Limits...
Generation of induced pluripotent stem cells from renal tubular cells of a patient with Alport syndrome.

Chen W, Huang J, Yu X, Lin X, Dai Y - Int J Nephrol Renovasc Dis (2015)

Bottom Line: Approximately 80% of AS is X-linked because of mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen.According to the human embryonic stem cell (hESC) charter, iPSC formation was confirmed by comparatively analyzing hESC markers via colony morphology, immunohistochemistry, qRT-PCR, flow cytometry, gene expression profiling of the three germ layers, and karyotyping.Our results demonstrated that iPSCs were similar to hESCs with regard to morphology, proliferation, hESC-specific surface marker expression, and differentiation into the cell types of the three germ layers.

View Article: PubMed Central - PubMed

Affiliation: The Clinical Medical Research Center, Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, Shenzhen, Guangdong, People's Republic of China.

ABSTRACT
Alport syndrome (AS) is a hereditary disease that leads to kidney failure and is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes that lead to the absence of collagen α3α4α5 (IV) networks in the mature kidney glomerular basement membrane. Approximately 80% of AS is X-linked because of mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen. To investigate the pathogenesis of AS at the genetic level, we generated induced pluripotent stem cells (iPSCs) from renal tubular cells of a patient with AS. The successful iPSC generation laid the foundation to master the repair of the COL4A5 gene and to evaluate the differentiation of iPSC into Sertoli cells and the accompanying epigenetic changes at each stage. The generation of iPSCs from AS patients not only confirms that iPSCs could be generated from renal tubular cells, but also provides a novel type of genetic therapy for AS patients. In this study, we generated iPSCs from renal tubular cells via ectopic expression of four transcription factors (Oct4, Sox2, c-myc, and Klf4). According to the human embryonic stem cell (hESC) charter, iPSC formation was confirmed by comparatively analyzing hESC markers via colony morphology, immunohistochemistry, qRT-PCR, flow cytometry, gene expression profiling of the three germ layers, and karyotyping. Our results demonstrated that iPSCs were similar to hESCs with regard to morphology, proliferation, hESC-specific surface marker expression, and differentiation into the cell types of the three germ layers. The efficient generation of iPSCs from the renal tubular cells of an AS patient would provide a novel model to investigate the mechanisms underlying AS and to develop new treatments for AS.

No MeSH data available.


Related in: MedlinePlus

qRT-PCR analysis of gene expression in iPSCs.Notes: 1, iPSCs; 2, hESCs; 3, renal tubular cells.Abbreviations: iPSC, induced pluripotent stem cells; hESC, human embryonic stem cell.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4551301&req=5

f6-ijnrd-8-101: qRT-PCR analysis of gene expression in iPSCs.Notes: 1, iPSCs; 2, hESCs; 3, renal tubular cells.Abbreviations: iPSC, induced pluripotent stem cells; hESC, human embryonic stem cell.

Mentions: Immunocytochemistry confirmed that iPSCs expressed several hESC-specific marker proteins (Oct4, SSAE-4, TRA-1-60, and TRA-1-81). However, the iPSCs were negative for SSAE-1 expression (Figure 5). qRT-PCR analyses of the total cellular RNA further confirmed the expression of pluripotency-associated genes (Figure 6), including Oct4 (endogenous), Sox2 (endogenous), Klf4 (endogenous), c-myc (endogenous), Nanog, Lin28, SALL4, and REX1. Figure 6 shows that the pluripotency-related genes in iPSCs had a much higher expression than those in tubular cells and a similar expression to those in hESCs. These genes were expressed at almost undetectable levels in tubular cells. The exogenous pluripotency genes were evident in most of the clones. In contrast, the vector transgenes were silenced in derived iPSC clones. We also used flow cytometry to verify cell surface markers, including Oct4, SSEA-4, TRA-1-60, and TRA-1-81. iPSCs highly expressed cell surface markers associated with Oct4, SSEA-4, TRA-1-60, and TRA-1-81 (Figure 7). The expression of the pluripotency-associated genes indicated the successful generation of iPSCs from renal tubular cells.


Generation of induced pluripotent stem cells from renal tubular cells of a patient with Alport syndrome.

Chen W, Huang J, Yu X, Lin X, Dai Y - Int J Nephrol Renovasc Dis (2015)

qRT-PCR analysis of gene expression in iPSCs.Notes: 1, iPSCs; 2, hESCs; 3, renal tubular cells.Abbreviations: iPSC, induced pluripotent stem cells; hESC, human embryonic stem cell.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551301&req=5

f6-ijnrd-8-101: qRT-PCR analysis of gene expression in iPSCs.Notes: 1, iPSCs; 2, hESCs; 3, renal tubular cells.Abbreviations: iPSC, induced pluripotent stem cells; hESC, human embryonic stem cell.
Mentions: Immunocytochemistry confirmed that iPSCs expressed several hESC-specific marker proteins (Oct4, SSAE-4, TRA-1-60, and TRA-1-81). However, the iPSCs were negative for SSAE-1 expression (Figure 5). qRT-PCR analyses of the total cellular RNA further confirmed the expression of pluripotency-associated genes (Figure 6), including Oct4 (endogenous), Sox2 (endogenous), Klf4 (endogenous), c-myc (endogenous), Nanog, Lin28, SALL4, and REX1. Figure 6 shows that the pluripotency-related genes in iPSCs had a much higher expression than those in tubular cells and a similar expression to those in hESCs. These genes were expressed at almost undetectable levels in tubular cells. The exogenous pluripotency genes were evident in most of the clones. In contrast, the vector transgenes were silenced in derived iPSC clones. We also used flow cytometry to verify cell surface markers, including Oct4, SSEA-4, TRA-1-60, and TRA-1-81. iPSCs highly expressed cell surface markers associated with Oct4, SSEA-4, TRA-1-60, and TRA-1-81 (Figure 7). The expression of the pluripotency-associated genes indicated the successful generation of iPSCs from renal tubular cells.

Bottom Line: Approximately 80% of AS is X-linked because of mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen.According to the human embryonic stem cell (hESC) charter, iPSC formation was confirmed by comparatively analyzing hESC markers via colony morphology, immunohistochemistry, qRT-PCR, flow cytometry, gene expression profiling of the three germ layers, and karyotyping.Our results demonstrated that iPSCs were similar to hESCs with regard to morphology, proliferation, hESC-specific surface marker expression, and differentiation into the cell types of the three germ layers.

View Article: PubMed Central - PubMed

Affiliation: The Clinical Medical Research Center, Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, Shenzhen, Guangdong, People's Republic of China.

ABSTRACT
Alport syndrome (AS) is a hereditary disease that leads to kidney failure and is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes that lead to the absence of collagen α3α4α5 (IV) networks in the mature kidney glomerular basement membrane. Approximately 80% of AS is X-linked because of mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen. To investigate the pathogenesis of AS at the genetic level, we generated induced pluripotent stem cells (iPSCs) from renal tubular cells of a patient with AS. The successful iPSC generation laid the foundation to master the repair of the COL4A5 gene and to evaluate the differentiation of iPSC into Sertoli cells and the accompanying epigenetic changes at each stage. The generation of iPSCs from AS patients not only confirms that iPSCs could be generated from renal tubular cells, but also provides a novel type of genetic therapy for AS patients. In this study, we generated iPSCs from renal tubular cells via ectopic expression of four transcription factors (Oct4, Sox2, c-myc, and Klf4). According to the human embryonic stem cell (hESC) charter, iPSC formation was confirmed by comparatively analyzing hESC markers via colony morphology, immunohistochemistry, qRT-PCR, flow cytometry, gene expression profiling of the three germ layers, and karyotyping. Our results demonstrated that iPSCs were similar to hESCs with regard to morphology, proliferation, hESC-specific surface marker expression, and differentiation into the cell types of the three germ layers. The efficient generation of iPSCs from the renal tubular cells of an AS patient would provide a novel model to investigate the mechanisms underlying AS and to develop new treatments for AS.

No MeSH data available.


Related in: MedlinePlus