Induction of hepatocyte-like cells from mouse embryonic stem cells by lentivirus-mediated constitutive expression of Foxa2/Hnf4a

Abstract

Hepatocytes can be generated from embryonic stem cells (ESCs) using inducers such as chemical compounds and cytokines, but issues related to low differentiation efficiencies remain to be resolved. Recent work has shown that overexpression of lineage-specific transcription factors can directly cause cells phenotypic changes, including differentiation, trans-differentiation, and de-differentiation. We hypothesized that lentivirus-mediated constitutive expression of forkhead box A2 (Foxa2) and hepatocyte nuclear factor 4 alpha (Hnf4a) could promote inducing mouse ESCs to hepatocyte-likes cells. First, ESC lines that stably expressed Foxa2, Hnf4a, or Foxa2/Hnf4a were constructed via lentiviral expression vectors. Second, observations of cell morphology changes were made during the cell culture process, followed by experiments examining teratoma formation. Then, the effects of constitutive expression of Foxa2 and Hnf4a on hepatic differentiation and maturation were determined by measuring the marker gene expression levels of Albumin, α-fetoprotein, Cytokeratin18, and α1-antitrypsin. The results indicate that constitutive expression of Foxa2 and Hnf4a does not affect ESCs culture, teratoma formation, or the expression levels of the specific hepatocyte genes under autonomous differentiation. However, with some assistance from inducing factors, Foxa2 significantly increased the hepatic differentiation of ESCs, whereas the expression of Hnf4a alone or Foxa2/Hnf4a could not. Differentiated CCE-Foxa2 cells were more superior in expressing several liver-specific markers and protein, storing glycogen than differentiated CCE cells. Therefore, our method employing the transduction of Foxa2 would be a valuable tool for the efficient generation of functional hepatocytes derived from ESCs.

Forced expression of Hnf4a induces hepatic gene activation through directed differentiation

“…Although we did not observe any compelling differences in Afp and Alb mRNA expressions, there was significant upregulation of Ttr and endogenous Hnf4a in the test group. These findings appeared to be consistent with previous reports that showed enforced expression of Hnf4a in hepatic medium that contained a number of assistant factors which could have positively influenced hepatic gene expressions [34,35].…”

“…The results from microarray data supported this hypothesis since the HGF and FGF receptor signaling considered crucial in embryonic liver development showed upregulation in the developing EBs [33]. A recent study reported that ectopic expression of Hnf4a during suspension differentiation did not activate hepatocyterelated genes in the absence of extrinsic inducers [34].…”

In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration

“…A combined treatment with Wnt3a and BMP4 efficiently differentiated human ESCs (Kim et al, 2013 ); after co-culture with STO feeder cells, human ESCs were able to differentiate into HLCs and cholangiocyte-like cells (Zhao et al, 2009 ). Forkhead box A2 (Liu et al, 2013 ) and synthesized basement membrane components (Shiraki et al, 2011 ) significantly increased the hepatic differentiation of ESCs.…”

“…The micro-cavitary hydrogel (MCG) system enhances nutrient exchange, permits greater living space for the encapsulated pluripotent stem cells to rapidly grow into colonies and results in significantly greater production of endoderm markers, hepatic markers, urea, and ALB by iPSCs compared to the typical non-MCG system, or monolayer culture (Lau et al, 2013 ). Zhang et al (Liu et al, 2013 ) reported three-dimensional clump culture collagen matrices compatible with high throughput screening resulted in significantly increased functional maturation of iPSC-Heps towards an adult phenotype when compared to conventional culture systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves the functional longevity when culturing in vitro over 75 days.…”

Forced expression of Hnf1b/Foxa3 promotes hepatic fate of embryonic stem cells

“…Therefore, it can be speculated that certain specific transcription factors or a combination of them have the capability to facilitate differentiation or direct reprogramming of ES cells into HLCs. Recently, it has been demonstrated that transcription factors or micRNAs which constitutively or transiently express in ES cells could induce hepatic differentiation in addition to conventional differentiation methods [13][14][15][16][17][18][19].…”