Abstract
Recent development of human three-dimensional organoid cultures has opened new doors and opportunities ranging from modelling human development in vitro to personalised cancer therapies. These new in vitro systems are opening new horizons to the classic understanding of human development and disease. However, the complexity and heterogeneity of these models requires cutting-edge techniques to capture and trace global changes in gene expression to enable identification of key players and uncover the underlying molecular mechanisms. Rapid development of sequencing approaches made possible global transcriptome analyses and epigenetic profiling. Despite challenges in organoid culture and handling, these techniques are now being adapted to embrace organoids derived from a wide range of human tissues. Here, we review current state-of-the-art multi-omics technologies, such as single-cell transcriptomics and chromatin accessibility assays, employed to study organoids as a model for development and a platform for precision medicine.
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Background
The use of animals in biomedical research [1] and in vitro cell cultures led to milestone discoveries and the development of lifesaving treatments [2]. Nonetheless, limitations of animal models have been an Achilles heel in research of human development and disease for decades [3] as multiple processes are human-specific and therefore cannot be completely recapitulated in other animals [4]. Furthermore, conventional 2D cell cultures do not resemble the physiological tissue architecture, which limits the study of complex processes in vitro [5].
The introduction of 3D cultures derived from adult or embryonic stem cells became a breakthrough in biomedical research [6, 7], for example, for liver, pancreas [8], prostate [9] and intestinal [10] tissues. Rapid development of the isolation of adult stem cells from biopsies allowed the establishment of tissue-specific three-dimensional systems, or organoids. These achievements enable modelling of human organ development in a Petri dish including lung [11], skeletal muscle [12], bile duct [13], heart [14], neurone system [15] and hair-bearing skin [16].
Furthermore, organoids can be used to mimic biological processes, such as infection by pathogens with restricted host tropism. For instance, intestinal and gastric organoids have been proposed as a model for Salmonella sp. [17] and H. pylori [18, 19] infection, while lung organoids can be used to recapitulate S. pneumoniae infection of human lungs [20]. Moreover, impact of human immunodeficiency virus [21] and cytomegalovirus virus [22] on central neuronal system has been studied in cerebral organoids, while liver organoids can be used to mimic human liver infection by hepatitis B virus [23]. Remarkably, 3D cultures enabled a study of a simultaneous co-infection with distinct pathogens such as Chlamydia and human papilloma virus in ectocervix organoids [24]. It is noteworthy that organoids of different lineages have been extensively used during the recent COVID-19 pandemic to understand the impact of SARS-CoV-2 on respiratory airways [25], kidney [26] or eyes [27] (Fig. 1).
Organoids for biomedical research. A Organoids can be established from single embryonic, adult stem cells or reprogrammed induced pluripotent stem cells. B Organoid model for multiple tissues and organs have been successfully established in recent years. C–D Organoids are widely used in basic and applied biomedical research
Multiple organoid systems rely on adult stem cells isolated from patients. More recently, the introduction of cell reprogramming [28] by using stemness factors Oct3/4 [29], Sox2 [30], c-Myc [29] or KLF4 [31] allowed the in vitro generation of induced pluripotent stem cells (iPSC) from somatic cells. iPSC soon became an attractive source of stem cells for organoid culture and enabled the generation and expansion of patient-derived organoids to study disorders that previously had no experimental models. The establishment of organoids from patient-derived iPSC opens new opportunities to study Parkinson disease [32] and epilepsy [33], heart chamber defects [34] or rare skin genetic disorders like epidermolysis bullosa [35]. Organoids based on iPSC can also serve as a platform for assessing drug toxicity, for example, in the liver [36], neurons [37] or retina [38].
Tumour organoids represent an attractive platform for personalised medicine as 3D cultures can be generated from patient-derived tumour samples, expanded in vitro and subjected to treatment with a panel of drugs in order to find an optimal therapeutic approach for a specific patient. In fact, Larsen et al. established a new pipeline for drug screening using tumour organoids [39, 40] which undoubtedly has a great potential in clinics as a platform for personalised medicine. Moreover, drug screening followed by phenotypic and multi-omics analyses provides mechanistic insights into tumour biology. For instance, recent studies employed tumour organoids to study origins of oesophageal [41], colorectal [42] and metastatic ovarian cancer [43], while other identified key epigenetic factors leading to drug resistance in colorectal [44] and breast cancer [45]. Furthermore, several studies proposed new treatment based on cancer organoids, for example targeting MEK or mTOR in colorectal cancer [46] or combined inhibition of TRAIL and CDK9 in pancreatic cancer [47].
The advances in organoid culture open the door of possibilities for mechanistical studies in very complex systems involved in multiple pathologies, such as, for example, redox balance [48,49,50,51], COVID-19 [52] or cell death [53, 54]. Therefore, employment of multi-omics poses an excellent opportunity to gain a deep understanding of physiological processes occurring in human organs [55]. Multi-omics allow global genetic, epigenetic, gene expression, and metabolic analyses. For instance, recent pancancer [56, 57] multi-omics studies have unveiled new putative targets for cancer therapy [58, 59]. In this review, we will focus on gene expression and its regulation on organoids as a research model, we will describe recent advances of sequencing techniques, and we will outline future direction in organoid research.
Single-cell transcriptomics of organoids
Using conventional techniques to quantify gene expression, such as RT-PCR, gene array, and bulk RNA sequencing, can only provide limited information and fail to discriminate between distinct cell types present within a sample. Therefore, analysis of complex 3D cultures containing multiple cell types requires state-of-the-art technology such as single-cell transcriptomics.
Single-cell RNA sequencing (scRNA-seq) [60, 61] combines whole transcriptome amplification with next-generation sequencing at single cell level. Currently, various improved platforms have been established, allowing for the analysis of a greater number of cells at significantly lower cost. Current strategies rely on single cell isolation by cell sorting or separation in microdroplets. Then, RNA within isolated single cells is converted into cDNA and barcoded, followed by sequencing. The sequencing data are then processed, normalised, and subject to clustering, which enables identifications of cell types within the sample. Further analyses can provide information about enrichment of molecular pathways, cell cycle state, cell‐cell communication, gene expression kinetics (Fig. 2), as recently described [62]. For instance, one of the pioneering studies in the field of single-cell transcriptomics in organoids assessed distribution of cell types within mouse intestine organoids. Grün et al. identified Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells [63]. In the past seven years, scRNA-seq has been significantly improved and is now widely applied in organoid research. In this chapter, we will provide an overview of four different applications of scRNA-seq in organoid-based research.
Single-cell transcriptomics of organoids. A Single-cell transcriptomics consists of isolation of single cells, followed by lysis, RNA conversion to cDNA by reverse transcription and barcoding. Barcoded cDNA is then used for preparation of cDNA library which can be sequenced and subsequently analysed. B Dimension reduction allows clustering of single cells based on transcriptome profile. Identification of highly expressed marker genes enables cell type annotation. Additional analyses can assess RNA kinetics (pseudotime analysis), cell cycle state, gene expression and pathway enrichment. C Recently, single-cell atlases of human organoid models corresponding different tissues have been established
Comparison of organoids with matched organs and tissues
Primary advantage of 3D culture is its broader specialisation of cells dictated by three-dimensional architecture compared to conventional 2D cultures. In fact, prostate organoids derived from primary prostate cells contain additional intermediate differentiation cell types compared to the same cells grown in a Petri dish [64]. scRNA-seq allowed to compare newly established organoid models with matched liver [65], intestine [66], endometrium [67], epididymis [68], biliary epithelium [69], salivary gland [70], and heart [34] tissues (Fig. 2). Of note, transcriptome analysis at single level allows investigation of complex 3D systems, for instance, in vitro neuromuscular network [71] or bronchioalveolar lung organoids co-cultured with mesenchymal cells [72]. A series of studies underlined high level of resemblance of retinal organoids which can be successfully differentiated into key retinal cell types: retinal pigment epithelium, retinal ganglion cells, cone and rod photoreceptors, and Müller glia [73,74,75]. scRNA-seq has been employed to ensure high degree of similarity between tumour samples and patient-derived organoids to model gastric cancer [76] and glioblastoma [77]. Recently, a biobank of different types of paediatric kidney cancer has been established [78]. This collection, along with normal kidney organoids [79], will become a powerful tool to study kidney homeostasis and cancer in future.
Organoids as a model for human disease
As mentioned in the introduction, organoids became an attractive platform for modelling rare diseases including genetic disorders. Importantly, correction of mutations in organoid cultures, followed by single-cell transcriptome analysis has recently showed promising results. For instance, culture of ear organoids can be used to study role of genetic alterations responsible for the hear loss [80] such as mutations in TMPRSS3 gene [81]. Aberrant transcription of FXN gene causes an autosomal-recessive neurodegenerative and cardiac disorder known as Friedreich's ataxia (FRDA). Experiments aimed to rescue transcription of FXN gene carried out in dorsal root ganglia organoids allowed to fully reverse pathological hallmarks in vitro, as assessed by single-cell transcriptomics [82]. A series of studies focused on establishment of organoid models for neurological diseases such as autism associated with mutation in SUV420H1, ARID1B, CHD8 [83] and CNTNAP2 genes [84] or Prader-Willi syndrome affecting hypothalamic arcuate nucleus [85]. Furthermore, cortical organoids were used to study malformations of human cortical development caused by EML1 gene mutations [86]. Interestingly, single-cell transcriptomics allowed to identify specific depletion of neuronal programming factors in progenitor cells within cerebral organoids derived from patients suffering from Schizophrenia [87]. Organoids bearing specific genetic alteration introduced into DNA of stem cells can be a useful tool to study initial steps of tumorigenesis [88,89,90], opening up new clarification on the underlying molecular mechanisms [91,92,93,94,95]. This approach has been adapted to model progression of colorectal cancer [96], retinoblastoma [97] as well as invasive glioblastoma [98]. Single-cell-based identification of clusters allows to detect molecular pathways behind cancerous transformation. This concept has proven useful to study tissue-specific and cell-type-specific transmission of SARS-CoV-19 infection in eyes [27], intestine [99] and kidney [100] during COVID-19 pandemic.
Developmental studies in vitro using organoids
Refined transcriptome analyses of differentiating organoids made possible investigation of organ and tissue development in vitro. Recent studies provided transcriptional profiling for human embryonic liver [101], glandular epithelia [102], kidney basal membrane [103], mammary gland [104], and mesoderm [105] development. Importantly, Kim and colleagues reported establishment of embryonic body development in vitro followed by temporal single-cell analysis [106].
Development in vitro enables identification of key pathways orchestrating developmental processes. For instance, during intestinal organoids differentiation, BMP signalling controls expression of zonated genes in enterocytes [107] and Exportin 1 expression leads to an increased abundancy of Paneth cells [108], while FGF2 pathway has an essential role in salivary gland development [109]. Moreover, Motazedian et al.showed an important role for RAG1 in development of human T-cells originating from hemogenic endothelium [110]. Detailed analysis of multiple stages of retinal differentiation identified novel role of ATOH7 and Neurog2 in regulation of retinogenesis [111, 112] while temporally controlled overexpression of CCND1 led to promotion of early retinal neurogenesis [113].
Multiple studies have focused on the development of brain using 3D cultures and single-cell transcriptomics. Mouse and human cerebral organoids proved to be an excellent model for brain development in vitro [114]. 3D cultures recapitulate brain development from pluripotency, through neuroectoderm and neuroepithelial stages. Single-cell transcriptomics and pseudotime alignment allow generation of a temporal transcriptome atlas of human brain development at single-cell level [115]. For instance, this approach led to identification of three molecularly distinct subtypes of human dopamine neurons [116] as well as investigation of maturation of cerebral electrophysiologic properties [117]. Of note, inducible cell division labelling enables tracking of cell division and differentiation related pathways [118]. Furthermore, CRISPR-Cas9-based lineage tracing can be used to assess cell fate decisions during cerebral organoid development [119].
Organoids as a platform to study tumorigenesis
As a major gene involved in tumorigenesis as well as in cancer progression, TP53 regulates distinct structures at the level of nuclear envelope [120], N6-methyladenosine methylation profile [121], reticulons [122, 123] and distinct nodes of the tumorigenic network [124,125,126,127,128]. Accordingly, very recent advances on the p53 biology [129,130,131] indicated a significant role for p53 in DNA damage response and apoptotic cell death [132, 133], ferroptosis [134], ribosome biogenesis [135] as well as ncRNA [136,137,138,139]. This complex network is clearly highly relevant for individual treatment [140] to understand the molecular mechanisms at the bases of malignant progression [141, 142] and therefore to identify specific cluster of prognostic markers [143,144,145], hence constituting the scientific bases for precision medicine [146,147,148]. In this framework, organoids play an essential role.
Lung organoids have been used to model early-stage lung adenocarcinoma [149] and to identify differentially expressed genes in alveolar epithelial progenitor cells of patients affected by smoking-associated disease [150]. Organoid model of PDAC allowed identification of new intermediate pancreatic ductal adenocarcinoma (PDAC) transcriptional cell states [151], while co-culture of PDAC organoids with endothelial cells shed light into the role of JAG1 and NOTCH pathways in cancer cell plasticity [152]. Single-cell transcriptomics of patient-derived prostate organoids unveiled presence of heterogeneous populations of prostate epithelial cells characterised by enhanced androgen signalling along with a cluster of tumour-associated club cells that may be linked to prostate carcinogenesis [153]. Analysis of colorectal cancer organoids revealed that enteroendocrine progenitor cells are enriched in BRAF-mutant samples and their differentiative capacities are inhibited [154]. Moreover, a cluster of stem-like cells with high expression of OLFM4 has been identified [155].
Spatial transcriptomics of organoids
A new development of single-cell multi-omics is spatial transcriptomics approach which enables analysis of gene expression in situ within a tissue sample. Technically, spatial transcriptomics can be performed in two ways. In sequencing-based techniques, the position of transcripts is labelled in situ followed by sequencing and subsequential reconstruction of the tissue map of transcription. A tissue section is placed on a slide prelabelled with RNA probes, followed by release of RNA and sequencing. Sequencing approach first reported in 2016 producing sequencing of two-dimensional sequencing map of mouse brain and human breast cancer [156]. On contrast, imaging-based approaches use the amplification of transcripts as well as sequencing directly within tissue followed by imaging. These techniques include ISH-based methods where a complementary fluorescent probe is used to label the transcript. Recent developments have introduced sequential rounds of hybridisation [157], which enable reconstruction of large tissue maps such as an atlas of mouse hypothalamic preoptic region [158] (Fig. 3).
Spatial transcriptomics of organoids. In spatial transcriptomics, a tissue section is positioned on a slide covered with an array of cells containing reagents for a subsequential cell lysis and barcoding. This allows spatial reconstruction of clusters of cells followed by analysis of gene expression
Due to high cost of this technology, it is not widely used on organoids field. However, several recent studies showed promising results. The use of spatial and single-cell transcriptomics highlighted strong similarities between gastruloids and mammalian embryos [159]. These observations were further confirmed by tomo-seq approach which consists of the tissue embedding in cryopreservation medium and consecutive sectioning followed by RNA sequencing [160]. These data establish gastruloids as an attractive model to study development of mammalian embryos, allowing to overcome ethical limitation.
Furthermore, spatial and single-cell transcriptomics were used to assess whether endometrial organoids resemble physiological pathways in vivo. Hormone treatment of organoids resulted in creation of clusters of cells expressing secretory and ciliated populations of cells, confirming that organoids respond very similarly to the in vivo counterpart. A further pseudotime analysis revealed that organoids can be indeed used to determine cell fate decisions. These single cell transcriptomics data can be used to deconvolute bulk sequencing data from samples of endometrial tumours [161].
Of note, Fleck and colleagues developed a new platform which enables characterisation of regional composition of organoids as well as deconvolution of bulk RNA-seq of cortical organoids by using existing atlas of gene expression of developing brain, spatial expression map and accessible chromatin landscape [162]. This platform can help to integrate existing multi-omics datasets with complex organoids studies and can be used in future as a reference to establish new models.
Organoids as a model to study regulation of expression
Epigenetic modifications of chromatin are crucial for regulation of gene expression and are precisely regulated by a complex network of interaction between transcription factors, chromatin remodellers [163] and even non-coding RNAs [57, 164, 165]. Identification of genomic regulatory elements and study of epigenetic state of chromatin historically has been achieved through analyse of DNA occupancy by distinct transcription factors (TFs) and histone modifications. A chromatin immunoprecipitation followed by sequencing (ChIP-seq) assay allows identification of specific regions occupied by a transcription factor genome-wide (Fig. 4).
Epigenomics of organoids. (Top) RNA-seq, ChIP-seq and ATAC-seq techniques allow analysis of gene expression and its regulation at epigenetic level. Actively transcribed genes are characterised by a higher accessibility of regulatory elements (promoters and enhancers) and binding of specific transcription factors. (Bottom) Organoids have been used to study genome-wide occupancy of several transcription factors
Despite ChIP-seq being widely used in 2D cultures, there are only a handful of studies involving organoids as it remains technically challenging [166]. For instance, mouse prostate organoids were used to unveil the role of different FOXA1 mutations found in prostate cancer patients. Analysis of FOXA1 genome occupancy uncovered new genomic regions bound exclusively by mutant FOXA1, which alter normal program of wild-type FOXA1 and thus its role in luminal differentiation [167]. Another study comprehensively assessed the impact of distinct isoforms of the bile acid receptor (FXR) on transcription of genes involved in bile acid, fat, sugar, and amino acid metabolism in mouse liver organoids as a model. Only two isoforms, FXRα2 or FXRα4, were found to bind FXR loci, primarily by occupying ER2-motif containing regions [168] (Fig. 4).
ChIP-seq analysis of specific histone modifications allows the identification of open (active) or inactive chromatin regions genome-wide [169]. Among commonly used approaches are ChIP-seq for methylated lysine K4 and acetylated lysine K27 of histone H3 which are markers of active transcription [170]. As an alternative to mapping chromatin state by ChIP, multiple assays have been developed based on DNA accessibility for different enzymes which is correlated with chromatin state. For instance, approaches involving deoxyribonuclease (DNase) or micrococcal nuclease (MNase) have been used for long time to assess chromatin accessibility [171, 172]. DNase and MNase cleave DNA regions which are not protected by nucleosomes or occupied by TFs. The introduction of Tn5-transposase revolutionised the field. A new assay for transposase- accessible chromatin assay (ATAC) is based on identification of nucleosome-free regions which are simultaneously fragmented and labelled for further sequencing by pre-loaded transposase enzyme. The new technology allows to overcome previous obstacles, reducing the cost and requiring lower amount of material [173].
As discussed in the previous chapter, global analysis of chromatin accessibility is used to assess resemblance of organoids to original tissue at epigenetic level and thus reliability as a research model. For instance, ChIP-seq analysis of H3K4 and H3K27 methylation in mouse intestinal organoids revealed that long-term culture leads to global transcriptional changes via accumulation of H3K4me3 and loss of H3K27me3 [174]. Moreover, multi-epigenomics allow comparison of organoid culture with conventional cell cultures, such as Caco2 colorectal carcinoma cell lines. Combined RNA-seq and ATAC-seq carried out in human intestinal organoids allowed to identify transcriptional and open chromatin signatures governed by transcription factor caudal type homeobox 2 (CDX2), which are specific for organoids but not Caco2 cells grown in a Petri dish [175].
Study of chromatin accessibility in cancer organoids is becoming a new powerful tool for understanding the molecular mechanisms of tumorigenesis and development of precision medicine. For instance, using ethanol-treated colon organoids as model of alcohol-induced damage, Devall and colleagues integrated RNA-seq with ATAC-seq and identified new differentially accessibly regions of chromatin in colon organoids upon treatment with ethanol. Importantly, activation of these response factors was not found in 2D cultures, underlining the importance of three-dimensional growth conditions in recapitulating physiological environment [176]. Additionally, human colon organoids were used to study the role of vitamin D on colon homeostasis. Transcriptomics combined with ATAC-seq allowed identification of regions with increased accessibility containing VDR binding motif [177]. ATAC-seq has been proposed as a prognostic platform to profile chromatin accessibility in pancreatic cancer samples. Preliminary data revealed a subset of differentially accessible regions based on patient’s’ survival [178]. In depth analysis using ATAC-seq and H3K27ac ChIP-seq complemented by transcriptomics, led to identification of MNX1-HNF1B transcriptional axis specifically upregulated in pancreatic cancer organoids. Activation of this pathway regulated the expression of key genes responsible for maintenance of stemness of gastrointestinal cells including MYC, SOX9, and OLFM4. Moreover, high-throughput chromosome conformation capture demonstrated that expression of identified target genes was supported by specific three-dimensional chromatin architecture [179]. Interestingly, when comparing chromatin accessibility changes in colorectal cancer organoids treated with oxaliplatin, fibroblast growth factor receptor 1 (FGFR1) and oxytocin receptor (OXTR) were identified among upregulated genes, however these results were observed only in a subset of patients, highlighting complex heterogeneity of epigenetic and transcriptional response to cancer treatment [180]. Similarly, in prostate cancer organoids ATAC-seq integrated with transcriptomics allowed to identify new cancer subtypes such as stem cell–like (SCL) subtype. Interestingly, in SCL tumour cells AP-1 complex interacts with the YAP/TAZ and TEAD proteins to allow subtype-specific chromatin accessibility [181].
Single-cell epigenomics in organoids for developmental research
Chromatin accessibility assays performed in organoids have been recently applied to developmental research. For instance, transcriptional and chromatin accessibility dynamics of human medial ganglionic eminence and generated cortex-specific organoids from human pluripotent stem allowed to confirm that proposed model can be used as a platform for generating domain-specific brain organoids to study development [182].
Furter breakthroughs are expected, as Kanton et al. have recently established a protocol for simultaneous RNA-seq and ATAC-seq at single-cell level in human cerebral organoids. This refined technology allows sequential transposase-based labelling of DNA and reverse transcription in isolated single nuclei [183]. Combined single-cell multi-omics can allow an in-depth analysis of cell-type specific features during organ development [184]. A recent study used combined single-cell RNA and ATAC sequencing in the developing and adult human retina and in retinal organoids derived from induced pluripotent stem cells. This comprehensive analysis revealed existence of cell type specific cis-regulatory elements (CREs) [185].
Conclusions
The establishment of organoids as a research model is still in its early stages; still it is already evident their contribution to clarify the highly complex network of events leading to disease progression. As we have outlined in this review, distinct sequencing approaches have been recently employed to compare organoids with conventional cell lines and tissues. Multiple studies have successfully proved that three-dimensional cultures can in fact recapitulate tissue architecture with high degree of fidelity. These efforts have been complemented by technical optimization of omics techniques and adaptation of standard protocols to specific settings dictated by organoid growth conditions. Undoubtedly, next milestone in organoid research will be a broader, well-established application of multi-omics for basic and translational research. For instance, further improvement of spatial transcriptomics will reveal new insights into development of human organs, previously impossible due to the lack of an appropriate model. Combination of chromatin accessibility assays with chromatin conformation capture will uncover complex spatiotemporal architecture of chromatin in tissue-specific manner. Improvements of ChIP-seq technology will bring study of transcription factors to a new a level allowing to globally analyse binding profiles within virtually physiological conditions (Fig. 5). Furthermore, transcriptional analyses can be complimented by emerging single-cell proteomics to get a wider picture of gene and protein expression. Finally, application of omics to patient-derived organoids will enable prediction of treatment response for malignancies such as cancer or neurogenerative disorders, a step forward towards life-saving precision medicine.
Future directions. Future developments will enable a comprehensive analysis of gene expression and chromatin state, including three-dimensional chromatin architecture, in organoids
References
Russo I, Sartor E, Fagotto L, Colombo A, Tiso N, Alaibac M. The Zebrafish model in dermatology: an update for clinicians. Discov Oncol. 2022;13(1):48.
Jota Baptista CV, Faustino-Rocha AI, Oliveira PA. Animal models in pharmacology: a brief history awarding the nobel prizes for physiology or medicine. Pharmacology. 2021;106(7–8):356–68.
Van Norman GA. Limitations of animal studies for predicting toxicity in clinical trials: part 2: potential alternatives to the use of animals in preclinical trials. JACC Basic Transl Sci. 2020;5(4):387–97.
Doncheva NT, Palasca O, Yarani R, Litman T, Anthon C, Groenen MAM, et al. Human pathways in animal models: possibilities and limitations. Nucleic Acids Res. 2021;49(4):1859–71.
Kapalczynska M, Kolenda T, Przybyla W, Zajaczkowska M, Teresiak A, Filas V, et al. 2D and 3D cell cultures—a comparison of different types of cancer cell cultures. Arch Med Sci. 2018;14(4):910–9.
Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell. 2008;3(5):519–32.
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262–5.
Broutier L, Andersson-Rolf A, Hindley CJ, Boj SF, Clevers H, Koo BK, et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc. 2016;11(9):1724–43.
Drost J, Karthaus WR, Gao D, Driehuis E, Sawyers CL, Chen Y, et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat Protoc. 2016;11(2):347–58.
Sprangers J, Zaalberg IC, Maurice MM. Organoid-based modeling of intestinal development, regeneration, and repair. Cell Death Differ. 2021;28(1):95–107.
Hein RFC, Wu JH, Holloway EM, Frum T, Conchola AS, Tsai YH, et al. R-SPONDIN2(+) mesenchymal cells form the bud tip progenitor niche during human lung development. Dev Cell. 2022;129:4831.
Shin MK, Bang JS, Lee JE, Tran HD, Park G, Lee DR, et al. Generation of skeletal muscle organoids from human pluripotent stem cells to model Myogenesis and muscle regeneration. Int J Mol Sci. 2022. https://doi.org/10.3390/ijms23095108.
Roos FJM, van Tienderen GS, Wu H, Bordeu I, Vinke D, Albarinos LM, et al. Human branching cholangiocyte organoids recapitulate functional bile duct formation. Cell Stem Cell. 2022;29(5):776-94 e13.
Lewis-Israeli YR, Wasserman AH, Gabalski MA, Volmert BD, Ming Y, Ball KA, et al. Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease. Nat Commun. 2021;12(1):5142.
Rosebrock D, Arora S, Mutukula N, Volkman R, Gralinska E, Balaskas A, et al. Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells. Nat Cell Biol. 2022;24(6):981–95.
Lee J, Rabbani CC, Gao H, Steinhart MR, Woodruff BM, Pflum ZE, et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature. 2020;582(7812):399–404.
Boyle EC, Wunschel EJ, Grassl GA. Salmonella enterica infection of human and mouse colon organoid-derived monolayers. Methods Mol Biol. 2022;2427:149–63.
Boccellato F, Woelffling S, Imai-Matsushima A, Sanchez G, Goosmann C, Schmid M, et al. Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection. Gut. 2019;68(3):400–13.
Buti L, Ruiz-Puig C, Sangberg D, Leissing TM, Brewer RC, Owen RP, et al. CagA-ASPP2 complex mediates loss of cell polarity and favors H. pylori colonization of human gastric organoids. Proc Natl Acad Sci U S A. 2020;117(5):2645–55.
Sempere J, Rossi SA, Chamorro-Herrero I, Gonzalez-Camacho F, de Lucas MP, Rojas-Cabaneros JM, et al. Minilungs from Human Embryonic Stem Cells to Study the Interaction of Streptococcus pneumoniae with the Respiratory Tract. Microbiol Spectr. 2022:e0045322.
Gumbs SBH, Berdenis van Berlekom A, Kubler R, Schipper PJ, Gharu L, Boks MP, et al. Characterization of HIV-1 infection in microglia-containing human cerebral organoids. Viruses. 2022. https://doi.org/10.3390/v14040829.
O’Brien BS, Mokry RL, Schumacher ML, Pulakanti K, Rao S, Terhune SS, et al. Downregulation of neurodevelopmental gene expression in iPSC-derived cerebral organoids upon infection by human cytomegalovirus. iScience. 2022;25(4): 104098.
Romal S, Hossain T, Mahmoudi T. Generation, maintenance and HBV infection of human liver organoids. Bio Protoc. 2022;12(6): e4358.
Koster S, Gurumurthy RK, Kumar N, Prakash PG, Dhanraj J, Bayer S, et al. Modelling chlamydia and HPV co-infection in patient-derived ectocervix organoids reveals distinct cellular reprogramming. Nat Commun. 2022;13(1):1030.
Sano E, Suzuki T, Hashimoto R, Itoh Y, Sakamoto A, Sakai Y, et al. Cell response analysis in SARS-CoV-2 infected bronchial organoids. Commun Biol. 2022;5(1):516.
Garreta E, Prado P, Stanifer ML, Monteil V, Marco A, Ullate-Agote A, et al. A diabetic milieu increases ACE2 expression and cellular susceptibility to SARS-CoV-2 infections in human kidney organoids and patient cells. Cell Metab. 2022;34(6):857-73 e9.
Eriksen AZ, Moller R, Makovoz B, tenOever BR, Blenkinsop TA. Protocols for SARS-CoV-2 infection in primary ocular cells and eye organoids. STAR Protoc. 2022;3(2): 101383.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.
Prieto J, Garcia-Canaveras JC, Leon M, Sendra R, Ponsoda X, Izpisua Belmonte JC, et al. c-MYC triggers lipid remodelling during early somatic cell reprogramming to pluripotency. Stem Cell Rev Rep. 2021;17(6):2245–61.
Schaefer T, Lengerke C. SOX2 protein biochemistry in stemness, reprogramming, and cancer: the PI3K/AKT/SOX2 axis and beyond. Oncogene. 2020;39(2):278–92.
Panatta E, Lena AM, Mancini M, Affinati M, Smirnov A, Annicchiarico-Petruzzelli M, et al. Kruppel-like factor 4 regulates keratinocyte senescence. Biochem Biophys Res Commun. 2018;499(2):389–95.
Bolognin S, Fossepre M, Qing X, Jarazo J, Scancar J, Moreno EL, et al. 3D cultures of Parkinson’s disease-specific dopaminergic neurons for high content phenotyping and drug testing. Adv Sci (Weinh). 2019;6(1):1800927.
Samarasinghe RA, Miranda OA, Buth JE, Mitchell S, Ferando I, Watanabe M, et al. Identification of neural oscillations and epileptiform changes in human brain organoids. Nat Neurosci. 2021;24(10):1488–500.
Feng W, Schriever H, Jiang S, Bais A, Wu H, Kostka D, et al. Computational profiling of hiPSC-derived heart organoids reveals chamber defects associated with NKX2-5 deficiency. Commun Biol. 2022;5(1):399.
Ramovs V, Janssen H, Fuentes I, Pitaval A, Rachidi W, de Sousa Lopes SMC, et al. Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids. Stem Cell Rep. 2022;17(6):1279–88.
Kim H, Im I, Jeon JS, Kang EH, Lee HA, Jo S, et al. Development of human pluripotent stem cell-derived hepatic organoids as an alternative model for drug safety assessment. Biomaterials. 2022;286: 121575.
Pamies D, Wiersma D, Katt ME, Zhao L, Burtscher J, Harris G, et al. Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity. Neurobiol Dis. 2022;169: 105719.
Dorgau B, Georgiou M, Chaudhary A, Moya-Molina M, Collin J, Queen R, et al. Human retinal organoids provide a suitable tool for toxicological investigations: a comprehensive validation using drugs and compounds affecting the retina. Stem Cells Transl Med. 2022;11(2):159–77.
Larsen BM, Kannan M, Langer LF, Leibowitz BD, Bentaieb A, Cancino A, et al. A pan-cancer organoid platform for precision medicine. Cell Rep. 2021;36(4): 109429.
Larsen BM, Cancino A, Shaxted JM, Salahudeen AA. Protocol for drug screening of patient-derived tumor organoids using high-content fluorescent imaging. STAR Protoc. 2022;3(2): 101407.
Scott SJ, Li X, Jammula S, Devonshire G, Lindon C, Fitzgerald RC, et al. Evidence that polyploidy in esophageal adenocarcinoma originates from mitotic slippage caused by defective chromosome attachments. Cell Death Differ. 2021;28(7):2179–93.
Ramesh P, Lannagan TRM, Jackstadt R, Atencia Taboada L, Lansu N, Wirapati P, et al. BCL-XL is crucial for progression through the adenoma-to-carcinoma sequence of colorectal cancer. Cell Death Differ. 2021;28(12):3282–96.
Velletri T, Villa CE, Cilli D, Barzaghi B, Lo Riso P, Lupia M, et al. Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer. Cell Death Differ. 2022;29(3):614–26.
Toshimitsu K, Takano A, Fujii M, Togasaki K, Matano M, Takahashi S, et al. Organoid screening reveals epigenetic vulnerabilities in human colorectal cancer. Nat Chem Biol. 2022;18(6):605–14.
Tsai KK, Huang SS, Northey JJ, Liao WY, Hsu CC, Cheng LH, et al. Screening of organoids derived from patients with breast cancer implicates the repressor NCOR2 in cytotoxic stress response and antitumor immunity. Nat Cancer. 2022. https://doi.org/10.1038/s43018-022-00375-0.
Betge J, Rindtorff N, Sauer J, Rauscher B, Dingert C, Gaitantzi H, et al. The drug-induced phenotypic landscape of colorectal cancer organoids. Nat Commun. 2022;13(1):3135.
Montinaro A, Areso Zubiaur I, Saggau J, Kretz AL, Ferreira RMM, Hassan O, et al. Potent pro-apoptotic combination therapy is highly effective in a broad range of cancers. Cell Death Differ. 2022;29(3):492–503.
Favaloro B, Tamburro A, Angelucci S, Luca AD, Melino S, di Ilio C, et al. Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi. Biochem J. 1998;335:573–9.
Angelucci S, Sacchetta P, Moio P, Melino S, Petruzzelli R, Gervasi P, et al. Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver. Arch Biochem Biophys. 2000;373(2):435–41.
Xiong X, Hasani S, Young LEA, Rivas DR, Skaggs AT, Martinez R, et al. Activation of Drp1 promotes fatty acids-induced metabolic reprograming to potentiate Wnt signaling in colon cancer. Cell Death Differ. 2022;29(10):1913–27.
Ilacqua N, Anastasia I, Aloshyn D, Ghandehari-Alavijeh R, Peluso EA, Brearley-Sholto MC, et al. Expression of Synj2bp in mouse liver regulates the extent of wrappER-mitochondria contact to maintain hepatic lipid homeostasis. Biol Direct. 2022;17(1):37.
Verkhratsky A, Li Q, Melino S, Melino G, Shi Y. Can COVID-19 pandemic boost the epidemic of neurodegenerative diseases? Biol Direct. 2020;15(1):28.
Melino G, Knight RA, Nicotera P. How many ways to die? How many different models of cell death? Cell Death Differ. 2005;12(Suppl 2):1457–62.
Muller F, Lim JKM, Bebber CM, Seidel E, Tishina S, Dahlhaus A, et al. Elevated FSP1 protects KRAS-mutated cells from ferroptosis during tumor initiation. Cell Death Differ. 2022. https://doi.org/10.1038/s41418-022-01096-8.
Michaletti A, Mancini M, Smirnov A, Candi E, Melino G, Zolla L. Multi-omics profiling of calcium-induced human keratinocytes differentiation reveals modulation of unfolded protein response signaling pathways. Cell Cycle. 2019;18(17):2124–40.
Cancer Genome Atlas Research N, Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA, et al. The cancer genome atlas pan-cancer analysis project. Nat Genet. 2013;45(10):1113–20.
Ma J, Jin Y, Gong B, Li L, Zhao Q. Pan-cancer analysis of necroptosis-related gene signature for the identification of prognosis and immune significance. Discov Oncol. 2022;13(1):17.
Liang W, Mo C, Wei J, Chen W, Gong W, Shi J, et al. FAM65A as a novel prognostic biomarker in human tumors reveal by a pan-cancer analysis. Discov Oncol. 2021;12(1):60.
Jahangiri L, Pucci P, Ishola T, Pereira J, Cavanagh ML, Turner SD. Deep analysis of neuroblastoma core regulatory circuitries using online databases and integrated bioinformatics shows their pan-cancer roles as prognostic predictors. Discov Oncol. 2021;12(1):56.
Lao KQ, Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, et al. mRNA-sequencing whole transcriptome analysis of a single cell on the SOLiD system. J Biomol Tech. 2009;20(5):266–71.
Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009;6(5):377–82.
Jovic D, Liang X, Zeng H, Lin L, Xu F, Luo Y. Single-cell RNA sequencing technologies and applications: a brief overview. Clin Transl Med. 2022;12(3): e694.
Grun D, Lyubimova A, Kester L, Wiebrands K, Basak O, Sasaki N, et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature. 2015;525(7568):251–5.
McCray T, Moline D, Baumann B, Vander Griend DJ, Nonn L. Single-cell RNA-Seq analysis identifies a putative epithelial stem cell population in human primary prostate cells in monolayer and organoid culture conditions. Am J Clin Exp Urol. 2019;7(3):123–38.
Peng WC, Logan CY, Fish M, Anbarchian T, Aguisanda F, Alvarez-Varela A, et al. Inflammatory cytokine TNFalpha promotes the long-term expansion of primary hepatocytes in 3D culture. Cell. 2018;175(6):1607-19 e15.
Fujii M, Matano M, Toshimitsu K, Takano A, Mikami Y, Nishikori S, et al. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell. 2018;23(6):787-93 e6.
Fitzgerald HC, Dhakal P, Behura SK, Schust DJ, Spencer TE. Self-renewing endometrial epithelial organoids of the human uterus. Proc Natl Acad Sci U S A. 2019;116(46):23132–42.
Leir SH, Yin S, Kerschner JL, Xia S, Ahmadi S, Bear C, et al. An organoid model to assay the role of CFTR in the human epididymis epithelium. Cell Tissue Res. 2020;381(2):327–36.
Sampaziotis F, Muraro D, Tysoe OC, Sawiak S, Beach TE, Godfrey EM, et al. Cholangiocyte organoids can repair bile ducts after transplantation in the human liver. Science. 2021;371(6531):839–46.
Yoon YJ, Kim D, Tak KY, Hwang S, Kim J, Sim NS, et al. Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glands. Nat Commun. 2022;13(1):3291.
Faustino Martins JM, Fischer C, Urzi A, Vidal R, Kunz S, Ruffault PL, et al. Self-organizing 3D human trunk neuromuscular organoids. Cell Stem Cell. 2020;27(3):498.
Vazquez-Armendariz AI, Heiner M, El Agha E, Salwig I, Hoek A, Hessler MC, et al. Multilineage murine stem cells generate complex organoids to model distal lung development and disease. EMBO J. 2020;39(21): e103476.
Collin J, Queen R, Zerti D, Dorgau B, Hussain R, Coxhead J, et al. Deconstructing retinal organoids: single cell RNA-seq reveals the cellular components of human pluripotent stem cell-derived retina. Stem Cells. 2019;37(5):593–8.
Cowan CS, Renner M, De Gennaro M, Gross-Scherf B, Goldblum D, Hou Y, et al. Cell types of the human retina and its organoids at single-cell resolution. Cell. 2020;182(6):1623-40 e34.
Kim S, Lowe A, Dharmat R, Lee S, Owen LA, Wang J, et al. Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids. Proc Natl Acad Sci U S A. 2019;116(22):10824–33.
Kumar V, Ramnarayanan K, Sundar R, Padmanabhan N, Srivastava S, Koiwa M, et al. Single-cell atlas of lineage states, tumor microenvironment, and subtype-specific expression programs in gastric cancer. Cancer Discov. 2022;12(3):670–91.
LeBlanc VG, Trinh DL, Aslanpour S, Hughes M, Livingstone D, Jin D, et al. Single-cell landscapes of primary glioblastomas and matched explants and cell lines show variable retention of inter- and intratumor heterogeneity. Cancer Cell. 2022;40(4):379-92 e9.
Calandrini C, Schutgens F, Oka R, Margaritis T, Candelli T, Mathijsen L, et al. An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity. Nat Commun. 2020;11(1):1310.
Shankar AS, Du Z, Mora HT, van den Bosch TPP, Korevaar SS, Van den Berg-Garrelds IM, et al. Human kidney organoids produce functional renin. Kidney Int. 2021;99(1):134–47.
Kubota M, Scheibinger M, Jan TA, Heller S. Greater epithelial ridge cells are the principal organoid-forming progenitors of the mouse cochlea. Cell Rep. 2021;34(3): 108646.
Tang PC, Alex AL, Nie J, Lee J, Roth AA, Booth KT, et al. Defective Tmprss3-associated hair cell degeneration in inner ear organoids. Stem Cell Rep. 2019;13(1):147–62.
Mazzara PG, Muggeo S, Luoni M, Massimino L, Zaghi M, Valverde PT, et al. Frataxin gene editing rescues Friedreich’s ataxia pathology in dorsal root ganglia organoid-derived sensory neurons. Nat Commun. 2020;11(1):4178.
Paulsen B, Velasco S, Kedaigle AJ, Pigoni M, Quadrato G, Deo AJ, et al. Autism genes converge on asynchronous development of shared neuron classes. Nature. 2022;602(7896):268–73.
de Jong JO, Llapashtica C, Genestine M, Strauss K, Provenzano F, Sun Y, et al. Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder. Nat Commun. 2021;12(1):4087.
Huang WK, Wong SZH, Pather SR, Nguyen PTT, Zhang F, Zhang DY, et al. Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells. Cell Stem Cell. 2021;28(9):1657-70 e10.
Jabali A, Hoffrichter A, Uzquiano A, Marsoner F, Wilkens R, Siekmann M, et al. Human cerebral organoids reveal progenitor pathology in EML1-linked cortical malformation. EMBO Rep. 2022;23(5): e54027.
Notaras M, Lodhi A, Dundar F, Collier P, Sayles NM, Tilgner H, et al. Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids. Mol Psychiatry. 2022;27(3):1416–34.
Kato T Jr, Muotri AR. Mapping the hotspots for DNA repair synthesis in human brain organoids. Cell Death Differ. 2021;28(11):3193–5.
Priami C, Montariello D, De Michele G, Ruscitto F, Polazzi A, Ronzoni S, et al. Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells. Cell Death Differ. 2022;29(12):2429–44.
Aqeilan RI. Engineering organoids: a promising platform to understand biology and treat diseases. Cell Death Differ. 2021;28(1):1–4.
Candi E, Cipollone R, Rivetti di Val Cervo P, Gonfloni S, Melino G, Knight R. p63 in epithelial development. Cell Mol Life Sci. 2008;65(20):3126–33.
Strubel A, Munick P, Chaikuad A, Dreier B, Schaefer J, Gebel J, et al. Designed Ankyrin repeat proteins as a tool box for analyzing p63. Cell Death Differ. 2022;29(12):2445–58.
Gallo M, Paludi D, Cicero DO, Chiovitti K, Millo E, Salis A, et al. Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation. Int J Immunopathol Pharmacol. 2005;18(1):95–112.
Bizen N, Bepari AK, Zhou L, Abe M, Sakimura K, Ono K, et al. Ddx20, an Olig2 binding factor, governs the survival of neural and oligodendrocyte progenitor cells via proper Mdm2 splicing and p53 suppression. Cell Death Differ. 2022;29(5):1028–41.
Lampis A, Hahne JC, Gasparini P, Cascione L, Hedayat S, Vlachogiannis G, et al. MIR21-induced loss of junctional adhesion molecule A promotes activation of oncogenic pathways, progression and metastasis in colorectal cancer. Cell Death Differ. 2021;28(10):2970–82.
Yan HHN, Siu HC, Ho SL, Yue SSK, Gao Y, Tsui WY, et al. Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles. Gut. 2020;69(12):2165–79.
Liu H, Zhang Y, Zhang YY, Li YP, Hua ZQ, Zhang CJ, et al. Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin. Proc Natl Acad Sci U S A. 2020;117(52):33628–38.
Krieger TG, Tirier SM, Park J, Jechow K, Eisemann T, Peterziel H, et al. Modeling glioblastoma invasion using human brain organoids and single-cell transcriptomics. Neuro Oncol. 2020;22(8):1138–49.
Triana S, Metz-Zumaran C, Ramirez C, Kee C, Doldan P, Shahraz M, et al. Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut. Mol Syst Biol. 2021;17(4): e10232.
Jansen J, Reimer KC, Nagai JS, Varghese FS, Overheul GJ, de Beer M, et al. SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids. Cell Stem Cell. 2022;29(2):217-31 e8.
Guan Y, Chen X, Wu M, Zhu W, Arslan A, Takeda S, et al. The phosphatidylethanolamine biosynthesis pathway provides a new target for cancer chemotherapy. J Hepatol. 2020;72(4):746–60.
Centonze A, Lin S, Tika E, Sifrim A, Fioramonti M, Malfait M, et al. Heterotypic cell-cell communication regulates glandular stem cell multipotency. Nature. 2020;584(7822):608–13.
Morais M, Tian P, Lawless C, Murtuza-Baker S, Hopkinson L, Woods S, et al. Kidney organoids recapitulate human basement membrane assembly in health and disease. Elife. 2022. https://doi.org/10.7554/eLife.73486.
Gutierrez G, Sun P, Han Y, Dai X. Defining mammary basal cell transcriptional states using single-cell RNA-sequencing. Sci Rep. 2022;12(1):4893.
Budjan C, Liu S, Ranga A, Gayen S, Pourquie O, Hormoz S. Paraxial mesoderm organoids model development of human somites. Elife. 2022. https://doi.org/10.7554/eLife.68925.
Kim IS, Wu J, Rahme GJ, Battaglia S, Dixit A, Gaskell E, et al. Parallel single-Cell RNA-seq and genetic recording reveals lineage decisions in developing embryoid bodies. Cell Rep. 2020;33(1): 108222.
Beumer J, Puschhof J, Yengej FY, Zhao L, Martinez-Silgado A, Blotenburg M, et al. BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states. Cell Rep. 2022;38(9): 110438.
Mead BE, Hattori K, Levy L, Imada S, Goto N, Vukovic M, et al. Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation. Nat Biomed Eng. 2022;6(4):476–94.
Moskwa N, Mahmood A, Nelson DA, Altrieth AL, Forni PE, Larsen M. Single-cell RNA sequencing reveals PDFGRalpha+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids. Development. 2022. https://doi.org/10.1242/dev.200167.
Motazedian A, Bruveris FF, Kumar SV, Schiesser JV, Chen T, Ng ES, et al. Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids. Nat Cell Biol. 2020;22(1):60–73.
Lu Y, Shiau F, Yi W, Lu S, Wu Q, Pearson JD, et al. Single-cell analysis of human retina identifies evolutionarily conserved and species-specific mechanisms controlling development. Dev Cell. 2020;53(4):473-91 e9.
Zhang X, Mandric I, Nguyen KH, Nguyen TTT, Pellegrini M, Grove JCR, et al. Single cell transcriptomic analyses reveal the impact of bHLH factors on human retinal organoid development. Front Cell Dev Biol. 2021;9: 653305.
Mao X, An Q, Xi H, Yang XJ, Zhang X, Yuan S, et al. Single-Cell RNA sequencing of hESC-derived 3D retinal organoids reveals novel genes regulating RPC commitment in early human retinogenesis. Stem Cell Rep. 2019;13(4):747–60.
Velasco S, Kedaigle AJ, Simmons SK, Nash A, Rocha M, Quadrato G, et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature. 2019;570(7762):523–7.
Kanton S, Boyle MJ, He Z, Santel M, Weigert A, Sanchis-Calleja F, et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature. 2019;574(7778):418–22.
Fiorenzano A, Sozzi E, Birtele M, Kajtez J, Giacomoni J, Nilsson F, et al. Author Correction: Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids. Nat Commun. 2022;13(1):3312.
Fair SR, Julian D, Hartlaub AM, Pusuluri ST, Malik G, Summerfied TL, et al. Electrophysiological maturation of cerebral organoids correlates with dynamic morphological and cellular development. Stem Cell Rep. 2020;15(4):855–68.
Denoth-Lippuner A, Jaeger BN, Liang T, Royall LN, Chie SE, Buthey K, et al. Visualization of individual cell division history in complex tissues using iCOUNT. Cell Stem Cell. 2021;28(11):2020-34 e12.
He Z, Maynard A, Jain A, Gerber T, Petri R, Lin HC, et al. Lineage recording in human cerebral organoids. Nat Methods. 2022;19(1):90–9.
Panatta E, Butera A, Celardo I, Leist M, Melino G, Amelio I. p53 regulates expression of nuclear envelope components in cancer cells. Biol Direct. 2022;17(1):38.
Peng T, Liu M, Hu L, Guo D, Wang D, Qi B, et al. LncRNA Airn alleviates diabetic cardiac fibrosis by inhibiting activation of cardiac fibroblasts via a m6A-IMP2-p53 axis. Biol Direct. 2022;17(1):32.
Fazi B, Melino S, De Rubeis S, Bagni C, Paci M, Piacentini M, et al. Acetylation of RTN-1C regulates the induction of ER stress by the inhibition of HDAC activity in neuroectodermal tumors. Oncogene. 2009;28(43):3814–24.
Melino S, Nepravishta R, Bellomaria A, Di Marco S, Paci M. Nucleic acid binding of the RTN1-C C-terminal region: toward the functional role of a reticulon protein. Biochemistry. 2009;48(2):242–53.
Rozenberg JM, Zvereva S, Dalina A, Blatov I, Zubarev I, Luppov D, et al. The p53 family member p73 in the regulation of cell stress response. Biol Direct. 2021;16(1):23.
Panatta E, Zampieri C, Melino G, Amelio I. Understanding p53 tumour suppressor network. Biol Direct. 2021;16(1):14.
Thomas AF, Kelly GL, Strasser A. Of the many cellular responses activated by TP53, which ones are critical for tumour suppression? Cell Death Differ. 2022;29(5):961–71.
Hoyos D, Greenbaum B, Levine AJ. The genotypes and phenotypes of missense mutations in the proline domain of the p53 protein. Cell Death Differ. 2022;29(5):938–45.
Mammarella E, Zampieri C, Panatta E, Melino G, Amelio I. NUAK2 and RCan2 participate in the p53 mutant pro-tumorigenic network. Biol Direct. 2021;16(1):11.
Levine AJ. Exploring the future of research in the Tp53 field. Cell Death Differ. 2022;29(5):893–4.
Kennedy MC, Lowe SW. Mutant p53: it’s not all one and the same. Cell Death Differ. 2022;29(5):983–7.
de Andrade KC, Lee EE, Tookmanian EM, Kesserwan CA, Manfredi JJ, Hatton JN, et al. The TP53 database: transition from the international agency for research on cancer to the US national cancer institute. Cell Death Differ. 2022;29(5):1071–3.
El-Saafin F, Bergamasco MI, Chen Y, May RE, Esakky P, Hediyeh-Zadeh S, et al. Loss of TAF8 causes TFIID dysfunction and p53-mediated apoptotic neuronal cell death. Cell Death Differ. 2022;29(5):1013–27.
Smirnov A, Cappello A, Lena AM, Anemona L, Mauriello A, Di Daniele N, et al. ZNF185 is a p53 target gene following DNA damage. Aging. 2018;10(11):3308–26.
Liu Y, Gu W. p53 in ferroptosis regulation: the new weapon for the old guardian. Cell Death Differ. 2022;29(5):895–910.
Lindstrom MS, Bartek J, Maya-Mendoza A. p53 at the crossroad of DNA replication and ribosome biogenesis stress pathways. Cell Death Differ. 2022;29(5):972–82.
Yuan J, Zhu Q, Zhang X, Wen Z, Zhang G, Li N, et al. Ezh2 competes with p53 to license lncRNA Neat1 transcription for inflammasome activation. Cell Death Differ. 2022;29(10):2009–23.
Agostini M, Mancini M, Candi E. Long non-coding RNAs affecting cell metabolism in cancer. Biol Direct. 2022;17(1):26.
Liang J, Li G, Liao J, Huang Z, Wen J, Wang Y, et al. Non-coding small nucleolar RNA SNORD17 promotes the progression of hepatocellular carcinoma through a positive feedback loop upon p53 inactivation. Cell Death Differ. 2022;29(5):988–1003.
Lu Y, Meng Q, Bai L, Wang R, Sun Y, Li J, et al. LINC00858 stabilizes RAN expression and promotes metastasis of gastric cancer. Biol Direct. 2022;17(1):41.
Wang Z, Strasser A, Kelly GL. Should mutant TP53 be targeted for cancer therapy? Cell Death Differ. 2022;29(5):911–20.
Butera A, Roy M, Zampieri C, Mammarella E, Panatta E, Melino G, et al. p53-driven lipidome influences non-cell-autonomous lysophospholipids in pancreatic cancer. Biol Direct. 2022;17(1):6.
Mukherji R, Yin C, Hameed R, Alqahtani AZ, Kulasekaran M, He AR, et al. The current state of molecular profiling in gastrointestinal malignancies. Biol Direct. 2022;17(1):15.
Wang D, Liufu J, Yang Q, Dai S, Wang J, Xie B. Identification and validation of a novel signature as a diagnostic and prognostic biomarker in colorectal cancer. Biol Direct. 2022;17(1):29.
Lin C, Wang Y, Dong Y, Lai S, Wang L, Weng S, et al. N6-methyladenosine-mediated SH3BP5-AS1 upregulation promotes GEM chemoresistance in pancreatic cancer by activating the Wnt signaling pathway. Biol Direct. 2022;17(1):33.
Zhuo C, Ruan Q, Zhao X, Shen Y, Lin R. CXCL1 promotes colon cancer progression through activation of NF-kappaB/P300 signaling pathway. Biol Direct. 2022;17(1):34.
Amelio I, Bertolo R, Bove P, Candi E, Chiocchi M, Cipriani C, et al. Cancer predictive studies. Biol Direct. 2020;15(1):18.
Amelio I, Bertolo R, Bove P, Buonomo OC, Candi E, Chiocchi M, et al. Liquid biopsies and cancer omics. Cell Death Discov. 2020;6(1):131.
Ganini C, Amelio I, Bertolo R, Bove P, Buonomo OC, Candi E, et al. Global mapping of cancers: the cancer genome atlas and beyond. Mol Oncol. 2021;15(11):2823–40.
Dost AFM, Moye AL, Vedaie M, Tran LM, Fung E, Heinze D, et al. Organoids model transcriptional hallmarks of oncogenic KRAS activation in lung epithelial progenitor cells. Cell Stem Cell. 2020;27(4):663-78 e8.
Wu X, Bos IST, Conlon TM, Ansari M, Verschut V, van der Koog L, et al. A transcriptomics-guided drug target discovery strategy identifies receptor ligands for lung regeneration. Sci Adv. 2022;8(12):eabj9949.
Raghavan S, Winter PS, Navia AW, Williams HL, DenAdel A, Lowder KE, et al. Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer. Cell. 2021;184(25):6119-37 e26.
Choi JI, Rim JH, Jang SI, Park JS, Park H, Cho JH, et al. The role of Jagged1 as a dynamic switch of cancer cell plasticity in PDAC assembloids. Theranostics. 2022;12(9):4431–45.
Song H, Weinstein HNW, Allegakoen P, Wadsworth MH 2nd, Xie J, Yang H, et al. Single-cell analysis of human primary prostate cancer reveals the heterogeneity of tumor-associated epithelial cell states. Nat Commun. 2022;13(1):141.
Miller SA, Policastro RA, Sriramkumar S, Lai T, Huntington TD, Ladaika CA, et al. LSD1 and aberrant DNA methylation mediate persistence of enteroendocrine progenitors that support BRAF-mutant colorectal cancer. Cancer Res. 2021;81(14):3791–805.
Okamoto T, duVerle D, Yaginuma K, Natsume Y, Yamanaka H, Kusama D, et al. Comparative analysis of patient-matched PDOs revealed a reduction in OLFM4-associated clusters in metastatic lesions in colorectal cancer. Stem Cell Rep. 2021;16(4):954–67.
Stahl PL, Salmen F, Vickovic S, Lundmark A, Navarro JF, Magnusson J, et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 2016;353(6294):78–82.
Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science. 2015;348(6233):aaa6090.
Moffitt JR, Bambah-Mukku D, Eichhorn SW, Vaughn E, Shekhar K, Perez JD, et al. Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region. Science. 2018;362(6416):eaau5324.
van den Brink SC, Alemany A, van Batenburg V, Moris N, Blotenburg M, Vivie J, et al. Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature. 2020;582(7812):405–9.
Moris N, Anlas K, van den Brink SC, Alemany A, Schroder J, Ghimire S, et al. An in vitro model of early anteroposterior organization during human development. Nature. 2020;582(7812):410–5.
Garcia-Alonso L, Handfield LF, Roberts K, Nikolakopoulou K, Fernando RC, Gardner L, et al. Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro. Nat Genet. 2021;53(12):1698–711.
Fleck JS, Sanchis-Calleja F, He Z, Santel M, Boyle MJ, Camp JG, et al. Resolving organoid brain region identities by mapping single-cell genomic data to reference atlases. Cell Stem Cell. 2021;28(6):1148-59 e8.
Park J, Lee K, Kim K, Yi SJ. The role of histone modifications: from neurodevelopment to neurodiseases. Signal Transduct Target Ther. 2022;7(1):217.
Mancini M, Cappello A, Pecorari R, Lena AM, Montanaro M, Fania L, et al. Involvement of transcribed lncRNA uc.291 and SWI/SNF complex in cutaneous squamous cell carcinoma. Discov Oncol. 2021;12(1):14.
Panatta E, Lena AM, Mancini M, Smirnov A, Marini A, Delli Ponti R, et al. Long non-coding RNA uc.291 controls epithelial differentiation by interfering with the ACTL6A/BAF complex. EMBO Rep. 2020;21(3):e46734.
Tan WX, Bok CM, Ng NHJ, Teo AKK. Chromatin immunoprecipitation in human pluripotent stem cell-derived 3D organoids to analyze DNA-protein interactions. Methods Mol Biol. 2022;2429:215–32.
Adams EJ, Karthaus WR, Hoover E, Liu D, Gruet A, Zhang Z, et al. FOXA1 mutations alter pioneering activity, differentiation and prostate cancer phenotypes. Nature. 2019;571(7765):408–12.
Ramos Pittol JM, Milona A, Morris I, Willemsen ECL, van der Veen SW, Kalkhoven E, et al. FXR isoforms control different metabolic functions in liver cells via binding to specific DNA motifs. Gastroenterology. 2020;159(5):1853-65 e10.
Ma S, Zhang Y. Profiling chromatin regulatory landscape: insights into the development of ChIP-seq and ATAC-seq. Mol Biomed. 2020;1(1):9.
Beacon TH, Delcuve GP, Lopez C, Nardocci G, Kovalchuk I, van Wijnen AJ, et al. The dynamic broad epigenetic (H3K4me3, H3K27ac) domain as a mark of essential genes. Clin Epigenetics. 2021;13(1):138.
Chen A, Chen D, Chen Y. Advances of DNase-seq for mapping active gene regulatory elements across the genome in animals. Gene. 2018;667:83–94.
Deng WH, Li XH. Resolving nucleosomal positioning and occupancy with MNase-seq. Yi Chuan. 2020;42(12):1143–55.
Grandi FC, Modi H, Kampman L, Corces MR. Chromatin accessibility profiling by ATAC-seq. Nat Protoc. 2022;17(6):1518–52.
Thalheim T, Siebert S, Quaas M, Herberg M, Schweiger MR, Aust G, et al. Epigenetic drifts during long-term intestinal organoid culture. Cells. 2021. https://doi.org/10.3390/cells10071718.
Yin S, Ray G, Kerschner JL, Hao S, Perez A, Drumm ML, et al. Functional genomics analysis of human colon organoids identifies key transcription factors. Physiol Genomics. 2020;52(6):234–44.
Devall M, Jennelle LT, Bryant J, Bien S, Peters U, Powell S, et al. Modeling the effect of prolonged ethanol exposure on global gene expression and chromatin accessibility in normal 3D colon organoids. PLoS ONE. 2020;15(1): e0227116.
Li J, Witonsky D, Sprague E, Alleyne D, Bielski MC, Lawrence KM, et al. Genomic and epigenomic active vitamin D responses in human colonic organoids. Physiol Genomics. 2021;53(6):235–48.
Dhara S, Chhangawala S, Chintalapudi H, Askan G, Aveson V, Massa AL, et al. Pancreatic cancer prognosis is predicted by an ATAC-array technology for assessing chromatin accessibility. Nat Commun. 2021;12(1):3044.
Kato H, Tateishi K, Fujiwara H, Nakatsuka T, Yamamoto K, Kudo Y, et al. MNX1-HNF1B axis is indispensable for intraductal papillary mucinous neoplasm lineages. Gastroenterology. 2022;162(4):1272-87 e16.
Tung KL, Chen KY, Negrete M, Chen T, Safi A, Aljamal AA, et al. Integrated chromatin and transcriptomic profiling of patient-derived colon cancer organoids identifies personalized drug targets to overcome oxaliplatin resistance. Genes Dis. 2021;8(2):203–14.
Tang F, Xu D, Wang S, Wong CK, Martinez-Fundichely A, Lee CJ, et al. Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets. Science. 2022;376(6596):eabe1505.
Xiang Y, Tanaka Y, Patterson B, Kang YJ, Govindaiah G, Roselaar N, et al. Fusion of regionally specified hPSC-derived organoids models human brain development and interneuron migration. Cell Stem Cell. 2017;21(3):383-98 e7.
Chen S, Lake BB, Zhang K. High-throughput sequencing of the transcriptome and chromatin accessibility in the same cell. Nat Biotechnol. 2019;37(12):1452–7.
Kanton S, Treutlein B, Camp JG. Single-cell genomic analysis of human cerebral organoids. Methods Cell Biol. 2020;159:229–56.
Thomas ED, Timms AE, Giles S, Harkins-Perry S, Lyu P, Hoang T, et al. Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids. Dev Cell. 2022;57(6):820-36 e6.
Funding
This work has been supported by the PNRR-PE6 Heal Italia by the Ministry of University, by Associazione Italiana per la Ricerca contro il Cancro (AIRC) to EC (IG#22206; 2019–2023) and Ministry of Health IDI-IRCCS (RC to EC). AS was supported by REACT-EU PON "Ricerca e Innovazione 2014–2020" (DM 1062/2021).
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Smirnov, A., Melino, G. & Candi, E. Gene expression in organoids: an expanding horizon. Biol Direct 18, 11 (2023). https://doi.org/10.1186/s13062-023-00360-2
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DOI: https://doi.org/10.1186/s13062-023-00360-2