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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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Characterization of Clones Generated by Human BMSCs after HK2 Cell-Extract Treatment(A) Relative expression of ENG, AQP1, and GGT1 mRNA by qRT-PCR in five selected clones and in HK2 cells compared to BMSCs. BMSC mRNA expression was used as the reference sample. Analyses were performed in triplicate. See also Figure S3A.(B) Expression of TJP1, AQP1, and GGT1 proteins in clone 17 (CL17) and in HK2 cells. Scale bars, 50 μm.(C) SEM analysis showed no microvilli in BMSCs. Instead, CL17 cells displayed microvilli similar to the HK2 cells. Representative images of three independent experiments.(D) Duplication time (hours) in BMSCs, CL17 and HK2 cells (n = 5). Data are expressed as mean ± SD. ∗p < 0.01 versus BMSCs.(E–G) RNA sequencing analysis of BMSCs and CL17 and HK2 cells. (E) Multidimensional scaling plot based on pairwise distances between global gene transcriptome profiles of six analyzed samples (two biological replicates for each cell lineages). (F) Workflow of transcriptome analysis showing the differentially expressed genes (DEGs) in BMSCs, CL17, and HK2 cells. At the intersection of the Venn diagrams, up- or downregulated genes both in CL17 and HK2 cells compared to BMSCs (top) and those up- or downregulated both in CL17 and BMSCs compared to HK2 cells (bottom) are reported. See also Table S3, which contains the full list of DEGs. The top 20 down- and upregulated genes and functionally characterized DEGs for selected pathways are reported in Tables S1, S2, and S3, respectively.(G) Heatmap of the differentially expressed genes of the tight junction, brush border, focal adhesion, and EGFR networks. Columns represent biological replicates, and rows represent each gene. Green and red indicate high and low expression, respectively. The dendrogram of the unsupervised hierarchical clustering of the analyzed samples is shown in the upper part of the heatmap.See also Figures S3–S5 and Tables S1, S2, and S3.
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fig3: Characterization of Clones Generated by Human BMSCs after HK2 Cell-Extract Treatment(A) Relative expression of ENG, AQP1, and GGT1 mRNA by qRT-PCR in five selected clones and in HK2 cells compared to BMSCs. BMSC mRNA expression was used as the reference sample. Analyses were performed in triplicate. See also Figure S3A.(B) Expression of TJP1, AQP1, and GGT1 proteins in clone 17 (CL17) and in HK2 cells. Scale bars, 50 μm.(C) SEM analysis showed no microvilli in BMSCs. Instead, CL17 cells displayed microvilli similar to the HK2 cells. Representative images of three independent experiments.(D) Duplication time (hours) in BMSCs, CL17 and HK2 cells (n = 5). Data are expressed as mean ± SD. ∗p < 0.01 versus BMSCs.(E–G) RNA sequencing analysis of BMSCs and CL17 and HK2 cells. (E) Multidimensional scaling plot based on pairwise distances between global gene transcriptome profiles of six analyzed samples (two biological replicates for each cell lineages). (F) Workflow of transcriptome analysis showing the differentially expressed genes (DEGs) in BMSCs, CL17, and HK2 cells. At the intersection of the Venn diagrams, up- or downregulated genes both in CL17 and HK2 cells compared to BMSCs (top) and those up- or downregulated both in CL17 and BMSCs compared to HK2 cells (bottom) are reported. See also Table S3, which contains the full list of DEGs. The top 20 down- and upregulated genes and functionally characterized DEGs for selected pathways are reported in Tables S1, S2, and S3, respectively.(G) Heatmap of the differentially expressed genes of the tight junction, brush border, focal adhesion, and EGFR networks. Columns represent biological replicates, and rows represent each gene. Green and red indicate high and low expression, respectively. The dendrogram of the unsupervised hierarchical clustering of the analyzed samples is shown in the upper part of the heatmap.See also Figures S3–S5 and Tables S1, S2, and S3.

Mentions: To better characterize the human BMSCs treated with HK2 cell extracts, we next generated clones using a limiting-dilution approach. Of the 50 clones derived from one reprogrammed human BMSC experiment, the five that showed the most epithelial-like morphology were subjected to further analysis using qRT-PCR to examine their mesenchymal and epithelial marker profile. The expression of ENG, AQP1, and gamma glutamyl transferase 1 (GGT1) in these five clones is depicted in Figure 3A. The difference in the expression of these markers reflects heterogeneity in reprogramming efficiency. Cells from clone 17 (CL17) were the most similar to HK2 cells and maintained a stable phenotype and morphology throughout passages. Expression of AQP1 and GGT1 mRNA has been also evaluated in primary proximal tubular epithelial cells (PTECs; Figure S3A). Consistent with the mRNA data, immunocytochemistry revealed that CL17 cells expressed TJP1, AQP1, and GGT1 proteins (Figure 3B). Scanning electron microscopy (SEM) revealed the presence of microvilli in CL17 similar to HK2 cells (Figure 3C). As expected, BMSCs did not show any microvilli (Figure 3C). Moreover, CL17 exhibited a duplication time comparable to that of HK2 cells (CL17: 20.4 ± 1.9 versus HK2: 18.9 ± 1.9 hr) and significantly shorter than BMSCs (55.2 ± 4.2 hr, Figure 3D). Altogether, these data indicated that the CL17, generated by BMSCs treated with the HK2 cell extracts, acquired the renal tubular epithelial phenotype.


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)

Characterization of Clones Generated by Human BMSCs after HK2 Cell-Extract Treatment(A) Relative expression of ENG, AQP1, and GGT1 mRNA by qRT-PCR in five selected clones and in HK2 cells compared to BMSCs. BMSC mRNA expression was used as the reference sample. Analyses were performed in triplicate. See also Figure S3A.(B) Expression of TJP1, AQP1, and GGT1 proteins in clone 17 (CL17) and in HK2 cells. Scale bars, 50 μm.(C) SEM analysis showed no microvilli in BMSCs. Instead, CL17 cells displayed microvilli similar to the HK2 cells. Representative images of three independent experiments.(D) Duplication time (hours) in BMSCs, CL17 and HK2 cells (n = 5). Data are expressed as mean ± SD. ∗p < 0.01 versus BMSCs.(E–G) RNA sequencing analysis of BMSCs and CL17 and HK2 cells. (E) Multidimensional scaling plot based on pairwise distances between global gene transcriptome profiles of six analyzed samples (two biological replicates for each cell lineages). (F) Workflow of transcriptome analysis showing the differentially expressed genes (DEGs) in BMSCs, CL17, and HK2 cells. At the intersection of the Venn diagrams, up- or downregulated genes both in CL17 and HK2 cells compared to BMSCs (top) and those up- or downregulated both in CL17 and BMSCs compared to HK2 cells (bottom) are reported. See also Table S3, which contains the full list of DEGs. The top 20 down- and upregulated genes and functionally characterized DEGs for selected pathways are reported in Tables S1, S2, and S3, respectively.(G) Heatmap of the differentially expressed genes of the tight junction, brush border, focal adhesion, and EGFR networks. Columns represent biological replicates, and rows represent each gene. Green and red indicate high and low expression, respectively. The dendrogram of the unsupervised hierarchical clustering of the analyzed samples is shown in the upper part of the heatmap.See also Figures S3–S5 and Tables S1, S2, and S3.
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fig3: Characterization of Clones Generated by Human BMSCs after HK2 Cell-Extract Treatment(A) Relative expression of ENG, AQP1, and GGT1 mRNA by qRT-PCR in five selected clones and in HK2 cells compared to BMSCs. BMSC mRNA expression was used as the reference sample. Analyses were performed in triplicate. See also Figure S3A.(B) Expression of TJP1, AQP1, and GGT1 proteins in clone 17 (CL17) and in HK2 cells. Scale bars, 50 μm.(C) SEM analysis showed no microvilli in BMSCs. Instead, CL17 cells displayed microvilli similar to the HK2 cells. Representative images of three independent experiments.(D) Duplication time (hours) in BMSCs, CL17 and HK2 cells (n = 5). Data are expressed as mean ± SD. ∗p < 0.01 versus BMSCs.(E–G) RNA sequencing analysis of BMSCs and CL17 and HK2 cells. (E) Multidimensional scaling plot based on pairwise distances between global gene transcriptome profiles of six analyzed samples (two biological replicates for each cell lineages). (F) Workflow of transcriptome analysis showing the differentially expressed genes (DEGs) in BMSCs, CL17, and HK2 cells. At the intersection of the Venn diagrams, up- or downregulated genes both in CL17 and HK2 cells compared to BMSCs (top) and those up- or downregulated both in CL17 and BMSCs compared to HK2 cells (bottom) are reported. See also Table S3, which contains the full list of DEGs. The top 20 down- and upregulated genes and functionally characterized DEGs for selected pathways are reported in Tables S1, S2, and S3, respectively.(G) Heatmap of the differentially expressed genes of the tight junction, brush border, focal adhesion, and EGFR networks. Columns represent biological replicates, and rows represent each gene. Green and red indicate high and low expression, respectively. The dendrogram of the unsupervised hierarchical clustering of the analyzed samples is shown in the upper part of the heatmap.See also Figures S3–S5 and Tables S1, S2, and S3.
Mentions: To better characterize the human BMSCs treated with HK2 cell extracts, we next generated clones using a limiting-dilution approach. Of the 50 clones derived from one reprogrammed human BMSC experiment, the five that showed the most epithelial-like morphology were subjected to further analysis using qRT-PCR to examine their mesenchymal and epithelial marker profile. The expression of ENG, AQP1, and gamma glutamyl transferase 1 (GGT1) in these five clones is depicted in Figure 3A. The difference in the expression of these markers reflects heterogeneity in reprogramming efficiency. Cells from clone 17 (CL17) were the most similar to HK2 cells and maintained a stable phenotype and morphology throughout passages. Expression of AQP1 and GGT1 mRNA has been also evaluated in primary proximal tubular epithelial cells (PTECs; Figure S3A). Consistent with the mRNA data, immunocytochemistry revealed that CL17 cells expressed TJP1, AQP1, and GGT1 proteins (Figure 3B). Scanning electron microscopy (SEM) revealed the presence of microvilli in CL17 similar to HK2 cells (Figure 3C). As expected, BMSCs did not show any microvilli (Figure 3C). Moreover, CL17 exhibited a duplication time comparable to that of HK2 cells (CL17: 20.4 ± 1.9 versus HK2: 18.9 ± 1.9 hr) and significantly shorter than BMSCs (55.2 ± 4.2 hr, Figure 3D). Altogether, these data indicated that the CL17, generated by BMSCs treated with the HK2 cell extracts, acquired the renal tubular epithelial phenotype.

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