Hierarchy and Plasticity in the Intestinal Stem Cell Compartment

Supplementary Material

Hierarchy and Plasticity in the Intestinal Stem Cell Compartment

Maryam Yousefi1, Linheng Li2 and Christopher J. Lengner1

1Department of Biomedical Sciences, School of Veterinary Medicine and Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104

2Stowers Institute for Medical Research, Kansas City, Missouri, 64110, USA

Corresponding author: Lengner, C.J. (); Li, L. ()

Table S1. Proxy Reporter Alleles Marking Intestinal Stem Cell Populations, Their Specificity, and Contribution toIntestinal Regeneration and Oncogenesis.

Marker / Reporter Type / Marked Cell Position / Marked Cell Function (Assay) / Regeneration under basal conditions / Function during regeneration (Injury model) / Tumor initiation capacity (model) / Ref
Lgr5
Lgr5-eGFP-IRES-CreER (CreER) / Targeted knock-in / Crypt base / CBCs (Lineage tracing) / Frequent lineage tracing / Gene ablation in Lgr5 ISCs impairs regeneration (12 Gy gamma IR), lineage tracing after etiposide treatment / hyperplasia uponAPCflox/flox deletion / [1–5]
Lgr5-eGFP-IRES-CreER (eGFP) / Targeted knock-in / Crypt base and above / CBCs &T/A cells (Organoid formation) / N/A / Lgr5-eGFPHigh cells are radiosensitive (12 Gy gamma IR) / N/A / [1,2,6,7]
Lgr5-LacZ / Targeted knock-in / Crypt base and above / Undetermined / N/A / N/A / N/A / [1]
Lgr5-dsRED-IRES-CreERT2 / Targeted knock-in / Crypt base and above / CBCs (Lineage tracing) / Lineage tracing up to 60 days after CreER induction / N/A / N/A / [8]
Lgr5-DTR-eGFP / Targeted knock-in / Crypt base and above / CBCs (Organoid formation) / Cell ablation is tolerated in vivo and in vitro / Cell ablation impairs regeneration after 10 Gy radiation, but not following DSS / APC loss in the epithelium drives hyperplasia in the absence of Lgr5+ cells / [8,9]
mRNA / Endogenous / Crypt base and above / Undetermined / N/A / N/A / N/A / [8,10,11]
Protein / Endogenous / Crypt base (Human) / Undetermined / N/A / N/A / N/A / [12]
Bmi1
Bmi1-CreER / Targeted knock-in / Crypt cells from the +2 to +8 position and rare cells within villi / Reserve ISC (Lineage Tracing/Organoid formation/Cell ablation) / Lineage tracing up to 12 months, Bmi1-Cre+ cell ablation results in crypt loss / Contributes to regeneration following irradiation (12 Gy gamma IR) and etiposide treatment / Adenomas upon deletion of Ctnnb1exon3flox / [2,5,13]
Bmi1-CreER / Random integrant BAC transgenic / Single cells at position +3 to +6 / Reserve ISC (Lineage Tracing) / CBC generation under basal conditions / Increased CBC generation and lineage tracing after CBC cell ablation / N/A / [8]
Bmi1-GFP / Targeted knock-in / Position +3 to +6 (IF for GFP), undetectable by flow cytometry / Undetermined / N/A / Increase in frequency upon CBC ablation / N/A / [2,8]
mRNA / Endogenous / Crypt base and above / Undetermined / N/A / N/A / N/A / [2,11,14]
Protein / Endogenous / Around the +4 position / Undetermined / N/A / N/A / N/A / [15]
Hopx
Hopx-CreER / Targeted knock-in / Rare cells at or near the +4 position / Reserve ISC (Lineage Tracing/Organoid formation) / Lineage tracing with no clonal extinction 6 months after induction / Gene ablation in Hopx ISCs impairs regeneration (12 Gy gamma IR), lineage tracing after etiposide treatment / N/A / [2,5,16]
Hopx-eGFP / Targeted knock-in / Crypt base and above / Reserve ISCs, Active CBCs, Secretory cells (Organoid formation) / N/A / N/A / N/A / [2]
mRNA / Endogenous / Crypt base and above / Undetermined / N/A / N/A / N/A / [2,11]
Protein / Endogenous / Present in Lgr5-eGFP+ cells (proteomic analysis) / Undetermined / N/A / N/A / N/A / [10]
Lrig1
Lrig1-CreER / Targeted knock-in / Scattered single cells throughout crypt / Reserve and active ISCs (lineage tracing) / Lineage tracing up to 90 days, decreasing tracing events over time / Increased lineage tracing after 8 Gy x-ray injury / APC loss drives intestinal adenomas and colonal tumors / [17]
Lrig1-mApple / Random integrant BAC transgenic / Crypt base and above / Undetermined / N/A / N/A / N/A / [18]
mRNA / Endogenous / Crypt base and above (more Lrig1 mRNA in Lgr5 cells than Lrig-CreER cells) / Undetermined / N/A / N/A / N/A / [2,11,17,19]
Protein / Endogenous
Lrig1- antibody / Recognizes glycosylated & non-glycosylated Lrig1 / Crypt base / Undetermined / N/A / N/A / N/A / [10,18,19]
Lrig1-VU antibody / Recognizes non-glycosylated form of Lrig1 / One to three cells at the crypt base and occasionally cells higher up the crypt / Some overlap with Lgr5 CBCs (gene expression) / N/A / N/A / N/A / [17,18]
mTert
mTert–CreER / Random integrant transgenic / Single or small clusters of cells around +4 position / Reserve ISs (Lineage tracing) / Lineage tracing is detected even after one year / Tracing following 1, 10, and 15 Gy gamma-IR / N/A / [20]
mTert-eGFP / Random integrant transgenic / Rare cells at position +5 to +8, one marked cell per 150 crypts / Undetermined / N/A / Resistant to 10 Gy gamma-IR / N/A / [20,21]
mRNA / Endogenous / Crypt base / Undetermined / N/A / N/A / N/A / [2,10,14]
Protein / Endogenous / N/A / Undetermined / N/A / N/A / N/A
Sox9
Sox9-CreERT2 / Targeted knock-in / Crypt base / Reserve ISCs, CBCs, Paneth cells (lineage tracing) / Frequent lineage tracing / Tracing following 14 Gy x-ray radiation / N/A / [22]
Sox9-eGFP / Random integrant BAC transgenic / Crypt base / Reserve ISCs, CBCs, secretory progenitors (organoid formation, expression) / N/A / N/A / N/A / [22,23]
mRNA / Endogenous / Crypt base / N/A / N/A / N/A / N/A / [2,11,14]
Protein / Endogenous / Crypt base and some cells in the villi / N/A / N/A / N/A / N/A / [24,25]
Msi1
Msi1-eGFP / Random integrant transgenic / Crypt base and above / N/A / N/A / N/A / N/A / [26]
mRNA / Endogenous / Crypt base and above / N/A / N/A / N/A / N/A / [2,11,14]
Protein / Endogenous / Crypt base and above / N/A / N/A / Msi1 protein is upregulated during regeneration / N/A / [2,3,27,28]
DCLK1
DCKL1-CreER / Random integrant BAC transgenic / Rare cells around +4 position in the crypt / Tuft cells (Immunostaining), rarely long-lived (lineage tracing) / Rare lineage tracing. Cell ablation tolerated in vivo. / Cell ablation impairs regeneration (12 Gy radiation or 3% DSS; tracing is rare) / Colon tumor only after both deletion of APC and DSS treatment / [29]
DCKL1-CreER / Targeted knock-in / Scattered cells, mostly in the lower crypt / Tuft cells(Immunostaining) / No lineage tracing, cell ablation tolerated in vivo. / Rare tracing after radiation 8Gy or DSS (2%) / Polyp collapse after cell ablation, no adenoma in Ctnnb1exon3flox model / [30]
mRNA / Endogenous / Isolated cells from the base of the crypts up to villi / Tuft cells (Transcript counting) / N/A / N/A / N/A / [14]
Protein / Endogenous / Rare cells at crypt base, +4 position, and villi / Some CBCs and Tuft cells (Immunostaining for Tuft cell markers) / N/A / Protein not detected during regeneration following 12 Gy gamma IR / Limited expression pattern in APC min tumors / [29,31,32]
Axin2
Axin2-CreERT2-tdTomato(CreER) / Targeted knock-in / Crypt base and above / CBCs, Reserve ISCs, and TA cells / Frequent lineage tracing events / Frequent lineage tracing following 12 Gy gamma-IR / N/A / [33]
Axin2-CreERT2-tdTomato (Tomato) / Targeted knock-in / Crypt base and above / CBCs, Reserve ISCs, and TA cells / N/A / N/A / N/A / [34]
Axin2-LacZ / Targeted knock-in / Crypt base and above / Crypt cells except Paneth cells / N/A / N/A / N/A / [25,35]
mRNA / Endogenous / Crypt base / CBCS and Reserve ISCs (single cell gene expression analysis) / N/A / N/A / N/A / [34,36]
Protein / Endogenous / Crypt base / Undetermined / N/A / N/A / N/A / [36]
Dll1
Dll1-GFP-IRES-CreERT (CreER) / Targeted knock-in / Single cells around +5 position / Secretory progenitors (lineage tracing) / Rare lineage tracing (~10 per mouse) / Rare lineage tracing after injury (6 Gy gamma-IR) / N/A / [37]
Dll1-GFP-IRES-CreERT(GFP) / Targeted knock-in / Paneth cells and some cells higher in the crypt and villi / Paneth cells, secretory progenitors, and goblet cells (histology, expression) / Rarely generate Lgr5+ ISCs. Organoid formation only with Wnt3a / N/A / [37]
mRNA / Endogenous / Paneth cells and some cells higher in the crypt and villi / N/A / N/A / N/A / N/A / [37]
Protein / Endogenous / N/A / N/A / N/A / N/A / N/A
Alpi
Alpi-IRES-CreERT2 / Targeted knock-in / Cells in the villi / Enterocytes / No tracing under basal condition / Tracing after ablation of CBCs / No tumor formation after APC/Kras mutation / [38]
mRNA / Endogenous / Cells in the villi / Enterocytes / N/A / N/A / N/A / [38]
Protein / Endogenous / Cells in the villi / Enterocytes / N/A / N/A / N/A / [39]
Label retention
TetO-H2B-eGFP / Targeted knock-in (safe haven chromatin) / Crypt base and above at +3 to +5 positions / Paneth Cells, enteroendocrine cells, secretory progenitors, and rare reserve ISCs / N/A / No EdU incorporation post-12Gy gamma-IR / N/A / [11]
Cyp1a1-H2B-YFP / Random integrant transgenic / Crypt base and above at +3 to +5 positions / Paneth Cells, secretory progenitors / 2 day lineage tracing / Very rare lineage tracing (~10 events per mouse) after injury / N/A / [40]

N/A: Not applicable

Reference

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