# Using a new regenerative model system to elucidate mechanisms for stem cell regulation

> **NIH NIH R35** · HARVARD UNIVERSITY · 2020 · $421,903

## Abstract

PROJECT SUMMARY
Though great advances have been made in uncovering molecular pathways that maintain pluripotency and
regulate differentiation in vertebrate cells in culture, the control of pluripotent stem cells and their progeny in
the context of whole animals is poorly understood. The long-term goal of our work is to obtain a
mechanistic understanding of how pluripotent stem cells are controlled in vivo such that they can
regenerate any missing cell type in an adult animal. The overall objective in this application is to identify the
cellular and molecular mechanisms that control the specification, maintenance, and response to regeneration
of pluripotent adult stem cells called “neoblasts” in the acoel Hofstenia miamia. Hofstenia can regenerate any
missing cell type and is amenable to high-throughput functional studies of regeneration. The rationale for
choosing a new model system over planarians, the more established system, is that Hofstenia produces
manipulable embryos in large numbers, allowing the use of methods currently unavailable in planarians to
answer outstanding questions about neoblast biology. The experiments proposed here will combine
lineage-tracing methods with functional genomic approaches to discover and characterize critical
regulators of pluripotent cells. This project will ask three major questions about neoblasts: 1) What are the
developmental origins of Hofstenia neoblasts and which molecular pathways are required for the formation of
these pluripotent cells, 2) Which gene regulatory networks mediate the maintenance of Hofstenia neoblasts,
and 3) How do individual neoblasts respond to amputation, i.e., proliferate, migrate, and/or differentiate during
regeneration. Lineage tracing and in situ hybridization in Hofstenia embryos, techniques that have been
established as feasible in the applicants' hands, will be utilized to identify the developmental origins of
pluripotent cells. Transcriptome profiling will then be used to identify candidate genes that control the
specification of neoblasts during development; the functions of these candidates will be studied by RNAi and
CRISPR/Cas9-mediated genome editing. Genome-wide assays for assessing chromatin state will be used to
identify regulatory DNA for known neoblast genes and the upstream transcription factors that putatively
regulate the genes. The functions of the DNA and the associated transcription factors in maintenance of
pluripotent stem cells will be assayed via CRISPR-Cas9 genome editing and RNAi respectively. Individual
neoblasts will be labeled by photoconverting fluorescent proteins that label neoblasts and their proliferation,
migration, and differentiation will be monitored via time lapse confocal microscopy. This proposal is
innovative in its use of a new model system that enables the study of long-standing questions about
stem cell biology by using approaches that cannot be achieved by studying established model
systems. This project will reveal basic cellula...

## Key facts

- **NIH application ID:** 9944592
- **Project number:** 5R35GM128817-03
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** Mansi Srivastava
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $421,903
- **Award type:** 5
- **Project period:** 2018-07-13 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9944592

## Citation

> US National Institutes of Health, RePORTER application 9944592, Using a new regenerative model system to elucidate mechanisms for stem cell regulation (5R35GM128817-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9944592. Licensed CC0.

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