# Laboratory Opossum iPSC Lines for Biomedical Research

> **NIH NIH R21** · UNIVERSITY OF TEXAS RIO GRANDE VALLEY · 2020 · $181,625

## Abstract

PROJECT SUMMARY/ABSTRACT
The laboratory opossum is the only marsupial that is available in large numbers for biomedical research; it is a
unique or specialized model for research on many human diseases and developmental processes, as well as
for comparative biology and comparative genomics purposes. The long-term objective is to establish induced
pluripotent stem cell (iPSC) lines from this species for use in those research areas, and particularly for developing
iPSC therapies. The specific aims are 1) to optimize iPSC reprogramming methodology and establish iPSC lines
from two inbred strains, 2) to validate the method while reprogramming iPSCs from four partially inbred strains,
and 3) to demonstrate the functional potential of reprogrammed iPSC lines. Using rapidly dividing skin fibroblasts
derived from embryos, we will adapt a method developed by one of us in creating the first iPSC lines from any
marsupial species, the Tasmanian devil. We will use lentivirus-based delivery of human reprogramming factors
in the initial experiments, and we will determine the reprogramming efficiency in MEF feeder-based and feeder-
free culture conditions. We will then optimize a transgene integration-free reprogramming protocol using
OriP/EBNA episomal plasmid based delivery of the human iPSC reprogramming factors. In the third set of
experiments, we will test various culture conditions by modulating growth factors and small molecules to improve
reprogramming efficiency. The established iPSCs will be characterized by immunocytochemistry and differential
gene expression analysis of the pluripotency markers. For Aim 2, the developed methodology will be applied to
fibroblasts of animals from four partially inbred strains. For Aim 3, the established iPSC lines from all six animals
will be differentiated into neurons, cardiomyocytes, and hepatocytes (terminally differentiated cells of all three
germ layers). Fibroblasts from the six animals, their reprogrammed iPSCs (2 clones/animal), and differentiated
cells (from 1 iPSC clone/animal) will undergo genome-wide RNA sequencing (RNASeq) to better understand the
gene/transcript expression profiles and functional potential of the iPSCs and their differentiated cells. The
genomic integrity of the generated cells will be determined from RNASeq data by performing “location enrichment
analysis” and “CGH-PCF analysis.” In addition, iPSC expression profiles will be compared with existing human
and mouse iPSC RNASeq data in a comparative approach to develop a better understanding of pluripotency.
The established iPSC lines will be used by us (beyond the term of this project) for the development of iPSC
therapies for hypercholesterolemia and non-alcoholic fatty liver disease. They also will be available to
collaborators for research in diverse areas of biomedicine such as therapies for spinal cord injury, and prevention
of birth defects caused by drugs such as thalidomide; as well as for the development of fundamental knowledge
...

## Key facts

- **NIH application ID:** 9990867
- **Project number:** 5R21OD026625-02
- **Recipient organization:** UNIVERSITY OF TEXAS RIO GRANDE VALLEY
- **Principal Investigator:** Satish Kumar
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $181,625
- **Award type:** 5
- **Project period:** 2019-08-15 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9990867

## Citation

> US National Institutes of Health, RePORTER application 9990867, Laboratory Opossum iPSC Lines for Biomedical Research (5R21OD026625-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9990867. Licensed CC0.

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