# Phylogenetic reconstruction of the intestinal epithelium via evolving barcodes

> **NIH NIH F30** · UNIVERSITY OF PENNSYLVANIA · 2020 · $32,610

## Abstract

Project Summary
 The intestine is the highest turnover tissue in the body, making it an excellent model of adult stem cell
biology. Constant renewal makes the gut susceptible to colorectal cancer and injury, such as radiation toxicity.
Intestinal stem cells (ISCs) are integral to the renewal of the intestine and hence to many of its numerous
pathologies. Despite the clinical significance of ISCs, two unreconciled models of their identity and behavior
exist. The hierarchical model argues that two distinct ISC types exist – rare quiescent stem cells (QSCs) and
more abundant, rapidly-dividing crypt base columnar stem cells (CBCs). Both CBCs and QSCs self-renew and
differentiate, but QSCs can only come from other QSCs. On the other hand, the continuum model argues that
CBCs are the only true stem cells and that they can exist in a continuum of states, including quiescence.
Extensive efforts to resolve these two models have been unsuccessful and contradictory. The primary source
of confusion has been an overreliance on purported cell type-specific promoters, which mark overlapping and
heterogeneous cell populations.
 A novel, unbiased, and precision approach is required to probe the existence of a distinct QSC
population. With this in mind, we will combine phylogenetic inference with CRISPR/Cas9 genome editing to
thoroughly map the intestinal cellular division tree without the use of biased promoters. We will generate
transgenic mice that contain the elements of the CRISPR/Cas9 system, as well as a short synthetic DNA
barcode sequence that is targeted by Cas9. As intestinal cells divide, their barcodes are cut and repaired by
the mutation-prone pathway of nonhomologous end-joining (NHEJ), producing an enormous diversity of
heritable mutations. Barcodes are then sequenced from single cells, along with the transcriptome, providing a
complete picture of both cellular lineage history and identity.
 By applying this system to the intestine, we hypothesize that we will observe lineage trees matching the
predictions of the hierarchical model, namely a continuous QSC lineage that all other cells branch from. The
existence of a continuous QSC lineage would act to maintain genome integrity in the face of replicative and
metabolic stress, which carries implications for carcinogenesis and aging biology. More broadly, our approach
will serve as a template for probing tissue development, maintenance, injury, and neoplasia across a variety of
tissues with unprecedented depth.

## Key facts

- **NIH application ID:** 9895428
- **Project number:** 5F30DK120135-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Kamen P Simeonov
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $32,610
- **Award type:** 5
- **Project period:** 2019-03-13 → 2022-03-13

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9895428

## Citation

> US National Institutes of Health, RePORTER application 9895428, Phylogenetic reconstruction of the intestinal epithelium via evolving barcodes (5F30DK120135-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9895428. Licensed CC0.

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