Radiation Induces Diffusible Feeder Cell Factor(s) That Cooperate with ROCK Inhibitor to Conditionally Reprogram and Immortalize Epithelial Cells

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells. Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells. The combination of irradiated feeder cells and a Rho kinase (ROCK) inhibitor Y-27632 conditionally reprograms adult keratinocytes and nonkeratinocyte epithelial cells to an indefinite proliferative state without the use of exogenous viral or cellular gene expression.1Chapman S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar, 2Liu X. Ory V. Chapman S. Yuan H. Albanese C. Kallakury B. Timofeeva O.A. Nealon C. Dakic A. Simic V. Haddad B.R. Rhim J.S. Dritschilo A. Riegel A. McBride A. Schlegel R. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.Am J Pathol. 2012; 180: 599-607Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar Even epithelial cells that are entering senescence proliferate immediately when transferred to the inductive conditions, which consist of F medium containing the ROCK inhibitor Y-27632 and irradiated Swiss 3T3-J2 mouse fibroblasts.3Guerra L. Capurro S. Melchi F. Primavera G. Bondanza S. Cancedda R. Luci A. De Luca M. Pellegrini G. Treatment of “stable” vitiligo by timed surgery and transplantation of cultured epidermal autografts.Arch Dermatol. 2000; 136: 1380-1389Crossref PubMed Scopus (126) Google Scholar, 4Pellegrini G. Ranno R. Stracuzzi G. Bondanza S. Guerra L. Zambruno G. Micali G. De Luca M. 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A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.Mol Cell Biol. 2002; 22: 5157-5172Crossref PubMed Scopus (288) Google Scholar Our research group recently demonstrated that these culture conditions induce an undifferentiated, adult stem cell-like state and that this transition reflects a reprogramming of all cells in the culture population, rather than the selective outgrowth of a small subpopulation.7Suprynowicz F.A. Upadhyay G. Krawczyk E. Kramer S.C. Hebert J.D. Liu X. Yuan H. Cheluvaraju C. Clapp P.W. Boucher Jr., R.C. Kamonjoh C.M. Randell S.H. Schlegel R. Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells.Proc Natl Acad Sci USA. 2012; 109: 20035-20040Crossref PubMed Scopus (215) Google Scholar Perhaps equally important, the conditionally reprogrammed cells (CRCs) exhibited normal differentiation when the feeder cells and Y-27632 were removed, which demonstrates their maintenance of lineage commitment.1Chapman S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar, 2Liu X. Ory V. Chapman S. Yuan H. Albanese C. Kallakury B. Timofeeva O.A. Nealon C. Dakic A. Simic V. Haddad B.R. Rhim J.S. Dritschilo A. Riegel A. McBride A. Schlegel R. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.Am J Pathol. 2012; 180: 599-607Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 7Suprynowicz F.A. Upadhyay G. Krawczyk E. Kramer S.C. Hebert J.D. Liu X. Yuan H. Cheluvaraju C. Clapp P.W. Boucher Jr., R.C. Kamonjoh C.M. Randell S.H. Schlegel R. Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells.Proc Natl Acad Sci USA. 2012; 109: 20035-20040Crossref PubMed Scopus (215) Google Scholar Although the mechanism for the generation of CRCs is still unclear, the combination of feeder cells and Y-27632 appears to provide two distinct activities that promote unrestricted cell proliferation: induction of telomerase and cytoskeletal remodeling and/or interference with the p16/Rb pathway.1Chapman S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar, 2Liu X. Ory V. Chapman S. Yuan H. Albanese C. Kallakury B. Timofeeva O.A. Nealon C. Dakic A. Simic V. Haddad B.R. Rhim J.S. Dritschilo A. Riegel A. McBride A. Schlegel R. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.Am J Pathol. 2012; 180: 599-607Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar Calcium- and serum-containing medium rapidly induces terminal differentiation in keratinocytes.8Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1568) Google Scholar, 9Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (400) Google Scholar, 10Sermadiras S. Dumas M. Joly-Berville R. Bonté F. Meybeck A. Ratinaud M.H. Expression of Bcl-2 and Bax in cultured normal human keratinocytes and melanocytes: relationship to differentiation and melanogenesis.Br J Dermatol. 1997; 137: 883-889Crossref PubMed Scopus (24) Google Scholar, 11Vicanová J. Boelsma E. Mommaas A.M. Kempenaar J.A. Forslind B. Pallon J. Egelrud T. Koerten H.K. Ponec M. Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.J Invest Dermatol. 1998; 111: 97-106Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar However, coculturing keratinocytes with feeder cells allows the keratinocytes to bypass these signals for terminal differentiation and to proliferate until they reach cell crisis. Including Y-27632 in the coculture enables the keratinocytes to bypass cell crisis and proliferate indefinitely. In the present study, we showed that Y-27632 contributes to the suppression of keratinocyte differentiation in the presence of calcium and serum. Moreover, we used both a Transwell culture system and conditioned medium to demonstrate that direct physical contact between the feeder cells and keratinocytes is not required for the induction of conditional reprogramming and immortalization. In general laboratory practice, fibroblast feeder cells are mitotically inactivated by irradiation to prevent their overgrowth of keratinocytes in coculture.5Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (4047) Google Scholar, 12Cuono C. Langdon R. McGuire J. Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury.Lancet. 1986; 327: 1123-1124Abstract Scopus (396) Google Scholar, 13Compton C.C. Hickerson W. Nadire K. Press W. Acceleration of skin regeneration from cultured epithelial autografts by transplantation to homograft dermis.J Burn Care Rehabil. 1993; 14: 653-662Crossref PubMed Scopus (126) Google Scholar, 14Hickerson W.L. Compton C. Fletchall S. Smith L.R. Cultured epidermal autografts and allodermis combination for permanent burn wound coverage.Burns. 1994; 20 (discussion S55–S56): S52-S55Abstract Full Text PDF PubMed Scopus (111) Google Scholar Here, we demonstrate that, in addition to preventing fibroblast overgrowth, irradiation of the feeder cells is critical for the production and/or release of one or more diffusible factors that are essential for conditional reprogramming and immortalization. (Hereafter, reference to factors in the plural incorporates the possibility of a single factor.) Swiss 3T3-J2 mouse fibroblasts7Suprynowicz F.A. Upadhyay G. Krawczyk E. Kramer S.C. Hebert J.D. Liu X. Yuan H. Cheluvaraju C. Clapp P.W. Boucher Jr., R.C. Kamonjoh C.M. Randell S.H. Schlegel R. Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells.Proc Natl Acad Sci USA. 2012; 109: 20035-20040Crossref PubMed Scopus (215) Google Scholar were maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, and 100 μg/mL glutamine (complete DMEM; Life Technologies, Carlsbad, CA). Cultures were passaged when 90% confluent using a 1:4, 1:8, or 1:16 dilution and were given fresh medium every 2 to 3 days. Suspensions of cells in complete DMEM were irradiated at 30 Gy (3000 rads). After irradiation, the cells were plated at a density of approximately 70% in complete DMEM and were allowed to attach for at least 2 hours before addition of keratinocytes. Primary human foreskin keratinocytes (HFKs) were isolated from neonatal foreskins as described previously.15Schlegel R. Phelps W.C. Zhang Y.L. Barbosa M. Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma.EMBO J. 1988; 7: 3181-3187PubMed Google Scholar For direct-contact coculture, HFKs were seeded on a feeder layer of lethally irradiated J2 fibroblasts5Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (4047) Google Scholar in F medium. The F medium consisted of 25% Ham’s F-12 nutrient mix (Life Technologies) and 75% complete DMEM, supplemented with 25 ng/mL hydrocortisone, 5 μg/mL insulin, 0.1 nmol/L cholera toxin (Sigma-Aldrich, St. Louis, MO), 250 ng/mL Fungizone (Thermo Fisher Scientific, Waltham, MA), 0.125 ng/mL epidermal growth factor, and 10 μg/mL gentamicin (Life In experiments, cells were cultured in the presence of the ROCK inhibitor S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar at a of 5 Life In the absence of feeder cells, HFKs were in F medium containing Y-27632 or in keratinocyte growth medium (Life For irradiated or J2 feeder cells were seeded on Transwell and HFKs were on the cell culture Life were by to Cell and and Scholar were by HFKs in culture in medium with or without After to the were with and with as described P. R. D. A. J. D. H. S. M.R. assay for Natl PubMed Scopus Google Scholar were by and were in 10 for of the at P. R. D. A. J. D. H. S. M.R. assay for Natl PubMed Scopus Google Scholar Cell was using a cell (Life Technologies) to the J2 feeder cells, keratinocyte were with Dulbecco’s and with (Life Technologies) for 30 at The culture was until the feeder cells and the feeder cells were by The keratinocytes were with Dulbecco’s and with for 3 to 5 at The keratinocytes were by and the was by Dulbecco’s containing 10% fetal bovine serum. After the keratinocytes were in F medium and plated on irradiated feeder cells. feeder cells to were plated in culture in 30 of F medium. The medium was 3 and was at for 5 at The was a F medium was and at of conditioned F medium were with one of fresh F this was supplemented with 5 Y-27632 before was isolated using (Life Technologies) and with an (Life Technologies) to the and were as described X. J. Dakic A. Zhang Schlegel R. contributes to the telomerase activity of immortalized and cells and PubMed Scopus Google Scholar using and Green CA). The and were and and and and was using and X. J. Dakic A. Zhang Schlegel R. contributes to the telomerase activity of immortalized and cells and PubMed Scopus Google Scholar were to of irradiated feeder cells were cultured in for or 3 using DMEM Life assay the activity of 3 and 100 of was to the at for 30 and for 30 at of the cell were transferred to a for of using a and CA). was using the from or are cultured in S. M. Elias P.M. and of growth and differentiation of normal human neonatal keratinocytes in a Cell 1988; PubMed Scopus Google Scholar or are in serum-containing F medium with lethally irradiated feeder J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (4047) Google Scholar shown that the ROCK inhibitor Y-27632 keratinocyte and differentiation in the presence of F medium and feeder S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar the of Y-27632 on the and differentiation of keratinocytes in the absence of feeder cells, HFKs were cultured for in or in F medium in the presence or absence of Y-27632 and feeder cells. was a in in F medium without feeder cells or Y-27632 in cells from the the addition of Y-27632 which was to the in the presence of Y-27632 and feeder cells. that Y-27632 directly on the HFKs to promote suppression of differentiation by calcium and serum. The of Y-27632 to keratinocyte differentiation was in more by the of associated with keratinocyte differentiation in F and F medium containing Y-27632 for the keratinocyte differentiation of and in human keratinocytes by hydrocortisone, and growth Google Scholar, S. and of the human gene in an epidermal 1993; PubMed Scopus Google Scholar, J. of human keratinocyte Cell PubMed Scopus Google Scholar P.M. The and small and 2 are of the human epidermal cell PubMed Scopus Google Scholar, J.A. barrier formation and in skin Clin Invest. PubMed Scopus Google Scholar A. A. E. L. M. S. F. R. Expression of an of in keratinocytes of the J PubMed Scopus Google Scholar, S. C. B. S. The factor is a of and in keratinocytes: of in wound and PubMed Scopus Google Scholar K. A. D. V. G. Zhang Z. J. A. J. G. C. between and in keratinocyte to PubMed Scopus (320) Google Scholar, A. for kinase in keratinocyte growth Invest Dermatol. in J Invest Full Text Full Text PDF PubMed Scopus Google Scholar and A. C. R. 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Y-27632 in the F medium and in these Y-27632 keratinocyte we HFKs in F medium in the absence of feeder cells. this we cell culture with two strains of HFKs at 2 and 3 and in in F medium containing Y-27632, or in F medium containing Y-27632 and feeder cells HFKs cultured in for the and Y-27632 this proliferative and HFKs with feeder cells in the presence of Y-27632 with in the of Y-27632 terminal differentiation and cell but bypass signals of senescence or crisis. direct contact between feeder cells and HFKs is for conditional we used an coculture system that use of Transwell to the feeder cells from the HFKs the HFKs to the factors released by the feeder cells The two strains of HFKs passaged least 100 and to in F medium containing Y-27632, of they were in direct contact with irradiated feeder cells Moreover, the HFKs exhibited of in the two culture HFKs cultured in which as control for conditional after demonstrate that the conditional immortalization of HFKs does not direct physical contact with feeder cells, and that the feeder cells release one or more diffusible factors that with Y-27632 to promote keratinocyte and The use of irradiated fibroblast feeder cells to maintain keratinocytes in coculture is J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (4047) Google Scholar, 6Rheinwald J.G. Hahn W.C. Ramsey M.R. Wu J.Y. Guo Z. Tsao H. De Luca M. Catricalà C. O’Toole K.M. A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.Mol Cell Biol. 2002; 22: 5157-5172Crossref PubMed Scopus (288) Google Scholar is to prevent the from the more epithelial cells. coculture system the feeder cells and we used this to irradiation of the feeder cells is required for the production and/or release of diffusible factors that conditional immortalization of the keratinocytes. HFKs at were with irradiated or feeder cells plated on Transwell for HFKs with irradiated feeder cells to proliferate at a the cultured with feeder cells their proliferative and senescence after days. In a using HFKs at irradiated feeder cells indefinite the growth of HFKs cultured with feeder cells immediately and after the that irradiation is a in conditional immortalization and is required to the feeder cells to and/or release one or more essential that diffusible factors to conditional we to demonstrate that conditioned medium from of irradiated J2 feeder cells for feeder cells in immortalization was to using a Y-27632 is for the release of factors by the feeder cells. HFKs were cultured for in conditioned F medium that was from irradiated feeder cells after 3 in the presence or absence of Y-27632 was after to a of the conditioned medium in absence medium without Y-27632 to small colonies of In both the conditioned medium in the presence of Y-27632 and the conditioned medium to which Y-27632 was after Y-27632 does not the release of feeder factors that keratinocyte differentiation and promote the of conditioned medium to conditionally keratinocytes in the presence of Y-27632 by and HFKs for using F medium containing Y-27632, direct coculture in F medium with irradiated feeder cells and Y-27632, and conditioned F medium containing Y-27632 medium was as as direct coculture with irradiated feeder cells for these HFKs from senescence and inducing indefinite in the presence of both conditioned medium and direct coculture with irradiated feeder cells conditionally immortalized HFKs and of the presence of a of and that physical contact with irradiated feeder is not required for the conditional immortalization of but that diffusible factors released by the feeder cells are essential to this that telomerase is a critical in cell immortalization and, that telomerase is in HFKs with feeder B. J. C.C. growth conditions telomerase and immortalization by human papillomavirus and Google Scholar but not in HFKs to Y-27632 in the absence of feeder S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar we shown that irradiated feeder cells release diffusible factors that are essential for conditional immortalization of we these factors telomerase expression. was used to of the telomerase in HFKs cultured in in F medium with irradiated feeder cells, or in conditioned F medium with and without Y-27632 The of was in but increased approximately both in the presence of irradiated feeder cells and in conditioned of was not a of the HFKs to F was in conditioned F with F medium diffusible factors released by irradiated feeder cells induce telomerase a mechanism by which the feeder cells promote conditional immortalization. Although the conditioned medium used in immortalization was 3 after irradiation of the feeder cells, was to the of medium from of J2 cells at and hours after irradiation was for to a of in the presence of Y-27632 activity was at hours after irradiation, but activity increased by hours and to at hours and at The general was medium was at from a single culture of irradiated J2 cells with replacement of fresh medium after Thus, the of not use of 3 for the of conditioned but that is a after irradiation before essential factors are released by the feeder cells. irradiation DNA and F. Liu X. W. W. Liu B. Z. L. J. G. J. A. A. D. J.S. of cell PubMed Scopus Google Scholar, A.A. J. of apoptosis by and in a human cell of and J 22: PubMed Scopus Google Scholar, H. C. is by of in a radiation-induced PubMed Scopus Google Scholar we the of apoptosis in irradiated feeder cells to the activity of conditioned medium. Using the activity of 3 and as a for we that the of apoptosis the release of of 3 and activity increased between and hours after irradiation and increased from hours to hours The between apoptosis of irradiated feeder cells and the release of feeder factors that promote conditional immortalization that irradiation induces these factors and/or that the factors are released from cells. shown that irradiated feeder cells and the ROCK inhibitor Y-27632 human keratinocytes to proliferate S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (245) Google Scholar, 2Liu X. Ory V. Chapman S. Yuan H. Albanese C. Kallakury B. Timofeeva O.A. Nealon C. Dakic A. Simic V. Haddad B.R. Rhim J.S. Dritschilo A. Riegel A. McBride A. Schlegel R. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.Am J Pathol. 2012; 180: 599-607Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar the present study, we that this cellular reprogramming is a direct of one or more diffusible factors released from the irradiated feeder cells. Moreover, we that these factors induce telomerase in the a mechanism by which feeder cells promote conditional immortalization. that, in the absence of feeder cells, Y-27632 and differentiation and but cell crisis and senescence and keratinocytes to indefinitely. epithelial cells of in coculture is a the irradiation and of feeder cells by of the with epithelial cells. the feeder cells to they are and not than 3 to days. using Transwell culture is that direct physical contact between epithelial cells and feeder cells is not required for conditional immortalization. the coculture system by feeder cells with medium that conditioned by irradiated feeder cells. is to that irradiation of the feeder cells is essential for the production and/or release of the factors that activity to conditioned medium. factors are released after a after irradiation and to released for the The of this release with the of radiation-induced apoptosis of the feeder cells, as by a in activity of 3 and activity after is that apoptotic cells release factors that promote in cells wound and regeneration in F. J. J.S. cells the to promote wound and 2010; Scopus Google Scholar D. P. M. is required of regeneration in Biol. PubMed Scopus Google Scholar J.S. C. K. K. T. of apoptosis regeneration by the of and PubMed Scopus Google Scholar S. L. K. C. W. B. cells provide an of to Full Text Full Text PDF PubMed Scopus Google Scholar and Guo M. by cell in the activity of the cell in a Biol. 14: Full Text Full Text PDF PubMed Scopus Google Scholar mouse the of and cells in irradiated in 3 and are for this F. J. J.S. cells the to promote wound and 2010; Scopus Google Scholar Moreover, both and of skin and significantly regeneration after F. J. J.S. cells the to promote wound and 2010; Scopus Google Scholar Although to of the to the J2 feeder cells to they the production of the these from the conditioned medium and with the of the Our research group is to J2 feeder cells with of the which their potential in this that the use of conditioned medium significantly the of CRCs to maintenance of but use of conditioned medium the use of CRCs to the of and for and to in the absence of but feeder cells. and for the of this