Generation of induced pluripotent stem cells from human kidney mesangial cells - PubMed (original) (raw)

Generation of induced pluripotent stem cells from human kidney mesangial cells

Bi Song et al. J Am Soc Nephrol. 2011 Jul.

Abstract

Glomerular injury and podocyte loss leads to secondary tubulointerstitial damage and the development of fibrosis. The possibility of genetically reprogramming adult cells, termed induced pluripotent stem cells (iPS), may pave the way for patient-specific stem-cell-based therapies. Here, we reprogrammed normal human mesangial cells to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4 and c-Myc). The kidney iPS (kiPS) cells resembled human embryonic stem-cell-like colonies in morphology and gene expression: They were alkaline phosphatase-positive; expressed OCT3/4, TRA-1 to 60 and TRA-1 to 81 proteins; and showed downregulation of mesangial cell markers. Quantitative (qPCR) showed that kiPS cells expressed genes analogous to embryonic stem cells and exhibited silencing of the retroviral transgenes by the fourth passage of differentiation. Furthermore, kiPS cells formed embryoid bodies and expressed markers of all three germ layers. The injection of undifferentiated kiPS colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. These results suggest that reprogrammed kidney induced pluripotent stem cells may aid the study of genetic kidney diseases and lead to the development of novel therapies.

Copyright © 2011 by the American Society of Nephrology

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Figures

Figure 1.

Figure 1.

Kidney mesangial cell-derived iPS cells express stem cell markers. (A) The timeline of induction of iPS cells from human mesangial cells following retroviral transduction. (B) Representative images of normal cultured human mesangial cells reprogrammed to generate iPS colonies (C). (D) Alkaline phosphatase-positive iPS colonies (arrows). Immunofluorescence staining of mesangial cell-derived iPS colonies shows localization of OCT3/4 protein (E; green), with corresponding DAPI-stained nuclei (F; blue) and a merged image (G). TRA-1-60 (H–J) and TRA-1-81 (K–M) proteins are also expressed. Original magnifications (B, E–M ×400, C ×10). The graph in the lower panel shows qPCR of mesangial cell markers in NHMCs, with a downregulated expression in iPS cells from 3 separate colonies at passage 4. 120 × 189mm (600 × 600 DPI).

Figure 2.

Figure 2.

(A) The iPS colonies display a normal 46XY karyotype. (B) PCR of genomic DNA shows the integration of the target vectors in mesangial cell-derived iPS cells at passage 4. Lanes 1–7 show representative iPS cells following retroviral transduction, confirming the expression of OCT3/4, c-Myc, KLF4 and SOX2, compared to human embryonic stem (hES) cells (Lane 8) and normal cultured mesangial cells (NHMC; Lane 9). β-actin is shown as a loading control and for positive amplification. 154x218mm (600 × 600 DPI).

Figure 3.

Figure 3.

(A) RT-PCR analysis of stem cell marker genes in kiPS cells for expression of OCT3/4, NANOG, SOX2, FGF4, REX1, TERT, DPPA2, DPPA4, DPPA5, c-Myc and KLF4. Lanes 1–7 represent mesangial cell-derived iPS (kiPS) cells compared to hES cells (Lane 8) and the NHMC target cells at day 0 (Lane 9). The β-actin housekeeping gene is also shown. Panel (B) shows quantitative PCR using primers specific for the transgenes, and not detecting endogenous gene expression levels (shown in Panel A), which confirms retroviral transgene silencing in most kips colonies (passage 4). This is evidenced by a loss of OCT3/4, SOX2, KLF4 and c-Myc expression analogous to expression levels in hES cells. In comparison, an upregulated expression of OCT3/4, SOX2, KLF4 and c-Myc is observed in NHMCS at 6 days following retroviral induction. 112×196mm (150 × 150 DPI).

Figure 4.

Figure 4.

Kidney-derived iPS cells demonstrate pluripotency. (A) qPCR of stem cell markers OCT3/4, SOX2 and NANOG in kidney-iPS cells (kiPS) relative to human embryonic stem cells (hES), with an absence of expression in normal human mesangial cells (NHMCs). (B) RT-PCR shows undifferentiated iPS cells (U; Lanes 1, 3, 5) and hES expressed stem cell marker genes OCT3/4, SOX2 and NANOG. In comparison, differentiated embryoid bodies (EBs) (B; Lanes 2, 4, 6) expressed markers from all three germ layers, including α-fetoprotein (AFP; endoderm), α-smooth muscle actin (α-SMA; mesoderm) and neural cell adhesion molecule (NCAM; ectoderm), but did not express undifferentiated stem cell markers. kips formed EBs (C; arrow) with protein localization evident for desmin (D; mesoderm) and Foxa2 (E; endoderm) by immunofluorescence staining. Brightfield images of neural-directed EBs (NDEBs) from hES cells (F) and kiPS cells (G) at day 14. Immunofluorescence staining of NDEBs from hES cells show expression of nestin (H), β-iii-tubulin and MAP2ab (merged, J). In Panels D–K, DAPI-stained nuclei are evident in blue. Nestin (I), β-iii-tubulin and MAP2ab (merged, K) are also observed in NDEBs from kiPS. Original Magnifications C ×10; D, E ×400; F–K ×200. Teratomas are evident following the injection of undifferentiated kiPS cells into immunodeficient mice (L; arrow). Panels M–S show hematoxylin and eosin staining of tissues from all three germ layers, including endoderm; (M) pseudostratified ciliated respiratory epithelium with goblet cells (arrow inset Mag ×1000), (N) serous glands (acini shown in inset Mag ×1000), mesoderm; (O) muscle, (P) nucleated red blood cells (arrows); and endoderm: (Q) neural tissue. At lower power (Mag ×100), panels R and S show glandular tissue (arrows) in cystic teratomas adjacent to adipocytes (asterisk; R) and immature cartilage (asterisk; S). Original magnifications (K × 400; H–J, L ×200; M, N ×100). 120 × 173mm (600 × 600 DPI).

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