Isolation and characterization of human fetal liver progenitor stem cells

Abstract

Fetal liver progenitor cells are the most natural precursors of hepatocytes and bile duct cells. They hold tremendous promise in their ability to provide a continual source of cells for cellular transplantation, bioartificial assisted liver devices, gene therapy for metabolic disorders, drug testing in pharmaceutical industries and even for studies in understanding mechanisms regulating normal liver development, specification and maturation. We thus aimed to isolate and characterize the population of progenitor cells from fetal liver and determine their role as a stem cell for liver repair and regeneration. Mid-trimester human fetal livers were cultured up to 3 months to allow enrichment of progenitor cells. A novel technique combining chemical selection with GS418 and mechanical harvesting was designed and successfully isolated these cells. Progenitor cells are small cells (10um) with high nuclear-cytoplasmic ratio of 46 hour doubling time. In optimized cultures on NIH 3T3 (mouse embryonal fibroblasts cells) feeder layers, clonal colonies validated by Humara X assay could be maintained up to 100 population doublings or 20 passages. Progenitor cells were then characterized by immunofluorescence, western blot and RT-PCR. They had markers consistent with a stem cell signature including CD34, thy-1, c-kit and SSEA-4. They appeared to have a mixed epithelial-mesenchymal phenotype being positive for EPCAM, CK18, CK19 as well as vimentin and CD44. Interestingly they had yet to specify into hepatic fate as they were negative for albumin and alphafetoprotein, and do not express HNF transcription factors specific for hepatic lineage.(HNF1a,3ß and 4a). Progenitor cells were able to differentiate to become hepatocytes and cholangioytes via mesenchymal-epithelial transition. Hepatocytes were positive for albumin, Hep-par and alpha-1-antitrypsin and showed consistent features on electron microscopy. Functional studies including albumin secretion, glycogen storage, P450 enzymes inducibility and glucose 6 phosphastase activity were also positive. Cholangiocyte differentiation was correspondingly determined with positivity for CK7, CK19, GGT and characteristic microvilli on electron microscopy. Differentiation into mesodermal lineages was also successful with lineage confirmed with at least 2 modalitites. Fat differentiation was positive for oil globules and PPARgamma RTPCR; cartilage differentiation was confirmed with identification of collagen II and mucopolysaccharides; bone differentiation was confirmed with calcium deposition and osteopontin RTPCR and endothelium differentiation confirmed with identification of VWF and CD31. Progenitor cells were transplanted into Rag2¿c double knockout mice treated with retrorsine and carbon tetracholoride. Successful engraftment, integration and function of transplanted human cells was determined with human specific albumin, Hep-Par and ZO-1 immunofluorescence, RT PCR of liver samples, ELISA of mice serum and in situ hybridization for human chromosome. Transplantation up to 9 months showed prolonged though variable survival with no evidence of fusion or aneuploidy. These progenitor cells exist in fetal liver de novo and using lineage tracing, do not appear to have been derived from hepatocytes, MSC or HSC cells. Given their mixed phenotype, they may have arisen from mesendoderm This is the first reported long term culture of clonal population of human fetal liver progenitor cells fulfilling stem cell properties of self-regneration, plasticity and functional offsprings both in vitro and in vivo.