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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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CL17 Cells Engrafted in Proximal Tubuli in Mice with Cisplatin-Induced AKI(A) Incorporation of CL17 cells into kidney tubuli of mice with cisplatin-induced AKI detected by human mitochondrial (h-Mito, upper-left panel; brown) and CENP-A (upper-right panel and insets; red) staining. Arrow indicates an h-Mito-positive cell in the proximal tubule connected to the glomerulus. No h-Mito- and CENP-A-positive cells are present in renal tissue of AKI animals with saline (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with wheat germ agglutinin (WGA, green). Scale bars represent 10 μm for h-Mito staining and 20 μm for CENP-A staining.(B) Renal localization of PKH26/positive CL17 cells (red) in AKI mice within proximal tubuli (top) or in peritubular areas (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with WGA (green). Scale bars, 20 μm.(C) Blood urea nitrogen (BUN) (mg/dl) levels in AKI mice treated with CL17 cells (n = 6) or saline (n = 5) at 4 days. Data are expressed as mean ± SD.(D) Quantification of tubular casts and necrotic tubuli (n/high-power field [HPF]) in kidneys of AKI mice treated with CL17 cells or saline at 4 days. ∗p < 0.05 versus cisplatin + saline.(E) Representative histological micrographs of the kidney of the AKI animals treated with CL17 cells (left) or saline (right) at 4 days. Scale bar, 50 μm.See also Figure S6.
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fig6: CL17 Cells Engrafted in Proximal Tubuli in Mice with Cisplatin-Induced AKI(A) Incorporation of CL17 cells into kidney tubuli of mice with cisplatin-induced AKI detected by human mitochondrial (h-Mito, upper-left panel; brown) and CENP-A (upper-right panel and insets; red) staining. Arrow indicates an h-Mito-positive cell in the proximal tubule connected to the glomerulus. No h-Mito- and CENP-A-positive cells are present in renal tissue of AKI animals with saline (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with wheat germ agglutinin (WGA, green). Scale bars represent 10 μm for h-Mito staining and 20 μm for CENP-A staining.(B) Renal localization of PKH26/positive CL17 cells (red) in AKI mice within proximal tubuli (top) or in peritubular areas (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with WGA (green). Scale bars, 20 μm.(C) Blood urea nitrogen (BUN) (mg/dl) levels in AKI mice treated with CL17 cells (n = 6) or saline (n = 5) at 4 days. Data are expressed as mean ± SD.(D) Quantification of tubular casts and necrotic tubuli (n/high-power field [HPF]) in kidneys of AKI mice treated with CL17 cells or saline at 4 days. ∗p < 0.05 versus cisplatin + saline.(E) Representative histological micrographs of the kidney of the AKI animals treated with CL17 cells (left) or saline (right) at 4 days. Scale bar, 50 μm.See also Figure S6.

Mentions: The renoprotective potential of the reprogrammed cells was investigated in an experimental murine model of cisplatin-induced AKI (Morigi et al., 2008). After intravenous injection of CL17 cells, 50% were recovered in the lung, 1% in the spleen, 0.47% in the liver, and 21% in the kidney; no cells were found in the heart (Figure S6A). Engrafted CL17 cells, stained for two human markers for mitochondria and centromere protein-A (CENP-A), in the renal parenchyma of immunodeficient AKI mice, were localized in the tubular compartment at 4 days post-cisplatin injection (Figure 6A) with a frequency averaging 213 PKH26+ve cells/100,000 renal cells. CL17 cells predominantly localized within proximal tubuli (80%) and, to a lesser extent (20%), in the peritubular areas (Figure 6B). As a positive control for human markers, we used human BMSCs that were mainly found at the peritubular level in tissues of cisplatin-treated mice (Figure S6B). Negative controls with non-immune immunoglobulin of the same isotype as the primary antibody are shown in Figure S6C. Next, we studied the effect of CL17 cell treatment on renal function impairment and tubular damage. In cisplatin-treated mice given CL17 cells, renal function was ameliorated (Figure 6C), although blood urea nitrogen (BUN) levels were not significantly different from those observed in untreated animals (22 ± 2 mg/dl) at 4 days. In parallel, renal damage was significantly improved in AKI animals infused with CL17 cells as demonstrated by the significant reduction of hyaline casts and necrotic tubuli (Figures 6D and 6E). Control mice did not show any signs of tubular damage. Altogether, these data indicate that the reprogrammed BMSCs directly incorporated into proximal tubuli and improved renal damage and function in AKI mice.


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)

CL17 Cells Engrafted in Proximal Tubuli in Mice with Cisplatin-Induced AKI(A) Incorporation of CL17 cells into kidney tubuli of mice with cisplatin-induced AKI detected by human mitochondrial (h-Mito, upper-left panel; brown) and CENP-A (upper-right panel and insets; red) staining. Arrow indicates an h-Mito-positive cell in the proximal tubule connected to the glomerulus. No h-Mito- and CENP-A-positive cells are present in renal tissue of AKI animals with saline (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with wheat germ agglutinin (WGA, green). Scale bars represent 10 μm for h-Mito staining and 20 μm for CENP-A staining.(B) Renal localization of PKH26/positive CL17 cells (red) in AKI mice within proximal tubuli (top) or in peritubular areas (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with WGA (green). Scale bars, 20 μm.(C) Blood urea nitrogen (BUN) (mg/dl) levels in AKI mice treated with CL17 cells (n = 6) or saline (n = 5) at 4 days. Data are expressed as mean ± SD.(D) Quantification of tubular casts and necrotic tubuli (n/high-power field [HPF]) in kidneys of AKI mice treated with CL17 cells or saline at 4 days. ∗p < 0.05 versus cisplatin + saline.(E) Representative histological micrographs of the kidney of the AKI animals treated with CL17 cells (left) or saline (right) at 4 days. Scale bar, 50 μm.See also Figure S6.
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fig6: CL17 Cells Engrafted in Proximal Tubuli in Mice with Cisplatin-Induced AKI(A) Incorporation of CL17 cells into kidney tubuli of mice with cisplatin-induced AKI detected by human mitochondrial (h-Mito, upper-left panel; brown) and CENP-A (upper-right panel and insets; red) staining. Arrow indicates an h-Mito-positive cell in the proximal tubule connected to the glomerulus. No h-Mito- and CENP-A-positive cells are present in renal tissue of AKI animals with saline (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with wheat germ agglutinin (WGA, green). Scale bars represent 10 μm for h-Mito staining and 20 μm for CENP-A staining.(B) Renal localization of PKH26/positive CL17 cells (red) in AKI mice within proximal tubuli (top) or in peritubular areas (bottom). DAPI (blue) stains nuclei, and renal structures are labeled with WGA (green). Scale bars, 20 μm.(C) Blood urea nitrogen (BUN) (mg/dl) levels in AKI mice treated with CL17 cells (n = 6) or saline (n = 5) at 4 days. Data are expressed as mean ± SD.(D) Quantification of tubular casts and necrotic tubuli (n/high-power field [HPF]) in kidneys of AKI mice treated with CL17 cells or saline at 4 days. ∗p < 0.05 versus cisplatin + saline.(E) Representative histological micrographs of the kidney of the AKI animals treated with CL17 cells (left) or saline (right) at 4 days. Scale bar, 50 μm.See also Figure S6.
Mentions: The renoprotective potential of the reprogrammed cells was investigated in an experimental murine model of cisplatin-induced AKI (Morigi et al., 2008). After intravenous injection of CL17 cells, 50% were recovered in the lung, 1% in the spleen, 0.47% in the liver, and 21% in the kidney; no cells were found in the heart (Figure S6A). Engrafted CL17 cells, stained for two human markers for mitochondria and centromere protein-A (CENP-A), in the renal parenchyma of immunodeficient AKI mice, were localized in the tubular compartment at 4 days post-cisplatin injection (Figure 6A) with a frequency averaging 213 PKH26+ve cells/100,000 renal cells. CL17 cells predominantly localized within proximal tubuli (80%) and, to a lesser extent (20%), in the peritubular areas (Figure 6B). As a positive control for human markers, we used human BMSCs that were mainly found at the peritubular level in tissues of cisplatin-treated mice (Figure S6B). Negative controls with non-immune immunoglobulin of the same isotype as the primary antibody are shown in Figure S6C. Next, we studied the effect of CL17 cell treatment on renal function impairment and tubular damage. In cisplatin-treated mice given CL17 cells, renal function was ameliorated (Figure 6C), although blood urea nitrogen (BUN) levels were not significantly different from those observed in untreated animals (22 ± 2 mg/dl) at 4 days. In parallel, renal damage was significantly improved in AKI animals infused with CL17 cells as demonstrated by the significant reduction of hyaline casts and necrotic tubuli (Figures 6D and 6E). Control mice did not show any signs of tubular damage. Altogether, these data indicate that the reprogrammed BMSCs directly incorporated into proximal tubuli and improved renal damage and function in AKI mice.

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