Abstract
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.
Highlights
Somatic cell fates determined during development can be reversed into an embryonic stem cell-like state by the forced expression of Oct4, Klf4, Sox2, and cMyc (i.e., OKSM), resulting in the generation of induced pluripotent stem cells [1, 2]
We previously described that the hepatic factor Hnf1a could convert mouse somatic cells into induced hepatocyte-like cells, which represent cells of a more mature hepatic state compared with iHeps generated by hepatic reprogramming cocktails consisting of multiple hepatic transcription factors [31]
IPSC technology has long been considered as a patient-specific cell source for transplantable hepatocytes [41], safety issues concerning mainly the tumorigenic risk of induced pluripotent stem cells (iPSCs)-derived hepatocytes still preclude the clinical translation of these cells [42]
Summary
Somatic cell fates determined during development can be reversed into an embryonic stem cell-like state by the forced expression of Oct, Klf, Sox, and cMyc (i.e., OKSM), resulting in the generation of induced pluripotent stem cells (iPSCs) [1, 2]. Converting a differentiated state into cellular pluripotency is a highly orchestrated process in which both exogenous OKSM factors and their endogenous counterparts play a distinct role in a stage-specific manner [3,4,5]. For initiating the reprogramming process, each reprogramming factor plays an essential and distinct role, such as erasing somatic identity and activating the endogenous counterpart. After the successful reprogramming of differentiated cells into an iPSC state, the transgenes are typically silenced due to high levels of DNA methyltransferases in iPSCs [3]. This result indicates that the transgenes are dispensable in the maintenance of an iPSC state [8] and that the endogenous pluripotential network is sufficient for maintaining
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