PROJECT SUMMARY
The plasticity of cell identity is evident through nuclear transfer and transcription factor (TF)-mediated
reprogramming. Despite this, current reprogramming methods often yield developmentally immature and
heterogeneous cell populations and therefore are unsuitable for therapeutic application or disease modeling. We
seek to address the fundamental questions of why direct reprogramming is inefficient, representing a critical
gap in knowledge that will be widely applicable across many cell fate engineering strategies and has
broader significance for understanding how cell identity is regulated. Over the past five years of NIGMS-
supported research, we have developed and applied innovative single-cell multiomic lineage tracing and novel
computational technologies to dissect the mechanisms of pioneer TF-mediated reprogramming. Our proposed
research builds on this work, focusing on two primary objectives:
1. Current evidence supports the hypothesis that successful reprogramming events arise from rare
'reprogramming permissive' cell types or states in which normally inaccessible target genes are engaged by
ectopic TFs to drive fate change. However, the lack of understanding about the origins of reprogramming
presents a significant challenge in characterizing reprogramming permissive states. We will develop and
apply multiomic lineage tracing to identify the origins of successfully reprogrammed cells across various cell
fate conversion strategies. Elucidating reprogramming initiation mechanisms will identify new avenues to
enhance reprogramming efficiency, uncovering common and cell-type specific regulation of cell identity.
2. The identification of new, more effective reprogramming cocktails represents a current gap in cell
engineering. We hypothesize that expanded cocktails of precisely delivered TFs targeting the gene
regulatory networks controlling terminal cell identity will more faithfully reprogram fate. However, it is currently
experimentally and computationally intractable to predict these cocktails de novo. We will use cell fusion in
combination with our genomic technologies to empirically deconstruct gene regulation, providing unique
insights into efficient and accurate reprogramming, informing the development of novel TF cocktails.
The outcomes of this research will have significant impacts on multiple levels: a) It will facilitate improved
conversion efficiency and fidelity across different cell engineering strategies, overcoming a current barrier in
regenerative medicine; b) The experimental manipulation of cell fate offers a valuable model system to
deconstruct and model dynamic changes in cell identity. Our study of diverse reprogramming strategies will
uncover general and cell type-specific rules for cell fate specification and maintenance, providing broad biological
relevance beyond cell engineering; c) We will continue to develop our innovative genomic technologies, which
provide insight into the regulation of cell identity across diverse cell biology paradigms.