Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning
2018; Springer Nature; Volume: 20; Issue: 2 Linguagem: Inglês
10.15252/embr.201846221
ISSN1469-3178
AutoresMarit Navis, Tânia Martins Garcia, Ingrid B. Renes, Jacqueline L.M. Vermeulen, Sander Meisner, Manon E. Wildenberg, Gijs R. van den Brink, Ruurd M. van Elburg, Vanesa Muncan,
Tópico(s)Renal and related cancers
ResumoScientific Report10 December 2018Open Access Transparent process Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning Marit Navis Marit Navis Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Tânia Martins Garcia Tânia Martins Garcia Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Ingrid B Renes Ingrid B Renes Department of Pediatrics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Danone Nutricia Research, Utrecht, The Netherlands Search for more papers by this author Jacqueline LM Vermeulen Jacqueline LM Vermeulen Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Sander Meisner Sander Meisner Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Manon E Wildenberg Manon E Wildenberg Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Gijs R van den Brink Gijs R van den Brink Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands GlaxoSmithKline, Medicines Research Center, London, UK Search for more papers by this author Ruurd M van Elburg Ruurd M van Elburg Department of Pediatrics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Danone Nutricia Research, Utrecht, The Netherlands Search for more papers by this author Vanesa Muncan Corresponding Author Vanesa Muncan [email protected] orcid.org/0000-0003-4062-6430 Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Marit Navis Marit Navis Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Tânia Martins Garcia Tânia Martins Garcia Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Ingrid B Renes Ingrid B Renes Department of Pediatrics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Danone Nutricia Research, Utrecht, The Netherlands Search for more papers by this author Jacqueline LM Vermeulen Jacqueline LM Vermeulen Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Sander Meisner Sander Meisner Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Manon E Wildenberg Manon E Wildenberg Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Gijs R van den Brink Gijs R van den Brink Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands GlaxoSmithKline, Medicines Research Center, London, UK Search for more papers by this author Ruurd M van Elburg Ruurd M van Elburg Department of Pediatrics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Danone Nutricia Research, Utrecht, The Netherlands Search for more papers by this author Vanesa Muncan Corresponding Author Vanesa Muncan [email protected] orcid.org/0000-0003-4062-6430 Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Author Information Marit Navis1,‡, Tânia Martins Garcia1,‡, Ingrid B Renes2,3, Jacqueline LM Vermeulen1, Sander Meisner1, Manon E Wildenberg1, Gijs R van den Brink1,4, Ruurd M van Elburg2,3 and Vanesa Muncan *,1 1Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, AG&M, University of Amsterdam, Amsterdam, The Netherlands 2Department of Pediatrics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 3Danone Nutricia Research, Utrecht, The Netherlands 4GlaxoSmithKline, Medicines Research Center, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +31 20 5668159; E-mail: [email protected] EMBO Reports (2019)20:e46221https://doi.org/10.15252/embr.201846221 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo. In vitro postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo. Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation. Synopsis Organoids derived from mouse primary fetal intestinal epithelial cells undergo suckling-to-weaning transition independent of stromal cells and can be used to model the molecular mechanisms of postnatal epithelial development. Mouse fetal intestinal organoids undergo the suckling-to-weaning transition in vitro. Fetal intestinal organoids express functional adult brush border enzymes over time. Dexamethasone can accelerate intestinal epithelial maturation in vitro. Fetal intestinal organoids can be used to model postnatal epithelial development. Introduction At birth, the mouse small intestinal epithelium consists of a single layer of epithelium that covers finger-like projections into the intestinal lumen called villi. At this time, the villi are populated with three intestinal cell types: the enterocyte, goblet cell, and enteroendocrine cell. Proliferating epithelial cells are localized at the base of the villi in so-called "intervillus pockets" 1. In the first month after birth, the epithelium undergoes major structural and functional changes. The most apparent structural changes occur around 2 weeks after birth, when crypts form at the base of the villi and the Paneth cells start to populate the bottom of the crypts 2, 3. Concurrently, major functional changes are initiated in a process called suckling-to-weaning transition, which is completed within two following weeks. This transition consists of a number of highly specific enzymatic and metabolic changes that allow diet change from milk that is rich in fat and has lactose as a major carbohydrate, to solid food that is rich in complex carbohydrates 4. Many of the major changes occur at the epithelial brush border, which expresses various proteins involved in the digestion of food, such as enzymes needed for processing of carbohydrates. More specifically, in the first two postnatal weeks the principal carbohydrate is lactose, and its digestion is dependent on the enzyme lactase-phlorizin hydrolase (Lct) 5. Around postnatal day 14 in mice (P14), the adaptation to digest complex carbohydrates from solid food is initiated. This is accompanied by gradual increase in expression of the brush border disaccharides sucrase-isomaltase (Sis) and trehalase (Treh). Expression levels of these enzymes rise rapidly to adult levels in the third postnatal week 4, 6. One of the key metabolic changes during postnatal development involves arginine biosynthesis. Arginine is a semi-essential amino acid that is only present in limited amounts in milk and therefore synthesized in the neonatal enterocytes 7. Argininosuccinate synthetase 1 (Ass1) is the rate-limiting enzyme in arginine biosynthesis and in mice exclusively expressed in the first two postnatal weeks. During the suckling-to-weaning transition, epithelial cells lose the expression of Ass1 and switch to expressing arginase 2 (Arg2) allowing catabolism of arginine, which is abundantly present in solid food 7, 8. Another characterized occurrence of the suckling-to-weaning transition in mice is loss of expression of the neonatal Fc receptor for immunoglobulin (FcRn) 9, 10. Neonatal intestinal epithelium expresses high levels of FcRn, which mediates the transfer of maternal IgG from the milk across the intestinal epithelial membrane to facilitate passive immunity. In addition, loss of cathelicidin-related antimicrobial peptide (CRAMP) during the suckling-to-weaning transition of mouse intestinal epithelium has been described as well 11. Transplantation studies of fetal intestinal segments into subcutaneous tissues of nude adult mice revealed that these segments developed normally in the absence of luminal signals 12-14. These experiments established that information needed for appropriate intestinal epithelial development, including the suckling-to-weaning transition, is driven by a genetic program that is intrinsic to the intestinal mucosa and specified in early development. We and others have previously shown that the intestinal transcription factor Blimp-1 is selectively expressed in the mouse intestinal epithelium during embryonic and postnatal development, and that its expression is lost at the suckling-to-weaning transition 15, 16. Conditional deletion of Blimp-1 from the mouse intestinal epithelium resulted in an adult-type epithelium at birth, with complete absence of ultrastructural and molecular characteristics of postnatal phase of development and severe growth impairment and death of newborn pups 15, 16. These data showed that Blimp-1 in the mouse intestinal epithelium is a critical driver of the postnatal epithelial phenotype and that its loss of expression in the third postnatal week is likely required for maturation from neonatal to adult epithelium. The factors driving Blimp-1 expression in the first 2 weeks and its loss of expression in the third postnatal week are not known. The suckling-to-weaning transition might be completely intrinsically regulated, but epithelial gut maturation can to some extent be modulated by hormonal status and extrinsic luminal signals like microbiota and nutrition. Changes in endogenous and exogenous circulating hormones in the developing neonate, such as glucocorticoids, can precociously induce intestinal maturation in vivo 17, 18. Luminal signals, such as microbiota, regulate developmental-dependent expression of epithelial glycosyl transferases, enzymes necessary for glycosylation of epithelial-specific proteins during gut maturation 19. Finally, dietary factors such as growth factors, human milk oligosaccharides, and hormones that are present in human milk have been shown to influence gut growth and maturation in cell lines and/or rodent models 20, 21. It has been an ongoing discussion to which extent and which specific aspects of suckling-to-weaning transition are intrinsically programmed. Here, we use cultures of primary fetal epithelial cells to examine whether the epithelial suckling-to-weaning transition also occurs in vitro, in a stable culture medium, and in the absence of stromal cells. Results and Discussion Primary mouse fetal intestinal epithelium matures in vitro To study whether the intestinal epithelium matures and undergoes the suckling-to-weaning transition in vitro, intestinal epithelium from developmental stage E19 was chosen as starting material for the organoid cultures. Organoids were cultured for 1 month, in a stable culture medium, following the passage scheme and harvesting of organoids on the same day after each passage (Fig 1A). As isolated intestinal epithelial cells need 24–48 h to establish in vitro growth, this developmental stage translates in vitro just prior to birth. We performed genomewide gene expression analyses on fetal and adult organoids at days 3 and 30 of culture and mouse intestinal tissues at birth (day 0) and adult (day 42). Principal component analysis (PCA) of this multi-dimensional dataset revealed that four clusters can be distinguished based on gene expression profiles: (i) fetal organoids day 3; (ii) fetal organoids day 30 together with adult organoids (days 3 and 30); (iii) fetal tissue; and (iv) adult tissue (Fig 1B). Along the first component (PC1 34%), the organoids (epithelium) are clearly separated from the whole tissue, indicating that the gene expression profile of organoids differs substantially from intestinal tissues. Along PC2 (PC2 16.2%), the day 3 fetal organoids separate from day 30 fetal and days 3 and 30 adult organoids, as is also the case for fetal and adult tissue. Of note, no significant difference in the global gene expression profile between day 30 fetal organoids and days 3 or 30 adult organoids assessed by Pearson correlation is observed (Fig EV1A and B). The direction of separation along PC2 for organoids and tissue is the same, suggesting that the maturation state contributes to this separation. Figure 1. Gene expression analyses of E19 organoids at early and late culture time points A. Fetal organoids isolated from fetal intestine at embryonic day 19 were cultured for 30 days in ENR medium and analyzed 3 days after indicated passage. B. PCA was conducted on global gene activity in mouse fetal tissue at days 0 and 42, mouse E19 organoids at days 3 and 30 of culture, mouse adult organoids at days 3 and 30 of culture (n = 4 independent intestinal tissue specimens and n = 4 independent organoid cultures). C, D. Lists of differentially expressed genes between adult and fetal tissue, days 3 and 30 organoids were generated for (C) up- and (D) downregulated genes. Results are shown as Venn diagram. E. Curated heat maps of top 76 up- and 72 downregulated genes. Highlighted genes were chosen based on biological interest. The colored bar represents the z-score transformed expression level from low (green) to high (red). F. The most significantly changed canonical pathways between fetal organoid at days 3 and 30 of culture. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Comparison of organoid with tissue maturation A. Pearson's correlation matrix including hierarchical clustering for whole-genome normalized microarray probes expressed in organoids/tissues indicated in the colored figure legend. b. Pearson's correlation coefficient for the comparison of whole-genome normalized microarray probes between each of the sample listed on the x-axis and adult tissue. P-values indicate the results of Student's t-test. NS is not significant. n = 4. C, D. Gene set enrichment analyses of 200 most (C) up- and (D) downregulated genes from mouse primary fetal versus adult epithelium (GSE35596) across fetal organoid maturation dataset. Vertical lines below x-axis display relative distribution of expression per gene included in the geneset. Download figure Download PowerPoint To assess differences and similarities between organoids and tissues and to examine these differences relative to those that exist when comparing fetal intestines and adult intestines, we performed a differential gene expression analysis. Out of 178 genes (Table EV1) that were upregulated 4-fold or more in fetal organoids cultured for 30 days, 115 genes (65%) were in common with genes upregulated in the adult tissue (Fig 1C). Similarly, 105 out of 310 genes (35%) overlapped between downregulated genes in fetal organoids cultured for 3 days and fetal tissue (Fig 1D). Heat map of the top 76 upregulated genes after 30 days of E19 organoid cultures clustered with adult (day 42) tissue (Fig 1E). Moreover, the majority of the most downregulated genes in 30-day-old fetal organoid cultures were similarly expressed in fetal tissue. Additionally, comparison of our fetal organoid dataset to previously published transcription profiles of primary isolated intestinal epithelial cells of neonatal versus adult mice (GEO GSE35596) 22, 23 revealed that 200 upregulated genes in neonatal mouse epithelium were significantly enriched in the fetal organoids cultured for 3 days, whereas 200 downregulated genes correlated with fetal organoids after 30 days of culture (Fig EV1C and D). We next performed Ingenuity Pathway Analyses (IPA) using as input the list of differentially expressed genes between day 3 and day 30 fetal organoid cultures. Predominant changes in canonical pathways involved metabolic alterations indicative for epithelial maturation that is associated with a change in diet from mother's milk to solid food (Fig 1F). Together, these analyses suggest that fetal organoids mature over time and undergo the suckling-to-weaning transition in vitro. In vivo maturation process of mouse intestinal epithelium We first examined the intestinal epithelial maturation in vivo in detail, using a panel of maturation markers that are described in literature as markers for fetal/neonatal, suckling-to-weaning, and adult epithelium. With this approach, we aimed to obtain a standard for temporal comparison with the in vitro maturation process of the E19 fetal organoids. In the fetal phase (E18.5), we observed a strong expression of the neonatal enzyme argininosuccinate synthetase 1 (Ass1) (Fig EV2A and D), transcription factor Blimp-1 (Fig EV2B and E), and neonatal Fc receptor (FcRn) (Fig EV2F) throughout the whole epithelium. Histological assessment of tissues from the first two postnatal weeks (P7.5 and P14) showed that expression of these markers gradually disappeared from the proliferative intervillus regions but remained in the differentiated cells of the villi. In the adult gut (P42), expression of Ass1 was completely lost (Fig EV2A), whereas Blimp-1 was restricted to a limited number of cells at the villus tips (Fig EV2B). In vivo, Lct was highly expressed in rodent neonatal epithelium and declined after weaning, however still present in the adult intestine, mainly in the jejunum (Fig EV2C and G) 24, 25. Activity of Lct peaked at weaning, yet it remained present in adult albeit at lower level (Fig EV2H). Although Lct expression pattern differed from Ass1 and Blimp-1, it is characteristic for neonatal development and is associated with milk diet 24, 25. Click here to expand this figure. Figure EV2. In vivo expression of neonatal intestinal epithelial markers A–C. Immunohistochemistry of neonatal markers: (A) Ass1, (B) Blimp-1, and (C) Lct. Insets represent higher magnification of the rectangle. White arrowheads indicate negative cells, and black arrowheads indicate positive cells. Scale bars: 50 μm. D–G. Whole tissue real-time qPCR on (D) Ass1, (E) Blimp-1, (F) FcRn, and (G) Lct (n = 5–8 individual intestinal specimens generated from offspring of single pregnant mice for E17 and P0-21, n = 8 independent intestines of adult mice for P42). H. Enzyme activity assay of fetal and adult whole tissue lysates for lactase (n = 3–5 individual intestinal specimens, generated from offspring of single pregnant mice for E17-19, n = 5 independent intestines of adult mice for P42). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant, in (D–G) relative to expression level at E17 (one-way ANOVA). Download figure Download PowerPoint In contrast to the neonatal enterocyte markers, the adult α-glucosidases sucrase-isomaltase (Sis) (Fig EV3A and C) and arginase-2 (Arg2) (Fig EV3B and D) were absent from the small intestine epithelium until the second week after birth, around the suckling-to-weaning transition, when a gradual increase in both enzymes was observed exclusively in the villi. The same pattern was observed for trehalase (Treh) (Fig EV3E). These results were confirmed at enzyme activity level (Fig EV3I–L). In addition, expression of the Paneth cell markers lysozyme-1 (Lyz1) and α-defensins (Defa1, Defa5) was detected from day 14 onwards (Fig EV3F–H). This correlates with the maturation of this secretory cell type at 2 weeks after birth, concurrently with the development of the crypts. The in vivo maturation pattern described here was subsequently used and compared with the time course of maturation of the fetal small intestinal organoids in vitro as described below. Click here to expand this figure. Figure EV3. In vivo expression of adult intestinal epithelial markers A, B. Immunohistochemistry of adult markers (A) Sis and (B) Arg2. Insets represent higher magnification of the rectangle. White arrowheads indicate negative cells, and black arrowheads indicate positive cells. Scale bars: 50 μm. C–H. Whole tissue real-time qPCR on (C) Sis, (D) Arg2, (E) Treh, (F) Lyz1, (G) Defcr1, and (H) Defcr5 (n = 5–8 individual intestinal specimens generated from offspring of single pregnant mice for E17 and P0-21, n = 4–8 independent intestines of adult mice for P42). I–L. Enzyme activity assay of fetal and adult whole tissue lysates for (I) sucrase, (J) maltase, (K) trehalase, and (L) arginase activities (n = 3–5 individual intestinal specimens, generated from offspring of single pregnant mice for E17–19, n = 5 independent intestines of adult mice for P42). Data information: Data are presented as mean ± SEM. ND not detected, *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant, in (C–H) relative to expression level at E17 (one-way ANOVA). Download figure Download PowerPoint Small intestinal fetal organoids mature and recapitulate suckling-to-weaning transition in vitro We first analyzed the expression pattern of the fetal/neonatal enterocyte markers in the cultured fetal organoids by qRT–PCR. Both Ass1 and Blimp-1 were expressed during the first week of culture and nearly absent after 3 weeks (Fig 2A and B). Similarly, FcRn and CRAMP (Figs 2C and EV4A) followed the same expression pattern. Likewise, Lct (Fig 2D) expression was similar to the in vivo expression pattern (Fig EV2G). In contrast, markers of the suckling-to-weaning transition and adult intestine Sis and Treh were only detected in organoids as of 2 weeks of culture (Figs 2E and F). Arg2 was expressed at 1 week of culture (Fig 2G) and progressively increased thereafter. Development of a functional brush border was confirmed on enzyme activity level (Figs 2H–L). Comparing the in vitro maturation from suckling-to-weaning with the in vivo maturation process revealed that the time frame of epithelial maturation is similar between the fetal organoids in vitro and the intestinal tissue in vivo (compare Figs 2A–D and EV2 for neonatal markers and Figs 2E–G and EV3 for suckling-to-weaning/adult markers). Previously, it has been reported that different fetal intestinal segments, i.e., proximal versus distal, generate different organoids 26. We therefore separated proximal and distal parts of E19 intestinal epithelium and studied the in vitro maturation course of the fetal organoids originating from these segments (Appendix Fig S1). Although relative expression levels of some of the maturation markers differed among the segments, the timing and the course of maturation were similar (Appendix Fig S1). Figure 2. Small intestinal fetal organoids recapitulate suckling-to-weaning transition in vitro A–G. Real-time qPCR analysis of fetal organoids cultured for 1 month showing decrease in relative expression of indicated neonatal markers (A) Ass1, (B) Blimp-1, (C) FcRn, and (D) Lct and increase in the adult markers (E) Sis, (F) Treh, and (G) Arg2 (n = 3 individual wells from single organoid culture (see Materials and Methods); experiment was repeated in four to eight independent organoid cultures with similar results). H–L. Enzyme activity assay of fetal organoids for (H) lactase, (I) sucrase, (J) maltase, (K) trehalase, and (L) arginase. Activity is given in μM glucose/μg protein/min (experiment was generated from single organoid culture (see Materials and Methods) and repeated in three independent organoid cultures with similar results). Data information: Data are presented as mean ± SEM. ND not detected, *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant, relative to expression or activity level at day 3 (one-way ANOVA). Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Fetal organoids mature in vitro A–D. Real-time qPCR analysis of fetal organoids cultured for 1 month showing decrease in relative expression of (A) CRAMP and increase in the Paneth cell markers (B) Lyz1, (C) Defcr1, and (D) Defcr5 (n = 3 individual wells from single organoid culture (see Materials and Methods); experiment was repeated four times with similar results). E. Microscopic images of fetal and adult organoids at days 6, 17, and 28 of culture. Scale bars: 250 μm. F–H. Real-time qPCR of (F) Villin1, (G) ChgA, and (H) Muc2 (n = 3 individual wells from single organoid culture (see Materials and Methods); experiment was repeated four times with similar results). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant, relative to expression level at day 3 (one-way ANOVA). Download figure Download PowerPoint Lgr5, the best described adult intestinal stem cell marker under homeostatic condition, is expressed by intercolumnar cells residing inbetween the Paneth cells. We next investigated the presence of Lgr5-expressing cells throughout the course of our culture by means of RNAscope in situ hybridization complemented with immunofluorescence for Lyz1, a marker for Paneth cells (Appendix Fig S2). At the start of E19 culture, Lgr5 was expressed at low levels throughout the organoid epithelium and Paneth cells were absent. Coinciding with the appearance of Paneth cells, Lgr5 signals increase and become confined to the crypt region. Finally, Paneth cell-specific markers Lyz1, Defcr1, and Defcr5 were detected at 2 weeks of culture (Fig EV4B–D), again similar to the intestinal tissue in vivo (Fig EV3F–H). Together, these findings demonstrate that fetal intestinal organoids, when cultured in vitro, follow an intrinsic epithelial maturation pattern characteristic for the in vivo maturation program in a similar time frame. Fetal organoids resemble adult organoids after 1 month in culture At 1 month after birth, the mouse intestinal epithelium in vivo reaches its adult functional state. We cultured E19 fetal and adult intestinal organoids simultaneously for 1 month (Fig EV4E). Indeed, at day 30 of culture, fetal and adult organoids expressed similar levels of the maturation markers (Fig 3A–G). Importantly, expression levels of neonatal markers remained absent in adult organoids, while adult markers were stably expressed throughout the 30 days of culture. This was further confirmed at enzyme activity level for all enzymes analyzed (Fig 3H–L). Moreover, except for the Paneth cells which became evident as of 14 days of culture, the main epithelial cell types, i.e., enterocytes, enteroendocrine, and goblet cells, were present in the fetal cultures from the start-up until 30 days of culture (Appendix Figs S2 and S3). Expression levels of markers for enterocytes, enteroendocrine, and goblet cells were relatively stable over time in fetal organoids (Fig EV4F–H). This demonstrates that fetal organoid maturation in vitro, as described here, is not a consequence of the long-term culturing process of organoids. These findings, alongside with virtually no difference in transcription profiles in prolonged culture of adult organoids (Figs 1B and EV1A), impose intrinsic transition from fetal to adult features in vitro and exclude prolonged culture as a contributor to this process. Figure 3. Fetal organoids resemble adult organoids after 1 month in culture A–G. Relative expression detected by real-time qPCR in fetal organoids (black circles) and adult organoids (blue triangles). Neonatal markers (A) Ass1, (B) Blimp-1, (C) FcRn, and (D) Lct in fetal organoids cultured for 1 month compared to levels adult organoids. Mature markers (E) Sis, (F) T
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