Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol

Abstract

Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. Conclusion: We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases.

Menstrual blood-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells

“…2b). Undifferentiated cells showed no hepatocyte-specific gene expression except for CK18 and CYP1A1, which is consistent with previous studies (Lee et al, 2004;Chen et al, 2012).…”

“…It is attractive to develop stem-cell-based therapy or transplanting hepatocytes generated in vitro from stem cells for treatment of liver diseases. Many types of stem cells from different sources have been investigated for hepatic differentiation ability (Yamamoto et al, 2003;Banas et al, 2007;Chen et al, 2012). In addition, the in vivo transplantation of stem cell-derived HLCs improved liver functions of animal models undergoing liver damage (Yamamoto et al, 2003;Chen et al, 2012).…”

“…Many types of stem cells from different sources have been investigated for hepatic differentiation ability (Yamamoto et al, 2003;Banas et al, 2007;Chen et al, 2012). In addition, the in vivo transplantation of stem cell-derived HLCs improved liver functions of animal models undergoing liver damage (Yamamoto et al, 2003;Chen et al, 2012). Embryonic stem cells have enormous differentiation ability, but many limitations including teratoma formation after transplantation, immunogenicity and ethical issues are arresting their clinical application (Lin et al, 2011).…”

“…Mesenchymal stem cells (MSCs) obtained from human bone marrow (BM), adipose tissue (AT), placenta, and umbilical cord blood (UCB) have been reported to differentiate in vitro into a variety of lineages, such as osteoblasts, adipocytes, chondrocytes, myoblasts and cardiomyocytes, neural cells, depending on the microenvironment in which they reside (Pittenger et al, 1999). Although MSCs from BW, UCB, and AT have been induced into a hepatic lineage, the question of whether these cells are the best sources for liver regeneration remains (Bieback et al, 2004;Banas et al, 2007;Chen et al, 2012).…”

Human induced pluripotent stem cells derived hepatocytes: rising promise for disease modeling, drug development and cell therapy

“…Meanwhile, the persistent expression of alpha-fetoprotein (a fetal stage hepatocyte marker) implies an immature or fetal-stage status. More importantly, iPSC-hepatocytes have demonstrated a capacity to pass stringent in vivo test as engrafting and maturing in animal models, as an ideal proof for their identity and functionality (Si-Tayeb et al, 2010b;Chen et al, 2011). The advantages of using iPSC-hepatocytes over their ESC counterparts in both basic and clinical studies are obvious: (1) the generation of iPSC from patient bearing inherited pathological background provides a more objective platform for disease modeling and drug development; (2) the cell reprogramming using patient own cells makes iPSC-hepatocytes a preferred source for autologous cell therapy; (3) the somatic origin of iPSC avoids controversies surrounding the use of human embryonic cells and erases many ethical concerns in application.…”