Hepatocyte Differentiation by Highly Purified CD34+/CD34− Blast Cells from G-CSF and SCF Primed Bone Marrow from Rhesus Monkey.

Hematopoietic stem cells and hepatic stem cells share characteristic markers such as CD34, CD117(c-kit) and CD90 (Thy-1). In this study, we investigated the potential of primitive CD34+/CD34− blast cells to differentiate into hepatocytes in vitro. Rhesus monkeys were administered granulocyte colony stimulating factor, G-CSF, combined with stem cell factor, SCF, the ligand for c-kit receptor, for 3 days. This novel G-CSF and SCF combined cytokine treatment is believed to stimulate the self renewal of primitive blast cells in the bone marrow. Blast cells were purified by a novel technique that combines purging of T-cells and other immune-competent cells that express the Fc receptor. The immuno-depleted mononuclear fraction was labeled with biotin-conjugated SKH6.1 anti-CD34 monoclonal antibody belonging to the class-I epitope. The SKH 6.1 recognizes a heterogeneous blast cell population in rhesus bone marrow that consists mainly of approximately 80% CD34+ and a CD34− small blast cell minor population that also shares low forward and side scatter properties. Blast cells were cultured on Matrigel in a defined medium containing hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor-4(FGF-4), and fed every 3–4 days. Conditioned medium was analyzed at weekly intervals for four weeks. ELISA assays were used to detect de-novo synthesis of rhesus monkey specific albumin, and urea production was measured using a quantitative colorimetric assay. Peak production of albumin occurred at 7-days and then began to continuously decrease through week 4, while urea was detected at 7-days but continued to increase through the end of the 4th week. The hepatocyte-like cells were also shown to produce significant intra-cellular glycogen as detected by periodic acid-schiff staining by their 4th week in culture. Hepatocyte function was also characterized by their ability to uptake Dil-Ac-LDL as detected by their strong fluorescence on the 4th week of culture. Our results demonstrated that G-CSF and SCF stimulate the bone marrow by increasing the frequency CD34+ / CD34− blast cells that are able to differentiate into hepatocyte-like cells.