Intestinal Subepithelial Myofibroblasts Support the Growth of Intestinal Epithelial Stem Cells

<div><p>Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current <i>in vitro</i> models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs <i>in vitro</i> and allows for their successful <i>in vivo</i> implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown <i>in vitro</i> into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium <i>in vitro</i> in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful <i>in vivo</i> engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.</p></div

Histology of co-cultures implanted <i>in vivo</i> for 28 days.

<p>(A) H&E. (B) Immunohistochemical staining for (B) α-SMA, (C) E-Cadherin, (D) Cdx2, (E) lysozyme, (F) synaptophysin, and (G) PAS. The scale bar represents 50 µm.</p

Histology of <i>in vitro</i> co-cultures on top of ISEMFs after 7 days.

<p>(A) Hematoxylin and eosin (H&E). (B–G) Immunohistochemical staining for (B) α-SMA, (C) E-Cadherin, (D) caudal type homeobox 2 (Cdx2), (E) lysozyme, (F) synaptophysin, and (G) periodic acid-Schiff (PAS). The scale bar represents 50 µm.</p

Enteroid forming efficiency from crypts after <i>in vitro</i> growth with and without ISEMFs.

<p>Enteroid forming efficiency was measured in mono- and co-cultures with ISEMFs, with and without exogenous Rspo1 in the media (n = 3). Asterisk indicates p<0.05 between the designated groups.</p

Epithelial characterization after <i>in vitro</i> culture with and without ISEMFs, in intimate contact.

<p>Crypts with and without ISEMFs were grown in culture media that contained Rspo1 and assessed after 7 days of culture. (A) Average area of an enteroid in co-culture was measured and compared with monoculture (n = 5). (B) eGFP DNA qPCR was used as a measurement of total epithelial expansion, normalized to the degree of expansion in monoculture (n = 7). (C) mRNA expression of Lgr5 and differentiated epithelial markers was assessed by qPCR and normalized to whole small bowel (n = 3). (D, E) Representative micrographs of an enteroid without ISEMFs (D) and with ISEMFs (E) were captured at day 7. The scale bar represents 100 µm. Asterisk indicates p<0.05 when compared to <i>No ISEMF</i>.</p

Characterization of intestinal subepithelial myofibroblasts (ISEMFs).

<p>Isolated ISEMFs were characterized through immunofluorescent staining and reverse transcriptase qPCR. (A) The cells were stained for α-smooth muscle actin (α-SMA, Acta2), vimentin (Vim), and desmin (Des). (B) Reverse transcriptase qPCR was performed on ISEMFs mRNA lysates using the same markers and normalized to small bowel (n = 4). The scale bar represents 200 µm.</p

Supportive versus Non-Supportive ISEMFs.

<p>(A) Rspo1 and Rspo2 mRNA expression was measured by qPCR in ISEMFs that were supportive or non-supportive of crypt growth without exogenous Rspo1 (n = 3). (B) Enteroid forming efficiency was measured in co-cultures with supportive or non-supportive ISEMFs, with and without exogenous Rspo2 in the culture media. Asterisk indicates p<0.05 when compared to <i>Supportive ISEMF</i> in (A) and between the indicated groups in (B).</p

Epithelial characterization after <i>in vitro</i> culture with and without ISEMFs, separated by a trans-well membrane.

<p>Crypts with and without ISEMFs were grown in culture media that contained Rspo1 and assessed after 7 days of culture. (A) Average enteroid area was measured on cultures grown on a semi-permeable membrane, with or without ISEMFs in the well 0.8 mm beneath it (n = 5). (B) eGFP DNA was quantified with qPCR to measure epithelial growth (n = 7). (C) mRNA expression of Lgr5 and differentiated epithelial markers was assessed by qPCR and normalized to whole small bowel (n = 3). (D, E) Representative micrographs of an enteroid without ISEMFs (D) and with ISEMFs (E) below the membrane were captured at day 7. The scale bar represents 100 µm. Asterisk indicates p<0.05 when compared to <i>No ISEMF</i>.</p

Epithelial growth after <i>in vitro</i> culture with and without ISEMF conditioned media (CM).

<p>Crypts were grown in culture media containing Rspo1, with or without ISEMF CM, and assessed after 7 days of culture. (A) Average area of an enteroid grown with ISEMF CM was compared to without conditioned media (n = 4). (B,C) Representative micrographs of an enteroid grown without CM (B) and with ISEMF CM (B). The scale bar represents 100 µm. Asterisk indicates p<0.05 when compared to <i>No CM</i>.</p

<i>In vitro</i> culture and <i>in vivo</i> histology of enteroids derived from single sorted stem cells.

<p>(A) The total cross-sectional area of the enteroids per well was measured to quantify the growth of the stem cells with and without ISEMFs (n = 2). (B) Time lapse images of the growth up to day 14. (C–F) Immunohistochemical staining of single ISC-derived enteroids and ISEMFs implanted <i>in vivo</i>. (C) H&E, (D) α-SMA, (E) Cdx2, and (F) PAS. The scale bar represents 100 µm in B and 50 µm in C through F. Asterisk indicates p<0.05 when compared to <i>No ISEMF</i>.</p