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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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Morphological and Ultrastructural Changes in Human BMSCs after HK2 Cell-Extract Treatment(A) Human BMSC morphology 24 hr after extract treatment (D1).(B) Cobblestone morphology of the BMSCs 13 days (D13) after extract treatment.(C) Epithelial colony 15 days (D15) after extract treatment.(D) High magnification of the epithelial colony showing the cobblestone cell morphology.(E and F) Dome formation (E, arrows) and tubular structures (F) after 25- and 35-day BMSC extract treatment (D25 and D35), respectively.(G) Control HK2 cell morphology on the day of the extract preparation.(H) Dome formation (arrows) under overconfluence culture conditions.(I and J) BMSCs cultured in HK2 cell culture media at 24 hr (I, D1) and 35 days (J, D35).Human BMSCs treated with HK2 cell extract form microvilli and tight intercellular contacts.(K and L) Details of adjacent BMSCs treated with extracts grown on Thermanox for 35 days. Tight intercellular contacts are formed in BMSCs treated with HK2 cell extracts at D35 (arrowheads).(M) Brush border formed in a BMSC treated with extract after 35 days.(N–P) TEM images of HK2 cells (N and O) and BMSCs (P) grown in HK2 cell medium for 35 days (D35). Images are representative of two independent experiments.Scale bars represent 200 μm (A), 50 μm (B and D–J), and 100 μm (C). Images are representative of three independent experiments.
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fig1: Morphological and Ultrastructural Changes in Human BMSCs after HK2 Cell-Extract Treatment(A) Human BMSC morphology 24 hr after extract treatment (D1).(B) Cobblestone morphology of the BMSCs 13 days (D13) after extract treatment.(C) Epithelial colony 15 days (D15) after extract treatment.(D) High magnification of the epithelial colony showing the cobblestone cell morphology.(E and F) Dome formation (E, arrows) and tubular structures (F) after 25- and 35-day BMSC extract treatment (D25 and D35), respectively.(G) Control HK2 cell morphology on the day of the extract preparation.(H) Dome formation (arrows) under overconfluence culture conditions.(I and J) BMSCs cultured in HK2 cell culture media at 24 hr (I, D1) and 35 days (J, D35).Human BMSCs treated with HK2 cell extract form microvilli and tight intercellular contacts.(K and L) Details of adjacent BMSCs treated with extracts grown on Thermanox for 35 days. Tight intercellular contacts are formed in BMSCs treated with HK2 cell extracts at D35 (arrowheads).(M) Brush border formed in a BMSC treated with extract after 35 days.(N–P) TEM images of HK2 cells (N and O) and BMSCs (P) grown in HK2 cell medium for 35 days (D35). Images are representative of two independent experiments.Scale bars represent 200 μm (A), 50 μm (B and D–J), and 100 μm (C). Images are representative of three independent experiments.

Mentions: Human BMSCs were permeabilized with 400 ng/ml streptolysin O (SLO), a concentration that did not affect cell viability. Permeabilized BMSCs exposed to the extract of human proximal tubular epithelial (HK2) cells changed from their usual spindle-shape appearance (Figure 1A) to cobblestone islands within 13–15 days (Figures 1B–1D). During the subsequent 2 weeks, these islands expanded and the formation of “domes” and tubular-like structures (Figures 1E and 1F) similar to HK2 cells (Figure 1H) occurred. This morphological transition did not occur in BMSCs grown in epithelial-specific culture medium, even after 35 days (Figures 1I and 1J). The observed change in morphological appearance of BMSCs treated with epithelial cell extract suggested a phenotypic switch in the cells.


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)

Morphological and Ultrastructural Changes in Human BMSCs after HK2 Cell-Extract Treatment(A) Human BMSC morphology 24 hr after extract treatment (D1).(B) Cobblestone morphology of the BMSCs 13 days (D13) after extract treatment.(C) Epithelial colony 15 days (D15) after extract treatment.(D) High magnification of the epithelial colony showing the cobblestone cell morphology.(E and F) Dome formation (E, arrows) and tubular structures (F) after 25- and 35-day BMSC extract treatment (D25 and D35), respectively.(G) Control HK2 cell morphology on the day of the extract preparation.(H) Dome formation (arrows) under overconfluence culture conditions.(I and J) BMSCs cultured in HK2 cell culture media at 24 hr (I, D1) and 35 days (J, D35).Human BMSCs treated with HK2 cell extract form microvilli and tight intercellular contacts.(K and L) Details of adjacent BMSCs treated with extracts grown on Thermanox for 35 days. Tight intercellular contacts are formed in BMSCs treated with HK2 cell extracts at D35 (arrowheads).(M) Brush border formed in a BMSC treated with extract after 35 days.(N–P) TEM images of HK2 cells (N and O) and BMSCs (P) grown in HK2 cell medium for 35 days (D35). Images are representative of two independent experiments.Scale bars represent 200 μm (A), 50 μm (B and D–J), and 100 μm (C). Images are representative of three independent experiments.
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fig1: Morphological and Ultrastructural Changes in Human BMSCs after HK2 Cell-Extract Treatment(A) Human BMSC morphology 24 hr after extract treatment (D1).(B) Cobblestone morphology of the BMSCs 13 days (D13) after extract treatment.(C) Epithelial colony 15 days (D15) after extract treatment.(D) High magnification of the epithelial colony showing the cobblestone cell morphology.(E and F) Dome formation (E, arrows) and tubular structures (F) after 25- and 35-day BMSC extract treatment (D25 and D35), respectively.(G) Control HK2 cell morphology on the day of the extract preparation.(H) Dome formation (arrows) under overconfluence culture conditions.(I and J) BMSCs cultured in HK2 cell culture media at 24 hr (I, D1) and 35 days (J, D35).Human BMSCs treated with HK2 cell extract form microvilli and tight intercellular contacts.(K and L) Details of adjacent BMSCs treated with extracts grown on Thermanox for 35 days. Tight intercellular contacts are formed in BMSCs treated with HK2 cell extracts at D35 (arrowheads).(M) Brush border formed in a BMSC treated with extract after 35 days.(N–P) TEM images of HK2 cells (N and O) and BMSCs (P) grown in HK2 cell medium for 35 days (D35). Images are representative of two independent experiments.Scale bars represent 200 μm (A), 50 μm (B and D–J), and 100 μm (C). Images are representative of three independent experiments.
Mentions: Human BMSCs were permeabilized with 400 ng/ml streptolysin O (SLO), a concentration that did not affect cell viability. Permeabilized BMSCs exposed to the extract of human proximal tubular epithelial (HK2) cells changed from their usual spindle-shape appearance (Figure 1A) to cobblestone islands within 13–15 days (Figures 1B–1D). During the subsequent 2 weeks, these islands expanded and the formation of “domes” and tubular-like structures (Figures 1E and 1F) similar to HK2 cells (Figure 1H) occurred. This morphological transition did not occur in BMSCs grown in epithelial-specific culture medium, even after 35 days (Figures 1I and 1J). The observed change in morphological appearance of BMSCs treated with epithelial cell extract suggested a phenotypic switch in the cells.

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