ER stress reliever enhances functionalities of in vitro cultured hepatocytes.

Endoplasmic reticulum stress (ER stress) leads an unfolded protein response (UPR) which results in internal cellular responses such as proteostasis and protein clearance. Recently, several reports demonstrated that the ER stress in stem cells could affect their stemness and fates to differentiate into certain lineages. However, the potential for controlling differentiation and function of cells by regulating ER stress needs to be further addressed. Here, we demonstrated that relieving the ER stress in cell cultures enhances the functionalities of hPSC-derived hepatocytes and other hepatic cells to be used in various research fields. Firstly, we found that UPR genes were up-regulated during hepatic differentiation of hPSCs and treatment of ER stress reliever at the hepatic induction stage of the differentiation resulted the enhanced mature marker expressions and glycogen storage of the differentiated hepatocytes. The treatment of ER stress reliever also improved the maintenance of hepatic characteristics in long-term culture of hPSC-derived hepatocytes. Furthermore, relieving ER stress increased the hepatic marker expression and CYP3A4 activity in hepatoma cell lines and human primary hepatocytes. Taken together, our findings indicate that regulating ER stress of in vitro cultured hepatocytes might be a crucial factor for enhancing differentiation, function and maintaining hepatic identity.

Metastable Reprogramming State of Single Transcription Factor-Derived Induced Hepatocyte-Like Cells.

We previously described the generation of induced hepatocyte-like cells (iHeps) using the hepatic transcription factor Hnf1a together with small molecules. These iHeps represent a hepatic state that is more mature compared with iHeps generated with multiple hepatic factors. However, the underlying mechanism of hepatic conversion involving transgene dependence of the established iHeps is largely unknown. Here, we describe the generation of transgene-independent iHeps by inducing the ectopic expression of Hnf1a using both an episomal vector and a doxycycline-inducible lentivirus. In contrast to iHeps with sustained expression of Hnf1a, transgene-independent Hnf1a iHeps lose their typical morphology and in vitro functionality with rapid downregulation of hepatic markers upon withdrawal of small molecules. Taken together, our data indicates that the reprogramming state of single factor Hnf1a-derived iHeps is metastable and that the hepatic identity of these cells could be maintained only by the continuous supply of either small molecules or the master hepatic factor Hnf1a. Our findings emphasize the importance of a factor screening strategy for inducing specific cellular identities with a stable reprogramming state in order to eventually translate direct conversion technology to the clinic.

Direct Conversion of Mouse Fibroblasts into Cholangiocyte Progenitor Cells.

Disorders of the biliary epithelium, known as cholangiopathies, cause severe and irreversible liver diseases. The limited accessibility of bile duct precludes modeling of several cholangiocyte-mediated diseases. Therefore, novel approaches for obtaining functional cholangiocytes with high purity are needed. Previous work has shown that the combination of Hnf1ß and Foxa3 could directly convert mouse fibroblasts into bipotential hepatic stem cell-like cells, termed iHepSCs. However, the efficiency of converting fibroblasts into iHepSCs is low, and these iHepSCs exhibit extremely low differentiation potential into cholangiocytes, thus hindering the translation of iHepSCs to the clinic. Here, we describe that the expression of Hnf1α and Foxa3 dramatically facilitates the robust generation of iHepSCs. Notably, prolonged in vitro culture of Hnf1α- and Foxa3-derived iHepSCs induces a Notch signaling-mediated secondary conversion into cholangiocyte progenitor-like cells that display dramatically enhanced differentiation capacity into mature cholangiocytes. Our study provides a robust two-step approach for obtaining cholangiocyte progenitor-like cells using defined factors.

Ligand-Dependent Interaction of PPARδ With T-Cell Protein Tyrosine Phosphatase 45 Enhances Insulin Signaling.

Peroxisome proliferator-activated receptor (PPAR) δ plays a pivotal role in metabolic homeostasis through its effect on insulin signaling. Although diverse genomic actions of PPARδ are postulated, the specific molecular mechanisms whereby PPARδ controls insulin signaling have not been fully elucidated. We demonstrate here that short-term activation of PPARδ results in the formation of a stable complex with nuclear T-cell protein tyrosine phosphatase 45 (TCPTP45) isoform. This interaction of PPARδ with TCPTP45 blocked translocation of TCPTP45 into the cytoplasm, thereby preventing its interaction with the insulin receptor, which inhibits insulin signaling. Interaction of PPARδ with TCPTP45 blunted interleukin 6-induced insulin resistance, leading to retention of TCPTP45 in the nucleus, thereby facilitating deactivation of the signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3) signal. Finally, GW501516-activated PPARδ improved insulin signaling and glucose intolerance in mice fed a high-fat diet through its interaction with TCPTP45. This novel interaction of PPARδ constitutes the most upstream component identified of the mechanism downregulating insulin signaling.

Inhibition of BET selectively eliminates undifferentiated pluripotent stem cells.

Embryonic stem cells (ESCs) maintain their cellular identity through the systematic regulation of master transcription factors and chromatin remodeling complexes. Recent work has shown that the unusually large-scale enhancers-namely super-enhancers (SEs), on which BRD4, a member of the bromodomain and extraterminal domain (BET) family is highly enriched-could regulate pluripotency-related transcription factors. Moreover, inhibition of BRD4 binding on SEs has been shown to induce the differentiation of ESCs. However, the underlying mechanism of BRD4 inhibition-mediated stem cell differentiation remains elusive. Here we show that both mouse and human ESCs lose their capacity for self-renewal upon treatment with JQ1, a selective inhibitor of BET family including BRD4, with rapid suppression of pluripotency-associated genes. Notably, a high concentration of JQ1 could selectively eliminate ESCs via apoptosis, without affecting the functionality of differentiated somatic cells from ESCs, suggesting that inhibition of BET may have a beneficial effect on the development of pluripotent stem cell-based cell therapy.

Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming.

Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps). However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK) dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.

Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.

The ability to generate integration-free induced hepatocyte-like cells (iHeps) from somatic fibroblasts has the potential to advance their clinical application. Here, we have generated integration-free, functional, and expandable iHeps from mouse somatic fibroblasts. To elicit this direct conversion, we took advantage of an oriP/EBNA1-based episomal system to deliver a set of transcription factors, Gata4, Hnf1a, and Foxa3, to the fibroblasts. The established iHeps exhibit similar morphology, marker expression, and functional properties to primary hepatocytes. Furthermore, integration-free iHeps prolong the survival of fumarylacetoacetate-hydrolase-deficient (Fah(-/-)) mice after cell transplantation. Our study provides a novel concept for generating functional and expandable iHeps using a non-viral, non-integrating, plasmid-based system that could facilitate their pharmaceutical and biomedical application.