Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells

Kowolik, Claudia; Liang, Sophie Hsin-Yi; Yu, Ying; Yee, Jiing‐Kuan

doi:10.1038/sj.onc.1207801

Cited by 28 publications

(19 citation statements)

References 42 publications

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“…Lentiviral vectors with human telomerase (hTERT) or SV40 large T antigen (TAg) were produced by transfection of 293 cells. Plasmids containing TAg (pHIV7-CNPO-TAg) and hTERT (pHIV7-CNPO-hTERT) have been described in detail (14) and were kindly provided by Dr. Jiing-Kuan Yee (City of Hope National Medical Center, Duarte, CA). After 48 h, viral supernatant was collected and added to HPSCs.…”

Section: Methodsmentioning

confidence: 99%

Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression

et al. 2008

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Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer. [Cancer Res 2008;68(3):918-26]

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Section: Methodsmentioning

confidence: 99%

Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression

et al. 2008

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“…This example was described by Kowolik et al ( 2004 ) where primary human renal proximal tubule epithelial cells (RPTECs) were transduced with two lentiviral vectors each containing one of these " fl oxed" immortalizing genes (Kowolik et al 2004 ) . Transient Cre expression in such transduced cells led to ef fi cient proviral deletion and decreased growth rates, therefore allowing reversible immortalization of human RPTECs and large-scale production of RPTECs that retain most tissuespeci fi c properties.…”

Section: Cell Immortalizationmentioning

confidence: 99%

Bio-applications Derived from Site-Directed Genome Modification Technologies

Delenda¹,

Paris²,

Arnould

et al. 2012

Site-Directed Insertion of Transgenes

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This review summarizes the overall downstream applications of currently available genome customization systems, based on cell-based assays ( in cellulo ) or animal models ( in vivo ). These include (i) functional genomics, (ii) drug discovery, (iii) bioproduction, (iv) cell transformation (i.e. immortalization, reprogrammation and differentiation), as well as (v) molecular biology and microbiology tools. The different enzymatic systems that exist to speci fi cally modify ( insertional or sitedirected mutagenesis ), modulate ( knock-down ) and disrupt ( constitutive or conditional knock-out ) cellular genes, or to integrate transgenic elements ( knock-in ) at speci fi c chromosomal loci will be discussed herein.

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“…With this approach, immortalization genes such as those encoding human telomerase reverse transcriptase (hTERT), SV40 large T antigen (SV40LT), and Bmi-1 are expressed alone or in combination in primary cells and then removed typically with the Cre-loxP system [6][7][8][9]. However, a 34-bp fragment is left that may disrupt the functions of other genes [6,8,10].…”

Section: Introductionmentioning

confidence: 98%

Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells

Xie

Gong

et al. 2016

Stem Cells and Development

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Fibroblasts can be transdifferentiated directly into other somatic cells such as cardiomyocytes, hematopoietic cells, and neurons. An advantage of somatic cell differentiation without first generating induced pluripotent stem cells (iPSCs) is that it avoids contamination of the differentiated cells with residual iPSCs, which may cause teratoma. However, since primary fibroblasts from biopsy undergo senescence during repeated culture, it may be difficult to grow transdifferentiated cells in sufficient numbers for future therapeutic purposes. To circumvent this problem, we reversibly immortalized primary fibroblasts by using the piggyBac transposon to deliver the human telomerase reverse transcriptase (hTERT) gene hTERT plus SV40 Large T. Both approaches enabled fibroblasts to grow continuously without senescence, and neither caused teratoma formation in immunodeficient mice. However, fibroblasts immortalized with hTERT plus SV40 large T antigen accumulated chromosomal rearrangements, whereas fibroblasts immortalized with hTERT retained the normal karyotype. To transdifferentiate hTERT-immortalized fibroblasts into other somatic lineage cells, we transiently transfected them with episomal OCT4 and cultured them under neural cell growth condition with transposase to remove the transposon. Tripotent neural progenitor cells were seamlessly and efficiently generated. Thus, reversible immortalization of primary fibroblasts with hTERT will allow potential autologous cell-based therapeutics that bypass and simulate iPSC generation.

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Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells

Cited by 28 publications

References 42 publications

Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression

Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression

Bio-applications Derived from Site-Directed Genome Modification Technologies

Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells

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