Human Cord Blood Hematopoietic Stem Cells Differentiate Into Hepatocyte-like Cells In Vitro

Umbilical cord blood (UCB) contains circulating stem/progenitor cells, and the cellular contents UCB are know to be quite distinct from those of bone marrow (BM) and adult peripheral blood. Over the past two decades, the presence and characteristics of hematopoietic stem cells in UCB have been clarified. The frequency of UCB hematopoietic stem/progenitor cells equals or exceeds that of BM and greatly surpasses that of adult peripheral blood. Compared with adult cells, UCB hematopoietic stem cells produce larger hematopoietic colonies in vitro, have different growth factor requirements, are able to expand in long-term culture in vitro. UCB transplantation for various hematopoietic diseases has resulted in successful hematopoietic reconstitution and a lower incidence of graft-versus-host disease than expected with conventional therapies. Furthermore, UCB provides fewer ethical problems for basic studies and clinical applications. UCB cells can be collected without any harm to the newborn infant, and UCB hematopoietic stem cell grafts can be cryopreserved and transplanted to a host after thawing without losing their repopulating ability. For these reasons, UCB could be a prominent source of cells for transplantation in various diseases.In the present study, in vitro and in vivo, whether UCB is a potential source of cells for transplantation for the support of liver injury. Human UCB cells generated hepatocyte lineage cells in culture system supplemented with a combination of growth/differentiation factors. In the cell-transplantation model for the support of liver injury, human UCB cells displayed the characteristics of functionally differentiated hepatocytes and released human albumin(ALB) into the recipient's peripheral blood after engraftment in liver-injured severe combined immunodeficient(SCID) mice, indicating that UCB-derived hepatocytes may play a role in the support for liver injury.Our objective is to investigate the possibility of the human cord blood hematopoietic stem cells differentiate into hepatocyle-like cells with hepatocyte growth factor(HGF), stem cell factor(SCF) and leukemia inhibitory factor(LIF) which provide us a potential cell source for liver tissue engieering in vitro. The hematopoietic stem cells were obtained through density gradient centrifugation and magnetic activated cell sorting(MACS). The induced HSCs were cultured in the DMEM with HGF(10ng/ml)+SCF(10ng/ml)+LIF(10ng/ml), and the Undifferentiated HSCs acted as the control group without HGF, SCF and LIF. The morphology of cells was observed by an inverted phase contrast microscope. The expression of albumin(ALB), human hepatocyte of cytokeratin (CK18) and alpha-fetoprotein (AFP) were detected withimmunofluorescence, immunohistochemistry and RT-PCR assay in the 21-day culture. The ALB product in the medium was determined by radioimmunoassay (RIA) at 7,14,21,23 and 25 days.The shapes of HSCs changed and their sizes and numbers increased in the course of the culturing. Induced for three weeks, the cells became round shaped and resembled hepatocyle-like cells. The mRNA of ALB was detected by RT-PCR in the differentiated HSCs, and the mRNA of AFP was poorly detected by RT-PCR at 21 days. ALB and CK18 were positive through immunofluorescence and immunohistochemistry at 21 days compared with the Undifferentiated HSCs. The ALB product in the medium significantly increased compared with control groups and increased highest at 21 days, then decreased at 23 days.Under some definite inducing conditions, hHSCs can differentiate into hepatocyte-like cells.