166 expression in shRNA CD90 MSCs from different tissue sources. An isotype control (shaded histogram) was used to establish the boundary between negative and positive fluorescent regions. Median fluorescence intensities (MFI) of b CD44 and c CD166 markers on MSCs are shown (MFI = MFI marker – MFI isotype); n = 7; *p < 0.05. ADSC adipose tissue mesenchymal stromal cell, AF-MSC amniotic fluid mesenchymal stromal cell, DPSC dental pulp mesenchymal stromal cell, sh short hairpin
Fig. 6
Fig. 6
Reduction of CD90 expression stimulates MSC osteogenesis. MSCs, shRNA control MSCs, shRNA CD90 MSCs, and CD90-negative MSCs from dental pulp (DPSC), amniotic fluid (AF-MSC) and adipose tissue (ADSC) were tested in parallel for their ability to differentiate in vitro into osteogenic lineages. Calcified deposits were evidenced by Alizarin Red Staining (AR) in cells after 4 weeks of growth in osteogenic induction medium. Calcification was assessed by gross appearance (a) and light microscopy (b). Data shown are representative of multiple replicates. OS osteogenic induction, sh short hairpin
Fig. 7
Fig. 7
Quantitative evaluation of osteogenesis. a Quantification of Alizarin Red staining by dissolving the dye and subsequent absorption measurement. b Alkaline phosphatase (ALP) activity in cells cultured in osteogenic medium. ALP activity (mU μmol p-nitrophenol released per min) was normalized for protein. c Calcium concentration determinations were possible only for DPSC samples and AF-MSC samples. The data are expressed as mean ± SD and are representative of two independent experiments, each performed in triplicate (DPSC = 2 donors, ADSC = 2 donors, AF-MSC = 2 donors). *p < 0.05; **p < 0.01; ***p < 0.001. ADSC adipose tissue mesenchymal stromal cell, AF-MSC amniotic fluid mesenchymal stromal cell, DPSC dental pulp mesenchymal stromal cell, MSC mesenchymal stromal cell, sh short hairpin
Fig. 8
Fig. 8
Reduction of CD90 expression stimulates the adipogenesis of mesenchymal stromal cells (MSCs). MSCs, short hairpin (sh)RNA control MSCs, shRNA CD90 MSCs, and CD90-negative MSCs were tested for their ability to differentiate into adipogenic lineages. a Representative photomicrograph images show oil red staining indicative of adipogenic differentiation. MSCs from dental pulp (DPSC), amniotic fluid (AF-MSCs), and lipoaspirate (ADSC) were cultured in the non-differentiation medium MSCs (control) and adipogenic differentiation medium (AD). The images shown are representative of two independent experiments. b Oil red dye retained in the lipid vacuoles was measured by determining the optical density (OD) at 510 nm. Data shown represent the mean ± SD of one experiment performed in triplicate (n = 7). *p < 0.05; **p < 0.01; ***p < 0.001

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