In vitro generated hepatocyte-like cells: a novel tool in regenerative medicine and drug discovery

Hepatocyte-like cells [HLCs] are generated from either various human pluripotent stem cells [hPSCs] including induced pluripotent stem cells [iPSCs] and embryonic stem cells [ESCs], or direct cell conversion, mesenchymal stem cells as well as other stem cells like gestational tissues. They provide potential cell sources for biomedical applications. Liver transplantation is the gold standard treatment for the patients with end stage liver disease, but there are many obstacles limiting this process, like insufficient number of donated healthy livers. Meanwhile, the number of patients receiving a liver organ transplant for a better life is increasing. In this regard, HLCs may provide an adequate cell source to overcome these shortages. New molecular engineering approaches such as CRISPR/Cas system applying in iPSCs technology provide the basic principles of gene correction for monogenic inherited metabolic liver diseases, as another application of HLCs. It has been shown that HLCs could replace primary human hepatocytes in drug discovery and hepatotoxicity tests. However, generation of fully functional HLCs is still a big challenge; several research groups have been trying to improve current differentiation protocols to achieve better HLCs according to morphology and function of cells. Large-scale generation of functional HLCs in bioreactors could make a new opportunity in producing enough hepatocytes for treating end-stage liver patients as well as other biomedical applications such as drug studies. In this review, regarding the biomedical value of HLCs, we focus on the current and efficient approaches for generating hepatocyte-like cells in vitro and discuss about their applications in regenerative medicine and drug discovery

Site-directed mutagenesis, expression and biological activity of E. coli 5-enolpyruvylshikimate 3-phosphate synthase gene

Site-directed mutagenesis [SDM] as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase [EPSPS] gene of E. coli. The mutations changed glycine 96 to alanine and alanine 183 to threonine. These two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to EPSP synthase enzymes. By designing mutagen primers and overlapping extension method, three kinds of altered bacterial EPSPS enzymes with first, second and both mutations were produced. These modified enzymes are expected to show decreased affinity for herbicide, with least alteration in their enzymatic activity. These altered genes were cloned under the control of chemically inducible T7 promoter and over expressed in E. coli. Biological activity analyses in the presence of glyphosate show that the bacteria containing the mutated enzymes, especially the enzyme with two mutations, were more tolerant to glyphosate