Faculty Opinions recommendation of Distinct Levels of Radioresistance in Lgr5(+) Colonic Epithelial Stem Cells versus Lgr5(+) Small Intestinal Stem Cells.

Current views of the identity, distribution, and regulation of small intestinal epithelial stem cells and their immediate progeny are discussed. Recent works implicating Wnt signaling in stem and progenitor proliferation, the involvement of Notch signaling in epithelial lineage specification, and the role of hedgehog and bone morphogenetic protein families in crypt formation are integrated. We had the good fortune that many of these papers came in pairs from independent groups. We attempt to identify points of agreement, reinterpret each in the context of the other, and indicate directions for continued progress.

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Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells

10.1101/2021.09.08.459297 ◽

2021 ◽

Author(s):

Keith A. Breau ◽

Meryem T. Ok ◽

Ismael Gomez-Martinez ◽

Joseph Burclaff ◽

Nathan P. Kohn ◽

...

Keyword(s):

Stem Cells ◽

Intestinal Stem Cells ◽

Epithelial Stem Cells ◽

Culture Methods ◽

Antibiotic Resistant ◽

Conventional Culture ◽

Transgenic Lines ◽

Small Intestinal ◽

Electroporation Efficiency

AbstractBackground & Aims2D monolayers of primary intestinal and colonic epithelial cells represent next-generation in vitro models of the gut. Efficient transgenesis and gene-editing in human intestinal stem cells (hISCs) would significantly improve utility of these models by enabling generation of reporter and loss/gain-of-function hISCs, but no published methods exist for transfecting 2D hISC monolayers. Electroporation has proven effective in other difficult-to- transfect cells; thus we applied this method to hISCs.MethodsTwenty-four electroporation parameters were tested, and the optimal condition for efficiency and viability was validated on hISCs from six anatomical regions along the small intestine and colon. PiggyBac™ transposase and Cas9 ribonucleoprotein (RNP) complexes were used for stable genomic integration of reporter genes. High-throughput methods for clone isolation, expansion, and screening were developed. An hISC OLFM4-emGFP reporter was generated and validated by qPCR, organoid assays, and hISC compartmentalization on a planar crypt-microarray (PCM) device.ResultsMaximum electroporation efficiency was 79.9% with a mean survival of 65%. Transfection of 105 hISCs produced ∼142 (0.14%) stable transposase-mediated clones. Transfection of OLFM4-targetting RNPs yielded ∼35% editing and 99/220 (45%) of antibiotic-resistant colonies analyzed expressed emGFP. OLFM4-emGFP hISCs applied to PCMs remained emGFP+ and proliferative in high-Wnt3a/R-spondin3/Noggin zones yet differentiated to emGFP-/KRT20+ cells outside engineered crypt zones. OLFM4-emGFP levels correlated with endogenous OLFM4. Olfm4-emGFPhigh cells were LGR5high/KRT20low, and demonstrated high organoid-forming potential.ConclusionsElectroporation of hISCs is highly efficient for stable transgenesis and transgenic lines can be generated in 3-4 weeks. Workflows mirror conventional culture methods, facilitating rapid integration into established tissue-culture operations. OLFM4high is a robust hISC marker with functional properties in culture.

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A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation

Journal of Radiation Research ◽

10.1093/jrr/rrt123 ◽

2014 ◽

Vol 55 (2) ◽

pp. 381-390 ◽

Cited By ~ 11

Author(s):

Motohiro Yamauchi ◽

Kensuke Otsuka ◽

Hisayoshi Kondo ◽

Nobuyuki Hamada ◽

Masanori Tomita ◽

...

Keyword(s):

Stem Cells ◽

Ionizing Radiation ◽

Intestinal Stem Cells ◽

Survival Assay ◽

Small Intestinal ◽

In Vitro Survival

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Heterogeneity in histone 2B-green fluorescent protein-retaining putative small intestinal stem cells at cell position 4 and their absence in the colon

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00080.2012 ◽

2012 ◽

Vol 303 (11) ◽

pp. G1188-G1201 ◽

Cited By ~ 20

Author(s):

Kevin R. Hughes ◽

Ricardo M. C. Gândara ◽

Tanvi Javkar ◽

Fred Sablitzky ◽

Hanno Hock ◽

...

Keyword(s):

Stem Cells ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Paneth Cells ◽

Epithelial Stem Cells ◽

Epithelial Regeneration ◽

Cell Position ◽

Small Intestinal ◽

Green Fluorescent ◽

Dose Radiation

Stem cells have been identified in two locations in small intestinal crypts; those intercalated between Paneth cells and another population (which retains DNA label) are located above the Paneth cell zone, at cell position 4. Because of disadvantages associated with the use of DNA label, doxycycline-induced transient transgenic expression of histone 2B (H2B)-green fluorescent protein (GFP) was investigated. H2B-GFP-retaining putative stem cells were consistently seen, with a peak at cell position 4, over chase periods of up to 112 days. After a 28-day chase, a subpopulation of the H2B-GFP-retaining cells was cycling, but the slow cycling status of the majority was illustrated by lack of expression of pHistone H3 and Ki67. Although some H2B-GFP-retaining cells were sensitive to low-dose radiation, the majority was resistant to low- and high-dose radiation-induced cell death, and a proportion of the surviving cells proliferated during subsequent epithelial regeneration. Long-term retention of H2B-GFP in a subpopulation of small intestinal Paneth cells was also seen, implying that they are long lived. In contrast to the small intestine, H2B-GFP-retaining epithelial cells were not seen in the colon from 28-day chase onward. This implies important differences in stem cell function between these two regions of the gastrointestinal tract, which may have implications for region-specific susceptibility to diseases (such as cancer and ulcerative colitis), in which epithelial stem cells and their progeny are involved.

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Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

The FASEB Journal ◽

10.1096/fasebj.31.1_supplement.1090.1 ◽

2017 ◽

Vol 31 (S1) ◽

Author(s):

Kunihiro Kishida ◽

Sarah Pearce ◽

Shiyan Yu ◽

Nan Gao ◽

Ronaldo Ferraris

Keyword(s):

Stem Cells ◽

Nutrient Sensing ◽

Intestinal Stem Cells ◽

Small Intestinal

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Unveiling role of Sphingosine-1-phosphate receptor 2 as a brake of epithelial stem cell proliferation and a tumor suppressor in colorectal cancer

10.21203/rs.3.rs-56319/v1 ◽

2020 ◽

Author(s):

Luciana Petti ◽

Giulia Rizzo ◽

Federica Rubbino ◽

Sudharshan Elangovan ◽

Piergiuseppe Colombo ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Normal Mucosa ◽

Genetically Engineered ◽

Colorectal Carcinogenesis ◽

Intestinal Stem Cells ◽

Cancer Information ◽

Intestinal Epithelial ◽

Epithelial Stem Cells ◽

Mucosal Regeneration

Abstract BackgroundSphingosine-1-phosphate receptor 2 (S1PR2) mediates pleiotropic functions encompassing cell proliferation, survival, and migration, which become collectively de-regulated in cancer. Information onto whether S1PR2 participates in colorectal carcinogenesis/cancer is scanty, and we set out to fill the gap.MethodsWe screened expression changes of S1PR2 in human CRC and matched normal mucosa specimens [N = 76]. We compared CRC arising in inflammation-driven and genetically engineered models in wild-type (S1PR2+/+) and S1PR2 deficient (S1PR2−/−) mice. We reconstituted S1PR2 expression in RKO cells and assessed their growth in xenografts. Functionally, we mimicked ablation of S1PR2 in normal mucosa by treating S1PR2+/+ organoids with JTE013, and characterized intestinal epithelial stem cells isolated from S1PR2−/−Lgr5-EGFP- mice.ResultsS1PR2 expression was lost in 33% of CRC; in 55%, it was significantly decreased, only 12% retaining expression comparable to normal mucosa. Both colitis-induced and genetic Apc+/min mouse models of CRC showed a higher incidence in size and number of carcinomas and/or high-grade adenomas, with increased cell proliferation in S1PR2−/− mice compared to S1PR2+/+ controls. Loss of S1PR2 impaired mucosal regeneration, ultimately promoting the expansion of intestinal stem cells. Whereas its overexpression attenuated cell cycle progression, it reduced the phosphorylation of AKT and augmented the levels of PTEN.ConclusionsIn normal colonic crypts, S1PR2 gains expression along with intestinal epithelial cells differentiation, but not in intestinal stem cells, and contrasts intestinal tumorigenesis by promoting epithelial differentiation, preventing the expansion of stem cells and braking their malignant transformation. Targeting of S1PR2 may be of therapeutic benefit for CRC expressing high Lgr5.

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ALX-0600, a glucagon-like peptide-2 analogue, protects small intestinal stem cells from radiation damage

Gastroenterology ◽

10.1016/s0016-5085(03)83089-5 ◽

2003 ◽

Vol 124 (4) ◽

pp. A609-A610 ◽

Cited By ~ 3

Author(s):

Christopher S. Potten ◽

Sarah Williamsom ◽

Demchyshyn Lidia L. ◽

Catherine S. Booth

Keyword(s):

Stem Cells ◽

Radiation Damage ◽

Intestinal Stem Cells ◽

Glucagon Like Peptide ◽

Small Intestinal

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Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00416.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. G592-G605 ◽

Cited By ~ 13

Author(s):

Kunihiro Kishida ◽

Sarah C. Pearce ◽

Shiyan Yu ◽

Nan Gao ◽

Ronaldo P. Ferraris

Keyword(s):

Stem Cells ◽

Cell Types ◽

Paneth Cells ◽

Nutrient Sensing ◽

Intestinal Stem Cells ◽

Intestinal Cell ◽

Cell Type ◽

Extended Lifespan ◽

Small Intestinal ◽

Single Cell Type

Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing. NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.

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