# Inferring Gene Regulatory Networks Governing Definitive Endoderm Differentiation from Single Cell RNA Velocity Measurements

> **NIH NIH F31** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2021 · $37,550

## Abstract

Project Summary
The World Health Organization estimates that over 65 million people suffer from moderate to severe chronic
obstructive pulmonary disease, a condition characterized by poor airflow and restricted breathing 1. The ability
to regenerate damaged lung tissue would dramatically improve the quality of life for these individuals while
reducing the prevalence and burden of pulmonary diseases worldwide. A promising approach to this problem
is to use human pluripotent stem cells to produce lung and airway progenitor cells. Indeed, specialized
protocols have been developed to convert stem cells into definitive endoderm, a lung precursor cell type 10-19.
These protocols use small molecules to modulate the expression of key regulators of lung development
including WNT, TGFβ, BMP, and FGF; however, these protocols are limited by the inability to generate a
homogeneous population of definitive endoderm cells 11,15. This problem necessitates a better mechanistic
understanding of how individual cells transition from their pluripotent cell state into definitive endoderm.
Specifically, there is a critical need to understand how the gene regulatory networks in a given cell control its
morphogenesis, proliferation, and differentiation decisions. Therefore, with the long-term goal of increasing
homogeneity in lung precursor cells, the research objective of this fellowship is to determine how
transcriptional heterogeneity in human embryonic stem cells influences their commitment to definitive
endoderm. I hypothesize that heterogeneity in the starting population of cells generates alternate trajectories to
definitive endoderm (or other cell types) and that these differences increase over time due to mutual inhibition
between specific pairs of transcription factors (e.g., OCT4/SOX17, NANOG/GATA6). To test this hypothesis, I
will first use single-cell RNA sequencing26 to define the transcriptional heterogeneity in human pluripotent stem
cells during differentiation to definitive endoderm. I will then quantify the time-dependent changes in gene
expression for each cell using RNA velocity, a computational method that uses spliced and unspliced transcript
counts to estimate future gene expression states 28-29. Using these single-cell measurements, I will then
develop a mechanistic model of the gene regulatory networks governing differentiation to DE and validate the
model using known gene-gene interactions. Model simulations will: (1) confirm major gene regulators that drive
differentiation; (2) identify novel gene networks that control heterogeneity before and during differentiation; and
(3) reveal crosstalk among gene regulatory networks governing differentiation and other ongoing cellular
processes such as proliferation and metabolism. The proposed experimental and computational studies
provide a general framework to systematically identify gene regulatory mechanisms controlling differentiation to
definitive endoderm and aid in the development of more effic...

## Key facts

- **NIH application ID:** 10140686
- **Project number:** 1F31HL156433-01
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Jolene Sarah Ranek
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $37,550
- **Award type:** 1
- **Project period:** 2021-04-02 → 2024-04-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10140686

## Citation

> US National Institutes of Health, RePORTER application 10140686, Inferring Gene Regulatory Networks Governing Definitive Endoderm Differentiation from Single Cell RNA Velocity Measurements (1F31HL156433-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10140686. Licensed CC0.

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