Tissue-Specific Cultured Human Pericytes: Perivascular Cells from Smooth Muscle Tissue Have Restricted Mesodermal Differentiation Ability

Abstract

Microvascular pericytes (PCs) are considered the adult counterpart of the embryonic mesoangioblasts, which represent a multipotent cell population that resides in the dorsal aorta of the developing embryo. Although PCs have been isolated from several adult organs and tissues, it is still controversial whether PCs from different tissues exhibit distinct differentiation potentials. To address this point, we investigated the differentiation potentials of isogenic human cultured PCs isolated from skeletal (sk-hPCs) and smooth muscle tissues (sm-hPCs). We found that both sk-hPCs and sm-hPCs expressed known pericytic markers and did not express endothelial, hematopoietic, and myogenic markers. Both sk-hPCs and sm-hPCs were able to differentiate into smooth muscle cells. In contrast, sk-hPCs, but not sm-hPCs, differentiated in skeletal muscle cells and osteocytes. Given the reported ability of the Notch pathway to regulate skeletal muscle and osteogenic differentiation, sk-hPCs and sm-hPCs were treated with N-[N-(3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a known inhibitor of Notch signaling. DAPT treatment, as assessed by histological and molecular analysis, enhanced myogenic differentiation and abolished osteogenic potential of sk-hPCs. In contrast, DAPT treatment did not affect either myogenic or osteogenic differentiation of sm-hPCs. In summary, these results indicate that, despite being isolated from the same anatomical niche, cultured PCs from skeletal muscle and smooth muscle tissues display distinct differentiation abilities.

FIG. 1.

Characterization of cultured PCs from skeletal and smooth muscle tissues. (A) Flow cytometry analysis on cultured skeletal muscle-derived human pericytes (sk-hPCs) and cultured smooth muscle-derived human pericytes (sm-hPCs). Representative plots of a panel of markers (green: CD13, CD31, CD34, CD44, CD45, CD146, CD73, CD90, CD105, and NG2) are shown for isogenic cell populations isolated from donors #3 and #5. Ig isotype-matched antibodies are shown in red. (B) Phase contrast images of isogenic sk-hPCs (a, b) and sm-hPCs (c, d) from donors #3 and #5. Scale bars: 100 μm. (C) RT-PCR analysis of the expression of PDGFRB, ALP, MYOD, and MYF5 genes in isogenic cultured PCs from skeletal (sk) and smooth (sm) muscle of donors #1 and #3. Total RNA from human bone marrow-derived mesenchymal stem cells (MSCs) and no-template samples were used as positive (+) and negative (−) controls, respectively, for PDGFRB and ALP. Total RNA from human myoblasts and terminal differentiated osteocytes were used as positive (+) and negative (−) controls, respectively, for MYF5 and MYOD. The expression of β-actin was used as control. β-actin expression of human bone marrow MSCs and no-template samples are not shown. (D) Western blot analysis of NG2 and desmin expression in isogenic cultured PCs from skeletal (sk) and smooth (sm) muscle of donors #2 and #4. Expression of β-actin was used as loading control. (E) Immunofluorescence microscopy analysis with anti-desmin (a–d) and anti-α-SMA (e–h) antibodies (both revealed in green) in sk-hPCs and sm-hPCs from donors #2 and #6. Nuclei were counterstained with TO-PRO^®-3 Iodide. Scale bars: 20 μm. α-SMA, smooth muscle actin-α; ALP, alkaline phosphatase; NG2, neural/glial antigen 2; PCs, pericytes; PDGFR-β, platelet-derived growth factor receptor-β; RT-PCR, reverse transcription–polymerase chain reaction. Color images available online at www.liebertpub.com/scd

FIG. 2.

Smooth muscle, osteogenic, and adipogenic differentiation of sk-hPCs and sm-hPCs. (A) Immunofluorescence analysis with anti-SM22-α (red) of isogenic sk-hPCs and sm-hPCs from all donors (#1–#6), following 8 days of smooth muscle differentiation. Nuclei were counterstained with DAPI. (B) Western blot (a) and relative densitometric analysis (b) of the expression of α-SMA and SM22-α of isogenic sk-hPCs and sm-hPCs from donors #4 and #6 before (undifferentiated, u) and following 8 days of smooth muscle differentiation (differentiated, d). α-Tubulin was used as loading control. Densitometric values relative to undifferentiated cells that are arbitrarily set as one, are reported as mean ± SEM (n = 2). (C) Alizarin Red staining of calcified bone mineral matrix deposited by sk-hPCs and sm-hPCs from all donors (#1–#6). Sk-hPCs (a–f) and sm-hPCs (g–l) were stained 2 and 4 weeks after the induction of osteogenic differentiation, respectively. (D) Oil Red O staining of lipid droplets in sk-hPCs (a–f) and sm-hPCs (g–l) from all donors (#1–#6). All cell populations were stained 2 weeks after the induction of adipogenic differentiation. For all cell populations, three experimental replicates were performed in each differentiation assay. Scale bars: 20 μm (A), 4.35 mm (C), and 100 μm (D). DAPI, 4′,6-diamidino-2-phenylindole; SM22-α, smooth muscle protein-22 α. Color images available online at www.liebertpub.com/scd

FIG. 3.

Skeletal muscle (SKM) differentiation of sk-hPCs and sm-hPCs. (A) Immunofluorescence analysis of α-actinin expression in isogenic sk-hPCs (a–f) and sm-hPCs (g–l) from all donors (#1–#6), 10 days following induction of skeletal myogenic differentiation. Scale bars: 20 μm. (B) Detailed immunofluorescence analysis of SKM differentiation of sk-hPCs (a–c, g–i, m–o) and sm-hPCs (d–f, j–l, p–r) following 2 days (a–f), 7 days (g–l), and 15 days (m–r) of differentiation. MYOD (a, c, d, f; red) and Myogenin (b, c, e, f; green) double-positive nuclei were detected after 2 days of differentiation only in sk-hPCs. α-Actinin (h, i, k, l; violet)-positive myotubes that contain several myogenin-positive nuclei (g, i, j, l) were identified at 7 days of differentiation only in sk-hPCs. Fifteen days after the induction of differentiation, α-actinin-positive myotubes were detected in differentiating sk-hPCs (n, o), but not in sm-hPCs (q, r). To visualize cell morphology, F-actin cytoskeleton of 15 days differentiated cells was stained by rhodamine-conjugated phalloidin (m, o, p, r; red). All nuclei were counterstained with DAPI. For all cell populations three experimental replicates were performed in each differentiation assay. Scale bars: 20 μm. (C) Relative expression analyses of mir-1, mir-133b, and mir-206 in sk-hPCs and sm-hPCs following 3 days of SKM differentiation. The expression of each target microRNA in undifferentiated cells was used as internal reference and arbitrarily set as one. (D) Relative expression analysis of mir-1, mir-133b, and mir-206 in undifferentiated sk-hPCs and sm-hPCs, and in differentiating sk-hPCs and sm-hPCs 3 days after the induction of SKM differentiation. MyomiRs expression in sk-hPCs was used as internal reference and arbitrarily set as one in both undifferentiated and differentiating cells. (E) Quantitative analysis of PW1/Peg3 in undifferentiated sk-hPCs and sm-hPCs. For the analysis of relative expression, PW1/Peg3 expression in sk-hPCs was used as internal reference and arbitrarily set as one. (C–E) 2^−ΔΔCt method was used to calculate the relative fold change of target expression. Data reported in (C, D) are mean + SEM of six pairs of isogenic sk-hPCs and sm-hPCs. Data reported in E are the mean + SD of three different experiments performed on each cell population. Statistical analyses were performed by Wilcoxon matched pairs, nonparametric T-test (C) and Mann–Whitney unpaired, nonparametric T-test (D, E). Significant P value <0.05; *P < 0.05; **P < 0.01; ***P < 0.001. PW1/Peg3, PW1/paternally expressed gene 3. Color images available online at www.liebertpub.com/scd

FIG. 4.

Effects of Notch pathway inhibition on myogenic ability of sk-hPCs and sm-hPCs. Representative immunofluorescence analysis of α-Actinin expression in sk-hPCs (A) and sm-hPCs (B) cultured in standard myogenic differentiation medium (CTRL, a) or in myogenic differentiation medium supplemented with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, b), the γ-secretase inhibitor, for 12 days. Nuclei were stained with DAPI. Relative fusion index of CTRL and DAPT-treated cells are reported in the histograms in (A, c and B, c). Fusion index of CTRL cells was arbitrarily set as one. Plotted data are the mean ratio ± SEM of fusion index of six DAPT-treated cell populations over six CTRL cell populations. Fusion index was calculated as the number of nuclei inside α-actinin-positive multinucleated cells, divided by the total number of nuclei. ND, not detected. (C) Quantitative analysis by real-time PCR of MYOD and MYF5 expression in sk-hPCs from all donors (n = 6), 2 and 5 days after the induction of SKM differentiation. Relative expression in CTRL and DAPT-treated cells was calculated by 2^−ΔΔCt method. MYOD and MYF5 expression in CTRL cells was arbitrarily set as one. Reported data are mean ± SEM. (D) Relative expression analysis of mir-1, mir-133b, and mir-206 in CTRL and DAPT-treated undifferentiated sk-hPCs from all donors (n = 6). Relative expression between CTRL and DAPT-treated cells was calculated by 2^−ΔΔCt method. MyomiR expression in CTRL cells was arbitrarily set as one. Statistical analyses were performed by Mann–Whitney unpaired, nonparametric T-test. Reported data are mean ± SEM. (E) Relative myogenic differentiation efficiency of sk-hPCs cultured with (+) or without (−) DAPT administration during proliferation and/or SKM differentiation. Plotted data are the mean ratio ± SEM of fusion index of three treated sk-hPCs populations over three CTRL cell populations. Fusion index of CTRL cells (−/−) was arbitrarily set as one. Significant P value <0.05; *P < 0.05; **P < 0.01. Scale bars: 20 μm.

FIG. 5.

Effects of Notch pathway inhibition on osteogenic ability of sk-hPCs and sm-hPCs. Osteogenic differentiation ability of sk-hPCs (A) and sm-hPCs (B) was visualized by Alizarin Red staining of bone mineral matrix 4 weeks after the induction of differentiation. Representative micrographs of control (CTRL) and DAPT-treated sk-hPCs and sm-hPCs cultured in standard osteogenic medium (CTRL, a) or in osteogenic medium supplemented with DAPT (DAPT, b). Relative quantification analysis of osteogenesis by Alizarin Red extraction in CTRL and DAPT-treated cells are reported in the histograms in (A, c and B, c). The absorbance of extracted Alizarin Red was converted in μg and normalized to DNA content. Normalized Alizarin Red content of CTRL cells was arbitrarily set as one. Plotted data are the mean ratio ± SEM of three different cell populations per treatment. (C) Quantitative analysis by real-time PCR of TAZ, RUNX2, and OSX expression of three sk-hPCs populations, before induction of differentiation and 2 and 5 days after osteogenesis induction. Relative expression between CTRL and DAPT-treated cells was calculated by 2^−ΔΔCt method. TAZ, RUNX2, and OSX expression in control cells was arbitrarily set as one. Reported data are mean ± SEM. (D) Relative quantification analysis of sk-hPCs cultured with (+) or without (−) DAPT administration during proliferation and/or osteogenic differentiation. Plotted data are the mean ratio ± SEM of normalized Alizarin Red content of three treated sk-hPCs over three control cell populations. Significant P value <0.05; *P < 0.05; **P < 0.01. Scale bars: 100 μm. RUNX2, Runt-related transcription factor 2; TAZ, PDZ-binding motif. Color images available online at www.liebertpub.com/scd