Highly Efficient Generation of Human Hepatocyte-Like Cells from Induced Pluripotent Stem Cells

Department of Cell Biology, Division of Pediatric Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Hepatology (Impact Factor: 11.06). 01/2010; 51(1):297-305. DOI: 10.1002/hep.23354
Source: PubMed


There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient-specific human hepatocytes from induced pluripotent stem (iPS) cells. CONCLUSION: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo.

  • Source
    • "Therefore, we propose that the use of human induced pluripotent stem cells (hiPSCs) differentiated into hepatocyte-like cells (HLCs) could provide an adapted cellular environment to conduct such studies. Indeed, it has been shown that hiPSCs generated from skin fibroblasts of an individual carrying mutations in the LDLR gene and differentiated into HLCs were able to recapitulate the features of ADH (Cayo et al., 2012; Si-Tayeb et al., 2010). Here, we used a convenient source of patient-derived somatic "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (-38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (-89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19±0.77-fold in HLC-S127R compared to 1.38±0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.
    Disease Models and Mechanisms 11/2015; DOI:10.1242/dmm.022277 · 4.97 Impact Factor
  • Source
    • "Human iPSK3 cells were derived from human foreskin fibroblasts transfected with plasmid DNA encoding reprogramming factors OCT4, NANOG, SOX2 and LIN28 (kindly provided by Dr. Stephen Duncan, Medical College of Wisconsin) [27] [28]. Human iPSK3 cells were maintained in mTeSR serum-free medium (StemCell Technologies, Inc., Vancouver, Canada) or knockout serum replacement (SR) medium supplemented with FGF-2 (40 ng/ mL) on 6-well plates coated with Geltrex (Life Technologies) [29]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Extracellular matrices (ECM) derived from pluripotent stem cells (PSCs) provide a unique tissue microenvironment that can direct cellular differentiation and tissue regeneration, and rejuvenate aged progenitor cells. The unlimited growth capacity of PSCs allows for the scalable generation of PSC-secreted ECMs. Therefore, the derivation and characterization of PSC-derived ECMs is of critical importance in drug screening, disease modeling and tissue regeneration. In this study, 3-D ECMs were generated from decellularized undifferentiated embryonic stem cell (ESC) aggregates (AGG), spontaneously differentiated embryoid bodies (EB), and ESC-derived neural progenitor cell (NPC) aggregates. The capacities of different ECMs to direct proliferation and neural differentiation of the reseeded mouse ESCs and human induced pluripotent stem cells (iPSCs) were characterized. Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed protein expression profiles that reflected distinct niche properties for each tested ECM group. The reseeded mouse ESCs and human iPSCs responded to different types of ECMs with different cellular phenotypes. Cells grown on the AGG-ECM displayed high levels of pluripotent markers Oct-4 and Nanog, while the cells grown on the NPC-ECM showed increased expression of neural marker β-tubulin III. The expression levels of β-catenin were high for cells grown on the AGG-ECM and the EB-ECM, but reduced in cells grown on the NPC-ECM, indicating a possible role of Wnt/β-catenin signaling in the cell-matrix interactions. This study demonstrates that PSC-derived ECMs can influence stem cell fate decisions by providing a spectrum of stem cell niche microenvironments during tissue development.
    Biomaterials 09/2015; 73:231-242. DOI:10.1016/j.biomaterials.2015.09.020 · 8.56 Impact Factor
  • Source
    • "To overcome these challenges, human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have been considered as an alternative cell source for production of human hepatocytes. To date, there are many studies reporting hepatic differentiation of hiPSCs/hESCs (Ogawa et al., 2013; Si-Tayeb et al., 2010; Takayama et al., 2012). However , in most cases, differentiation of hepatocytes from hiPSCs is accomplished by a time-consuming culture protocol with multiple differentiation steps using expensive cytokines. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.
    Stem Cell Reports 09/2015; 5(4). DOI:10.1016/j.stemcr.2015.08.008 · 5.37 Impact Factor
Show more