# Derivation and characterization of induced trophoblast stem cells

> **NIH NIH R21** · UNIVERSITY OF GEORGIA · 2020 · $188,750

## Abstract

PROJECT DESCRIPTION
The trophectoderm (TE) is an extraembryonic tissue that supplies instructive signals required for embryo
patterning during gastrulation and gives rise to the placenta, an organ that connects the developing fetus to
the uterine wall to allow nutrient uptake, waste elimination, gas exchange and thermoregulation via the
mother's blood supply. In mice, trophoblast stem cells (mTSCs) derived from the preimplantation blastocyst or
from fibroblasts via direct lineage conversion have become an important tool with which to dissect the
mechanisms responsible for TE specification, differentiation and function. However, attempts to establish TSCs
with features of early human TE have been unsuccessful. Our proposal seeks to fill this gap in knowledge by
establishing induced human TSCs (i-hTSCs) through the process of lineage conversion. To better understand
how TSCs form in the human embryo we analyzed previously collected single cell RNA sequencing data from
human blastocyst stage embryos. We identified a set of transcription factors that are highly expressed in the
TE but not in the embryonic epiblast (EPI) cells. We also identified several signaling ligands expressed in the
EPI and/or in the TE itself that may be responsible for specification and/or maintenance of TSCs. To facilitate
lineage conversion experiments we have generated human embryonic stem cells (hESCs) that carry a
fluorescent mCherry reporter linked to the endogenous GATA2 or GATA3 genes which are highly active in all
cells of the TE. We have differentiated these cells to obtain ESC-derived human fibroblast-like cells.
Transduction of these reporter cells with the cocktail of doxycycline (Dox)-inducible TE-specific transcription
factors yielded several GATA2/3-mCherry positive clones. These clones could be passaged in the presence of
Dox and expressed the key genes known to regulate TSC self-renewal. Based on these data, we hypothesize
that hTSCs can be induced from fibroblasts and /or hESCs via direct lineage conversion with a cocktail of TE-
specific factors in the presence of maintenance cytokines and/or signaling inhibitors. This hypothesis will be
tested via two specific aims. In Aim 1, we will develop protocols for direct lineage conversion into i-hTSCs from
existing hESCs and/or fibroblasts: (1A) Define the minimal set of conversion factors required for induction of
hTSCs from hESCs or fibroblasts; and (1B) Define a set of cytokines and/or signaling inhibitors which allow
Dox-independent maintenance of i-hTSCs. In Aim 2, we will perform functional and molecular characterization
of i-hTSC lines. Our approach is innovative, because it will develop new biological reagents that would allow
derivation and maintenance of early i-hTSCs. The proposed research is significant, because it would enable
better understanding of fundamental biology of blastocyst lineages in humans. Such knowledge has the
potential to inform new treatments of pregnancy complications, infertility a...

## Key facts

- **NIH application ID:** 10017694
- **Project number:** 5R21HD099694-02
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** Natalia B Ivanova
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $188,750
- **Award type:** 5
- **Project period:** 2019-09-12 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10017694

## Citation

> US National Institutes of Health, RePORTER application 10017694, Derivation and characterization of induced trophoblast stem cells (5R21HD099694-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10017694. Licensed CC0.

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