Efficiency Of Hepatic Differentiation Research Articles

Generation of Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells for Cellular Medicine.

Liver transplantation and hepatocyte transplantation are effective treatments for severe liver injuries, but the donor shortage is a serious problem. Therefore, hepatocyte-like cells generated from human induced pluripotent stem (iPS) cells with unlimited proliferative ability are expected to be a promising new transplantation resource. The technology for hepatic differentiation from human iPS cells has made great progress in this decade. The efficiency of hepatic differentiation now exceeds 90%, making it possible to produce nearly homogeneous hepatocyte-like cells from human iPS cells. Because there is little contamination of undifferentiated cells, there is a lower risk of teratoma formation. To date, the transplantation of human iPS cell-derived hepatocyte-like cells has been shown to have therapeutic effects using various liver injury model mice. Currently, studies are underway using model animals larger than mice. The day when human iPS cell-derived hepatocyte-like cells can be used as cellular medicine is surely approaching. In this review, we introduce the forefront of regenerative medicine applications using human iPS cell-derived hepatocyte-like cells.

Recent Advances for Enhancing Drug Metabolizing Functions of Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells

Clinical treatment for chronic liver failure using human embryonic stem cell (hESC) and human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) is considered a promising alternative method to organ transplantation. In addition to their use for treatment in liver failure, stem cell-derived HLCs have been considered for in vitro drug screening and toxicology researches [1]. Therefore, HLCs directly induced from hESCs have been intensively studied, resulting in a significant improvement in the efficiency of hepatic differentiation using human pluripotent stem cells. Albumin-positive HLCs can now be produced at the end of in vitro hepatic differentiation at levels up to 90% [2,3]. However, in spite of efforts to induce further maturation of HLCs derived from human pluripotent stem cells, the phenotype of most HLCs is more similar to fetal hepatocytes rather than fully mature hepatocytes. Critical inducing mature hepatocyte functions, such as phase I and II enzyme activity, tend to be significantly reduced in 2D-cultured HLCs (approximately <1% of human primary hepatocytes) [4,5]. Furthermore, under in vitro culture conditions, hepatobiliary transporter expression rapidly decreases [6], and most HLCs are spontaneously differentiate into various cell lineages, regardless of the differentiation protocol. Thus, at the final stage of hepatic differentiation, purification is needed to obtain highly homogenous HLCs. These key differences between HLCs and human primary hepatocytes result in limited use of HLCs as Hanyang Med Rev 2015;35:196-206 http://dx.doi.org/10.7599/hmr.2015.35.4.196

Comparative Evaluation of Differentiation Potential of Menstrual Blood- Versus Bone Marrow- Derived Stem Cells into Hepatocyte-Like Cells

Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs) possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under protocols developed by different concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB), cytokeratin 18 (CK-18), tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1) at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1) compared to lower concentration of these factors (P2). Moreover, omission of serum during differentiation process (P3) caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

Activin A and Wnt-dependent specification of human definitive endoderm cells

Activin/Nodal and Wnt signaling are known to play important roles in the regional specification of endoderm. Here we have investigated the effect of the length of stimulation with Activin A plus Wnt3a on the development of hepatic and pancreatic progenitors from the definitive endoderm (DE) cells derived from human pluripotent stem cells (hPSC). We show that DE-cells derived from hPSC with 3 days high Activin A and Wnt3a treatment were able to differentiate further into both tested endodermal lineages. When prolonging the DE-induction protocol from 3 to 5 or 7 days, almost pure DE-marker positive cell populations were obtained. However, these cells had an impaired pancreatic differentiation capacity, while they still developed into hepatocyte-like cells. Further propagation of the DE-cells in the presence of Wnt3a and Activin A led to the complete loss of differentiation capacity into hepatic or pancreatic lineages. When Wnt3a was removed after 24h from the initiation of the differentiation, the cells were able to differentiate into PDX1+/NKX6.1+ pancreatic progenitors even with longer DE induction time while efficiency of hepatic differentiation was lower. Our results suggest that both the length and the timing of Wnt3a treatment during DE induction are crucial for the final differentiation outcome. Although it is possible to derive apparently pure DE cells with prolonged Activin A/Wnt-stimulation, their progenitor capacity is restricted to a limited time window.

Generation of functional hepatocytes from mouse induced pluripotent stem cells

Induced pluripotent stem cells are derived from somatic cells by forced expression of several transcriptional factors. Induced pluripotent stem cells resemble embryonic stem cells in many aspects, such as the expression of certain stem cell markers, chromatin methylation patterns, embryoid body formation and teratoma formation. Therefore, induced pluripotent stem cells provide a powerful tool for study of developmental biology and unlimited resources for transplantation therapy. Here we reported the successful induction of mouse induced pluripotent stem cells and a simple and efficient process for generation of functional hepatocytes from mouse induced pluripotent stem cells by sequential addition of inducing factors. These induced pluripotent stem cell-derived hepatocytes, just as mouse embryonic stem cell-derived hepatocytes, expressed hepatic lineage markers including CK7, CK8, CK18, CK19, alpha-fetoprotein, albumin, Cyp7a1, and exhibited functional hepatic characteristics, including glycogen storage, indocyanine green (ICG) uptake and release, low-density lipoprotein (LDL) uptake and urea secretion. Although we observed some variations in the efficiency of hepatic differentiation between induced pluripotent stem cells and common mouse embryonic stem cell lines, our results indicate that mouse induced pluripotent stem cells can efficiently differentiate into functional hepatocytes in vitro, which may be helpful for the study of liver development and regenerative medicine.

What Came First: Fully Functional or Metabolically Mature Liver?

Hepatocytes, which constitute about 70% of the liver cell population, perform complex metabolic functions such as plasma protein synthesis and transport, xenobiotic metabolism, glucose homeostasis, urea synthesis, and ketogenesis. The process of liver development is marked by distinct changes in mitochondrial mass, activity, and function, especially during the transition from the fetal to the adult phenotype. The identification of the biochemical mechanisms implicated in hepatic development in vitro using embryonic stem cells is critical because it can unravel the relationship between metabolic changes and cell-specific functional differentiation. Moreover, embryonic stem cell differentiation into hepatocytes can provide an unlimited source of cells for clinical applications in liver transplantation for cirrhosis and fulminant hepatic failure. The "chicken or the egg" problem of whether functional maturation precedes or follows metabolic maturation remains unanswered, but it might simply be that the two processes develop in a complex integrated mode for metabolic and functional homeostasis. The identification of the regulation of complex developments in liver organogenesis can provide interventions to improve the efficiency of hepatic differentiation and a better understanding of the developmental process. Finally, this reasoning can be extended to various cell specific differentiation systems, including the cardiac, pancreatic, and neuronal lineages.