Decoding the Molecular Mechanisms Underlying Reprogramming to Functional Hepatic and Intestinal Cell Types

Recent advances in stem cell research over the past decade have sparked considerable interest in their therapeutic potential for treating gastrointestinal and hepatic disorders. By reprogramming somatic cells to pluripotency and differentiating them or directly reprogramming cells between somatic states via transcription factor (TF) overexpression, we can produce clinically valuable cells from abundant sources like skin fibroblasts. Despite its potential, direct reprogramming often leads to low yield and incomplete specification of target cells, limiting its practical use for therapy or disease modeling. Further, while many protocols to derive hepatocyte-like cells in mouse and human exist, there is a current unmet need to derive intestinal-like cells via reprogramming. Building on our previous five years of R01-funded research, we aim to answer why reprogramming is inefficient and how we can guide engineered cells toward a more defined identity and functionality. The conversion of mouse embryonic fibroblasts to induced endoderm progenitors (iEPs) represents a prototypical reprogramming strategy, producing cells that can functionally engraft the liver and intestine. Our work to date has demonstrated heterogeneity in this reprogramming process, revealing defined conversion trajectories to distinct cell populations. In our proposed research, we aim to; 1) Characterize reprogramming origin gene regulatory state, charting exogenous TF engagement in successful fate conversion. By understanding a) where ectopic TFs bind upon reprogramming initiation and b) the genetic regulatory barriers to this binding, we will reveal fundamental mechanistic knowledge that can be used to enhance the fidelity and efficiency of reprogramming; 2) Characterize hepatic and intestinal potential of iEPs. Our current data suggest that iEPs are a heterogeneous population of cells harboring hepatic and intestinal potential. By employing multiomic single-cell lineage tracing to characterize the heterogeneity of iEPs, we will track their successful engraftment into the liver and intestine to define their cellular potential. Further, we will track iEP differentiation within these target organs to establish a comprehensive blueprint for in vitro cell maturation; 3) Assess and optimize human direct reprogramming to induced endoderm progenitors. While many TF cocktails directing cells to hepatic identity have been reported, there is currently an unmet need to derive reprogrammed intestinal identities. Our preliminary data suggest that akin to mouse iEPs, human hepatocyte-like cells harbor intestinal identity. We hypothesize that the pioneer TF-based cocktails used in human reprogramming install this broad potential. By applying our innovative genomic technologies, we aim to unlock unforeseen potential in these engineered cells, paving the way to produce functional human liver and intestine cells through reprogramming.