Human Fetal Gut Research Articles

RET enhancer haplotype-dependent remodeling of the human fetal gut development program.

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.

Virome and metagenomic analysis reveal the distinct distribution of microbiota in human fetal gut during gestation.

Studies have shown that fetal immune cell activation may result from potential exposure to microbes, although the presence of microbes in fetus has been a controversial topic. Here, we combined metagenomic and virome techniques to investigate the presence of bacteria and viruses in fetal tissues (small intestine, cecum, and rectum). We found that the fetal gut is not a sterile environment and has a low abundance but metabolically rich microbiome. Specifically, Proteobacteria and Actinobacteria were the dominant bacteria phyla of fetal gut. In total, 700 species viruses were detected, and Human betaherpesvirus 5 was the most abundant eukaryotic viruses. Especially, we first identified Methanobrevibacter smithii in fetal gut. Through the comparison with adults' gut microbiota we found that Firmicutes and Bacteroidetes gradually became the main force of gut microbiota during the process of growth and development. Interestingly, 6 antibiotic resistance genes were shared by the fetus and adults. Our results indicate the presence of microbes in the fetal gut and demonstrate the diversity of bacteria, archaea and viruses, which provide support for the studies related to early fetal immunity. This study further explores the specific composition of viruses in the fetal gut and the similarities between fetal and adults' gut microbiota, which is valuable for understanding human fetal immunity development during gestation.

Are microorganisms everywhere they can be?

Baas-Becking is famously attributed with the conjecture that 'everything is everywhere, but the environment selects'. Although this aphorism is largely challenged by microbial biogeographical data, even weak versions of the claim leave unanswered the question about whether all environments that could theoretically support life contain life. In the last decade, the discovery of thermally sterilized habitable environments disconnected from inhabited regions, and habitats within organisms such as the sterile, but habitable human fetal gut, suggest the existence of a diversity of macroscopic habitable environments apparently devoid of actively metabolizing or reproducing life. Less clear is the status of such environments at the micron scale, for example, between colonies of organisms within rock interstices or on and within other substrates. I discuss recent evidence for these types of environments. These environments have practical uses in: (i) being negative controls for understanding the role of microbial processes in geochemical cycles and geological processes, (ii) yielding insights into the extent to which the biosphere extends into all spaces it theoretically can, (iii) suggesting caution in interpreting the results of life detection instrumentation, and (iv) being useful for establishing the conditions for the origin of life and its prevalence on other planetary bodies.

Homeostasis of mucosal glial cells in human gut is independent of microbiota

In mammals, neural crest cells populate the gut and form the enteric nervous system (ENS) early in embryogenesis. Although the basic ENS structure is highly conserved across species, we show important differences between mice and humans relating to the prenatal and postnatal development of mucosal enteric glial cells (mEGC), which are essential ENS components. We confirm previous work showing that in the mouse mEGCs are absent at birth, and that their appearance and homeostasis depends on postnatal colonization by microbiota. In humans, by contrast, a network of glial cells is already present in the fetal gut. Moreover, in xenografts of human fetal gut maintained for months in immuno-compromised mice, mEGCs persist following treatment with antibiotics that lead to the disappearance of mEGCs from the gut of the murine host. Single cell RNAseq indicates that human and mouse mEGCs differ not only in their developmental dynamics, but also in their patterns of gene expression.

S0832 Characterization of Intestinal Subepithelial PDGFRα(high), DLL1+, F3+ Cells in Ulcerative Colitis

INTRODUCTION: A deeper understanding of the cellular features of disease represents an important step in unlocking the pathogenesis of ulcerative colitis (UC). We recently characterized a novel cell type in the human fetal gut located in the subepithelial compartment adjacent to the epithelium expressing a unique combination of genes and niche factors. Due to their gene expression, we refer to these cells as intestinal, Subepithelial, PDGFR-αHI, DLL1+, F3+ (iSPαD3). Data suggests iSPαD3 cells are involved in intestinal development and maintenance of the stem cell niche. We aimed to determine if these cells are present in the adult colon, and if they play a role in the pathogenesis of inflammation in UC. METHODS: Primary human colonic tissues from non-IBD control and UC patients were obtained via surgical resection. Patients provided informed consent and this study was approved by the University of Michigan Institutional Review Board. Tissues were formalin fixed and paraffin embedded. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed. Further, we analyzed publicly available single cell RNA sequencing data and performed differential expression analysis on an iSPαD3 population. Data were queried for gene ontology term enrichment using the DAVID database. RESULTS: A population analogous to human fetal small intestinal iSPαD3 cells are present in the adult colon. In health iSPαD3 cells encircle crypts with a high degree of proximity and continuity. There are fewer cells that exhibit morphological changes in inflammation (Figures 1 and 2). In healthy colon iSPαD3 cells are enriched for genes involved in Wnt and NOTCH signaling, immune response to bacteria, B-cell regulation, TGFβ signaling, and cell growth. In inflammation they are enriched for genes related to apoptotic pathways, neutrophil chemotaxis, extracellular matrix, and regulation of IL-6, tumor necrosis factor, and JAK-STAT (Figure 3). CONCLUSION: iSPαD3 cells appear to have an important supportive role in the intestinal epithelium and are involved in the pathogenesis of inflammation in UC. Our study suggests that iSPαD3 cells function in homeostatic maintenance and containment of luminal microbiota in health. In contrast, inflammation is associated with loss of iSPαD3 homeostatic functions and in favor of apoptotic and inflammatory pathways, implicating these cells as important modulators in colonic inflammation from UC. More work is needed to elucidate a potential role for iSPαD3 cells in inflammatory bowel disease.Figure 1Figure 2Figure 3

Homeostasis of Mucosal Glial Cells in Human Gut is Independent of Microbiota

In mammals, neural crest cells populate the gut and form the enteric nervous system (ENS) early in embryogenesis. Although the basic ENS structure is highly conserved across species, we show important differences between mice and humans relating to the prenatal and postnatal development of mucosal enteric glial cells (mEGC), which are essential ENS components. We confirm previous work showing that in the mouse, mEGCs are absent at birth, and that their appearance and homeostasis depends on postnatal colonization by microbiota. In humans, by contrast, a network of glial cells is already present in the fetal gut. Moreover, in xenografts of human fetal gut maintained for months in immuno-compromised mice, mEGCs persist following treatment with antibiotics that lead to the disappearance of mEGCs from the gut of the murine host. Single cell RNAseq indicates that human and mouse mEGCs differ not only in their developmental dynamics, but also in their patterns of gene expression.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System

The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 μmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.

Transplantation of Human Intestine Into the Mouse: A Novel Platform for Study of Inflammatory Enterocutaneous Fistulas.

Enteric fistulas represent a severe and medically challenging comorbidity commonly affecting Crohn's disease [CD] patients. Gut fistulas do not develop in animal models of the disease. We have used transplantation of the human fetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. Human fetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analysed the resident immune cells and inflammatory response elicited by systemic lipopolysaccharide [LPS] in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyse fully developed xenografts that spontaneously developed enterocutaneous fistulas. Resident human innate and adaptive immune cells were demonstrated in gut xenografts during steady state and inflammation. The expression of human IL-8, IL-1β, IL-6, TNF-α, A20, and IkBα was significantly elevated in response to LPS, with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas, revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with massive expansion of resident human CD4+ lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals.

Sea Lions Develop Human-like Vernix Caseosa Delivering Branched Fats and Squalene to the GI Tract

Vernix caseosa, the white waxy coating found on newborn human skin, is thought to be a uniquely human substance. Its signature characteristic is exceptional richness in saturated branched chain fatty acids (BCFA) and squalene. Vernix particles sloughed from the skin suspended in amniotic fluid are swallowed by the human fetus, depositing BCFA/squalene throughout the gastrointestinal (GI) tract, thereby establishing a unique microbial niche that influences development of nascent microbiota. Here we show that late-term California sea lion (Zalophus californianus) fetuses have true vernix caseosa, delivering BCFA and squalene to the fetal GI tract thereby recapitulating the human fetal gut microbial niche. These are the first data demonstrating the production of true vernix caseosa in a species other than Homo sapiens. Its presence in a marine mammal supports the hypothesis of an aquatic habituation period in the evolution of modern humans.

Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

In vivo transplantation of fetal human gut-derived enteric neural crest cells.

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75NTR were lentivirally labeled with either GFP or calcium‐sensitive GCaMP and transplanted into the hindgut of Rag2 − /γc − /C5 −‐immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut‐derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.

Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

Memory CD4+CCR5+ T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1

AbstractDespite potential clinical importance, target cells for mother-to-child transmission of HIV-1 have not yet been identified. Cord blood–derived CD4+ T cells are largely naive and do not express CCR5, the mandatory coreceptor for transmitted HIV-1 R5 strains in infants. In the present study, we demonstrate that in the human fetal and infant gut mucosa, there is already a large subset of mucosal memory CD4+CCR5+ T cells with predominantly a Th1 and Th17 phenotype. Using next-generation sequencing of the TCRβ chain, clonally expanded T cells as a hallmark for memory development predominated in the gut mucosa (30%), whereas few were found in the lymph nodes (1%) and none in cord blood (0%). The gut mucosal fetal and infant CD4+ T cells were highly susceptible to HIV-1 without any prestimulation; pol proviral DNA levels were similar to infected phytohemagglutinin-stimulated adult PBMCs. In conclusion, in the present study, we show that extensive adaptive immunity is present before birth and the gut mucosa is the preferential site for memory CD4+ T cells. These CD4+CCR5+ T cells in the infant mucosa provide a large pool of susceptible cells for ingested HIV-1 at birth and during breastfeeding, indicating a mucosal route of mother-to-child transmission that can be targeted in prevention strategies.

Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models

SUMMARYEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx) and the type III secretion system (T3SS), including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3–4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut.

Generation of Human-Induced Pluripotent Stem Cells from Gut Mesentery-Derived Cells by Ectopic Expression of OCT4/SOX2/NANOG

Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of defined transcription factors. Application of this approach in human cells may have enormous potential to generate patient-specific pluripotent stem cells. However, traditional methods of reprogramming in human somatic cells involve the use of oncogenes c-MYC and KLF4, which are not applicable to clinical translation. In the present study, we investigated whether human fetal gut mesentery-derived cells (hGMDCs) could be successfully reprogrammed into induced pluripotent stem (iPS) cells by OCT4, SOX2, and NANOG alone. We used lentiviruses to express OCT4, SOX2, NANOG, in hGMDCs, then generated iPS cells that were identified by morphology, presence of pluripotency markers, global gene expression profile, DNA methylation status, capacity to form embryoid bodies (EBs), and terotoma formation. iPS cells resulting from hGMDCs were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into cell types of all three germ layers both in vitro and in vivo, as shown by EB and teratoma formation assays. DNA fingerprinting showed that the human iPS cells were derived from the donor cells, and are not a result of contamination. Our results provide proof that hGMDCs can be reprogrammed into pluripotent cells by ectopic expression of three factors (OCT4, SOX2, and NANOG) without the use of oncogenes c-MYC and KLF4.

Expression of Intermediate Filament Proteins and Neuronal Markers in the Human Fetal Gut

The human enteric nervous system (ENS) derives from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. During development of the NCC, a rearrangement of various cytoskeletal intermediate filaments such as nestin, peripherin, or alpha-internexin takes place. Although all are related to developing neurons, nestin is also used to identify neural stem cells. Until now, information about the prenatal development of the human ENS has been very restricted, especially concerning potential stem cells. In this study the expression of nestin, peripherin, and alpha-internexin, but also of neuronal markers such as protein gene product (PGP) 9.5 and tyrosine hydroxylase, were investigated in human fetal and postnatal gut. The tissue samples were rapidly removed and subsequently processed for immunohistochemistry or immunoblotting. Nestin could be detected in all samples investigated with the exception of the 9th and the 12th week of gestation (WOG). Although the neuronal marker PGP9.5 was coexpressed with nestin at the 14th WOG, this could no longer be observed at later time points. Alpha-internexin and peripherin expression also did not appear before the 14th WOG, where they were coexpressed with PGP9.5. This study reveals that the intermediate filament markers investigated are not suitable to detect early neural crest stem cells.

Phenotypic Characterization of CD3−7+ Cells in Developing Human Intestine and an Analysis of Their Ability to Differentiate into T Cells

We have identified a large population of CD3(-)7(+) cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8alphaalpha homodimer. In contrast about half of CD3(+) cells expressed CD4 and half expressed CD8alpha. A large proportion of CD3(-)7(+) cells expressed CD56, CD94, and CD161, and whereas CD3(+) T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3(-)7(+) cells have the potential to differentiate into CD3(+) cells. About half of CD3(-)7(+) cells contain intracellular CD3epsilon. Rearranged TCR gamma-chains were detected in highly purified CD3(-)7(+) cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5' Jgamma segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3(-)7(+) cells also gave rise to CD3(+) T cells. Thus, we demonstrate that the CD3(-)7(+) cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3(+) T cells.

Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4-14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2-3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of alpha-smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of alpha-smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.

Conditionally immortalized human colorectal crypt cell line

To establish human colorectal crypt cell line. Colorectal crypt cells were separated from human fetal gut by dispase I digestion, AKP-negative cells from fetal colorectal crypt were collected and cultured on Matrigel matrix. Subsequently the primary cultured cells were transfected with recombinant retrovirus containing human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40 LT) in 48 h. The characterization of immortalized cells was identified after the transfection and cells were screened with antibiotics for 12 approximately 16 weeks and expanded. Mucin, cytokeratin-pan, 8, 19 were presented in immortalized cells by immunohistochemical staining; ectopic expressions of both hTERT and SV40 LT were also found in immortalized cells by Western blotting. Agarose electrophoresis showed that the cells expressed Musashi-1 mRNA. No evidence of carcinogenesis was found in nude mouse experiment and soft-agarose cloning test. The immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro.