title: Isolation and Expansion of Hepatic Stem-like Cells from a Healthy Rat Liver and their Efficient Hepatic Differentiation of under Well-defined Vivo Hepatic like Microenvironment in a Multiwell Bioreactor.
abstract: BACKGROUND
  Currently, undifferentiated cells are found in all tissue and term as local stem cells which are quiescent in nature and less in number under normal healthy conditions but activate upon injury and repair the tissue or organs via automated activating mechanism. Due to very scanty presence of local resident somatic local stem cells in healthy organs, isolation and expansion of these adult stems is an immense challenge for medical research and cell based therapy. Particularly organ like liver, there is an ongoing controversy about existence of liver stem cells.
  
  
  METHODS
  Herein, Hepatic stem cells population was identified during culture of primary hepatocyte cells upon immediate isolation of primary hepatocyte cells. These liver stem cells has been expanded extensively and differentiated into primary hepatocytes under defined culture conditions in a nanostructured self assembling peptides modular bioreactor that mimic the state of art of liver microenvironment and compared with Matrigel as a positive control. Nanostructured self assembling peptides were used a defined extracellular matrix and Matrigel was used for undefined extracellular matrix. Proliferation of hepatic stem cells was investigated by two strategies. First strategy is to provide high concentration of hepatocyte growth factor (HGF) and second strategy is to evaluate the role of recombinant human erythropoietin (rHuEPO) in presence of trauma/ischemia cytokines (IL-6, TNF-α). Expansion to hepatic differentiation is observed by morphological analysis and was evaluated for the expression of hepatocyte-specific genes using RT-PCR and biochemical methods.
  
  
  RESULTS
  Hepatocyte-specific genes are well expressed at final stage (day 21) of differentiation period. The differentiated hepatocytes exhibited functional hepatic characteristics such as albumin secretion, urea secretion and cytochrome P450 expression. Additionally, immunofluorescence analysis revealed that hepatic stem cells derived hepatocytes exhibited mature hepatocyte markers (albumin, CK-19, CPY3A1, alpha 1-antitrypsin). Expansion and hepatic differentiation was efficiently in nanostructured self assembling peptides without such batch to batch variation while there was much variation in Matrigel coated bioreactor. In conclusion, the results of the study suggest that the nanostructured self assembling peptides coated bioreactor supports expansion as well as hepatic differentiation of liver stem cells which is superior than Matrigel.
  
  
  CONCLUSIONS
  This defined microenvironment conditions in bioreactor module can be useful for research involving bioartificial liver system, stem cell research and engineered liver tissue which could contribute to regenerative cell therapies or drug discovery and development.
authors.affiliation: Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Shibashish
authors.initials: S
authors.lastname: Giri
authors.affiliation: Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany ; Department of Gastroenterology and Hepatology, Klinikum St Georg, Delitzscher Straße, Leipzig, Germany.
authors.forename: Ali
authors.initials: A
authors.lastname: Acikgöz
authors.affiliation: Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Augustinus
authors.initials: A
authors.lastname: Bader
keywords: A1AT, Alpha 1-antitrypsin; AFP, α-fetoprotein; CK 7, Cytokeratin 7; CK-19, Cytokeratin 19; CPY3A1, Cytochrome P450 3A 1; EROD, Ethoxyresorufin O-deethylase; GaIN, D-galactosamine; HGF, Hepatocyte growth factor; IL-6, Interleukin 6; MROD, Methoxyresorufin O-demethylase; Matrigel; PROD, Pentoxyresorufin O-depentylase; TNF-α, Tumor necrosis factor alpha; Thy1, Thy-1 cell surface antigen; bioreactor; defined culture conditions; hepatic stem cells; nanostructured self assembling peptides; rHuEPO, Recombinant human erythropoietin