Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.

Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro 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 in vitro and allows for their successful in vivo 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 in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro 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 in vivo 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.

Macroporosity enhances vascularization of electrospun scaffolds.

BACKGROUND: One of the greatest challenges in scaffold-based tissue engineering remains poor and inefficient penetration of cells into scaffolds to generate thick vascularized and cellular tissues. Electrospinning has emerged as a preferred method for producing scaffolds with high surface area-to-volume ratios and resemblance to extracellular matrix. However, cellular infiltration and vascular ingrowth are insufficient because of lack of macropore interconnectivity in electrospun scaffolds with high-fiber density. In this study, we report a novel two-step electrospinning and laser cutting fabrication method to enhance the macroporosity of electrospun scaffolds. MATERIALS AND METHODS: Polycaprolactone dissolved in hexafluoroisopropanol was electrospun at 25 kV to create uniform 100-120 µm sheets of polycaprolactone fiber mats (1- to 5-µm fiber diameter) with an array of pores created using VERSA LASER CUTTER 2.3. Three groups of fiber mats with three distinct pore diameters (300, 160, and 80 µm, all with 15% pore area) were fabricated and compared with a control group without laser cut pores. After laser cutting, all mats were collagen coated and manually wrapped around a catheter six times to form six concentric layers before implantation into the omentum of Lewis rats. Cellular infiltration and vascular ingrowth were examined after 2 wk. RESULTS: Histologic analysis of 14-d samples showed that scaffolds with laser cut pores had close to 40% more cellular infiltration and increased vascular ingrowth in the innermost layers of the construct compared with the control group. Despite keeping pore area percentage constant between the three groups, the sheets with the largest pore size performed better than those with the smallest pore sizes. CONCLUSIONS: Porosity is the primary factor limiting the extensive use of electrospun scaffolds in tissue engineering. Our method of LASER cutting pores in electrospun fibrous scaffolds ensures uniform pore sizes, easily controllable and customizable pores, and enhances cellular infiltration and vascular ingrowth, demonstrating significant advancement toward utility of electrospun scaffolds in tissue engineering.

Use of collagen gel as an alternative extracellular matrix for the in vitro and in vivo growth of murine small intestinal epithelium.

Methods for the in vitro culture of primary small intestinal epithelium have improved greatly in recent years. A critical barrier for the translation of this methodology to the patient's bedside is the ability to grow intestinal stem cells using a well-defined extracellular matrix. Current methods rely on the use of Matrigel(™), a proprietary basement membrane-enriched extracellular matrix gel produced in mice that is not approved for clinical use. We demonstrate for the first time the capacity to support the long-term in vitro growth of murine intestinal epithelium in monoculture, using type I collagen. We further demonstrate successful in vivo engraftment of enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel. Small intestinal crypts were isolated from 6 to 10 week old transgenic enhanced green fluorescent protein (eGFP+) mice and suspended within either Matrigel or collagen gel; cultures were supported using previously reported media and growth factors. After 1 week, cultures were either lysed for DNA or RNA extraction or were implanted subcutaneously in syngeneic host mice. Quantitative real-time polymerase chain reaction (qPCR) was performed to determine expansion of the transgenic eGFP-DNA and to determine the mRNA gene expression profile. Immunohistochemistry was performed on in vitro cultures and recovered in vivo explants. Small intestinal crypts reliably expanded to form enteroids in either Matrigel or collagen in both mono- and co-cultures as confirmed by microscopy and eGFP-DNA qPCR quantification. Collagen-based cultures yielded a distinct morphology with smooth enteroids and epithelial monolayer growth at the gel surface; both enteroid and monolayer cells demonstrated reactivity to Cdx2, E-cadherin, CD10, Periodic Acid-Schiff, and lysozyme. Collagen-based enteroids were successfully subcultured in vitro, whereas pure monolayer epithelial sheets did not survive passaging. Reverse transcriptase-polymerase chain reaction demonstrated evidence of Cdx2, villin 1, mucin 2, chromogranin A, lysozyme 1, and Lgr5 expression, suggesting a fully elaborated intestinal epithelium. Additionally, collagen-based enteroids co-cultured with myofibroblasts were successfully recovered after 5 weeks of in vivo implantation, with a preserved immunophenotype. These results indicate that collagen-based techniques have the capacity to eliminate the need for Matrigel in intestinal stem cell culture. This is a critical step towards producing neo-mucosa using good manufacturing practices for clinical applications in the future.