AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.
AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcin...AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.展开更多
Skin serves as the first-order protective barrier against the environment and any significant disruptions in skin integrity must be promptly restored.Despite significant advances in therapeutic strategies,effective ma...Skin serves as the first-order protective barrier against the environment and any significant disruptions in skin integrity must be promptly restored.Despite significant advances in therapeutic strategies,effective management of large chronic skin wounds remains a clinical challenge.Dermal fibroblasts are the primary cell type responsible for remodeling the extracellular matrix(ECM)in wound healing.Here,we investigated whether ECM derived from exogenous fibroblasts,in combination with keratinocytes,promoted scarless cutaneous wound healing.To overcome the limited lifespan of primary dermal fibroblasts,we established reversibly immortalized mouse dermal fibroblasts(imDFs),which were non-tumorigenic,expressed dermal fibroblast markers,and were responsive to TGF-β1 stimulation.The decellularized ECM prepared from both imDFs and primary dermal fi-broblasts shared similar expression profiles of extracellular matrix proteins and promoted the proliferation of keratinocyte(iKera)cells.The imDFs-derived ECM solicited no local immune response.While the ECM and to a lesser extent imDFs enhanced skin wound healing with excessive fibrosis,a combination of imDFs-derived ECM and iKera cells effectively promoted the re-epithelization and scarless healing of full-thickness skin wounds.These findings strongly suggest that dermal fibroblast-derived ECM,not fibroblasts themselves,may synergize with keratinocytes in regulating scarless healing and re-epithelialization of skin wounds.Given its low immu-nogenic nature,imDFs-derived ECM should be a valuable resource of skin-specific biomaterial for wound healing and skin tissue engineering.展开更多
Skin injury is repaired through a multi-phase wound healing process of tissue granulation and re-epithelialization.Any failure in the healing process may lead to chronic non-healing wounds or abnormal scar formation.A...Skin injury is repaired through a multi-phase wound healing process of tissue granulation and re-epithelialization.Any failure in the healing process may lead to chronic non-healing wounds or abnormal scar formation.Although significant progress has been made in developing novel scaffolds and/or cell-based therapeutic strategies to promote wound healing,effective management of large chronic skin wounds remains a clinical challenge.Keratinocytes are critical to re-epithelialization and wound healing.Here,we investigated whether exogenous keratinocytes,in combination with a citrate-based scaffold,enhanced skin wound healing.We first established reversibly immortalized mouse keratinocytes(iKera),and confirmed that the iKera cells expressed keratinocyte markers,and were responsive to UVB treatment,and were non-tumorigenic.In a proof-of-principle experiment,we demonstrated that iKera cells embedded in citrate-based scaffold PPCN provided more effective re-epithelialization and cutaneous wound healing than that of either PPCN or iKera cells alone,in a mouse skin wound model.Thus,these results demonstrate that iKera cells may serve as a valuable skin epithelial source when,combining with appropriate biocompatible scaffolds,to investigate cutaneous wound healing and skin regeneration.展开更多
基金Supported by Major Scientific and Technological Project of Shandong Province,No.201221019Cisco Clinical Oncology Research Fund and Bayer Schering Cancer Research Fund,No.Y-B2012-011
文摘AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.
基金Supported by The Major Scientific and Technological Project of Hubei Province, No. 2007ABD005
文摘AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.
基金supported in part by research grants from the Natural Science Foundation of China(82102696,JF)Chongqing Nat-ural Science Foundation(2024NSCQ-MSX0073,JF)+3 种基金the National Institutes of Health(CA226303 to TCH,and DE030480 to RRR)supported in part by The University of Chicago Comprehensive Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.
文摘Skin serves as the first-order protective barrier against the environment and any significant disruptions in skin integrity must be promptly restored.Despite significant advances in therapeutic strategies,effective management of large chronic skin wounds remains a clinical challenge.Dermal fibroblasts are the primary cell type responsible for remodeling the extracellular matrix(ECM)in wound healing.Here,we investigated whether ECM derived from exogenous fibroblasts,in combination with keratinocytes,promoted scarless cutaneous wound healing.To overcome the limited lifespan of primary dermal fibroblasts,we established reversibly immortalized mouse dermal fibroblasts(imDFs),which were non-tumorigenic,expressed dermal fibroblast markers,and were responsive to TGF-β1 stimulation.The decellularized ECM prepared from both imDFs and primary dermal fi-broblasts shared similar expression profiles of extracellular matrix proteins and promoted the proliferation of keratinocyte(iKera)cells.The imDFs-derived ECM solicited no local immune response.While the ECM and to a lesser extent imDFs enhanced skin wound healing with excessive fibrosis,a combination of imDFs-derived ECM and iKera cells effectively promoted the re-epithelization and scarless healing of full-thickness skin wounds.These findings strongly suggest that dermal fibroblast-derived ECM,not fibroblasts themselves,may synergize with keratinocytes in regulating scarless healing and re-epithelialization of skin wounds.Given its low immu-nogenic nature,imDFs-derived ECM should be a valuable resource of skin-specific biomaterial for wound healing and skin tissue engineering.
基金The reported study was supported in part by research grants from the 2019 Chongqing Support Program for Entrepreneurship and Innovation(No.cx2019113)(JF)the 2019 Science and Technology Research Plan Project of Chongqing Education Commission(KJQN201900410)(JF)+9 种基金the 2019 Youth Innovative Talent Training Program of Chongqing Education Commission(No.CY200409)(JF)the 2019 Funding for Postdoctoral Research(Chongqing Human Resources and Social Security Bureau No.298)(JF)and the National Key Research and Development Program of China(2016YFC1000803)RRR,TCH and GAA were partially funded by the National Institutes of Health(DE030480)WW was supported by the Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)This project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430TCH was also supported by the Mabel Green Myers Research Endowment Fund,The University of Chicago Orthopaedics Alumni Fund,and The University of Chicago SHOCK Fund.Funding sources were not involved in the study designin the collection,analysis and/or interpretation of datain the writing of the reportor in the decision to submit the paper for publication.
文摘Skin injury is repaired through a multi-phase wound healing process of tissue granulation and re-epithelialization.Any failure in the healing process may lead to chronic non-healing wounds or abnormal scar formation.Although significant progress has been made in developing novel scaffolds and/or cell-based therapeutic strategies to promote wound healing,effective management of large chronic skin wounds remains a clinical challenge.Keratinocytes are critical to re-epithelialization and wound healing.Here,we investigated whether exogenous keratinocytes,in combination with a citrate-based scaffold,enhanced skin wound healing.We first established reversibly immortalized mouse keratinocytes(iKera),and confirmed that the iKera cells expressed keratinocyte markers,and were responsive to UVB treatment,and were non-tumorigenic.In a proof-of-principle experiment,we demonstrated that iKera cells embedded in citrate-based scaffold PPCN provided more effective re-epithelialization and cutaneous wound healing than that of either PPCN or iKera cells alone,in a mouse skin wound model.Thus,these results demonstrate that iKera cells may serve as a valuable skin epithelial source when,combining with appropriate biocompatible scaffolds,to investigate cutaneous wound healing and skin regeneration.
