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CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury - PubMed

️Sun Jan 01 2017

CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury

Igor Stzepourginski et al. Proc Natl Acad Sci U S A. 2017.

Abstract

The intestinal epithelium is continuously renewed by intestinal epithelial stem cells (IESCs) positioned at the base of each crypt. Mesenchymal-derived factors are essential to maintain IESCs; however, the cellular composition and development of such mesenchymal niche remains unclear. Here, we identify pericryptal CD34⁺ Gp38⁺ αSMA^- mesenchymal cells closely associated with Lgr5⁺ IESCs. We demonstrate that CD34⁺ Gp38⁺ cells are the major intestinal producers of the niche factors Wnt2b, Gremlin1, and R-spondin1, and are sufficient to promote maintenance of Lgr5⁺ IESCs in intestinal organoids, an effect mainly mediated by Gremlin1. CD34⁺ Gp38⁺ cells develop after birth in the intestinal submucosa and expand around the crypts during the third week of life in mice, independently of the microbiota. We further show that pericryptal CD34⁺gp38⁺ cells are rapidly activated by intestinal injury, up-regulating niche factors Gremlin1 and R-spondin1 as well as chemokines, proinflammatory cytokines, and growth factors with key roles in gut immunity and tissue repair, including IL-7, Ccl2, Ptgs2, and Amphiregulin. Our results indicate that CD34⁺ Gp38⁺ mesenchymal cells are programmed to develop in the intestine after birth to constitute a specialized microenvironment that maintains IESCs at homeostasis and contribute to intestinal inflammation and repair after injury.

Keywords: CD34; inflammation; intestinal stem cells; mesenchymal niche.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig. 1.**
Coexpression of gp38 and CD34 identifies pericryptal αSMA^– mesenchymal cells. (A) FACS plot and percentage of nonhematopoietic (CD45^–) cells from the colonic lamina propria expressing gp38 and CD31 (*Left*) and analysis of CD34 expression (*Right*). BECs, blood endothelial cells; DNCs, double negative gp38^–CD31^– cells; LECs, lymphatic endothelial cells. A plot representative of 10 independent experiments is shown. (B) Immunofluorescence analysis of gp38 (green) and CD34 (red) expression in mice colon (*Left*) and ileum (*Right*). Arrows indicate double positive gp38⁺CD34⁺ cells (appearing in orange/yellow) in crypts, and arrowheads indicate gp38⁺ CD34^– cells (appearing in green) in the villi and colon tops. (C) Immunofluorescence analysis of Lgr5 (green), gp38 (red), and CD34 (blue) expression in colon (*Left*) and ileum (*Right*) of Lgr5-egfp mice. Arrows indicate gp38⁺CD34⁺ cells in close contact with Lgr5⁺ cells (arrowheads). (D and E) Immunofluorescence analysis of gp38 (green), αSMA (red), and CD34 (blue) expression in colon (D) and ileum (E). Arrows indicate gp38⁺CD34⁺αSMA^–cells (CD34⁺ CSCs; *Upper*); arrowheads indicate gp38⁺CD34^–αSMA⁺ cells (CD34^– MyoFs; *Lower*). (F) Expression of the indicated genes, as measured by qRT-PCR, in the indicated populations. n = 8 mice from three independent experiments. Values are mean ± SD. In B–E, representative images of n = 3–6 independent experiments are shown. DAPI stains nuclei. (Scale bars: B and C, 50 µm; D and E, 25 µm.) ***P < 0.001.

**Fig. S1.**
Characterization of intestinal stromal populations. (A) FACS plot and percentages of nonhematopoietic (CD45^–) cells from the ileum lamina propria expressing gp38 and CD31 (*Left*) and analysis of CD34 expression in indicated populations (*Right*). BEC, blood endothelial cell; DNC, double negative cell; LEC, lymphatic endothelial cell. A representative FACS plot of 10 independent experiments is shown. (B) MFI analysis of αSMA expression as measured by intracellular FACS. n = 4 mice from two independent experiments. Values are mean ± SD. (C) Immunofluorescence analysis of gp38 (green), CD34 (red), and CD31 (blue) expression in mouse colonic lamina propria. Arrows indicate gp38⁺CD34^–CD31⁺ lymphatic endothelial cells, and arrowheads indicate gp38^–CD34⁺CD31⁺ blood endothelial cells. DAPI stains nuclei. (Scale bar: 50 μm.) ****P < 0.0001.

**Fig. 2.**
CD34⁺ CSCs support IESCs proliferation. (A) Bright field microscopy analysis (*Left*) and percentage of organoids versus spheroids (*Right*) among intestinal structures grown in the indicated conditions for 5 d. Arrows indicate budding crypts, and an asterisk indicates organoid lumen; n = 4 from three independent experiments. (B) Confocal analysis of Ki-67 (red) expression in intestinal structures grown as described in A. (C) Intracellular FACS analysis of Ki-67 in organoids grown as indicated. n = 6 from two independent experiments. (D) Confocal analysis (*Left*) and quantification (*Right*) of WGA expression (green) in intestinal structures in indicated conditions. n = 8 from two independent experiments. (E) Percentage of GFP⁺ cells, as measured by FACS, in organoids harvested from *Lgr5-egfp* mice and grown as indicated; n = 6 from two independent experiments. (F) FACS analysis of GFP reporting Lgr5 expression in intestinal structures obtained from *Lgr5-egfp* mice and grown as indicated; n = 6 from two independent experiments. In A and *C–F*, values are mean ± SD. (Scale bars: 100 μm.) DAPI stains nuclei. ****P < 0.0001, ***P < 0.001, **P < 0.005, *P < 0.05.

**Fig. S2.**
Similar gene signature and spheroid inducing potential of CD34⁺ CSCs isolated from colon or ileum. (A) Percentage of spheroids vs. organoids obtained in a transwell experiment after 5 d of culture in the indicated conditions. n = 4 from two independent experiments. (B) Expression of the indicated genes, as measured by qRT-PCR, in the indicated populations isolated by FACS from mouse ileum. n = 4 mice from two independent experiments. Values are mean ± SD. ****P < 0.0001; ***P < 0.001; **P < 0.005; ns, not significant.

**Fig. 3.**
CD34⁺ CSCs induce spheroids through secretion of Grem1 and Rspo1. (A) Expression of the indicated genes, as measured by qRT-PCR, in the indicated intestinal populations isolated by FACS from mouse colon. n = 3–5 mice from two independent experiments. DNCs, double negative cells (gp38^– CD31^–); ECs, endothelial cells (CD31⁺); Leukos, hematopoietic cells (CD45⁺); ND, nondetected. (B) Percentage of spheroids vs. organoids obtained in 5-d cultures, supplemented with anti-gremlin1 blocking antibodies (α-Grem1); n = 4, from four independent experiments. (C) Total numbers of intestinal structures (organoids or spheroids) obtained after 2 d of culture without stromal cells, or with addition of CD34⁺ CSCs. n = 3, from two independent experiments. (D) Percentage of spheroids and organoids obtained in a transwell experiment after 5 d of culture in the indicated conditions. n = 4 from two independent experiments. (E) Percentage of spheroids and organoids obtained 48 h after transferring 5-d intestinal structures to the indicated conditions. CD34⁺, complete medium containing CD34⁺ CSCs; Ctrl, complete medium. n = 3 from two independent experiments. Values are mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.005, *P < 0.05. ns, not significant.

**Fig. S3.**
CD34⁺ CSCs express *Foxl1* but not *Myh11*. Expression of the indicated genes, as measured by qRT-PCR, in the indicated populations isolated by FACS. n = 3 mice from two independent experiments. *P < 0.05.

**Fig. 4.**
CD34⁺ CSCs expand in the submucosal layer and the pericryptal niche after weaning. (A) Immunofluorescence analysis of gp38 (green) and CD34 (red) expression by lamina propria stromal cells at E16.5 (embryonic age after conception), and 14 d (P14) or 21 d (P21) after birth. *Insets* show gp38⁺ CD34^– stromal cells (arrowheads) and gp38⁺ CD34⁺ stromal cells (arrows); images are representative of n = 4 mice from three independent experiments. C, crypt; L, lumen; SML, submucosal layer. Costaining with CD31 confirmed that fetal and postnatal gp38^– CD34⁺ cells are blood vessels (Fig. S4). (Scale bar: 50 μm.) (B) FACS analysis of CD34⁺ and CD34^– cells among CD45^–CD31^–gp38⁺ intestinal stromal cells at the indicated time after birth; n = 4 mice from two independent experiments. (C) Expression of the indicated genes, as measured by qRT-PCR, in CD34⁺ and CD34^– stromal cells isolated by FACS from colons of 2- and 8-wk-old mice. n = 3–5 mice from two independent experiments. DAPI stains nuclei. In B and C, values are mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.005, *P < 0.05. ns, not significant.

**Fig. S4.**
(A) CD34⁺ CSCs are not detected in neonatal intestines. Immunofluorescence analysis of gp38 (green) and CD34 (red) expression by intestinal stromal cells of mice at birth (P0). Arrowheads indicate gp38⁺ CD34^– stromal cells lining epithelial cells and blood vessels (red). Image is representative of n = 3 mice from two independent experiments. L, lumen. (B) Fetal and postnatal CD34⁺ gp38^– cells of the intestinal lamina propria are CD31⁺ endothelial cells. Immunofluorescence analysis of gp38 (green) CD34 (red) and CD31 (blue) expression in intestine of E16.5 embryo or 2-wk-old mouse (P14). Arrowheads show gp38^– CD34⁺CD31⁺ blood endothelial cells. DAPI stains nuclei. (Scale bar: 50 μm.)

**Fig. S5.**
(A) Development of CD34⁺ CSCs is not affected by the intestinal microbiota. Immunofluorescence analysis of gp38 (green) and CD34 (red) expression by colon lamina propria cells of 3-wk-old B6 mouse bred in SPF (*Left*) or GF (*Right*) conditions. Arrows indicate double positive CD34⁺ CSCs. Representative images of three independent experiments are shown. At *Right*, percentage of CD34⁺ CSCs, as measured by FACS, in the colon lamina propria of SPF, GF or *Myd88^−/−TRIF^−/−* mice. n = 4 from two independent experiments, values are mean ± SD. (Scale bars: 50 µm.) (B) Gremlin1 expression is detected in the SML and in proximity to crypts. Immunofluorescence analysis of Gremlin1 (green) and CD34 (red) expression by colon stromal cells of mice at the indicated age (E, days after conception; P, days postnatal). *Insets* show expression of gremlin1 in the SML at 2 wk (P14) and in the pericryptal region at 3 wk after birth (P21). Red signal in intestinal lamina propria of E16.5 embryos and 1-wk-old mice (P7) indicate CD34⁺ blood vessels. Images are representative of two independent experiments. C, crypt; L, lumen; ns, not significant; SML, submucosal layer. DAPI stains nuclei. (Scale bar: 50 μm.)

**Fig. 5.**
Intestinal inflammation induces remodeling and activation of CD34⁺ CSCs. (A) Immunofluorescence analysis of gp38 (green) and CD34 (red) expression by intestinal stromal cells after acute or chronic DSS-induced colitis. *Insets* show bigger magnification of CD34⁺ CSCs (arrow) or CD34^– MyoFs (arrowhead) in acute DSS. BV, blood vessels; C, crypts; L, lumen. (Scale bar: 50 μm.) (B) Percentage of CD34⁺ and CD34^– cells among total gp38⁺ stromal cells (gated CD45^– CD31^–) obtained by FACS at steady state, or after acute DSS and chronic DSS. (C) Mean fluorescence intensity (MFI) of Vcam1 expression by CD34⁺ CSCs and CD34^– MyoFs in the indicated conditions. (D) Clustered gene expression analysis, as measured by qRT-PCR, for CD34⁺ CSCs and CD34^– MyoFs in the indicated conditions. n = 4–6 for each condition from two to three independent experiments. DAPI stains nuclei. In B and C, values are mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.005, *P < 0.05.

**Fig. S6.**
CD34⁺ CSCs support survival of T cells. Percentage of T-cell survival, as measured by FACS, in the indicated conditions. ****P < 0.0001.

**Fig. S7.**
Proposed model for the role of CD34⁺ CSCs and CD34^– MyoFs in intestinal homeostasis.

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References

1. Barker N, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449(7165):1003–1007. - PubMed
1. van der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol. 2009;71:241–260. - PubMed
1. Pinto D, Gregorieff A, Begthel H, Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 2003;17(14):1709–1713. - PMC - PubMed
1. Kuhnert F, et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc Natl Acad Sci USA. 2004;101(1):266–271. - PMC - PubMed
1. Sato T, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 2011;469(7330):415–418. - PMC - PubMed

CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury - PubMed