Liver cell reprogramming: parallels with iPSC biology.

Tissue damage—through normal wear-and-tear, toxin exposure, or assaults and injuries caused by predators—is inevitable, and thus regeneration is essential for the continued existence of biological entities. Mechanisms of regeneration, including proliferation, stem cell differentiation, and cellular transdifferentiation or reprogramming, depend on the organism and the nature or site of injury. The notion that changes in cellular identity could contribute to regeneration dates back more than a century, but our appreciation of cellular plasticity increased dramatically with the emergence of induced pluripotent stem cell (iPSC) technology in 2006. Aside from its therapeutic implications, the ability to convert fibroblasts into pluripotent stem cells in vitro prompted a series of biological questions regarding the ability of terminally differentiated cells to adopt other adult fates and the extent to which such changes in cell identity occur under physiological conditions.

We have recently shown that reprogramming occurs in vivo in the mammalian liver. During development, the 2 major cell types of the liver, hepatocytes and biliary epithelial cells (BECs), are derived from a common progenitor cell, the hepatoblast. Despite their common origin, these 2 differentiated cell types exhibit distinctive morphologies, gene expression patterns, and functions. While hepatocyte and biliary identity is maintained throughout the adult life of the animal, prior studies involving transplantation have suggested that the adult liver may retain a significant amount of cellular plasticity. Using a lineage tracing approach, we observed hepatocytes can undergo a robust process of biliary reprogramming following injury with numerous toxins, a process that is dependent on Notch signaling. As Notch signaling plays an instructive role in assigning a biliary fate during development, this finding indicates that the mechanism of biliary specification during reprogramming is conserved from embryogenesis. In addition, deletion of the essential Notch co-factor, RBP-J, attenuates the rate of injury-induced reprogramming, while expression of a constitutively active form of Notch in hepatocytes is sufficient to drive the process.

Several features of liver cell reprogramming are reminiscent of the process of iPSC reprogramming. The route from hepatocyte to BEC is a gradual one involving a “reprogramming cascade,” with distinct and sequential changes in morphology and gene expression patterns. For example, hepatocytes acquire primitive biliary features (e.g., decrease in cell size and re-alignment of cell polarity) and express primitive biliary markers (e.g., OPN and Sox9) shortly after injury, well before the cells adopt “terminal” biliary features (forming tubules and cilia and expressing of CK19, cytokeratin 19) later during the injury process. These tubules are often created with contribution from the reprogrammed hepatocytes joining pre-existing CK19+ biliary cells. The presence of cilia on the reprogrammed cells strongly suggests their functionality as biliary cells.

Similarly, iPSC generation is a multistep process. During the early phase of reprogramming, cells decrease in size, obtain epithelial cell characteristics, and upregulate various proliferative genes, while during the late phase of iPSC reprogramming, cells form ESC-like colonies and lose repressive chromatin marks, leading to the expression of endogenous pluripotency genes, such as Oct4. Transcription factors that mediate the reprogramming process during iPSC generation act in a distinct and sequential manner, characterized by both early changes (e.g., upregulation of proliferative genes, decreased cell size, H3K4me2 gain on many pluripotent genes) and late changes (e.g., activation of endogenous Nanog expression, loss of various repressive chromatin marks). The gradual nature of this reprogramming cascade most likely reflects the vast changes in global chromatin structure that are required for successful reprogramming, a phenomenon that contributes to the inefficiency of the process. Thus, it is likely that both hepatocyte reprogramming and iPSC reprogramming rely on similar molecular mechanisms, occurring with similar kinetics (1–3 wk) and involving an accumulation of epigenetic changes.

Another common theme of hepatocyte and iPSC reprogramming is heterogeneity. During injury, hepatocytes located in “zone 1” of the liver lobule (which are closest to portal veins and bile ducts) undergo biliary reprogramming with a much higher efficiency than “zone 3” hepatocytes (which are centrally located within the liver lobule). Similarly, despite near-universal activation of the Notch pathway in the liver, only those hepatocytes located in zone 1 and zone 2 can undergo biliary conversion. This result suggests that heterogeneity in reprogramming is a result of either a lack of competence of zone 3 hepatocytes (due, for example, to a difference in epigenetic state) or a requirement for a second signal that is absent from zone 3. Likewise, iPSC reprogramming is heterogenous, as the expression of all 4 “Yamanaka factors” (Klf4, Oct-3/4, SOX2, and c-Myc) in fibroblasts leads to pluripotency in only a subset of infected cells.

Other adult tissues besides the liver can undergo cellular reprogramming as part of an in vivo response to injury, including pancreatic α cells, pulmonary Clara cells, and others. Given the apparent similarities that exist between iPSC and adult cell reprogramming, the latter may serve as a useful “physiological” adjunct for understanding the molecular events that drive changes in cell identity. As both iPSC and adult cell reprogramming hold promise for cell-based therapy of disease, such mechanistic insights will be essential for translating this important cellular phenomenon into clinical practice.

Figure 1.Parallels in the multistep reprogramming process of hepatocytes and induced pluripotent stem cells. Early steps in iPSC generation and hepatocyte reprogramming both involve morphological changes, with cells decreasing in size and the former becoming more rounded. At this stage, gene expression involves downregulation of the starting cell identity (hepatocytes and fibroblasts) and upregulation of early genes expressed by the final cell type (biliary cells and iPSC, respectively). As the reprogramming process continues, additional structural and morphological changes take place along with expression of mature markers. (MET, mesenchymal to epithelial transition).