Lgr5 + cell fate regulation by coordination of metabolic nuclear receptors during liver repair

Theranostics

2022 Aug 15

Qin, D;Liu, S;Lu, Y;Yan, Y;Zhang, J;Cao, S;Chen, M;Chen, N;Huang, W;Wang, L;Chen, X;Zhang, L;
PMID: 36168631 | DOI: 10.7150/thno.74194

Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a target gene of Wnt/β-Catenin which plays a vital role in hepatic development and regeneration. However, the regulation of Lgr5 gene and the fate of Lgr5 + cells in hepatic physiology and pathology are little known. This study aims to clarify the effect of metabolic nuclear receptors on Lgr5 + cell fate in liver. Methods: We performed cell experiments with primary hepatocytes, Hep 1-6, Hep G2, and Huh 7 cells, and animal studies with wild-type (WT), farnesoid X receptor (FXR) knockout mice, peroxisome proliferator-activated receptor α (PPARα) knockout mice and Lgr5-CreERT2; Rosa26-mTmG mice. GW4064 and CDCA were used to activate FXR. And GW7647 or Wy14643 was used for PPARα activation. Regulation of Lgr5 by FXR and PPARα was determined by QRT-PCR, western blot (WB) and RNAscope in situ hybridization (ISH) and immunofluorescence (IF), luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) diet was used to induce liver injury. Results: Pharmacologic activation of FXR induced Lgr5 expression, whereas activation of PPARα suppressed Lgr5 expression. Furthermore, FXR and PPARα competed for binding to shared site on Lgr5 promoter with opposite transcriptional outputs. DDC diet triggered the transition of Lgr5 + cells from resting state to proliferation. FXR activation enhanced Lgr5 + cell expansion mainly by symmetric cell division, but PPARα activation prevented Lgr5 + cell proliferation along with asymmetric cell division. Conclusion: Our findings unravel the opposite regulatory effects of FXR and PPARα on Lgr5 + cell fate in liver under physiological and pathological conditions, which will greatly assist novel therapeutic development targeting nuclear receptors.

Immunohistochemical Study of a Correlation between Pemphigus Vulgaris Activity Score and Stem Cell Control

The Egyptian Journal of Hospital Medicine

2021 Apr 01

Bazid, H;Seleit, I;Abo Hegazy, S;Samaka, R;
| DOI: 10.21608/ejhm.2021.165168

BACKGROUND: Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease. PV autoantibodies disrupt desmosomal adhesion and cause acantholysis. Previous researches have shown that stem cells are indirectly involved as a result of desmoglein deficiency. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) as a follicular stem cell marker was evaluated in aim to correlate its intensity of expression with disease severity. OBJECTIVE: To correlate LGR5 intensity of expression with disease severity. PATIENT AND METHODS: This prospective cross sectional study was carried out on 20 PV patients. Patients were subjected to complete history taking, general, dermatological examination and assessment of disease severity by the Pemphigus Vulgaris Activity Score (PVAS), histopathological and immunohistochemical expression of LGR5 were done. RESULTS: All studied cases showed positive cytoplasmic basal LGR5 expression in patchy manner. 75% of cases had mild intensity of expression, 15% had moderate intensity and their Histo (H) score ranged from 50-130 with Mean ±SD 110±18.92. There were no significant correlation between PVAS scores ''skin, mucosa and total involvement'' and H score of LGR5 expression. CONCLUSION: The current study could shed a new light on the disease and its correlation with stem cells, LGR5 as a stem cell marker could be related to the healing process in PV. However, it didn't correlate PVAS scores either in skin, mucosa or total involvement.

Non-thermal plasma promotes hair growth by improving the inter-follicular macroenvironment

RSC Advances

2021 Aug 17

Kim, H;Choi, E;Choi, E;Kim, H;Kim, J;Cho, G;Kim, H;Na, S;Shin, J;Do, S;Park, B;
| DOI: 10.1039/d1ra04625j

Non-thermal plasma (NTP) is widely used in the disinfection and surface modification of biomaterials.

Single-cell roadmap of human gonadal development

Nature

2022 Jul 01

Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis

Scientific reports

2022 Jun 01

Polkoff, KM;Gupta, NK;Green, AJ;Murphy, Y;Chung, J;Gleason, KL;Simpson, SG;Walker, DM;Collins, B;Piedrahita, JA;
PMID: 35650234 | DOI: 10.1038/s41598-022-13056-w

Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche, have remarkable regenerative capacity throughout hair cycling, and display fate plasticity during cutaneous wound healing. Due to the need for a transgenic reporter, essentially all observations related to LGR5-expressing hair follicle stem cells have been generated using transgenic mice, which have significant differences in anatomy and physiology from the human. Using a transgenic pig model, a widely accepted model for human skin and human skin repair, we demonstrate that LGR5 is a marker of hair follicle stem cells across species in homeostasis and development. We also report the strong similarities and important differences in expression patterns, gene expression profiles, and developmental processes between species. This information is important for understanding the fundamental differences and similarities across species, and ultimately improving human hair follicle regeneration, cutaneous wound healing, and skin cancer treatment.

Partial male-to-female reprogramming of mouse fetal testis by sertoli cell ablation

Development (Cambridge, England)

2023 Jun 28

Imaimatsu, K;Hiramatsu, R;Tomita, A;Itabashi, H;Kanai, Y;
PMID: 37376880 | DOI: 10.1242/dev.201660

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions-near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3βHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

Embryogenic stem cell-derived intestinal crypt fission directs de novo crypt genesis

Cell reports

2022 Dec 13

Huang, XT;Li, T;Li, T;Xing, S;Tian, JZ;Ding, YF;Cai, SL;Yang, YS;Wood, C;Yang, JS;Yang, WJ;
PMID: 36516755 | DOI: 10.1016/j.celrep.2022.111796

Intestinal epithelial replenishment is fueled by continuously dividing intestinal stem cells (ISCs) resident at the crypt niche. However, the cell type(s) enabling replenishment upon damage and subsequent loss of whole crypts remain largely unclear. Using Set domain-containing protein 4 (Setd4), we identify a small population with reserve stem cell characteristics in the mouse intestine. Upon irradiation-induced injury, Setd4-expressing (Setd4+) cells survive radiation exposure and then activate to produce Sca-1-expressing cell types to restore the epithelial wall and regenerate crypts de novo via crypt fission. Setd4+ cells are confirmed to originate from the early fetal period, subsequently contributing to the development of embryonic gut and the establishment of postnatal crypts. Setd4+ cells are therefore represented as both originators and key regenerators of the intestine.

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids

JCI insight

2023 Feb 23

Childs, CJ;Holloway, EM;Sweet, CW;Tsai, YH;Wu, A;Vallie, A;Eiken, MK;Capeling, MM;Zwick, RK;Palikuqi, B;Trentesaux, C;Wu, JH;Pellon-Cardenas, O;Zhang, CJ;Glass, IA;Loebel, C;Yu, Q;Camp, JG;Sexton, JZ;Klein, OD;Verzi, MP;Spence, JR;
PMID: 36821371 | DOI: 10.1172/jci.insight.165566

Epithelial organoids derived from intestinal tissue, called 'enteroids', recapitulate many aspects of the organ in vitro, and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identify an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells, feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown and EREG-grown enteroids show that EGF-enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine-like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Amino acid transporter SLC7A5 regulates Paneth cell function to affect the intestinal inflammatory response

bioRxiv : the preprint server for biology

2023 Jan 24

Bao, L;Fu, L;Su, Y;Chen, Z;Peng, Z;Sun, L;Gonzalez, FJ;Wu, C;Zhang, H;Shi, B;Shi, YB;
PMID: 36789439 | DOI: 10.1101/2023.01.24.524966

The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout ( ΔIEC ) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5 ΔIEC reduces mTORC1 signaling. Surprisingly, Slc7a5 ΔIEC mice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells in Slc7a5 ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show that Slc7a5 ΔIEC mice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.

Suspension culture in a rotating bioreactor for efficient generation of human intestinal organoids

Cell reports methods

2022 Nov 21

Takahashi, J;Mizutani, T;Sugihara, HY;Nagata, S;Kato, S;Hiraguri, Y;Takeoka, S;Tsuchiya, M;Kuno, R;Kakinuma, S;Watanabe, M;Okamoto, R;
PMID: 36452871 | DOI: 10.1016/j.crmeth.2022.100337

Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) hold great promise for translational medical applications. A common method to obtain HIOs has been to harvest floating hindgut spheroids arising from hPSCs. As this technique is elegant but burdensome due to the complex protocol and line-to-line variability, a more feasible method is desired. Here, we establish a robust differentiation method into suspension-cultured HIOs (s-HIOs) by seeding dissociated cells on a spheroid-forming plate. This protocol realizes the reliable generation of size-controllable spheroids. Under optimized conditions in a rotating bioreactor, the generated spheroids quickly grow and mature into large s-HIOs with supporting mesenchyme. Upon mesenteric transplantation, s-HIOs further mature and develop complex tissue architecture in vivo. This method demonstrates that intestinal tissue can be generated from iPSC-derived HIOs via suspension induction and bioreactor maturation, establishing a reliable culture platform with wide applications in regenerative medicine.

Mesenchymal-epithelial interaction regulates gastrointestinal tract development in mouse embryos

Cell reports

2022 Jul 12

Zhao, L;Song, W;Chen, YG;
PMID: 35830795 | DOI: 10.1016/j.celrep.2022.111053

After gut tube patterning in early embryos, the cellular and molecular changes of developing stomach and intestine remain largely unknown. Here, combining single-cell RNA sequencing and spatial RNA sequencing, we construct a spatiotemporal transcriptomic landscape of the mouse stomach and intestine during embryonic days E9.5-E15.5. Several subpopulations are identified, including Lox+ stomach mesenchyme, Aldh1a3+ small-intestinal mesenchyme, and Adamdec1+ large-intestinal mesenchyme. The regionalization and heterogeneity of both the epithelium and the mesenchyme can be traced back to E9.5. The spatiotemporal distributions of cell clusters and the mesenchymal-epithelial interaction analysis indicate that a coordinated development of the epithelium and mesenchyme contribute to the stomach regionalization, intestine segmentation, and villus formation. Using the gut tube-derived organoids, we find that the cell fate of the foregut and hindgut can be switched by the regional niche factors, including fibroblast growth factors (FGFs) and retinoic acid (RA). This work lays a foundation for further dissection of the mechanisms governing this process.

R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development

Developmental cell

2022 Jun 07

Hein, RFC;Wu, JH;Holloway, EM;Frum, T;Conchola, AS;Tsai, YH;Wu, A;Fine, AS;Miller, AJ;Szenker-Ravi, E;Yan, KS;Kuo, CJ;Glass, I;Reversade, B;Spence, JR;
PMID: 35679862 | DOI: 10.1016/j.devcel.2022.05.010

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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