| Stem cell research offers unprecedented opportunities for paving the way towards novel medical therapies and in revolutionizing our understanding of basic biology. Stem cells are unspecialized cells that exhibit virtually unlimited self-renewal and differentiation potential into several mature cells with specialized functions, such as hepatocytes. For generating large numbers of functional hepatocytes from stem cells, robust and reproducible processes for isolation of stem cells, optimal expansion and maintenance in culture and directed differentiation to functional hepatocytes, need to be established in parallel.;Two types of stem cells - Embryonic stem cells (ESCs), derived from blastocyst stage embryos, and Multipotent adult progenitor cells (MAPCs), derived from bone marrow were employed here as potential renewable sources to obtain functional hepatocytes. Multipotent Adult Progenitor Cells (MAPCs) are isolated from the bone marrow, and have remarkably broad capability to differentiate into multiple cell types. As the first step to using MAPC derived hepatocytes, multiple new lines of MAPCs with very similar phenotype and differentiation capabilities were isolated from the bone marrow.;To efficiently direct the differentiation of MAPCs and ESCs toward hepatocytes in-vitro, a differentiation scheme was designed to mimic the distinct sequential changes experienced by cells during the developing embryonic liver development. Our approach to reconstruct this in vivo microenvironment was based on the time dependent addition of soluble factors in the context of three-dimensional (3D) cell aggregate configuration to achieve optimal differentiation with respect to the yield and functional maturity of hepatocytes obtained. The progression and efficiency of differentiation was demonstrated by quantification of gene expression of stage specific markers, including definitive endoderm markers Goosecoid and CXCR4, hepatic precursor markers AFP and Transthyretin, and mature hepatocyte markers such as albumin, alpha-1-antitrypsin, among others. We also showed the expression of hepatocyte-specific protein products, such as albumin, asialoglycoproteinreceptor-1 (ASGPR-1) and PEPCK, as determined by immunohistochemistry or fluorescence-activated cells sorting (FACS). The functional attributes of the differentiated cells included albumin and urea secretion, glycogen storage, glutathione S-transferase activity, coagulation factor secretion, cytochrome P450 activity. From ultra-structural evaluation (TEM), the differentiated cells resembled hepatocytes in terms of numerous mitochondria and lipid droplets, golgi complex, endoplasmic reticulum along with polarized junctional complexes and bile-canaliculi structures.;In an effort to achieve the scale-up of expansion of MAPCs and their differentiation to hepatocytes in a controlled environment stirred suspension bioreactors of both cell aggregates and cells attached to microcarriers. Cultivation in both platforms in stirred bioreactor lead to between 70-85 fold expansion in 4-6 days with final cell densities of about 106 cells/ml. Importantly, these bioreactor expanded cells were subsequently differentiated to 'hepatocyte-like' cells with efficiencies comparable to that observed in static culture.;Thus, these studies provide a renewable, scalable source of hepatocyte-like cells that demonstrate phenotypic and functional properties of primary hepatocytes for therapeutic applications, such as hepatocyte transplantation or bioartificial liver devices, and experimental studies, such as drug toxicity screening and as model systems for developmental studies. |
