Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.
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.
Affiliation: IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri," Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy. Electronic address: email@example.com.Show MeSH
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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.
Affiliation: IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri," Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy. Electronic address: firstname.lastname@example.org.