Formation Of Intestinal Crypts Research Articles

P043 ALI culture derived from human organoids recapitulates cell composition during intestinal crypt formation

Abstract Background The ALI (air liquid-interface) culture derived from human primary intestinal epithelium is generating considerable interest for closely reproducing the mature cell features of the differentiated epithelium in a 2D configuration. However, the phenotypic changes occurring in the human ALI culture during cell differentiation have thus far not been explored in depth. Here, we aimed to characterize the cell composition of human ALI culture over time. Methods 3D epithelial organoids (n=3) were expanded from human colonic samples and organoid-derived single cells were seeded on a Transwell. After reaching confluence, the submerged 2D was switched to ALI and maintained for 7 days. The ALI culture was analysed every 24h by qPCR, membrane staining (H&E and immunofluorescence) and trans-epithelial resistance (TER) (Figure 1). Results The confluent pre-ALI (TER>200 Ω*cm2) showed high mRNA expression of stem cell markers (i.e., LGR5, AXIN2), while several differentiation markers (i.e., ZG16, CLCA1, ANPEP, AQP8, BEST4, CHGA) were almost undetectable, thus resembling the stem cell niche compartment. Air exposure of the 2D culture induced an early and transient (limited to ALI-day 1) transcriptional up-regulation of proliferation markers (i.e., MKI67, CDCA7) and OLFM4, followed by a concomitant marked downregulation of stem cell markers, thus mimicking the Transit Amplifying compartment. OLFM4 protein, as observed by immunofluorescence, showed an early cytoplasmic accumulation followed by secretion at later time points (from ALI-day 5). Differentiation towards absorptive and secretory lineages increased gradually from ALI-day 5 and ALI-day 7 onward, respectively, at both the transcriptional and protein level. Expression changes were accompanied by the progressive transition of the ALI monolayer from a squamous to a columnar morphology. In parallel, TER promptly increased upon ALI switch, thus indicating the early establishment of cell-cell junctions. Conclusion By recapitulating the cell composition identified along the crypt, the ALI culture holds promise to be a versatile in vitro model for studying how the different epithelial cell compartments respond to specific environmental stimuli, like those present during an inflammatory flare.

The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult mouse gut.

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. We set out to systematically compare the effect of two ECM classes, the interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In contrast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture resembled gut development in vivo. The dramatic ECM remodelling was accompanied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse resulted in a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.

Translation initiation factor eIF2Bε promotes Wnt-mediated clonogenicity and global translation in intestinal epithelial cells

Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3β inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.

Senescence-accelerated mouse prone 8 mice exhibit specific morphological changes in the small intestine during senescence and after pectin supplemented diet.

Impairment of gastrointestinal function and reduction of nutrient absorption associated with aging contribute to increased risk of malnutrition in the elderly population, resulting in physical weakness and vulnerability to disease. The present study was performed to examine the relationships between aging-associated morphological changes of the small intestine and nutrient malabsorption using senescence-accelerated mouse prone 8 (SAMP8) mice. Comparison of the morphology of the small intestine of young (22-week-old) and senescent (43-week-old) SAMP8 mice showed no significant changes in villus length, while the mRNA expression levels of secretory cell marker genes were significantly reduced in senescent mice. In addition, crypts recovered from the small intestine of senescent mice showed a good capacity to form intestinal organoids ex vivo, suggesting that the regenerative capacity of intestinal stem cells (ISCs) was unaffected by accelerated senescence. These results indicated that changes induced by accelerated senescence in the small intestine of SAMP8 mice are different from changes reported previously in normal aging mouse models. Biochemical analyses of serum before and during senescence also indicated that senescent SAMP8 mice are not in a malabsorption state. Furthermore, a diet supplemented with persimmon pectin had a mild effect on the small intestine of senescent SAMP8 mice. Intestinal villus length was slightly increased in the medial part of the small intestine of pectin-fed mice. In contrast, intestinal crypt formation capacity was enhanced by the pectin diet. Organoid culture derived from the small intestine of mice fed pectin exhibited a greater number of lobes per organoid compared with those from mice fed a control diet, and Lyz1 and Olfm4 mRNA levels were significantly increased. In conclusion, accelerated senescence induced exclusive changes in the small intestine, which were not related to nutrient malabsorption. Therefore, the SAMP8 strain may not be a suitable model to evaluate the effects of aging on intestinal homeostasis and nutrient absorption impairment.

Translation Initiation Factor eIF2Bε Promotes Stemness of Intestinal Epithelial Cells

Modulation of global mRNA translation is essential for intestinal stem cell function. In colorectal cancer, proliferating stem-like cells are defined by high WNT signaling and display dysregulated translational control. It is currently unclear how factors that coordinate global translation affect stem cell behavior. Here we identified nucleotide exchange factor eIF2Be as a translation initiation factor involved in intestinal epithelial stemness. In eIF2Be Arg191His mice, which have a homozygous point mutation that intrinsically impairs eIF2Be’s enzymatic function, we demonstrate that dysfunctional eIF2Be affects small intestinal crypt formation, proliferation and stemness marker expression. By hyperactivating stem cell activity in ex vivo organoids, using a GSK3β inhibitor to mimic loss of tumor suppressor Apc , we demonstrate that eIF2Be is essential for clonogenic potential and associated with an increase in global translation. Finally, we observe high eIF2Be expression in human colonic adenoma tissues, exposing eIF2Be as a potential target in modulation of stemness.

Abstract 5931: PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo

Abstract Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed, all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. PdgfRα is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating for the first time that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα. [G.G. and Z.K. contributed equally to this work.] Citation Format: Gediminas Greicius, Zahra Kabiri, Kristmundur Sigmundsson, Chao Liang, Ralph Bunte, Manvendra K. Singh, David M. Virshup. PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5931.

PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα.

Wnt, RSPO and Hippo Signalling in the Intestine and Intestinal Stem Cells.

In this review, we address aspects of Wnt, R-Spondin (RSPO) and Hippo signalling, in both healthy and transformed intestinal epithelium. In intestinal stem cells (ISCs), the Wnt pathway is essential for intestinal crypt formation and renewal, whereas RSPO-mediated signalling mainly affects ISC numbers. In human colorectal cancer (CRC), aberrant Wnt signalling is the driving mechanism initiating this type of neoplasia. The signalling role of the RSPO-binding transmembrane proteins, the leucine-rich-repeat-containing G-protein-coupled receptors (LGRs), is possibly more pleiotropic and not only limited to the enhancement of Wnt signalling. There is growing evidence for multiple crosstalk between Hippo and Wnt/β-catenin signalling. In the ON state, Hippo signalling results in serine/threonine phosphorylation of Yes-associated protein (YAP1) and tafazzin (TAZ), promoting formation of the β-catenin destruction complex. In contrast, YAP1 or TAZ dephosphorylation (and YAP1 methylation) results in β-catenin destruction complex deactivation and β-catenin nuclear localization. In the Hippo OFF state, YAP1 and TAZ are engaged with the nuclear β-catenin and participate in the β-catenin-dependent transcription program. Interestingly, YAP1/TAZ are dispensable for intestinal homeostasis; however, upon Wnt pathway hyperactivation, the proteins together with TEA domain (TEAD) transcription factors drive the transcriptional program essential for intestinal cell transformation. In addition, in many CRC cells, YAP1 phosphorylation by YES proto-oncogene 1 tyrosine kinase (YES1) leads to the formation of a transcriptional complex that includes YAP1, β-catenin and T-box 5 (TBX5) DNA-binding protein. YAP1/β-catenin/T-box 5-mediated transcription is necessary for CRC cell proliferation and survival. Interestingly, dishevelled (DVL) appears to be an important mediator involved in both Wnt and Hippo (YAP1/TAZ) signalling and some of the DVL functions were assigned to the nuclear DVL pool. Wnt ligands can trigger alternative signalling that directly involves some of the Hippo pathway components such as YAP1, TAZ and TEADs. By upregulating Wnt pathway agonists, the alternative Wnt signalling can inhibit the canonical Wnt pathway activity.

Identification and characterization of functional single nucleotide polymorphisms (SNPs) in Axin 1 gene: a molecular dynamics approach.

Wnt signaling pathway has been reported to play crucial role in intestinal crypt formation and deregulation of this pathway is responsible for colorectal cancer initiation and progression. Axin 1, a scaffold protein, play pivotal role in the regulation of Wnt/β-catenin signaling pathway and has been found to be mutated in several cancers; primarily in colon cancer. Considering its crucial role, a structural and functional analysis of missense mutations in Axin 1 gene was performed in this study. Initially, one hundred non-synonymous single nucleotide polymorphisms in the coding regions of Axin 1 gene were selected for in silico analysis. Six variants (G820S, G856S, E830K, L811V, L847V, and R767C) were predicted to be deleterious by combinatorial prediction. Further investigation of structural attributes confirmed two highly deleterious single nucleotide polymorphisms (G820S and G856S). Molecular dynamics simulation demonstrated variation in different structural attributes between native and two highly deleterious Axin 1 mutant models. Finally, docking analysis showed variation in binding affinity of mutant Axin 1 proteins with two destruction complex members, GSK3β and adenomatous polyposis. The results collectively showed the deleterious effect of the above predicted single nucleotide polymorphisms on the Axin 1 protein structure and could prove to be an adjunct in the disease genotype-phenotype correlation studies.

Abstract 4274: Porcine ovarian matrix supports maintenance of human intestinal organoid cultures

Abstract Currently, extracellular matrix hydrogels derived from the Engelbreth-Holmes-Swarm murine tumor (Cultrex BME, Matrigel) are used for stem cell maintenance and differentiation of both normal and tumor organoid culture models. While this matrix is suitable for many in vitro applications, there is increasing demand for a 3D organoid culture platform that is more suitable for clinical applications. Here, we have developed a soluble extracellular matrix derived from normal porcine ovaries that forms a hydrogel polymer at physiological temperature and that supports maintenance of human small intestine organoids in culture. This matrix supports expansion of LGR5+ human stem cell populations using Wnt, EGF, Noggin, and R-Spondin 1 cell culture medium. The matrix also supports differentiation of stem cells into mini organs by removing Wnt which is characterized by the formation of intestinal crypts which exhibit immunostaining for lineage specific markers. This new extracellular matrix hydrogel represents a non-tumor sourced, normal extracellular matrix for evaluating both development and cancer progression. Citation Format: Jay George, Sol De Gese, Gabriel Benton. Porcine ovarian matrix supports maintenance of human intestinal organoid cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4274.

C-Myc Is Required for the Formation of Intestinal Crypts but Dispensable for Homeostasis of the Adult Intestinal Epithelium

In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-myc(flox) allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreER(T2) recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.

Fibroblast growth factor receptor-3 is expressed in undifferentiated intestinal epithelial cells during murine crypt morphogenesis.

Prior studies have demonstrated that fibroblast growth factor receptor-3 (FGFR-3) regulates proliferation of undifferentiated intestinal epithelial cells in vitro. However, the function(s) of FGFR-3-mediated signaling during intestinal development and epithelial differentiation in vivo remain unknown. The goal of this study was to define the temporal, regional, and cell-specific patterns of FGFR-3 expression and its ligands during normal intestinal ontogeny and epithelial regeneration. Both the IIIb and IIIc isoforms of FGFR-3 mRNA, which result from differential splicing of the FGFR-3 primary transcript, were detected in mouse small intestine as early as embryonic day 16. FGFR-3 levels peaked in the small intestine from 7 to 21 days after birth and decreased thereafter to reach the low levels observed in adult mice. FGFR-3 IIIb and IIIc mRNA levels were highest in the duodenum and proximal jejunum with lower levels of both seen in the distal jejunum, ileum, and colon. FGFR-3 was expressed in a subset of proliferating undifferentiated crypt epithelial cells located in the intervillous epithelium and in the lower half of nascently forming crypts but not in differentiated epithelial cell types. FGFR-3 IIIb was the dominant isoform expressed in both small intestinal and colonic crypts. Expression of FGF1, FGF2, and FGF9, known ligands of FGFR-3, paralleled patterns of FGFR-3 expression during gut development. These data suggest that signaling through FGFR-3 plays a role in regulating morphogenic events involved in formation of intestinal crypts and/or the fate of epithelial stem cells.

Immunolocalization of a mesenchymal antigen specific to the gastrointestinal tract.

The aim of the present study was to localize, at the fine structural level, a protein found by indirect immunofluorescence to be associated with the mesenchymal tissue 1) closely applied to the intervillus epithelium before the formation of intestinal crypts in the mouse fetus and 2) around intestinal crypts during and after their formation. We used a pre-embedding immunolabeling technique for extracellular matrix molecules, and a monoclonal antibody (Mab) directed against antigen MIM-1/130. Immunofluorescence disclosed the presence of antigen 1/130 in the connective tissue closely applied to the epithelium of the gallbladder, pyloric glands, and intestinal and colonic crypts in adult mice. The antigen was absent in all salivary glands, kidney, liver, lung, spleen, and pancreas. At the fine structural level, gold particles in positive organs were associated with the interstitial matrix around collagen fibrils underneath the epithelia; gold particles were completely absent in the basement membranes. In the small intestine, labeling was seen only around crypts from cell position 1 up to the crypt-villus junction; it was totally absent under the villus epithelium. In order to confirm this particular localization in vivo, Mab 1/130 was administered orogastrically to 9-day-old mice: after 3 hours the antibody was found lining the immediate periphery of duodenal crypts as seen by indirect immunofluorescence. In control animals, an anti-mouse laminin Mab of the same subclass as Mab 1/130 was orogastrically fed using the same protocol: basal laminae were labeled under the epithelium of duodenal villi and crypts and also in the lamina propria, with a decreasing gradient from the top of the villi to the bottom of the crypts. These observations indicate that the extracellular matrix associated with the epithelium of pyloric glands, of intestinal and colonic crypts, and of gallbladder contains a new antigen whose function remains to be determined. The neonatal mouse hence constitutes a good model to study the role of extracellular matrix components in determining organ differentiation in vivo.

An intestinal secretory protein is found in most glands associated with the gastrointestinal tract: von Ebner's and salivary glands, gallbladder, and pancreas.

We have previously shown that monoclonal antibodies (MAb) prepared against the duodenal mucosa of 4-day-old mice disclosed the presence of two antigens associated with the formation of intestinal crypts. One of these, MIM-1/39, was found in the apical cytoplasm of undifferentiated epithelial crypt cells of the duodenum and colon. We report here the immunolocalization of MIM-1/39 in different glands associated with the gastrointestinal tract, using a polyclonal antibody produced against antigen MIM-1/39. By indirect immunofluorescence on 1-micron thick Lowicryl K4M sections, MIM-1/39 was detected in secretory granules of serous cells in lingual (von Ebner's gland), sublingual, submandibular and parotid glands, and in pancreas; it was also found in epithelial cells of the gallbladder and in the secretory granules of chief cells of gastric glands. Liver, kidney, and Brunner's glands were not immunoreactive. Immunocytochemistry revealed the presence of antigen MIM-1/39 in small secretory granules of the gallbladder, duodenum, colon, and in the large secretory granules in serous cells of lingual and parotid glands, in pancreas, and in gastric chief cells. In Western blotting, the MIM-1/39 MAb revealed two bands (330 and 350 KD) in adult mouse duodenal mucosa, gallbladder and stomach, whereas only one (330 KD) was disclosed in pancreatic juice. However, two bands (330 and 350 KD) were detected in pancreatic juice with the polyclonal antibody. The distribution of MIM-1/39 was different from that reported for IgA, bound and free secretory components, cryptdin, and Tamm-Horsfall protein. Therefore, MIM-1/39 appears to be a unique protein. Its exact role remains to be elucidated.

Histogenesis of the mucosa of the descending colon in mouse fetuses.

The present study clarify intraepithelial events occurring during the formation of intestinal crypts in the descending colon. In early stage, the mucosa is lined with a simple columnar epithelium consisting of undifferentiated cells which are mutually joined by the junctional complex surrounding the intestinal lumen. The epithelium becomes thickened and stratified, and secondary junctional complexes are formed in the abluminal part of the epithelium. An intracellular cavity opens to the focal tight junction of the secondary junctional complex to form an intraepithelial (intercellular) cavity surrounded by the tight junction. The intraepithelial cavity grows by the fusion of intracellular cavities and vesicles as well as by the division of surrounding cells. Intraepithelial cavities fuse with each other and with the main intestinal lumen. This, together with mesenchymal invasion, results in the formation of intestinal crypts lined with a simple columnar epithelium, where various cell types begin to differentiate. These processes seem essentially similar to those occurring in the developing small intestine except that villi, not crypts, are formed in the small intestine. It is suggested that the formation site and elongation of the tight junction provide for the location and fusion of intraepithelial cavities and eventually result in the formation of villi or crypts, alternatively.

242 INDUCTION OF CRYPTS FORMATION IN CULTURED FETAL MOUSE DUODENUM

In the fetal mouse small intestine villi differentiate during the last four days of gestation; crypts will differentiate only after birth. We have shown that rat amniotic fluid (RAF) at 19 days of gestation contains a potent inducer of intestinal crypt formation (Anat. Rec. 205: 27–37, 1983). When an organic extract of RAF is added to Trowell T8 medium, well-developed villi differentiate and crypts are present after 48 h of culture, in duodenal explants taken from 15-day mouse fetuses. The existence of a crypt differentiation factor (CDF) was then postulate. We report here a partial characterization of this factor. The CDF activity in organic extract is well preserved alter heating at 60°C or 100°C during 10 min.: however after 1 h at 100°C the activity is considerably reduced. On reverse phase HPLC, CDF is poorly fractionated and shows a high polarily. With molecular seave chromatography by HPLC on Protein I 125P column, CDF is purified 10 times over original RAF dry weight. The apparent molecular weight of CDF is 13,000 daltons. When rat amniochorionic membranes are cocultured with 15-day duodenal explants, crypts also differentiate: these fetal membranes could be responsible for the production of CDF. Further investigation are needed in order to purify the CDF and to determine its chemical nature. “Supported by Grant No. MT6069 from the Medical Research Council of Canada.”

Extracts of rat amniotic fluid contain a potent inducer of intestinal crypt formation.

The effect of acid, basic, and organic extracts from Long Evans rat amniotic fluid (RAF) and from Swiss ICR mouse amniotic fluid (MAF) was studied on 15-day fetal mouse duodenal mucosa in organ culture. Amniotic fluids (20 ml) were lyophilized and extracted 1) with CHCl3:MeOH and the organic phase was evaporated; then 2), the residue was acidified with a solution of 0.1 N HCl in 10% acetic acid and the liquid phase was lyophilized; finally 3), 0.01 M NH4OH was added to the residue and the liquid phase was lyophilized. The product of each extraction was added to 20 ml of Trowell T8 medium. Acid and basic extracts of RAF and MAF have no effect on the formation of duodenal villi and crypts after 48 hours of culture. With the organic extract of MAF, small villi are present after 48 hours of culture and absorptive cells are poorly differentiated. With the organic extract of RAF, well-developed villi have differentiated after 48 hours of culture; moreover, crypts are present at the same stage and Paneth cells are identified within these crypts. During the 8-10 hour period, the explants cultured with the Trowell T8 medium supplemented with RAF or MAF organic extracts show a 35% increase in 3H-thymidine incorporation over the controls cultured with Trowell T8 medium alone. These results indicate that organic extracts from MAF and RAF are able to promote villus formation in undifferentiated explants from 15-day fetal mouse duodenum in organ culture. Furthermore, RAF organic extract contains a factor that can induce the formation of duodenal crypts and the differentiation of Paneth cells in culture at least 2 days before their normal appearance.