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Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

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Related in: MedlinePlus

Functional siRNA library screening of J2 cells for secreted factors.J2 cells were grown in the presence of Y, and transfected with a library of 332 siRNAs. The growth response of HFKs to the each conditioned medium (CM) resulting from each RNA ‘knockdown’ was evaluated two days after transfection by colony assay. Listed are genes whose ‘knockdown’ resulted in ≥50% reduction in growth.
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pone.0116755.g003: Functional siRNA library screening of J2 cells for secreted factors.J2 cells were grown in the presence of Y, and transfected with a library of 332 siRNAs. The growth response of HFKs to the each conditioned medium (CM) resulting from each RNA ‘knockdown’ was evaluated two days after transfection by colony assay. Listed are genes whose ‘knockdown’ resulted in ≥50% reduction in growth.

Mentions: J2 cells secreted factors into the ‘conditioned’ medium (CM) that were able to substitute for the use of feeder cells in conditional reprogramming [17]. To determine the nature of these factors, J2 cells were screened for their growth effects on HFKs using a siRNA library targeting 332 genes producing secreted factors that are expressed by 3T3 fibroblasts, the cell line from which J2 cells were originally derived (Table B in S1 File). The effect of gene ‘knockdown’ in J2 cells was determined by the effect of the CM corresponding to each siRNA on HFK colony formation. Knockdown of 14 genes was associated with growth suppression by their corresponding CM, indicating that in the absence of knockdown, these genes were responsible for growth stimulation (Fig. 3). These genes were associated with up-regulating cell adhesion, inflammation, invasion, motility, metabolism and proliferation (Fig. 3). Also noted as positive regulators of cell growth in the CM were negative regulators of TGF-β signaling (Fstl3, Lefty1, Lefty2) (Fig. 3). The net effect of these secreted factors affected signaling pathways associated with F-actin organization, cytoskeleton modification, Smads, GPCRs and Ephrin signaling (Fig. D in S1 File).


Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Functional siRNA library screening of J2 cells for secreted factors.J2 cells were grown in the presence of Y, and transfected with a library of 332 siRNAs. The growth response of HFKs to the each conditioned medium (CM) resulting from each RNA ‘knockdown’ was evaluated two days after transfection by colony assay. Listed are genes whose ‘knockdown’ resulted in ≥50% reduction in growth.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340869&req=5

pone.0116755.g003: Functional siRNA library screening of J2 cells for secreted factors.J2 cells were grown in the presence of Y, and transfected with a library of 332 siRNAs. The growth response of HFKs to the each conditioned medium (CM) resulting from each RNA ‘knockdown’ was evaluated two days after transfection by colony assay. Listed are genes whose ‘knockdown’ resulted in ≥50% reduction in growth.
Mentions: J2 cells secreted factors into the ‘conditioned’ medium (CM) that were able to substitute for the use of feeder cells in conditional reprogramming [17]. To determine the nature of these factors, J2 cells were screened for their growth effects on HFKs using a siRNA library targeting 332 genes producing secreted factors that are expressed by 3T3 fibroblasts, the cell line from which J2 cells were originally derived (Table B in S1 File). The effect of gene ‘knockdown’ in J2 cells was determined by the effect of the CM corresponding to each siRNA on HFK colony formation. Knockdown of 14 genes was associated with growth suppression by their corresponding CM, indicating that in the absence of knockdown, these genes were responsible for growth stimulation (Fig. 3). These genes were associated with up-regulating cell adhesion, inflammation, invasion, motility, metabolism and proliferation (Fig. 3). Also noted as positive regulators of cell growth in the CM were negative regulators of TGF-β signaling (Fstl3, Lefty1, Lefty2) (Fig. 3). The net effect of these secreted factors affected signaling pathways associated with F-actin organization, cytoskeleton modification, Smads, GPCRs and Ephrin signaling (Fig. D in S1 File).

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

Show MeSH
Related in: MedlinePlus