# Modifying regulators of heterochromatin to improve reprogramming to functional hepatocytes

> **NIH NIH K01** · UNIVERSITY OF PENNSYLVANIA · 2020 · $105,260

## Abstract

PROJECT SUMMMARY/ABSTRACT
Liver transplantation is the only curative treatment for many liver diseases, including hepatocellular carcinoma
and biliary atresia. However, the supply of donor livers is insufficient to meet the growing need. Cell identity
during embryogenesis is progressively established as cell fate choices are made and the potential to
differentiate into alternative lineages is restricted. With the discovery of induced pluripotency, wherein a
somatic cell is reprogrammed to an induced pluripotent stem cell (iPSC) through the ectopic expression of
transcription factors, it was revealed that it was possible to manipulate cell identity in ways that were previously
inconceivable. In addition to reprogramming to iPSC, multiple methods have been developed for trans-
differentiation where cells of one identity are converted to another, such as the induced conversion of
fibroblasts to hepatocytes. Although these methods exist, it has been well documented that the cells they
produce incompletely recapitulate the target cell both in gene expression and functionality. Accurate cell
reprogramming or conversion is dependent upon activating silent gene regulatory networks. We found that
H3K9me3 heterochromatin blocks binding of the reprogramming factors to target sites and impedes the
activation of the desired gene network both in reprogramming to iPSC and in direct conversion of fibroblasts to
human induced hepatocytes (hiHeps). We developed methodology to physically extract these recalcitrant
heterochromatin regions, based upon the criteria of sonication resistance, and determined their protein
composition; designating as sonication resistant heterochromatin (srHC) associated proteins. This process
found both well-known regulators of H3K9me3 and heterochromatin as well as novel proteins not previously
known to have roles in heterochromatin and cell identity maintenance. Our functional siRNA screen of 94 srHC
associated proteins identified many which repressed expression of heterochromatically silenced hepatocyte
genes in hiHeps. Based upon our extensive preliminary data I hypothesize that altering expression of these
heterochromatin associated proteins to selectively modify the epigenome can improve the accuracy and
functionality of hiHeps induced from fibroblasts. Functional assessment of the epigenetically modified hiHeps
will be determined through RNA-seq and serial transplantation studies in an immunodeficient mouse model
where liver humanization can be performed and liver function assessed. Utilizing this approach I will identify
how subtypes of heterochromatin form and are maintained, as well as how they can be selectively destabilized
to facilitate enhanced reprogramming to the hepatic lineage.

## Key facts

- **NIH application ID:** 9986758
- **Project number:** 5K01DK117970-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Ryan Lehman McCarthy
- **Activity code:** K01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $105,260
- **Award type:** 5
- **Project period:** 2018-08-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9986758

## Citation

> US National Institutes of Health, RePORTER application 9986758, Modifying regulators of heterochromatin to improve reprogramming to functional hepatocytes (5K01DK117970-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9986758. Licensed CC0.

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