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Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting neural signals, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled up to 11 types of cells from mucosa, muscularis and serosa to coexist and epithelial cells were stretched by the contracting muscularis cells. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the functional regeneration of the engineered intestine.

Background & aimsIntestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.MethodsHuman intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.ResultsFunctional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.ConclusionsHuman intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.

The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x10(5) (n = 14), 1x10(6) (n = 9), or 5x10(6) cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.

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.

BackgroundWe previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.MethodsHuman small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.ResultsCollagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.ConclusionType I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

BackgroundCommon single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity.Methodology/principal findingsA novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3.Conclusion/significanceOur findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

BACKGROUND:Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity. METHODOLOGY/PRINCIPAL FINDINGS:A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3. CONCLUSION/SIGNIFICANCE:Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.

Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting neural signals, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled up to 11 types of cells from mucosa, muscularis and serosa to coexist and epithelial cells were stretched by the contracting muscularis cells. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the functional regeneration of the engineered intestine.

BACKGROUND & AIMS:Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting. METHODS:Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium. RESULTS:Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells. CONCLUSIONS:Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.

The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x10(5) (n = 14), 1x10(6) (n = 9), or 5x10(6) cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.

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.

BACKGROUND:We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells. METHODS:Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry. RESULTS:Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages. CONCLUSION:Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

Pediatric epilepsies are a group of disorders with a broad phenotypic spectrum that are associated with great genetic heterogeneity, thus making sequential single-gene testing an impractical basis for diagnostic strategy. The advent of next-generation sequencing has increased the success rate of epilepsy diagnosis, and targeted resequencing using genetic panels is the a most cost-effective choice. We report the results found in a group of 87 patients with epilepsy and developmental delay using targeted next generation sequencing (custom-designed Haloplex panel). Using this gene panel, we were able to identify disease-causing variants in 17 out of 87 (19.5%) analyzed patients, all found in known epilepsy-associated genes (KCNQ2, CDKL5, STXBP1, SCN1A, PCDH19, POLG, SLC2A1, ARX, ALG13, CHD2, SYNGAP1, and GRIN1). Twelve of 18 variants arose de novo and 6 were novel. The highest yield was found in patients with onset in the first years of life, especially in patients classified as having early-onset epileptic encephalopathy. Knowledge of the underlying genetic cause provides essential information on prognosis and could be used to avoid unnecessary studies, which may result in a greater diagnostic cost-effectiveness.

Background: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood., Principal Findings: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II., Conclusions: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.