Epigenetic Drifts During Long-Term Intestinal Organoid Culture

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Article Epigenetic Drifts during Long-Term Intestinal Organoid Culture

Torsten Thalheim 1,*,†,‡ , Susann Siebert 2,3,†, Marianne Quaas 4, Maria Herberg 1, Michal R. Schweiger 2,3, Gabriela Aust 4,‡ and Joerg Galle 1,‡

1 Interdisciplinary Center for Bioinformatics (IZBI), Leipzig University, 04107 Leipzig, Germany; [email protected] (M.H.); [email protected] (J.G.) 2 Laboratory for Translational Epigenetics and Tumor Genetics, University Hospital Cologne, 50931 Cologne, Germany; [email protected] (S.S.); [email protected] (M.R.S.) 3 Center for Molecular Medicine Cologne (CMMC), 50931 Cologne, Germany 4 Research Laboratories, Department of Surgery, Leipzig University, 04103 Leipzig, Germany; [email protected] (M.Q.); [email protected] (G.A.) * Correspondence: [email protected] † Contributed equally. ‡ Shared senior authorship.

Abstract: Organoids retain the morphological and molecular patterns of their tissue of origin, are self- organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and Citation: Thalheim, T.; Siebert, S.; control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. Quaas, M.; Herberg, M.; Schweiger, We found that, besides the expected changes in short-term culture, the organoids showed profound M.R.; Aust, G.; Galle, J. Epigenetic changes in their epigenomes also during the long-term culture. The most prominent were epigenetic Drifts during Long-Term Intestinal gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss Organoid Culture. Cells 2021, 10, 1718. from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional https://doi.org/10.3390/cells10071718 changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Academic Editors: Kunlin Jin, Huanxing Su and Guo-Yuan Yang Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation Received: 18 June 2021 is disturbed. Accepted: 4 July 2021 Published: 7 July 2021 Keywords: age-related drifts; mouse small intestine; MMR-deficient mice; histone modification; H3K4me3; H3K27me3; ChIP-seq; RNA-seq Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction A long-term intestinal organoid culture enables the massive expansion of intestinal stem cells (ISCs) [1,2]. Accordingly, it offers a broad range of medical applications [3] and the chance to overcome the difficulties, as well as the ethical and legal concerns, associated Copyright: © 2021 by the authors. with the use of human fetal stem cells. However, massive in vitro expansion might come Licensee MDPI, Basel, Switzerland. with an increased frequency of cell transformation, as indicated by changed organoid This article is an open access article growth properties [4]. These growth changes suggest progressive activation of the Wnt distributed under the terms and pathway. They cannot be considered age-related, because aging of the intestine is associated conditions of the Creative Commons with reduced Wnt activity [5,6]. In a long-term tumor-derived organoid culture, genetic Attribution (CC BY) license (https:// profiles are preserved [7]. Thus, growth changes in long-term organoid cultures might creativecommons.org/licenses/by/ relate to epigenetic drifts, e.g., drifts of histone or DNA methylation. Such changes are 4.0/).

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reversible in principle. Indeed, adapted culture conditions, e.g., by supplementing aspirin, have been shown to suppress them [4]. Extrinsic Wnt activation is a general feature of intestinal organoid culture. Only after tumor-like transformation do organoids grow independently of Wnt [8]. Organoid culture media containing EGF, noggin and R-spondin (ENR culture) guarantee a tissue-like cell composition of the organoids and can be adapted to prefer either ISC maintenance or specifications [9]. Recently, we provided a computational model of organoid growth to explain cell compositions under altered Wnt activation [10]. The origin of progressive Wnt activation in organoid cultures, however, remains unresolved. Wnt activation in BrafV600E-activated colon organoids has been linked to CpG island methylator phenotype- like promoter DNA methylation [8]. The promoters of many genes of the Wnt pathway are known to be associated with bivalent modified nucleosomes [11], i.e., nucleosomes that carry trimethylation of lysine 4 and 27 at histone 3 (H3K4me3 and H3K27me3). In a theoretical approach, we demonstrated that these modifications are sensitive to the cell cycle length [12], providing a potential explanation for long-term modification changes during organoid expansion. As an alter- native explanation of epigenetic activation, one might consider stress-induced changes in the ISC pool, as observed following irradiation or in mismatch repair (MMR)-deficient VC+/?Msh2LoxP/LoxP (Msh2−/−) mice [13]. However, the changes described so far are not associated with gene expression differences in general or with Wnt pathway genes in particular [14]. Here, we study the epigenetic changes associated with the long-term organoid culture of macroscopic normal intestinal tissues. In order to capture the potential effects of genomic stress, we compare MMR-deficient (Msh2−/−) and control (Msh2+/+) mice. We focus on the changes of the H3K4me3 and H3K27me3 modifications known to be associated with transcriptional changes and, thus, with the potential regulatory input in the gene expression.

2. Materials and Methods 2.1. Mice The mouse strains B6.SJL-Tg(Villin-Cre)997Gum/J and B6.Cg-Msh2tm2.1Rak/J were obtained from The Jackson Laboratory (Bar Harbor, MA, USA). By crossing both, the conditional Msh2 allele was placed under the control of the Villin-Cre transgene. Mice were genotyped as described [15]. VC+/?Msh2LoxP/LoxP (Msh2−/−) and VC−/−Msh2flox/flox (Msh2+/+) mice were bred under specific pathogen-free conditions.

2.2. Organoid Preparation and Culture Six centimeters of the jejunum was used to generate intestinal organoids from 8-month-old Msh2−/− and Msh2+/+ mice [4]. About 50 isolated crypts were seeded into 10-µL Matrigel/well. Organoids were split once/week and cultured with or without 1-mM aspirin (Sigma Aldrich GmbH, Munich, Germany) for a further 18 weeks. After the ﬁrst and 18th weeks, organoids were stored in liquid nitrogen. These frozen organoids were re-cultured simultaneously in an IntestiCult™ mouse organoid growth medium (Stem- cell Technologies GmbH, Cologne, Germany) with or without 1-mM aspirin for a further two weeks and analyzed afterwards. As the culture time exceeded 72 h, close to 100% of the organoids show a branched growth pattern [4]. Finally, we compared organoids from Msh2−/− and Msh2+/+ mice at the following ages: 8 months plus 3 weeks of culture, 8 months plus 20 weeks of culture and 8 months plus 20 weeks of culture with aspirin.

2.3. Chromatin Immunoprecipitation (ChIP) Chromatin was prepared up to 50-mg jejunum using SDS shearing buffer and the truChlP Tissue Chromatin Shearing Kit (Covaris, Brighton, UK) according to the manufacturer’s instructions. ChIPs were run on the IP-Star compact system using the Auto iDeal Cells 2021, 10, 1718 3 of 15

ChIP-seq kit for histones (Diagenode, Seraing, Belgium) according to the manufacturer’s direct method for ChIP preparation.

2.4. Quantitative PCR and Illumina Sequencing As a quality control for ChIP precipitates, a quantitative real-time PCR analysis was performed in 384-well format in a 10-µL volume using GoTaq qPCR Master Mix (Promega, Mannheim, Germany) and run on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany). The primers used for qPCR are shown in Table S1. The oligonucleotides for Gapdh and the “background” were taken from references [16,17], respectively. For all the other primer pairs, publicly available ChIP-seq data were uploaded in UCSC, and the genomic regions showing an enrichment for the respective histone marks were selected for the primer design. The relative enrichment was calculated using the % input method. Library preparations were performed following the instructions of the TrueSeq LT PCR free Kit (Illumina, San Diego, CA, USA) or the TruSeq Nano DNA Kit (Illumina). Sequencing was performed on the Illumina HiSeq 4000 (Illumina) with 76-bp paired-end sequencings.

2.5. ChIP-Seq Data Preprocessing and Peak Calling The sequenced reads underwent a quality trimming using cutadapt [18] to ensure a minimum read length of 40 bases and a Phred quality score above 20. The quality reports were generated and analyzed using Fastqc [19]. The high-quality reads were mapped to the mouse reference genome NCBI37/mm9 using the software tool segemehl 0.2.0 [20] and its default parameters, except for enabled split/splice detection. Histone modification peaks, i.e., genomic regions in which modifications are enriched, were identified using MACS [21,22].

2.6. ChIP-Seq Data Analysis After the peak calling process, we generated summarized peak lists for each genotype and treatment. Each of the resulting lists contained the peaks that were either consistently detected in both of the replicates or that had a high reliability (MACS fold enrichment > 5.0). For peaks that were detected in both replicates, the maximum peak breadth and tag density were taken. Subsequently, we identified peaks within the promoter region of the genes (defined as the transcription start site, TSS, +/− 1000 bases) and the gene bodies (defined as the region between the TSS and the last base of the gene). The gene reference list containing 31,592 RefSeq genes was taken from the UCSC Table Browser. To avoid sex-specific artifacts, we excluded genes and peaks of the X- and Y-chromosomes from the analysis. If a peak had a minimum overlap of 5% with a promoter region and/or a gene body, the respective gene-associated histones were considered to carry the modification in a binary present or non-present manner. Thus, each gene received a three-digit histone modification code (0: modification absent and 1: modification present) for each sample. The order of this code was H3K4me3–H3K27me3.

2.7. RNA-Seq and Data Preprocessing The intestinal tissue was homogenized with a Tissue Lyser (20 s at 15 Hz). RNA was prepared following the manufacturer’s recommendations (Qiagen QIA AllPrep kit, Hilden (NRW), Germany), including DNase I digestion. RNA was sequenced on an Illumina HiSeq 4000 sequencer with 75-bp paired-end, strand-speciﬁc, random priming. Data preprocessing was performed as described for the ChIP-seq data (see above). Raw counts were normalized to the total number of reads, resulting in one read per million (RPM) values per gene. These RPM values were normalized to the gene lengths (RPKM: reads per kilobase million).

2.8. Self-Organizing Map (SOM) Analysis The data proﬁles over all the samples (ChIP-seq: histone codes and RNA-seq: RPKM data) were then clustered into metagene proﬁles using SOM machine learning, with each Cells 2021, 10, x FOR PEER REVIEW 4 of 15

2.8. Self-Organizing Map (SOM) Analysis The data profiles over all the samples (ChIP-seq: histone codes and RNA-seq: RPKM Cells 2021, 10, 1718 data) were then clustered into metagene profiles using SOM machine learning, with4 ofeach 15 metagene serving as a representative of a cluster of combined profiles. We used a grid of 30 × 30 metagenes and applied default parametrization as implemented in oposSOM [23]. metagene serving as a representative of a cluster of combined profiles. We used a grid of 2.9. Gene Set Enrichment Analysis 30 × 30 metagenes and applied default parametrization as implemented in oposSOM [23]. The analysis was performed using the GO enrichment analysis tool PANTHER, 2.9.Version Gene Set12.0 Enrichment [24]. Data Analysis and image analyses were conducted using statistics software R [25].The analysis was performed using the GO enrichment analysis tool PANTHER, Ver- sion 12.0 [24]. Data and image analyses were conducted using statistics software R [25]. 3. Results 3.3.1. Results Histone Modification Profiles in Organoids 3.1. HistoneTo characterize Modification the Profiles histone in Organoids modification profiles of intestinal organoids from 8- monthTo characterize-old mice cultured the histone for 3 modification (short-term) profiles and 20 of(long intestinal-term) organoidsweeks, we from measured 8-month- the oldhistone mice marks cultured H3K4me3 for 3 (short-term) and H3K27me3 and 20 and (long-term) the control weeks, H3pan we by measured histone ChIP the- histoneseq [26]. marksThe organoids H3K4me3 were and derived H3K27me3 from and two the Msh2 control+/+ and H3pan Msh2− by/− pairs histone of mice. ChIP-seq Based [26 on]. Thethese +/+ −/− organoidsdata, we wereconstructed derived summary from two Msh2lists of theand genomicMsh2 regionspairs of in mice. which Based modified on these histones data, wewere constructed detected summary for each modificationlists of the genomic and mouse regions pair in which[13]. The modified modified histones regions were are detectedhereafter for referred each modification to as peaks. and For mouse further pair analys [13]. Thees, we modified selected regions quality are peaks hereafter (see: referredMaterial to and as peaks.Methods). For The further number analyses, of quality we selected peaks measured quality peaks was similar (see: Material in all samples and Methods).(Figure 1A). The The number maximum of quality difference peaks measured we observed was betweensimilar in the all samplesshort- and (Figure long1-termA). Thecultures maximum was an difference increase we by observedabout 16% between in the H3K4me3 the short- andpeaks long-term in the long cultures-term wasculture. an increaseThis difference by about nearly 16% vanishe in the H3K4me3d when considering peaks in the gene long-term-associated culture. peaks This only. difference Each gene nearlyobtained vanished a 0/1-doublet when consideringcode (X Y) depending gene-associated on whether peaks the only. associated Each gene histones obtained carried a 0/1-doubleta particular codemodification (X Y) depending (1) or not on (0). whether The frequencies the associated of these histones modification carried a states particular were modification (1) or not (0). The frequencies of these modification states were also similar also similar among all the samples (Figure 1B). among all the samples (Figure1B).

FigureFigure 1. 1.Histone Histone ChIP-seqChIP-seq data.data. (A) Bar plots plots of of the the total total number number of of quality quality peaks peaks (left (left: H3K4me3;: H3K4me3; rightright: H3K27me3): H3K27me3) and +/+ −/− andthe the fraction fraction of these of these peaks peaks associated associated with with the the genes genes (colored (colored part parts)s) for for organoids organoids derived derived from from Msh2Msh2+/+ andand Msh2Msh2−/− mice. mice.Short Short-term-term (S) and (S) and long long-term-term (L) (L) organoids organoids are are compared. compared. (B (B) ) Distribution Distribution of of the the modiﬁcationmodification states states (H3K4me3 (H3K4me3 and and H3K27me3)H3K27me3) among among the the genes. genes.

3.2.3.2. Epigenetic Epigenetic Proﬁles Profiles of of Tissue Tissue and and Organoids Organoids Overlap Overlap Considerably Considerably WeWe compared compared these these organoid organoid data data with with those those of irradiatedof irradiated and and untreated untreated intestinal intestinal tis- +/+ −/− suestissues of Msh2 of Msh2and+/+ Msh2and Msh2mice−/− mice [13] to [13] identify to identify culture-speciﬁc culture changes-specific in changes the organoids. in the Fororganoids. this purpose, For this we calculated purpose, wea self-organizing calculated a selfmap-organizing (SOM) including map (SOM) all samples including of both all studiessamples (Figure of both2A). studies Within (Figure the SOM, 2A). genesWithin with the similarSOM, genes regulations with similar in all theregulation sampless in clustered together. In the following, we discuss selected clusters in detail (clusters a–f, Figure2B).

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all the samples clustered together. In the following, we discuss selected clusters in detail (clusters a–f, Figure 2B). The largest cluster (cluster a) comprised 11,627 genes that carried the (10) modification state with high probability in all the samples. The average K4 modification of all genes of the cluster within the individual samples was close to 1. Thus, an extensive overlap existed between the histone profiles of the organoids and tissues. In fact, 96% and 97% of the genes that carried the (10) state in all the organoids and all tissue samples, respectively, were part of cluster a. The GO enrichment analysis shows a high enrichment of the cluster a genes in the gene sets “protein transport”, “DNA repair” and “cell cycle”. Cluster b comprised 502 genes carrying the (01) modification state in all the samples. The Cells 2021, 10, 1718 average K27 modification of all genes within the individual samples, however, 5 only of 15 reached 0.8, indicating a substantial modification variance. These genes wer enriched in the gene sets “ion channel activity” and “G-protein-coupled receptor activity”.

Figure 2. SOM analysis of histone modification states. The analysis integrates the organoid ChIP-seq data presented in this Figure 2. SOM analysis of histone modification states. The analysis integrates the organoid ChIP-seq data presented in thisstudy study and and published published ChIP-seq ChIP-seq data data on on intestinal intestinal tissues tissue [s13 [13].]. (A ()A SOM) SOM portraits portraits of of all all samples samples (C (C == control,control, R = r radiated,adiated, SS = = s short-termhort-term and and L L = llong-termong-term culture). culture). ( (BB)) Gene Gene clusters clusters with with specific specific regulation regulations.s. Small Small letters letters (a (a–f)–f) indicate indicate the the correlationcorrelation clusters clusters discussed discussed in in the the text. text. The The modification modification states states of of the the cluster cluster-associated-associated gene geness in in the the tissue tissue and and organ organoidsoids areare indicated indicated in in parentheses parentheses..

3.3. DifferencesThe largest between cluster the (cluster Intestinala) comprised Tissue and 11,627 Organoid genes Culture that carried the (10) modification state with high probability in all the samples. The average K4 modification of all genes Besides the genes with similar modifications in the tissues and organoids, several of the cluster within the individual samples was close to 1. Thus, an extensive overlap subgroups of genes showed one modification profile in all the tissue samples and a existed between the histone profiles of the organoids and tissues. In fact, 96% and 97% of different one in all the organoid samples. This property refers to the genes of the clusters the genes that carried the (10) state in all the organoids and all tissue samples, respectively, c–f in the SOM (for other profiles, see Figures S1–S3). were part of cluster a. The GO enrichment analysis shows a high enrichment of the cluster Clusters c and d were associated with genes unmodified in the organoid or tissue a genes in the gene sets “protein transport”, “DNA repair” and “cell cycle”. Cluster b samples, respectively. While the genes of cluster c lost H3K4me3 in the organoid culture, comprised 502 genes carrying the (01) modification state in all the samples. The average those of cluster d gained H3K27me3 in the organoid culture (Figure 3A,B). To test whether K27 modification of all genes within the individual samples, however, only reached 0.8, the changes were due to the missing interactions of the epithelial cells with other cell types indicating a substantial modification variance. These genes wer enriched in the gene sets in“ion the channel organoids, activity” we compared and “G-protein-coupled our results with receptorpublished activity”. data on isolated ISCs and adult enterocytes (aECs) [27]. These ChIP-seq data strongly overlapped with the tissue data applied3.3. Differences in the SOM between [13]. the Isolated Intestinal ISCs Tissue and andaECs Organoid showed Culture the histone modification profiles seen Besides in the theorganoids genes with (Figure similar 3C,D). modifications This observation in the tissues provide andd organoids, evidence several for a nonepithelialsubgroups of genes origin showed of the onechanges modification in the histone profile in profiles. all the tissue A prediction samples and based a different on the one in all the organoid samples. This property refers to the genes of the clusters c–f in the SOM (for other profiles, see Figures S1–S3). Clusters c and d were associated with genes unmodified in the organoid or tissue samples, respectively. While the genes of cluster c lost H3K4me3 in the organoid culture,

those of cluster d gained H3K27me3 in the organoid culture (Figure3A,B). To test whether the changes were due to the missing interactions of the epithelial cells with other cell types in the organoids, we compared our results with published data on isolated ISCs and adult enterocytes (aECs) [27]. These ChIP-seq data strongly overlapped with the tissue data applied in the SOM [13]. Isolated ISCs and aECs showed the histone modification profiles seen in the organoids (Figure3C,D). This observation provided evidence for a nonepithelial origin of the changes in the histone profiles. A prediction based on the modification changes is that the genes of both clusters become transcriptionally downregulated in organoids. Cells 2021, 10, x FOR PEER REVIEW 6 of 15

modification changes is that the genes of both clusters become transcriptionally downregulated in organoids. We tested this prediction by performing RNA-seq on the short-term organoid cultures and compared the results with the RNA-seq data on the tissue samples published recently [14]. Most of the genes of cluster c, which lost H3K4me3, indeed became repressed in the organoids (Figure 3E). These genes were strongly enriched in the GO gene sets associated with the “immune response”. Such regulations can be expected in organoid cultures. In contrast, the genes of cluster d, which gained H3K27me3, showed a Cells 2021, 10, 1718 weak tendency for repression in the organoids (Figure 3F). These genes were 6not of 15 enriched in any GO gene set.

+/+ −/− Figure 3. EpigeneticFigure 3. Epigenetic repression repression of the ofcluster the cluster c andc and d dgenes.genes. Samples: Msh2 Msh2+/+: +/+,: +/+,Msh2 Msh2−:/−−: /−/−−;; blackblack letters: letters: tissue, tissue, C = control, R = Cirradiated = control, ,R blue = irradiated, letters: organoids, blue letters: organoids,S = short- Sterm = short-term and L = andlong L-term = long-term culture culture (error: (error: se). ( se).A) Cluster (A) Cluster c comprisesc comprises 401 genes in state (10) in all tissue samples that lose H3K4me3 in all organoid samples (00). The fraction of genes 401 genes in state (10) in all tissue samples that lose H3K4me3 in all organoid samples (00). The fraction of genes associated associated with the modification is shown. (B) Cluster d comprises 102 genes in state (00) in all tissue samples that gain with the modification is shown. (B) Cluster d comprises 102 genes in state (00) in all tissue samples that gain H3K27me3 H3K27me3 in all organoid samples (01). (C,D) In isolated ISCs and aECs (Kazakevych et al., 2017), genes of both clusters in all organoidshow samples the modification (01). (C state,D) ofIn the isolated organoids ISCs (error: and se). aECs (E,F )(Kazakevych The changes in et the al. organoids, 2017), aregenes expected of both to induce clusters gene show the modificationrepression. state of Athe significant organoids change (error: is seen se). for ( clusterE,F) Thec only. changes Box plots in includethe organoids expression are data expected of all genes to of induce the clusters gene in therepression. A significant3 tissuechange and is 3 seen short-term for cluster organoid c samples;only. Box *** plotsp-value include < 0.001 (expressiont-test). data of all genes of the clusters in the 3 tissue and 3 short-term organoid samples; *** p-value < 0.001 (t-test). We tested this prediction by performing RNA-seq on the short-term organoid cul- Thetures genes and comparedassociated the with results the with (11) the state, RNA-seq called data onbivalent the tissue genes, samples are published associated with recently [14]. Most of the genes of cluster c, which lost H3K4me3, indeed became repressed developmentalin the organoids and aging (Figure processes.3E). These Among genes were the strongly genes enrichedcarrying in the the (11) GO state gene setsin the tissue samplesassociated and changing with the “immune their histone response”. modification Such regulations state can in be the expected organoid in organoid cultures, one subgroupcultures. (cluster In contrast, e, Figure the genes 4A) of los clustert H3K4me3d, which in gained the H3K27me3,organoid showed culture a, weakand another subgrouptendency (cluster for repression f, Figure in 4B) the los organoidst H3K27me3. (Figure3 F).These These two genes subgroups were not enriched were enriched in any in the GO gene set. GO gene The sets genes“regulation associated of with ion the transport (11) state,” called and bivalent“morphogenesis genes, are associated of an epithelium with ”, respectively.developmental and aging processes. Among the genes carrying the (11) state in the tissue samples and changing their histone modification state in the organoid cultures, one subgroup (cluster e, Figure4A) lost H3K4me3 in the organoid culture, and another subgroup (cluster f, Figure4B) lost H3K27me3. These two subgroups were enriched in the GO gene sets “regulation of ion transport” and “morphogenesis of an epithelium”, respectively. The genes of cluster e, as those of clusters c and d, showed the same modification profiles in the organoids as in isolated ISCs and aECs, also indicating a nonepithelial origin of these changes (Figure4C). However, the genes of cluster f mainly showed the modification profile of the tissues in these isolated cells, suggesting a culture effect on the epithelium (Figure4D). As for the genes of cluster c, the loss of H3K4me3 in cluster e correlated with transcriptional repression (Figure4E), while a changed H3K27me3 status in cluster f was not linked to transcriptional changes on average (Figure4F). Among the cluster f genes were many regulators of the Wnt pathway (Lgr5, Fosl1, Wnt9a, Wnt10a, Wnt9b, Wnt6 and Fzd2). The transcription of Lgr5, Wnt9b and Fzd2, measured with sufficient reliability by RNA-seq, indicated a profound transcriptional activation (∆log(T): Lgr5 0.98, Wnt9b 0.82 and Fzd2 0.46), as expected for the organoid culture.

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FigureFigure 4. 4.EpigeneticEpigenetic changes changes in in the the bivalent bivalent genes genes of of clus clustersters e eandand f: f(:(AA) Cluster) Cluster e ecomprisescomprises 342 342 genes genes in in state state (11) (11) in in all all thethe tissue tissue samples samples that that lose lose H3K4me3 H3K4me3 in in all all the the organoid organoid samples samples (01). (01). (B ()B Cluster) Cluster f comprisesf comprises 147 147 genes genes in in state state (11) (11) in in all all thethe tissue tissue samples samples that that lose lose H3K27me3 H3K27me3 inin allall thethe organoid organoid samples samples (10). (10). (C (,CD,)D Modification) Modification states states in isolatedin isolated ISCs ISCs and and aECs aECsbased based on the onpublished the published data [data27]. ([27].C) Close (C) Close to 100% to of100% cluster of clustere genes e showgenes the show same the modification same modification state as instate the as organoids. in the organoids.(D) In contrast, (D) In aboutcontrast, 60% about of cluster 60% off genes cluster show f genes the modificationshow the modification state seen state in the seen tissue in samples.the tissue( Esamples.,F) Box plots(E,F) ofBox the plotsRNA-seq of the dataRNA of-seq all spotdata genesof all spot in the genes 3 tissue in the and 3 3tissue short-term and 3 organoidshort-term samples. organoid (E )samples. Cluster e (Egenes) Cluster that e lose genes H3K4me3 that lose are, H3K4me3as expected, are, transcriptionallyas expected, transcriptionally repressed. (F )repressed. The transcription (F) The t ofranscription cluster f genes of cluster is unchanged; f genes is *** unchanged;p-value < 0.001*** p-valu (t-test).e < 0.001 (t-test). 3.4. Epigenetic Activation during the Long-Term Organoid Culture The genes of cluster e, as those of clusters c and d, showed the same modification profilesIn in addition the organoids to the differences as in isolated between ISCs the and intestinal aECs, also tissue indicating and short-term a nonepithelial organoid originculture, of these we also changes observed (Figure changes 4C). that However, manifested the genes during of the cluster long-term f mainly culture show only.ed the Two modificationscenarios of profile epigenetic of the gene tissue activations in these wereisolated most cells, prominent: suggesting (i) a the culture loss ofeffect H3K27me3 on the epitheliumat genes bivalently (Figure 4D). modified As for in the the genes tissue of samples cluster (Figurec, the loss5A, of cluster H3K4me3g) and in (ii) cluster the gain e correlateof H3K4me3d with attranscriptional genes that were repression unmodified (Figure in the4E), tissue while samplesa changed (Figure H3K27me35B, cluster statush ). inThe cluster genes f was associated not linked with to thesetranscriptional regulation changes types clusteredon average in ( smallFigure spots 4F). Among of the SOMthe cluster(Figure f 5genesC,D), we whichre many were identifiedregulators byof anthe under-expression Wnt pathway (Lgr5, spot insteadFosl1, Wnt9a, of a correlation Wnt10a, Wnt9b,cluster Wnt6 analysis and [ 28Fzd2]. The). T observedhe transcription changes of occurred Lgr5, Wnt9b in both andMsh2 Fzd2-deficient, measured and control with sufficientorganoids. reliability Based by on RNA the published-seq, indicate datad [a27 profound], we found transcriptional that (Figure activation S4): (i) the ( geneslog(T): of cluster g and h did not change their modification states during ISC differentiation into Lgr5 0.98, Wnt9b 0.82 and Fzd2 0.46), as expected for the organoid culture. enterocytes. Thus, the observed modification changes could not originate during enterocyte 3.4.(de-)differentiation. Epigenetic Activation (ii) during The average the Long promoter-Term Organoid DNA C methylationulture of cluster g genes was similar to the other bivalent gene sets observed in normal intestinal tissue, while that of theIn cluster additionh genes to the was differences high, as typicallybetween foundthe intestinal for unmodified tissue and genes short that-term recruit organoid DNA culture,methylation we also to observed their promoter changes during that manifest intestinaled development during the long [29-].term In agreement, culture only. the Two CGs scenarioswere enriched of epigenetic in cluster geneg activationbut not in we clusterre mosth genes prominent: (Figure (i) S4). the Theloss genesof H3K27me3 themselves at geneswere bivalently not significantly modified enriched in the intissue the GOsamples sets. (Figure 5A, cluster g) and (ii) the gain of H3K4me3 at genes that were unmodified in the tissue samples (Figure 5B, cluster h). The genes associated with these regulation types clustered in small spots of the SOM (Figure 5C,D), which were identified by an under-expression spot instead of a correlation cluster analysis [28]. The observed changes occurred in both Msh2-deficient and control organoids. Based on the published data [27], we found that (Figure S4): (i) the genes of cluster g and h did not change their modification states during ISC differentiation into enterocytes. Thus, the observed modification changes could not originate during enterocyte (de-)differentiation. (ii) The average promoter DNA methylation of cluster g genes was similar to the other bivalent gene sets observed in normal intestinal tissue, while that of the cluster h genes was high, as typically found for unmodified genes that

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recruit DNA methylation to their promoter during intestinal development [29]. In Cells 2021 10 , , 1718 agreement, the CGs were enriched in cluster g but not in cluster h genes (Figure S4). 8The of 15 genes themselves were not significantly enriched in the GO sets.

FigureFigure 5. 5. EpigeneticEpigenetic activation activation during the long-termlong-term organoidorganoidculture. culture. ( A(A)) The The loss loss of of H3K27me3 H3K27me3 at at stable stable H3K4me3 H3K4me3 in oldin oldorganoids organoids occurs occurs in the in 86the genes 86 genes of cluster of clusterg.(B )g The. (B) gainThe ofgain H3K4me3 of H3K4me3 occurs occurs in the 233in the genes 233 ofgenes cluster of hcluster.(C,D )h Position. (C,D) Positionof the clusters of the gclustersand h in g theand SOM, h in respectively.the SOM, respectively. (E,F) Box plots (E,F of) Box the transcription plots of the oftranscription genes associated of genes with associated clusters g andwithh , clusterrespectively.s g and Theh, respectively. average transcription The average of bothtranscription clusters shows of both no clusters differences shows in no the differences short-term in (S) the culture, short- long-termterm (S) culture culture, +/+ −/− long(L) and-term long-term culture (L) cultures and long supplemented-term culture withs supplemented aspirin (LASP); withMsh2 aspirin+/+: (LASP); +/+ and Msh2Msh2−:/ −+/+: − and/−. Msh2 : −/−.

AspirinAspirin decreased decreased the the tumor frequencyfrequency ininMsh2 Msh2−−/−/− micemice [ [3030]].. In Msh2−−/−/− organoids,organoids, aspirinaspirin reduced reduced the the transient cyst-like cyst-like growth growth [4 [4].]. Based Based on on these these observations, observations, we we hy- hypothesizedpothesized that that aspirin aspirin has thehas potential the potential to suppress to suppress Wnt signaling. Wnt signaling. Thus, we Thus,performed we performedadditional RNA-seq additional analyses RNA-seq of organoids analyses from of organoids the long-term from culture the long supplemented-term culture with supplementedand without aspirin with for and both withoutMsh2 genotypes. aspirin for Comparing both Msh2 the genotypes. expression Comparing in the short- theand expressionlong-term in cultured the short organoids,- and long- weterm found cultured no differences organoids, inwe the found average no differenc transcriptiones in the of averagecluster gtranscriptionand, surprisingly, of cluster cluster g and,h genes surprisingly, (Figure5 E,F).cluster Notably, h genes the (Figure average 5E,F). transcription Notably, theof theaverage clusters transcriptiong and h genes of the was cluster highers g than and that h genes of similarly was higher modiﬁed than genethat setsof similarly (see: the modifiedtranscription gene of sets cluster (see:e, ftheand transcriptiond genes in the of tissue, cluster respectively). e, f and d genes in the tissue, respectively). 3.5. Long-Term Organoid Culture-Related Changes in the Transcription 3.5. LongTo- characterizeTerm Organoid the C transcriptionalulture-Related proﬁlesChanges ofin thethe long-termTranscription cultured organoids in more detail,To wecharacterize calculated the a “RNA transcriptional SOM”, including profiles theof the data long on all-term the cultured organoid organoids samples. Thein morecorrelation detail, spanningwe calculated tree baseda “RNA on SOM this SOM”, including showed the a cleardata separationon all the organoid between samples. the short- Theand correlation long-term organoidsspanning tree (Figure based6A). on One this branch SOM show (groupedG1) a clear contained separation all the between short-term the shortorganoid- and samples. long-term The organoids long-term (Figure organoid 6A). samples One branch formed (group two branches G1) contain (grouped all G2 the and G3). The organoids derived from Msh2+/+ and Msh2−/− mice did not separate into different short-term organoid samples. The long-term organoid samples formed two branches branches, although three out of four G2 samples were derived from Msh2−/− mice. The (group G2 and G3). The organoids derived from Msh2+/+ and Msh2−/− mice did not separate long-term organoid culture supplemented with aspirin tended to keep more similarities into different branches, although three out of four G2 samples were derived from Msh2−/− with the short-term culture organoids. mice. The long-term organoid culture supplemented with aspirin tended to keep more similarities with the short-term culture organoids. The entire area of the RNA SOM was divided into a set of K-means clusters [28] (Figure 6B). We analyzed these clusters in more detail. The long-term organoid culture induced both gene down- and upregulation, most consistently found in the cluster m and n genes, respectively. The cluster m genes were enriched in the GO set “anchored

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component of the membrane”. Among them were several genes that are typically expressed by enterocytes, such as Vnn1 and Dpep1. In addition, the hypoxia-inducible gene Ndufa4l2 [31] that regulates cellular oxygen consumption contributed to this cluster. The cluster n genes were enriched in the GO set “homophilic cell adhesion via plasma membrane adhesion molecules”. Among them was the protocadherin cluster Pcdhb [32].

Cells 2021, 10, 1718 In contrast to cluster m, cluster n comprised genes expressed by secretory lineages9 ofsuch 15 as Clca1, Zg16 and NeuroG3 [33]. Consistent with our results on the average transcription of cluster g and h genes, these genes did not accumulate in cluster n (Figure S5).

FigureFigure 6. 6.Transcriptional Transcriptional proﬁles profiles of of the the organoid organoid samples. samples. (A ()A Correlation) Correlation spanning spanning tree tree based based on on the the SOM SOM analysis analysis of of RNA-seqRNA-seq data data on the on short-the short andlong-term and long organoid-term organoid cultures culture(ASP: aspirin-supplementeds (ASP: aspirin-supplemented cultures). Unsupervised cultures). Unsupervised clustering identiﬁesclustering 3 groupsidentifies of 3 samples. groups of Group samples. 1 (G1, Group blue) 1 (G1, comprises blue) comprises all samples all from samples the short-termfrom the short organoids.-term organoids. Groups 2Groups (G2, green)2 (G2, and green) 3 (G3, and red) 3 (G3, comprise red) comprise samples of samples the long-term of the long organoids-term only.organoids (B) K-means only. (B cluster) K-means of the cluster RNA of SOM. the ClusterRNA SOM.m comprisesCluster m genes comprises that become genes downregulated that become downregulated during culture, while during cluster culture,n genes while become cluster upregulated. n genes become Selected upregulated. genes are indicated.Selected Thegenes clusters are indicated.o and p areThe associated clusters o withand p genes are associated exclusively with upregulated genes exclusively in group 2upregulated and group 3,in respectively. group 2 and Their group genes3, respectively. are enriched Their in the genes GOsets are relatedenriched to “responsein the GO to sets endoplasmic related to reticulum“response stress” to endoplasmic and “canonical reticulum Wnt signaling”. stress” and “canonical Wnt signaling”. Examples are provided for both sets. Genes of other clusters show intermediate regulation. Examples are provided for both sets. Genes of other clusters show intermediate regulation.

3.6.The Maturation entire areaand M ofetabolic the RNA Adaptation SOM was in L dividedong-Term into Organoid a set ofCulture K-means clusters [28] (FigureThe6B). o Webserved analyzed transcriptional these clusters changes in more might detail. be The associated long-term with organoid the culturechanging induceddifferentiation both gene during down- the and long upregulation,-term culture. most Thus, consistently we analyzed found ina gene the cluster set (setm andI: Lgr5n - genes,high, respectively.Figure S6) that The is cluster characteristicm genes for were cells enriched highly inexpressing the GO set the “anchored ISC marker component Lgr5 [34]. ofIn the the membrane”. G2 and Msh2 Among+/+ mouse them-derived were samples several of genes G3, this that set are was typically subjected expressed to activation by enterocytes,not seen in such the G1 as Vnn1 or Msh2and−/−Dpep1. mouseIn-derived addition, samples the hypoxia-inducible of G3 (Figure 7A). gene ThisNdufa4l2 activation[31] thatmay regulates refer to cellularmaturation oxygen of the consumption Lgr5-high contributedphenotype and/or to this cluster.an enrichment The cluster of Lgr5n genes-high werecells. enriched The parallel in the activation GO set “homophilic of marker genes cell adhesion of secretory via plasmacells argue membraned for a maturation adhesion molecules”.(Figure S6). Among A similar them activation was the as protocadherin of the set I genes cluster wasPcdhb seen for[32 ].another, In contrast largely to clusterdisjunct mgene, cluster set (setn comprised II: Fetal Long genes-Term expressed Culture by (FLTC) secretory-high, lineages Figure suchS6) that as Clca1characterize, Zg16 andd the NeuroG3maturation[33]. of Consistent mouse fetal with intestinalour results tissue on the-derived average organoids transcription during of cluster the longg and-termh genes,culture these [35]. genes As did gene not set accumulate II comprise in clusterd manyn (Figure genes S5). encoding P450 enzymes and transmembrane transporters, this showed, in parallel to maturation, a metabolic adaption 3.6. Maturation and Metabolic Adaptation in Long-Term Organoid Culture in the G2 and Msh2+/+ mouse-derived samples of G3. TheWhile observed the G2 transcriptionaland Msh2+/+ mous changese-derived might samples be associated of G3 share withd these the changing properties, differ- what entiationseparate duringd G2 and the G3? long-term The specific culture. activation Thus, in we G2 analyzed referred a to gene the genes set (set associated I: Lgr5-high, with Figure S6) that is characteristic for cells highly expressing the ISC marker Lgr5 [34]. In the cluster o of +/+the RNA SOM. In this cluster, genes of the GO set “response to endoplasmic G2reticulum and Msh2 stressmouse-derived” were enrich samplesed (Figure of G3, 6B). this In set contrast, was subjected the G3 to sample activation show noted seen the Msh2−/− inspecific the G1 or activation ofmouse-derived the genes associated samples of with G3 (Figure cluster 7pA).. Here, This activation genes of may the GO refer set to maturation of the Lgr5-high phenotype and/or an enrichment of Lgr5-high cells. The “canonical Wnt signaling pathway” were enriched. We analyzed the Wnt pathway gene parallel activation of marker genes of secretory cells argued for a maturation (Figure S6). transcriptional profile of G2 and G3 in more detail. While the inspection of these genes A similar activation as of the set I genes was seen for another, largely disjunct gene set (set II: Fetal Long-Term Culture (FLTC)-high, Figure S6) that characterized the maturation of mouse fetal intestinal tissue-derived organoids during the long-term culture [35]. As gene set II comprised many genes encoding P450 enzymes and transmembrane transporters, this showed, in parallel to maturation, a metabolic adaption in the G2 and Msh2+/+ mouse-derived samples of G3. Cells 2021, 10, x FOR PEER REVIEW 10 of 15

unveiled a general trend for activation in the long-term culture (Figure 7B), only the G2 samples showed uniform activation of the Wnt target gene Ccnd1, together with the activators of the canonical Wnt pathways Wnt3 and Sox4 and a strong repression of the Cells 2021, 10, 1718 Wnt suppressor Plpp3 (Figure 7B). In summary, only the G2 samples were characterized10 of 15 by ER stress and Wnt activation.

Figure 7. Evidence for maturation and metabolic changes in the long-term organoid culture. (A) The gene set Z (GSZ)-scores Figurefor Sets 7. IEvidence (Lgr5-high) for andmaturatio II (FLTC-high)n and metabolic separate changes the samples in the from long G1-3-term into organoid three regulatory culture. ( statesA) The (X, gene Y and set Z Z circles (GSZ) are- scoresto guide for theSets eyes). I (Lgr5 States-high) X and II Y are(FLTC occupied-high) byseparate G1 samples the samples from Msh2 from+/+ G1and-3 intoMsh2 three−/− mice,regulatory respectively. states (X, Samples Y and thatZ +/+ −/− circlesactivate are sets to guide I and IIthe during eyes). the States long-term X and culture Y are occupyoccupied state by Z.G1 This samples refers from to the Msh2 G2 samples and Msh2 and Msh2 mice,+/+ mouse-derivedrespectively. Samples that activate sets I and II during the long-term culture occupy state Z. This refers to the G2 samples and Msh2+/+ G3 samples. The G3 samples derived from Msh2−/− mice show no activation and occupy state Y. Brown labels indicate mouse-derived G3 samples. The G3 samples derived from Msh2−/− mice show no activation and occupy state Y. Brown 3 samples not obeying these rules. (B) Heatmap of the transcription of the genes of the canonical Wnt signaling pathway labels indicate 3 samples not obeying these rules. (B) Heatmap of the transcription of the genes of the canonical Wnt signaling(strongest pathway regulated (strongest genes) and regulated its target genes) gene andCcnd1 its. target Activation gene of Ccnd1 the Wnt. Activation pathway of genes the Wnt is seen pathway in the long-termgenes is seen culture in thein long general.-term However, culture in only general. the G2 However, sample showsonly the uniform G2 sample target show genes activation.uniform target gene activation.

+/+ 3.7. EpigeneticallyWhile the G2 Activated and Msh2 Genesmouse-derived Respond to Global samples Transcription of G3 shared these properties, what separated G2 and G3? The specific activation in G2 referred to the genes associated with We found clear differences when comparing the transcription of the epigenetically cluster o of the RNA SOM. In this cluster, genes of the GO set “response to endoplasmic activatedreticulum genes stress” sets were in G2 enriched and G3, (Figure (Figure6B). S5). In contrast, While a thelarge G3 number sample showedof cluster the g specificgenes weactivationre solely ofactivated the genes in associatedG2, the cluster with h cluster genes pwe. Here,re frequently genes of activated the GO set in “canonical G3 but not Wnt in G2.signaling Thus, the pathway” expected were transcriptional enriched. We activation analyzed was the seen Wnt in pathway one of genethe sample transcriptional groups only.profile The of genes G2 andof cluster G3 in f, morein general detail., show Whileed very the diverse inspection transcriptional of these genes changes unveiled in the a longgeneral-term trend culture, for includingactivation those in the contributing long-term culture to the(Figure GO set7B), “morphogenesis only the G2 samples of an epshowedithelium uniform” (Figure activation S7). The largest of the Wntregulated target group, gene Ccnd1 however,, together was activated with the activatorsin G2 only, of similarthe canonical to the cluster Wnt pathways g genes. Wnt3 and Sox4 and a strong repression of the Wnt suppressor Plpp3Notably,(Figure the7B). Wnt In summary, pathway onlygenes the of G2cluster samples f that were lostcharacterized H3K27me3 in by the ER short stress-term and organoidWnt activation. culture only weakly overlapped with the cluster j set of the Wnt pathway genes becoming upregulated in G3. The only common Wnt gene was Lgr5, which was weakly upregulated3.7. Epigenetically during Activated the long- Genesterm culture Respond (Figure to Global S7). Transcription This implicated that Wnt activation in the shortWe found-term clearculture differences was different when from comparing Wnt adaptation the transcription in the long of-term the epigenetically culture. activated genes sets in G2 and G3, (Figure S5). While a large number of cluster g genes 4.were Discussion solely activated in G2, the cluster h genes were frequently activated in G3 but not in G2.The Thus, long the-term expected culture transcriptional of organoids is activation associated was with seen different in one requirements of the sample; among groups themonly. is The thegenes adaptation of cluster to growthf, in general, factors showed and matrices very diverse [36] and transcriptional other environmental changes in changesthe long-term, such as culture, altered includingpartial oxygen those pressure contributing [33]. Moreover, to the GO under set “morphogenesis standard culture ofs an, organoidepithelium” epithelial (Figure cells S7). lack The largestcontact regulated to other cellgroup, types, however, including was activated mesenchymal in G2 and only, immunesimilar tocells the [37], cluster andg togenes. nonpathological microbes that support ISC maintenance [38]. Notably, the Wnt pathway genes of cluster f that lost H3K27me3 in the short-term organoid culture only weakly overlapped with the cluster j set of the Wnt pathway genes becoming upregulated in G3. The only common Wnt gene was Lgr5, which was weakly upregulated during the long-term culture (Figure S7). This implicated that Wnt activation in the short-term culture was different from Wnt adaptation in the long-term culture. Cells 2021, 10, x FOR PEER REVIEW 11 of 15

Cells 2021, 10, 1718 Here, we studied the epigenetic changes in organoids depending on the culture time11 and of 15 the degree of genetic instability, focusing on the H3K4me3 and H3K27me3 modifications. In addition to the expected changes in the short-term culture, organoids of both the Msh24. Discussion genotypes showed similar changes of their epigenomes during the long-term culture. These changes were mostly associated with gene activation, i.e., the affected genes The long-term culture of organoids is associated with different requirements; among showed a gain of H3K4me3 or loss of H3K27me3. them is the adaptation to growth factors and matrices [36] and other environmental Recently, we described the gain of H3K4me3 to H3K27me3-modified promoters as a changes, such as altered partial oxygen pressure [33]. Moreover, under standard cul- stress-related regulation [14]. Nevertheless, H3K4me3 recruitment to unmodified tures, organoid epithelial cells lack contact to other cell types, including mesenchymal and promotersimmune cells was [ 37seen], and under to nonpathologicalthe same settings, microbes although that with support lower ISCfrequency maintenance [13]. Thus, [38]. theHere, regulat we studiedion might the epigenetic represent achanges similar in stress organoids response depending that occurs on the during culture long time-term and culturethe degrees only. of geneticSimilar instability, changes, however, focusing onare the seen H3K4me3 for genes and of H3K27me3an ISC signature modifications. during maturationIn addition of the to fetal the expectedintestine [27]. changes A phase in the diagram short-term of the culture, H3K4me3 organoids and H3K27me3 of both the modificatiMsh2 genotypeson states showed as introduced similar changes by reference of their epigenomes [12] provides during an the explanation long-term for culture. the underlyingThese changes regulation were mostly (Figure associated 8A,B). with For gene strong activation, methylation i.e., the affected activity, genes four showed stable modificationa gain of H3K4me3 states exist: or loss (11), of H3K27me3. (10), (00) and (01). In the data presented, cluster h genes were Recently, originally we described unmodified the andgain of show H3K4me3ed strong to H3K27me3-modified promoter DNA promoters methylation, as characteristica stress-related for regulation state (00). This [14]. state Nevertheless, is globally H3K4me3 stable and, recruitment if attained, tocannot unmodified be left. This pro- becomesmoters was possible seen under following the same DNA settings, demethylation, although with as demonstrated lower frequency for [13 genes]. Thus, of the mentionedregulation might ISC signature represent [27]. a similar Reduced stress DNA response methylation that occurs activity during renderslong-term state cultures (00) instableonly. Similar and changes, can force however, a switch are to seen state for genes (10) (Figure of an ISC 8B, signatureC). For duringhighly maturation expressed, unmodifiedof the fetal intestine genes, DNA [27]. methylation A phase diagram is thereby of the reduced H3K4me3 considerably, and H3K27me3 while transcription modification remainsstates as largely introduced stable. by referenceAlthough [ 12being] provides present an in explanation general, such for thea regulation underlying seems regulation to be more(Figure effective8A,B). Forin G3, strong where methylation cluster h genes activity, become four transcriptionally stable modification activated states exist:compared (11), to(10), the (00)G2 samples. and (01). In the data presented, cluster h genes were originally unmodified andWhile showed DNA strong demethylation promoter DNA activates methylation, the ISC signature, characteristic the gain for stateof H3K27me3 (00). This is state the majoris globally epigenetic stable process and, if that attained, silences cannot developmental be left. This genes becomes [27]. In possible accordance following with DNAthese findings,demethylation, the loss as of demonstrated H3K27me3 from for genesgene promoters of the mentioned and subsequent ISC signature gene activation [27]. Reduced has beenDNA described methylation as a activityfrequent renders event in state tumorigenesis (00) instable [11]. and In can short force-term a switchorganoid to stateculture (10)s, it(Figure is a frequent8B,C). Forevent highly as well. expressed, Here, it unmodifiedis part of the genes, regulation DNA that methylation enabled ISC is thereby expansion re- byduced Wnt considerably, activation while (Lgr5 transcriptionactivation). remainsIn long- largelyterm organoid stable. Although cultures, being it continues, present in somewhatgeneral, such surprisingly, a regulation at seemshighly to transcribed be more effective genes. These in G3, genes where are cluster atypicalh genes H3K27me3 become targets,transcriptionally as this modification activated compared is preferentially to the G2associated samples. with gene silencing [39].

Figure 8. Hypothetical regulation regulation of of the the cluster ff,, g and h genes. ( A,,B)) Phase diagram of the H3K4me3 and H3K27me3 statesstates of of a a single single gene gene as introduced as introduced by reference by reference [12]. The[12]. line The shows line the shows states the that states are consistent that are with consistent histone with modification histone modificationdynamics for highdynamics DNA formethylation high DNA activity. methylation Each state activity. along the Each line state is associated along the with line a specific is associated transcription with a and specific DNA transcriptionmethylation (see:and colorDNA codes methylation in (A,B ),(see: respectively). color codes Only in ( aA few,B), statesrespectively are consistent). Only witha few the states transcriptional are consistent regulation with the of transcriptionalthe gene (circles); regulation among them, of the four gene are (circles); stable fixed among points them (red, four circles). are stable The transitions fixed points between (red circles). the fixed The points transitions due to between the fixed points due to fluctuations are common. For the cluster f and g genes, gene activation by the transcription fluctuations are common. For the cluster f and g genes, gene activation by the transcription factors results in a preference factors results in a preference for the H3K4me3 state (A). The unmodified state is globally stable. Accordingly, in order to for the H3K4me3 state (A). The unmodified state is globally stable. Accordingly, in order to leave it, cluster h genes require leave it, cluster h genes require DNA demethylation, rendering the state unstable (B). (C) Stable fixed points change for theDNA decreasing demethylation, DNA methylation rendering the activity state unstable(red: high; (B ).green: (C) Stable middle; fixed blue: points low changeactivity). for the decreasing DNA methylation activity (red: high; green: middle; blue: low activity).

While DNA demethylation activates the ISC signature, the gain of H3K27me3 is the major epigenetic process that silences developmental genes [27]. In accordance with these

Cells 2021, 10, 1718 12 of 15

findings, the loss of H3K27me3 from gene promoters and subsequent gene activation has been described as a frequent event in tumorigenesis [11]. In short-term organoid cultures, it is a frequent event as well. Here, it is part of the regulation that enabled ISC expansion by Wnt activation (Lgr5 activation). In long-term organoid cultures, it continues, somewhat surprisingly, at highly transcribed genes. These genes are atypical H3K27me3 targets, as this modification is preferentially associated with gene silencing [39]. There are several options to induce H3K27me3 loss, including DNA methylation changes, transcriptional gene activation and accelerated proliferation activity [12]. In short-term organoid cultures, it is most likely an effect of the transcriptional activation of genes by growth factors (switch from state (11) to state (10); Figure8A), e.g., activation of Lgr5 by R-spondin. We expect a loss of H3K27me3 to progress during a long-term culture whenever it is not impeded by maturation. This is most probable for genes originally highly expressed, in agreement with our observations and under conditions that suppress maturation. Our results suggest that the latter occurs in G2. The long-term organoid culture activated a transcriptional profile characteristic for intestinal maturation and metabolic adaptation to culture conditions. These changes were seen in all samples from the Msh2+/+ mice. They were partly suppressed in samples from the Msh2−/− mice. A potential explanation relates to the findings in a colonic monolayer culture [33]. Here, a prolonged culture induced similar changes from a regenerative, fetal- like transcriptional profile towards a mature one. These changes were reversed under hypoxic conditions. In the intestinal organoid culture, hypoxic conditions were induced by cellular oxygen consumption [40]. The intestinal culture from wild-type mice adapted to the hypoxic conditions and increased oxygenation in higher organoid passages [41]. We hypothesize that cells from aged Msh2−/− mice are less capable of culture adaptation. In this case, the hypoxic conditions remained and induced ER stress, as seen in the G2 samples. If this stress was high, it reversed the ongoing maturation, and the regenerative, fetal-like state of the short-term culture stabilized. This was in agreement with the observations in the colonic monolayer culture [33], where induced ER stress indeed reversed maturation. Moreover, a stabilized fetal-like state explained the increased cyst-like organoid growth particular in aged Msh2−/− mouse-derived organoid cultures [4]. There are several findings supporting this hypothesis. Ndufa4l2, which was transcribed in the short-term culture, can suppress oxidative phosphorylation [31]. Thus, its repression in the long-term culture likely increased the oxygen consumption and exacerbated the hypoxia. This explains why ER stress was observed in the G2 but not in the G1 samples. Vnn1 is known to control oxidative stress regulation. Actually, Vnn1 knockout mice have a much higher resistance to oxidative stress than wild-type mice [42]. Thus, the downregulation of Vnn1 in the long-term culture likely represented a growth advantage under the suggested scenario. The long-term culture activated components of the canonical Wnt-signaling pathway. However, pathway activity (in terms of Ccnd1 transcription) was increased in the G2 samples only. This can be explained by our hypothesis as well, as hypoxia can enforce Wnt activity [43]. Finally, hypoxia is known to abrogate Tet activity [44]. Thus, DNA demethylation, required to activate cluster h genes, was suppressed in G2. Our hypothesis has the following implications. The long-term epigenetic changes are a part of, and not the origin of, the observed maturation process. Thereby, gene activation by a gain of H3K4me3 can be considered as a consequence of DNA demethylation activity, known to be a driving mechanism during ISC maturation [27]. This activation was hindered in G2, where hypoxic conditions suppressed DNA demethylation. Under these conditions, gene activation by a loss of H3K27me3 was facilitated. The molecular profiles in the short-term organoid culture, compared to their tissues of origin, have been described in detail [45]. Here, we focused on the epigenetic histone H3 trimethylation changes associated with the long-term organoid culture and their conse- quences in gene transcription, focusing on the RNA-seq data of the gene sets with defined epigenetic regulation. Changes in the gene transcription not associated with these epigenetic gene sets reflected, among other functions, the immediate adaptation to culture Cells 2021, 10, 1718 13 of 15

conditions. These conditions vary in their supplements and matrices and may also im- pact the long-term effects. Therefore, the collagen matrix per se induces a stronger Lgr5 expression compared to Matrigel [46]. An ENR culture supplemented with CHIR99021 and valproic acid showed an even higher Lgr5 expression in the short-term culture [47]. Notably, this enrichment was lost in the long-term culture. The in vitro expansion of stem cells for therapeutic use came into focus more than 20 years ago. However, the risks associated with their rigorous expansion to achieve sufﬁcient cell numbers have been addressed only rarely. The activation of the regenerative potential in organoid cultures is associated with broad transcriptional and epigenetic changes. Our study suggests that long-term cultures are accompanied by maturation and adaptation processes that involve both regulatory layers as well. Prolonged genomic stress can suppress these processes. From our organoid studies, we can conclude that MMR deﬁciency disturbs the capa- bility of intestinal cells to control their metabolism. This potentially explains why profound transcriptional changes are seen in the macroscopic normal tissue of Lynch syndrome patients [48]. Whether such deregulations are associated with the onset of tumor formation requires further studies.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells10071718/s1, Figure S1: Recruitment of H3K4me3 to H3K27me3 targets. Figure S2: Recruitment of H3K4me3 to unmodified genes. Figure S3: Long-term culture-induced recruitment of H3K27me3 to H3K4me3 targets. Figure S4: Properties of gene sets that become epigenetically activated during long-term organoid culture. Figure S5: Genes epigenetically activated in long-term organoid culture show diverse transcriptional behavior. Figure S6: Details on the maturation and adaptation process in long-term organoid culture. Figure S7: Genes epigenetically activated in short-term organoid culture show diverse transcriptional behaviors. Table S1: The primers used for qPCR. Author Contributions: Conceptualization: G.A., J.G.; methodology: M.R.S., G.A., J.G.; formal analysis: T.T., M.H., J.G.; investigation: S.S., M.Q., T.T., M.H., J.G.; resources: M.Q., G.A., S.S., M.R.S.; software: T.T., M.H.; writing article—original draft: J.G. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Federal Ministry of Education and Research (BMBF) grants INDRA (grant number: 031A312 to T.T., M.H., M.Q. and J.G.) and by Volkswagenstiftung (Lichtenberg program; M.R.S.) and the German Research Foundation (DFG) Clinical Research Unit KFO286 (S.S. and M.R.S.) and DFG Collaborative Research Centre SFB1399 (S.S. and M.R.S.) and the Center for Molecular Medicine (CMMC). Institutional Review Board Statement: This research complied with the ethics guidelines of the Leipzig University. For the generation and breeding of transgenic mice we obtained ethics approval from the Landesdirektion Leipzig (protocol code TVV 12/15, date of approval 5 February 2016). Informed Consent Statement: Not applicable. Data Availability Statement: The mouse intestinal RNA-seq [14] and ChIP-seq data [13] can be obtained in the Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo, accessed on 30 Jun 2021) under the super series accession number GSE146520. The organoid ChIP-seq data were deposited in the GEO repository under accession number GSE179237. The RPKM data generated from RNA-seq and the gene cluster a–h can be obtained in the Leipzig Health Atlas (https://www.health-atlas.de, accessed on 5 July 2021) under the ID 87NG1KD2AT-1. Acknowledgments: We thank Ulrike Schmiedek for the technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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