AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated w...AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated with hepatic progenitor cells. METHODS： EpCAM ＋ve cells were isolated using magnetic cell sorting （MACS） from human fetuses （n = 10） at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein （AFP）, CD29 （integrin ~1）, CD49f （integrin c^6） and CD90 （Thy 1） was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ （A, B, C） and class Ⅱ （DR） expression was studied by flow cytometry only. RESULTS： FACS analysis indicated that EpCAM ＋ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class Ⅱ （DR） and CD45. RT PCR showed that EpCAM ＋ve cells expressed liver epithelial markers （CK18）, biliary specific marker （CK19） and hepatic markers （albumin, AFP）. On immunocytochemical staining, EpCAM ＋ve cells were shown positive signals for CK18 and albumin. CONCLUSION： Our study suggests that these EpCAM ＋ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.展开更多
BACKGROUND Chronic liver diseases(CLD)are the major public health burden due to the continuous increasing rate of global morbidity and mortality.The inherent limitations of organ transplantation have led to the develo...BACKGROUND Chronic liver diseases(CLD)are the major public health burden due to the continuous increasing rate of global morbidity and mortality.The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option.However,identifying the fate of transplanted cells in vivo represents a crucial obstacle.AIM To evaluate the potential applicability of DiD dye as a cell labeling agent for longterm,and non-invasive in vivo tracking of transplanted cells in the liver.METHODS Magnetically sorted,epithelial cell adhesion molecule positive(1×106 cells/mL)fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency(SCID)mice.Near-infrared(NIR)imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d.The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing.RESULTS NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7,15,30,45,60,and 80 post-transplantation.Furthermore,positive staining of cytokeratin,c-Met,and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells.Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase,glutamate-pyruvate transaminase,and bilirubin.The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80.CONCLUSION DiD-labeling is promising for long-term and non-invasive in vivo cell tracking,and understanding the regenerative mechanisms incurred by the transplanted cells.展开更多
基金Council of Scientific and Industrial Research Network Grant CMM002ICMR Grant (GAP 0215)
文摘AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated with hepatic progenitor cells. METHODS： EpCAM ＋ve cells were isolated using magnetic cell sorting （MACS） from human fetuses （n = 10） at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein （AFP）, CD29 （integrin ~1）, CD49f （integrin c^6） and CD90 （Thy 1） was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ （A, B, C） and class Ⅱ （DR） expression was studied by flow cytometry only. RESULTS： FACS analysis indicated that EpCAM ＋ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class Ⅱ （DR） and CD45. RT PCR showed that EpCAM ＋ve cells expressed liver epithelial markers （CK18）, biliary specific marker （CK19） and hepatic markers （albumin, AFP）. On immunocytochemical staining, EpCAM ＋ve cells were shown positive signals for CK18 and albumin. CONCLUSION： Our study suggests that these EpCAM ＋ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.
基金Supported by Department of Science and Technology(DST),Ministry of Science and Technology,Govt.of India and Indian Council of Medical Research(ICMR),New Delhi,Govt.of India Grants to GP,No.GAP-0220 and No.GAP-0383.
文摘BACKGROUND Chronic liver diseases(CLD)are the major public health burden due to the continuous increasing rate of global morbidity and mortality.The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option.However,identifying the fate of transplanted cells in vivo represents a crucial obstacle.AIM To evaluate the potential applicability of DiD dye as a cell labeling agent for longterm,and non-invasive in vivo tracking of transplanted cells in the liver.METHODS Magnetically sorted,epithelial cell adhesion molecule positive(1×106 cells/mL)fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency(SCID)mice.Near-infrared(NIR)imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d.The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing.RESULTS NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7,15,30,45,60,and 80 post-transplantation.Furthermore,positive staining of cytokeratin,c-Met,and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells.Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase,glutamate-pyruvate transaminase,and bilirubin.The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80.CONCLUSION DiD-labeling is promising for long-term and non-invasive in vivo cell tracking,and understanding the regenerative mechanisms incurred by the transplanted cells.
