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Identification and isolation of kidney-derived stem cells from transgenic rats with diphtheria toxin-induced kidney damage

View Article: PubMed Central - PubMed

ABSTRACT

Adult stem cells have been well characterized in numerous organs, with the exception of the kidneys. Therefore, the present study aimed to identify and isolate kidney-derived stem cells. A total of 12 Fischer 344 transgenic rats expressing the human diphtheria toxin receptor in podocyte cells of the kidney, were used in the present study. The rats were administered 5-bromo-2′-deoxyuridine (BrdU) in order to detect cellular proliferation. After 60 days, the rats were treated with the diphtheria toxin (DT), in order to induce kidney injury. Immunohistochemical analysis indicated that the number of BrdU-positive cells were increased following DT treatment. In addition, the expression of octamer-binding transcription factor 4 (Oct-4), a stem cell marker, was detected and suggested that kidney-specific stem cells were present in the DT-treated tissue samples. Furthermore, tissue samples exhibited repair of the DT-induced injury. Further cellular culturing was conducted in order to isolate the kidney-specific stem cells. After 5 weeks of culture, the majority of the cells were non-viable, with the exception of certain specialized, unique cell types, which were monomorphic and spindle-shaped in appearance. The unique cells were isolated and subjected to immunostaining and reverse transcription-polymerase chain reaction analyses in order to reconfirm the expression of Oct-4 and to detect the expression of Paired box 2 (Pax-2), which is necessary for the formation of kidney structures. The unique cells were positive for Oct-4 and Pax-2; thus suggesting that the identified cells were kidney-derived stem cells. The results of the present study suggested that the unique cell type identified in the kidneys of the DT-treated rats were kidney-specific stem cells that may have been involved in the repair of DT-induced tissue injury. In addition, these cells may provide a useful cell line for studying the fundamental characteristics of kidney stem cells, as well as identifying kidney-specific stem cell markers.

No MeSH data available.


Reverse transcription-polymerase chain reaction analysis. Expression profile of (A) Pax-2, (B) Oct-4 and (C) GAPDH in the isolated kidney-derived stem cells. The reactions were run in triplicate. PAX-2, paired box 2; Oct-4, octamer-binding transcription factor 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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f4-etm-0-0-3516: Reverse transcription-polymerase chain reaction analysis. Expression profile of (A) Pax-2, (B) Oct-4 and (C) GAPDH in the isolated kidney-derived stem cells. The reactions were run in triplicate. PAX-2, paired box 2; Oct-4, octamer-binding transcription factor 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Mentions: In order to confirm that the isolated cells were kidney-derived stem cells, immunostaining was conducted using the anti-Oct-4 antibody. The Oct-4 positive cells exhibited a dark brown coloration following staining with the DAB substrate (Fig. 3B); thus suggesting that the isolated cells were kidney-derived stem cells. Furthermore, RT-PCR was performed in order to confirm whether the Oct-4 positive cells had originated from the kidneys. Oct-4, Pax-2 and GAPDH mRNA expression was detected in the cultured cells (Fig. 4). These results suggested that the cultured unique cells were kidney-derived stem cells.


Identification and isolation of kidney-derived stem cells from transgenic rats with diphtheria toxin-induced kidney damage
Reverse transcription-polymerase chain reaction analysis. Expression profile of (A) Pax-2, (B) Oct-4 and (C) GAPDH in the isolated kidney-derived stem cells. The reactions were run in triplicate. PAX-2, paired box 2; Oct-4, octamer-binding transcription factor 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4998113&req=5

f4-etm-0-0-3516: Reverse transcription-polymerase chain reaction analysis. Expression profile of (A) Pax-2, (B) Oct-4 and (C) GAPDH in the isolated kidney-derived stem cells. The reactions were run in triplicate. PAX-2, paired box 2; Oct-4, octamer-binding transcription factor 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Mentions: In order to confirm that the isolated cells were kidney-derived stem cells, immunostaining was conducted using the anti-Oct-4 antibody. The Oct-4 positive cells exhibited a dark brown coloration following staining with the DAB substrate (Fig. 3B); thus suggesting that the isolated cells were kidney-derived stem cells. Furthermore, RT-PCR was performed in order to confirm whether the Oct-4 positive cells had originated from the kidneys. Oct-4, Pax-2 and GAPDH mRNA expression was detected in the cultured cells (Fig. 4). These results suggested that the cultured unique cells were kidney-derived stem cells.

View Article: PubMed Central - PubMed

ABSTRACT

Adult stem cells have been well characterized in numerous organs, with the exception of the kidneys. Therefore, the present study aimed to identify and isolate kidney-derived stem cells. A total of 12 Fischer 344 transgenic rats expressing the human diphtheria toxin receptor in podocyte cells of the kidney, were used in the present study. The rats were administered 5-bromo-2′-deoxyuridine (BrdU) in order to detect cellular proliferation. After 60 days, the rats were treated with the diphtheria toxin (DT), in order to induce kidney injury. Immunohistochemical analysis indicated that the number of BrdU-positive cells were increased following DT treatment. In addition, the expression of octamer-binding transcription factor 4 (Oct-4), a stem cell marker, was detected and suggested that kidney-specific stem cells were present in the DT-treated tissue samples. Furthermore, tissue samples exhibited repair of the DT-induced injury. Further cellular culturing was conducted in order to isolate the kidney-specific stem cells. After 5 weeks of culture, the majority of the cells were non-viable, with the exception of certain specialized, unique cell types, which were monomorphic and spindle-shaped in appearance. The unique cells were isolated and subjected to immunostaining and reverse transcription-polymerase chain reaction analyses in order to reconfirm the expression of Oct-4 and to detect the expression of Paired box 2 (Pax-2), which is necessary for the formation of kidney structures. The unique cells were positive for Oct-4 and Pax-2; thus suggesting that the identified cells were kidney-derived stem cells. The results of the present study suggested that the unique cell type identified in the kidneys of the DT-treated rats were kidney-specific stem cells that may have been involved in the repair of DT-induced tissue injury. In addition, these cells may provide a useful cell line for studying the fundamental characteristics of kidney stem cells, as well as identifying kidney-specific stem cell markers.

No MeSH data available.