Establishment of lentiviral-vector-mediated model of human alpha-1 antitrypsin delivery into hepatocyte-like cells differentiated from mesenchymal stem cells.
Alpha-1 antitrypsin (AAT) deficiency is a lethal hereditary disorder characterized by a severe diminution in plasma levels of AAT leading to progressive liver dysfunction. Since mesenchymal stem cells can differentiate into hepatocyte-like cells they offer a potential unlimited source in autologous transplant procedures. The transfer of genetically modified hepatocyte cells derived from hMSCs into the body constitutes a novel paradigm of coupling cell therapy with gene therapy for this disease. hMSCs were isolated by density gradient centrifugation and plastic adherence. Hepatic differentiation was induced by exposing hMSC to induction medium for up to 21 days. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR and immunocytochemistry. The chimeric AAT-Jred transgene was transferred to differentiated cells using a lentiviral vector and its expression was visualized by fluorescent microscopy. Flow cytometric analysis confirmed that hMSCs were obtained. Major hepatocyte marker genes expression were confirmed by RT-PCR and immunocytochemistry. AAT gene was successfully introduced into hepatocyte-like cells differentiated from hMSCs. This established system could be suitable for generation of hMSC derived hepatocyte-like cells containing the normal AAT gene, thus offering a potential in vitro source of cells for transplantation therapy of liver diseases in AAT-deficient patients.
Ghaedi
Mahboobe
M
Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
Lotfi
Abbas S
AS
Soleimani
Masoud
M
D002454:Cell Differentiation; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008954:Models, Biological; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism
D000515:alpha 1-Antitrypsin