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Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

Papadimou E, Morigi M, Iatropoulos P, Xinaris C, Tomasoni S, Benedetti V, Longaretti L, Rota C, Todeschini M, Rizzo P, Introna M, Grazia de Simoni M, Remuzzi G, Goligorsky MS, Benigni A - Stem Cell Reports (2015)

Bottom Line: Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts.RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway.Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

View Article: PubMed Central - PubMed

Affiliation: IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri," Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy. Electronic address: evangelia.papadimou@marionegri.it.

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Antigenic Profile of Human BMSCs after HK2 Cell-Extract Treatment(A–H) Expression of ENG, CDH1, TJP1, and AQP1 in BMSCs treated with extract at D8 (A–D) and D25 (E–H). (H) High-magnification view of the expression of ENG and AQP1 at the edge of the epithelial colony presented in (E). Scale bar, 50 μm. Representative images of three independent experiments.(I) Representative FACS analysis of BMSCs treated with HK2 cell extract at different time points (D8, D25, and D35), and of untreated BMSCs and HK2 cells. Values are % of fluorescent cells (mean ± SD, n = 3 independent experiments).(J) Representative images of CDH1/EGFP-positive cells 24 hr after transfection (days 6 and 12). Untreated BMSCs and HK2 cells are also shown. Scale bars represent 50 μm (BMSCs + extract [D6, D12] and BMSCs) and 100 μm (HK2). Transfection was performed in two independent reprogramming experiments (n = 2 for each time point).See also Figure S1.
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fig2: Antigenic Profile of Human BMSCs after HK2 Cell-Extract Treatment(A–H) Expression of ENG, CDH1, TJP1, and AQP1 in BMSCs treated with extract at D8 (A–D) and D25 (E–H). (H) High-magnification view of the expression of ENG and AQP1 at the edge of the epithelial colony presented in (E). Scale bar, 50 μm. Representative images of three independent experiments.(I) Representative FACS analysis of BMSCs treated with HK2 cell extract at different time points (D8, D25, and D35), and of untreated BMSCs and HK2 cells. Values are % of fluorescent cells (mean ± SD, n = 3 independent experiments).(J) Representative images of CDH1/EGFP-positive cells 24 hr after transfection (days 6 and 12). Untreated BMSCs and HK2 cells are also shown. Scale bars represent 50 μm (BMSCs + extract [D6, D12] and BMSCs) and 100 μm (HK2). Transfection was performed in two independent reprogramming experiments (n = 2 for each time point).See also Figure S1.

Mentions: To explore the renal identity of BMSCs treated with HK2 cell extracts, we studied candidate proximal tubular epithelial cell markers using immunofluorescence microscopy and flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. BMSCs consistently expressed endoglin (ENG; also known as CD105), characteristic of the mesenchymal lineage, and were devoid of the epithelial markers E-cadherin (CDH1) and aquaporin-1 (AQP1) (Figures S1A–S1D). After 8 days, BMSCs treated with HK2 cell extracts weakly expressed CDH1, TJP1 (tight junction protein 1; also known as zona occludens-1), and AQP1 in combination with BMSC markers (Figures 2A–2D). Moreover, after 25 days, BMSCs treated with HK2 cell extracts displayed robust expression of the epithelial markers but substantially reduced expression of ENG (Figures 2E and 2H), which is consistent with the expression profile of HK2 cells (Figures S1E–S1H). Notably, BMSCs that were not converted to cobblestone islands maintained the expression of mesenchymal markers (Figures 2E and 2H, area defined by the dotted line). BMSCs grown in HK2 cell medium, used as control, did not express epithelial markers and retained the expression of ENG (Figures S1I–S1L).


Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

Papadimou E, Morigi M, Iatropoulos P, Xinaris C, Tomasoni S, Benedetti V, Longaretti L, Rota C, Todeschini M, Rizzo P, Introna M, Grazia de Simoni M, Remuzzi G, Goligorsky MS, Benigni A - Stem Cell Reports (2015)

Antigenic Profile of Human BMSCs after HK2 Cell-Extract Treatment(A–H) Expression of ENG, CDH1, TJP1, and AQP1 in BMSCs treated with extract at D8 (A–D) and D25 (E–H). (H) High-magnification view of the expression of ENG and AQP1 at the edge of the epithelial colony presented in (E). Scale bar, 50 μm. Representative images of three independent experiments.(I) Representative FACS analysis of BMSCs treated with HK2 cell extract at different time points (D8, D25, and D35), and of untreated BMSCs and HK2 cells. Values are % of fluorescent cells (mean ± SD, n = 3 independent experiments).(J) Representative images of CDH1/EGFP-positive cells 24 hr after transfection (days 6 and 12). Untreated BMSCs and HK2 cells are also shown. Scale bars represent 50 μm (BMSCs + extract [D6, D12] and BMSCs) and 100 μm (HK2). Transfection was performed in two independent reprogramming experiments (n = 2 for each time point).See also Figure S1.
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Related In: Results  -  Collection

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fig2: Antigenic Profile of Human BMSCs after HK2 Cell-Extract Treatment(A–H) Expression of ENG, CDH1, TJP1, and AQP1 in BMSCs treated with extract at D8 (A–D) and D25 (E–H). (H) High-magnification view of the expression of ENG and AQP1 at the edge of the epithelial colony presented in (E). Scale bar, 50 μm. Representative images of three independent experiments.(I) Representative FACS analysis of BMSCs treated with HK2 cell extract at different time points (D8, D25, and D35), and of untreated BMSCs and HK2 cells. Values are % of fluorescent cells (mean ± SD, n = 3 independent experiments).(J) Representative images of CDH1/EGFP-positive cells 24 hr after transfection (days 6 and 12). Untreated BMSCs and HK2 cells are also shown. Scale bars represent 50 μm (BMSCs + extract [D6, D12] and BMSCs) and 100 μm (HK2). Transfection was performed in two independent reprogramming experiments (n = 2 for each time point).See also Figure S1.
Mentions: To explore the renal identity of BMSCs treated with HK2 cell extracts, we studied candidate proximal tubular epithelial cell markers using immunofluorescence microscopy and flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. BMSCs consistently expressed endoglin (ENG; also known as CD105), characteristic of the mesenchymal lineage, and were devoid of the epithelial markers E-cadherin (CDH1) and aquaporin-1 (AQP1) (Figures S1A–S1D). After 8 days, BMSCs treated with HK2 cell extracts weakly expressed CDH1, TJP1 (tight junction protein 1; also known as zona occludens-1), and AQP1 in combination with BMSC markers (Figures 2A–2D). Moreover, after 25 days, BMSCs treated with HK2 cell extracts displayed robust expression of the epithelial markers but substantially reduced expression of ENG (Figures 2E and 2H), which is consistent with the expression profile of HK2 cells (Figures S1E–S1H). Notably, BMSCs that were not converted to cobblestone islands maintained the expression of mesenchymal markers (Figures 2E and 2H, area defined by the dotted line). BMSCs grown in HK2 cell medium, used as control, did not express epithelial markers and retained the expression of ENG (Figures S1I–S1L).

Bottom Line: Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts.RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway.Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

View Article: PubMed Central - PubMed

Affiliation: IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri," Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy. Electronic address: evangelia.papadimou@marionegri.it.

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Related in: MedlinePlus