Overexpression of microRNA-375 and microRNA-122 promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

MicroRNAs (miRNAs) are involved in the regulation of gene expression. In this study, we evaluated the use of overexpression of microRNA-375 (miR-375) and miR-122 in differentiating the Human Induced Pluripotent Stem Cells (hiPSCs) into functional hepatocyte-like cells (HLCs) without growth factors. We also compared the differentiation by miRNAs versus growth factors. HiPSCs were divided into two main groups: 1- HiPSCs were induced using lentiviral overexpression of miR-375 to differentiate into definitive endoderm (DE) cells in seven days. Then lentiviral overexpression of miR-122 was applied to differentiate DE cells into HLCs in additional 14 days. 2- HiPSCs were differentiated into HLCs using growth factors in 21 days. DE and hepatocyte markers were investigated by qRT-PCR, immunofluorescence, secretion analysis and LDL uptake assay. In the produced cells of both groups: the expression levels of DE markers (FOXA2 and SOX17) and hepatocyte markers (albumin, CK18, and HNF4a) in comparison with the undifferentiated hiPSCs increased significantly in seven and 21 days respectively. The albumin and urea secretion and LDL uptake were also detected. These results weren't significantly different between two groups. Therefore, we demonstrated that the over expression of miR-375 and then miR-122 could differentiate hiPSCs into functional HLCs without growth factors for developing cell-based therapies.

Differentiation of Definitive Endoderm from Human Induced Pluripotent Stem Cells on hMSCs Feeder in a Defined Medium.

BACKGROUND: The Definitive Endoderm (DE) differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells (hiPSCs) on an appropriate feeder in a more defined medium. METHODS: Human Induced Pluripotent Stem Cells (hiPSCs) were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3-ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts (MEFs) and in the fifth method were human adult bone marrow Mesenchymal Stem Cells (hMSCs). DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry. RESULTS: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences. CONCLUSION: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine.

Efficient Differentiation of Human Induced Pluripotent Stem Cell (hiPSC) Derived Hepatocyte-Like Cells on hMSCs Feeder.

BACKGROUND: The use of stem cells is considered as an appropriate source in cell therapy and tissue engineering. Differentiation of human induced Pluripotent Stem Cells (hiPSCs) to Hepatocyte-like Cells (HLCs) on mouse embryonic fibroblasts (MEFs) feeders is confronted with several problems that hinder the clinical applications of these differentiated cells for the treatment of liver injuries. Safe appropriate cells for stem cell-based therapies could create new hopes for liver diseases. This work focused on the determination of a capacity/efficiency for the differentiation of the hiPSCs into Hepatocyte-like Cells on a novel human adult bone marrow mesenchymal stem cells (hMSCs) feeder. MATERIALS AND METHODS: Undifferentiated human iPSCs were cultured on mitotically inactivated human adult bone marrow mesenchymal stem cells. A three-step differentiation process has been performed in presence of activin A which added for 3 days to induce a definitive endoderm formation. In the second step, medium was exchanged for six days. Subsequently, cells were treated with oncostatin M plus dexamethasone for 9 days to generate hepatic cells. Endodermic and liver-specific genes were assessed via quantitative reverse transcription-polymerase chain reaction and RT-PCR, moreover, immunocytochemical staining for liver proteins including albumin and alpha-fetoprotein. In addition, functional tests for glycogen storage, oil red examination, urea production and alpha-fetoprotein synthesis, as well as, cells differentiated with a hepatocyte-like morphology was also performed. RESULTS: Our results show that inactivated human adult bone marrow mesenchymal stem cell feeders could support the efficient differentiation of hiPSCs into HLCs. This process induced differentiation of iPSCs into definitive endocrine cells that expressed sox17, foxa2 and expression of the specific genes profiles in hepatic-like cells. In addition, immunocytochemical analysis confirmed albumin and alpha-fetoprotein protein expression, as well as, the hiPSCs-derived Hepatocyte-like Cells on human feeder exhibited a typical morphology. CONCLUSIONS: we suggested a successful and efficient culture for differentiation and maturation of hepatocytes on an alternative human feeders; this is an important step to generate safe and functional hepatocytes that is vital for regenerative medicine and transplantation on the cell-based therapies.