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Objective To compare the potentiality and characteristics of different intestinal epithelial cell (IEC) lines to grow into enteroids when cultured in 3D system in order to provide experimental basis for the cultivation of enteroids by IECs. Methods Three IEC lines, IEC-6, CT26.WT and Caco-2, were cultured in a 3D system, and further characterized by morphology. Single Caco-2 cell was acquired by fluorescent activated cell sorting (FACS) and seeded in Matrigel. The growth speed of enteroids derived from single Caco-2 cell was determined. The expression levels of intestinal stem cell (ISC) genes, including Lgr5 and Olfm4, and proliferative marker, Ki67 were determined by qRT-PCR, and the results were compared between 2D and 3D cultured Caco-2 cells. The distribution of Ki67-positive cells and lamp1-positive lysosomes in 2D and 3D cultured Caco-2 cells were observed with aid of immunofluorescent staining. TUNEL staining was employed to analyze the apoptosis of Caco-2 cells after 5 and 15 Gy radiation, respectively. Results Among the 3 IEC lines, only Caco-2 cells could grow into enteroids when cultured in 3D system. Single Caco-2 cell acquired by FACS could also form enteroids. Compared to 2D culture, Caco-2 derived enteroids expressed lower mRNA levels for ISC markers Lgr5 and Olfm4, and also lower proliferative marker Ki67 (P < 0.05). There were less Ki67-positive cells in 3D than 2D cultured Caco-2 cells. Lysosomes in 3D cultured Caco-2 cells exhibited a polar distribution, which was similar to them in in vivo pattern. Radiation exposure caused apoptosis of IECs in Caco-2 derived enteroids, and the apoptotic rate was positively correlated to the dose of radiation. Conclusion Caco-2 cells could form enteroids when cultured in 3D system, which is similar to the structure of in vivo intestine in structure and function. This study provides reliable in vitro model for gastrointestinal radiation and pharmaceutical evaluation in the future.

Objective To observe the dynamic changes in autophagy during cutaneous wound healing and investigate its possible contributions to wound repair. Methods Two skin wound of 6 mm in size were inflicted on the back of GFP-LC3 transgenic mice. In 0, 3, 7 and 14 d after skin injury, the skin samples were harvested to prepare paraffin-embedded sections, and GFP immunohistochemical (IHC) staining with anit-LC3 antibody was performed for the dynamic changes of autophagy during wound healing. Double-labeling immunofluorescence assay was utilized for identifying cell types of autophgy in granulation tissues by staining both GFP and macrophage marker F4/80 or myofibroblasts marker α-SMA. The mice were divided into skin defect group and injured mice with 3-methyladenine (3-MA, 10 mg/kg, intra-peritoneal injection for 10 continuous days) treatment group. The dynamic changes of the wound were observed, and the skin tissues were collected in 0, 7 and 10 d after injury to evaluate the effect of autophagy on wound healing after fixation and staining. qRT-PCR was employed to detect the expression levels of α-SMA, TGF-β1, PAI-1, type Ⅰ and Ⅲ collagen in the wound samples. Results The autophagic activity was enhanced and then declined in the process of wound healing, and its strongest activity was observed in proliferative phase (7 d). In the granulation tissues, the activity was stronger in the keratinocytes than the interstitial cells, and the autophagic cells were mainly α-SMA-positive myofibroblasts. There were no significant differences in wound area and width, and epidermis thickness in 7 and 10 d after skin injury between the 3-MA treatment and untreated mice, but the former group had significantly increased proliferation activity of epidermal basal cells (P < 0.05) at 7 d after injury. However, no significant differences were seen in the mRNA levels of α-SMA, TGF-β1, PAI-1 and collagen molecules between the 2 groups of mice. Conclusion There exists stronger autophagic activity in the proliferative phase of cutaneous wound healing, but inhibiting autophagy shows little impact on the process.

Abstract: In 1900, Adami speculated that a sequence of context‐independent energetic and structural changes governed the reversion of differentiated cells to a proliferative, regenerative state. Accordingly, we show here that differentiated cells in diverse organs become proliferative via a shared program. Metaplasia‐inducing injury caused both gastric chief and pancreatic acinar cells to decrease mTORC1 activity and massively upregulate lysosomes/autophagosomes; then increase damage associated metaplastic genes such as Sox9; and finally reactivate mTORC1 and re‐enter the cell cycle. Blocking mTORC1 permitted autophagy and metaplastic gene induction but blocked cell cycle re‐entry at S‐phase. In kidney and liver regeneration and in human gastric metaplasia, mTORC1 also correlated with proliferation. In lysosome‐defective Gnptab−/− mice, both metaplasia‐associated gene expression changes and mTORC1‐mediated proliferation were deficient in pancreas and stomach. Our findings indicate differentiated cells become proliferative using a sequential program with intervening checkpoints: (i) differentiated cell structure degradation; (ii) metaplasia‐ or progenitor‐associated gene induction; (iii) cell cycle re‐entry. We propose this program, which we term “paligenosis”, is a fundamental process, like apoptosis, available to differentiated cells to fuel regeneration following injury. [ABSTRACT FROM AUTHOR]

Background: Bone marrow-derived cells (BMDCs) have the ability to differentiate into intestinal epithelial cells after transplantation and participate in the regeneration process of damaged epithelium.Aims: To investigate whether BMDCs could differentiate into intestinal epithelium long term in chimeric mice after transplantation and without special treatment.Methods: Forty irradiated C57BL/6 mice were used. Thirty of them (group A) received transplantation of BMDCs from GFP transgenic mice, and ten (group B) received PBS. The chimeric percentage at the 14th month was examined by flow cytometry. Engraftment of BMDCs was detected by immunohistochemistry in intestinal epithelium. Immunofluorescence observation was used to detect coexpression of PCK, CD45 and Chromogranin A with GFP. BMDCs in the epithelium were observed by an immune electron microscope. The percent of GFP(+) epithelial cells was also determined by flow cytometry.Results: Mice in group A had a survival rate of 93.3% 1 week after transplantation. BMDCs could engraft into recipients' intestinal epithelium. These cells expressed epithelial cell marker PCK, but could not express CD45. Some of them differentiated into enteroendocrine cells expressing Chromogranin A. GFP(+) villous epithelial cells ranged from 9.41 to 16.07% in different subgroups of group A. BMDCs in epithelium developed the characteristics of enterocytes and goblet cells. GFP(+)/PCK(+) epithelial cells at the 6th month made up a proportion of 16.11% among all the isolated epithelial cells.Conclusions: Long term, BMDCs could repopulate recipient's intestinal epithelium even without any special treatment, which suggests a novel insight into the maintenance of the intestinal epithelial constitution. [ABSTRACT FROM AUTHOR]

Significance: Intestinal stem cells (ISCs) are crucial for the continuous renewal and regeneration of the small intestinal epithelium. ISC fate decisions are strictly controlled by metabolism. Mitochondria act as the central hubs of energetic metabolism and dynamically remodel their morphology to perform required metabolic functions. Mitochondrial dysfunction is closely associated with a variety of gastrointestinal diseases. Recent Advances: In recent years, several studies have reported that mitochondria are potential therapeutic targets for regulating ISC function to alleviate intestinal diseases. However, how mitochondrial quality control mediates ISCs under physiological conditions and protects against intestinal injury remains to be comprehensively reviewed. Critical Issues: In this review, we summarize the available studies about how mitochondrial metabolism, redox state, dynamics, autophagy, and proteostasis impact ISC proliferation, differentiation, and regeneration, respectively. Future Directions: We propose that remodeling the function of mitochondria in ISCs may be a promising potential future direction for the treatment of intestinal diseases. This review may provide new strategies for therapeutically targeting the mitochondria of ISCs in intestinal diseases.

Mitochondria are critical for cellular energetic metabolism, intracellular signaling orchestration and programmed death regulation. Therefore, mitochondrial dysfunction is associated with various pathogeneses. The maintenance of mitochondrial homeostasis and functional recovery after injury are coordinated by mitochondrial biogenesis, dynamics and autophagy, which are collectively referred to as mitochondrial quality control. There is increasing evidence that mitochondria are important targets for melatonin to exert protective effects under pathological conditions. Melatonin, an evolutionarily conserved tryptophan metabolite, can be synthesized, transported and metabolized in mitochondria. In this review, we summarize the important role of melatonin in the damaged mitochondria elimination and mitochondrial energy supply recovery by regulating mitochondrial quality control, which may provide new strategies for clinical treatment of mitochondria-related diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lei, Xu, Huang, Huang, Tu, Xu and Liu.)

Objective: To summarize and review the heterogeneity of bone marrow derived stem cells (BMDSCs) and its formation mechanism and significance, and to analyze the possible roles and mechanisms in intestinal epithelial reconstruction., Methods: The related literature about BMDSCs heterogeneity and its role in intestinal epithelial repair was reviewed and analyzed., Results: The heterogeneity of BMDSCs provided better explanations for its multi-potency. The probable mechanisms of BMDSCs to repair intestinal epithelium included direct implantation into intestinal epithelium, fusion between BMDSCs and intestinal stem cells, and promotion of injury microcirculation reconstruction., Conclusion: BMDSCs have a bright future in gastrointestinal injury caused by inflammatory bowl disease and regeneration.