Effects of dose rates on radiation-induced replenishment of intestinal stem cells determined by Lgr5 lineage tracing

Abstract Background Radiation-induced rectal epithelial damage is a very common side effect of pelvic radiotherapy and often compromise the life quality and treatment outcome in patients with pelvic malignancies. Unlike small bowel and colon, effect of radiation in rectal stem cells has not been explored extensively. Here we demonstrate that Lgr5-positive rectal stem cells are radiosensitive and organoid-based transplantation of rectal stem cells mitigates radiation damage in rectum. Methods C57Bl6 male mice (JAX) at 24 h were exposed to pelvic irradiation (PIR) to determine the radiation effect in pelvic epithelium. Effect of PIR on Lgr5-positive rectal stem cells (RSCs) was determined in Lgr5-EGFP-Cre-ERT2 mice exposed to PIR. Effect of PIR or clinically relevant fractionated PIR on regenerative response of Lgr5-positive RSCs was examined by lineage tracing assay using Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice with tamoxifen administration to activate Cre recombinase and thereby marking the ISC and their respective progeny. Ex vivo three-dimensional organoid cultures were developed from Lgr5-EGFP-Cre-ERT2 mice. Organoid growth was determined by quantifying the budding crypt/total crypt ratio. Organoids from Lgr5-EGFP-ires-CreERT2-TdT mice were transplanted in C57Bl6 male mice exposed to PIR. Engraftment and repopulation of Lgr5-positive RSCs were determined after tamoxifen administration to activate Cre recombinase in recipient mice. Statistical analysis was performed using Log-rank (Mantel-Cox) test and paired two-tail t test. Result Exposure to pelvic irradiation significantly damaged rectal epithelium with the loss of Lgr5+ve rectal stem cells. Radiosensitivity of rectal epithelium was also observed with exposure to clinically relevant fractionated pelvic irradiation. Regenerative capacity of Lgr5+ve rectal stem cells was compromised in response to fractionated pelvic irradiation. Ex vivo organoid study demonstrated that Lgr5+ve rectal stem cells are sensitive to both single and fractionated radiation. Organoid-based transplantation of Lgr5+ve rectal stem cells promotes repair and regeneration of rectal epithelium. Conclusion Lgr5-positive rectal stem cells are radiosensitive and contribute to radiation-induced rectal epithelial toxicity. Transplantation of Lgr5-positive rectal stem cells mitigates radiation-induced rectal injury and promotes repair and regeneration process in rectum.

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Interplay among p21Waf1/Cip1, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury

Scientific Reports ◽

10.1038/s41598-020-75171-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Emilia J. Orzechowska ◽

Takahito Katano ◽

Agnieszka B. Bialkowska ◽

Vincent W. Yang

Keyword(s):

Stem Cells ◽

Gamma Radiation ◽

Therapeutic Potential ◽

Mrna Translation ◽

Negative Regulator ◽

Intestinal Stem Cells ◽

Quiescent State ◽

Γ Radiation ◽

Regenerative Response ◽

Radiation Induced

Abstract Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

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Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1013004108 ◽

2010 ◽

Vol 108 (1) ◽

pp. 179-184 ◽

Cited By ~ 337

Author(s):

Robert K. Montgomery ◽

Diana L. Carlone ◽

Camilla A. Richmond ◽

Loredana Farilla ◽

Mariette E. G. Kranendonk ◽

...

Keyword(s):

Stem Cells ◽

Reverse Transcriptase ◽

Tissue Injury ◽

Cell Types ◽

Lineage Tracing ◽

Intestinal Stem Cells ◽

Intestinal Cell ◽

Stem Cell Population ◽

Telomerase Reverse Transcriptase

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

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Correction to: The Role of Intestinal Stem Cells in Epithelial Regeneration Following Radiation-Induced Gut Injury

Current Stem Cell Reports ◽

10.1007/s40778-018-0121-0 ◽

2018 ◽

Vol 4 (1) ◽

pp. 95-95

Author(s):

Chang-Kyung Kim ◽

Vincent W. Yang ◽

Agnieszka B. Bialkowska

Keyword(s):

Stem Cells ◽

Intestinal Stem Cells ◽

Epithelial Regeneration ◽

Radiation Induced

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Growth factors protect intestinal stem cells from radiation-induced apoptosis by suppressing PUMA through the PI3K/AKT/p53 axis

Oncogene ◽

10.1038/onc.2009.451 ◽

2009 ◽

Vol 29 (11) ◽

pp. 1622-1632 ◽

Cited By ~ 89

Author(s):

W Qiu ◽

B Leibowitz ◽

L Zhang ◽

J Yu

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Intestinal Stem Cells ◽

Radiation Induced ◽

Induced Apoptosis

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Mo1065 - P21 Waf1/Cip1 and RNA Binding Protein Musashi-1 Regulate small Intestinal Epithelium Regeneration after Γ Radiation-Induced Injury by Activation of the Subpopulation of Reserve Intestinal Stem Cells In Vivo

Gastroenterology ◽

10.1016/s0016-5085(18)32426-0 ◽

2018 ◽

Vol 154 (6) ◽

pp. S-688 ◽

Cited By ~ 1

Author(s):

Emilia J. Orzechowska ◽

Shestruma Parajuli ◽

Agnieszka B. Bialkowska ◽

Vincent W. Yang

Keyword(s):

Stem Cells ◽

Intestinal Epithelium ◽

Rna Binding ◽

Rna Binding Protein ◽

Intestinal Stem Cells ◽

Γ Radiation ◽

Radiation Induced ◽

Small Intestinal ◽

Epithelium Regeneration

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Mature enteroendocrine cells contribute to basal and pathological stem cell dynamics in the small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00036.2018 ◽

2018 ◽

Vol 315 (4) ◽

pp. G495-G510 ◽

Cited By ~ 14

Author(s):

Yoshitatsu Sei ◽

Jianying Feng ◽

Leigh Samsel ◽

Ayla White ◽

Xilin Zhao ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Intestinal Epithelium ◽

Lineage Tracing ◽

Enteroendocrine Cells ◽

Intestinal Stem Cells ◽

Intestinal Tumors ◽

Cell Dynamics ◽

Neutral Drift ◽

Rapid Generation

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here, we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage-tracing studies identified a subset of EECs that reside at +4 position for more than 2 wk, most of which were BrdU-label-retaining cells. Under basal conditions, cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally, the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium, and in the development of EC-derived small intestinal tumors. NEW & NOTEWORTHY The current manuscript demonstrating that a subset of mature enteroendocrine cells (EECs), predominantly enterochromaffin cells, dedifferentiates to fully functional intestinal stem cells (ISCs) is novel, timely, and important. These cells dedifferentiate to ISCs not only in response to injury but also under basal homeostatic conditions. These novel findings provide a mechanism in which a specified cell can dedifferentiate and contribute to normal tissue plasticity as well as the development of EEC-derived intestinal tumors under pathologic conditions.

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Lineage tracing of Notch1-expressing cells in intestinal tumours reveals a distinct population of cancer stem cells

10.1101/364349 ◽

2018 ◽

Author(s):

Larissa Mourao ◽

Guillaume Jacquemin ◽

Mathilde Huyghe ◽

Wojciech J. Nawrocki ◽

Naoual Menssouri ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Critical Role ◽

Tumour Cells ◽

Lineage Tracing ◽

Intestinal Stem Cells ◽

Cellular Targets ◽

Transcriptional Signature ◽

Stem Cell Populations ◽

Notch1 Receptor

AbstractColon tumours are hierarchically organized and contain multipotent self-renewing cells, called Cancer Stem Cells (CSCs). We have previously shown that the Notch1 receptor is expressed in Intestinal Stem Cells (ISCs); given the critical role played by Notch signalling in promoting intestinal tumourigenesis, we explored Notch1 expression in tumours. Combining lineage tracing in two tumour models with transcriptomic analyses, we found that Notch1 + tumour cells are undifferentiated, proliferative and capable of indefinite self-renewal and of generating a heterogeneous clonal progeny. Molecularly, the transcriptional signature of Notch1+ tumour cells highly correlates with ISCs, suggestive of their origin from normal crypt cells. Surprisingly, Notch1+ expression labels a subset of CSCs that show reduced levels of Lgr5, a reported CSCs marker. The existence of distinct stem cell populations within intestinal tumours highlights the necessity of better understanding their hierarchy and behaviour, to identify the correct cellular targets for therapy.

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Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts

10.1101/2021.11.22.469515 ◽

2021 ◽

Author(s):

Aiguo Tian ◽

Virginia Morejon ◽

Sarah Kohoutek ◽

Yi-Chun Huang ◽

Wu-Min Deng ◽

...

Keyword(s):

Stem Cells ◽

Pathogenic Bacteria ◽

Growth Factor Receptor ◽

Lineage Tracing ◽

Intestinal Stem Cells ◽

Stem Cell Marker ◽

Secretory Cells ◽

Ras Signaling ◽

Epithelial Regeneration ◽

Resident Stem Cells

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.

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