interestingly, the results show that most psc - derived and some fsc - derived organoids contain a large proportion of cells resembling early fetal epithelial cells. this observation is consistent with our previous finding of undifferentiated cells in psc - and fsc - derived organoids. in summary, these data offer an integrated atlas for lung organoids ( hloca ) to complement the heoca for the study of lung 3d cultures at a single tissue level, providing insight into differences in cell - type composition, maturation state and resemblances to primary tissue from multiple stem cell sources. we developed a toolkit to incorporate organoid datasets and compare data with cell states in the integrated heoca ( fig. 5a ). this toolkit ( sc2heoca ) offers functions to compare samples with tissue references and assess ‘ on or off ’ target status and cell state maturation. in addition, it enables sample projection onto the integrated heoca through nearest neighbor analysis and cell annotation through label transfer. the mean expression of the nearest neighbors serves as paired reference cells for differential expression analysis and mean distance to nearest neighbors provides an estimate for the level of difference between sample and reference states. we applied this toolkit to assess organoid protocols, perturbations and disease models ( fig. 5a ). we provide several examples of how the heoca can be used to evaluate single - cell transcriptome datasets from recent organoid protocols ( fig. 5b – e ). first, we validate a finding that modulation of the tnf pathway promotes m cell abundance in intestinal organoids ( extended data fig. 6m ). we generated asc - derived ileal organoids in control media or media supplemented with tnf and receptor activator of nuclear factor - κb ligand ( rankl ), and performed scrna - seq after 6 days of treatment. reference comparison revealed that the majority of cells from both the control and tnf treatment samples ac curate ly matched the intended intestinal tissue cell types ( control, 98. 26 %; tnf, 95. 16 % ) ( extended data fig. 8a ). projection onto the heoca confirmed a notable increase in m cells in the tnf treatment versus control samples, rising from 0 % to 34. 92 %, with corresponding differential expression profiles ( fig. 5b and extended data fig. 8a ). second, we assessed colonic epithelial tissue generated by seeding human colon asc - derived organoids on a scaffolded hydrogel in a fluidic chip ( fig. 5c ). projection analysis demonstrated that this protocol led to colonocyte differentiation and maturation, as indicated by a substantially higher proportion of colonocytes compared with the control samples ( day 4, 20. 45 %; day 14, 54. 03 %; day 21, 66. 97 % ) ( fig. 5c and extended data fig. 8b ). this on - chip protocol offers advantages over conventional organoid protocols by providing access to the apical and basal sides of the epithelium and allowing the culture to be maintained for many weeks while sustaining both stem and differentiated cell types. third, we analyzed two lung datasets consisting of time courses of lung progenitor organoids differentiated into alveol ar or airway organoids ( fig. 5d, e ). in the alveol ar dataset, cells showed increased mapping to alveol ar epithelial identities ( at1 and at2 ) over the course of differentiation ( fig. 5d ). this increase was accompanied by a decrease in cells mapping to undifferentiated identities in the reference atlas ( extended data fig. 8c ). similarly, lung progenitor organoids differentiated toward the airway were ac curate ly mapped to airway - specific cell identities, including scgb3a2 airway progenitors, basal cells and secretory cells, consistent with previous descriptions of these organoids ( fig. 5e and extended data fig. 8d ). notably, these cells were minimally mapped to alveol ar epithelial identities, further validating the accuracy of the reference atlas in disting uishing different lung cell types ( fig. 5d, e ). finally, we incorporated four additional asc - derived intestinal organoid datasets ( two published and two unpublished ) including condition versus control ileum organoids treated with il - 4 and il - 13 and colon organoids treated with il - 22, and time course data of ileum and colon organoids in a medium to promote differentiation. for each dataset we projected to the heoca, annotate d cell types, assessed cell - type proportion, mapped to adult and fetal references, and performed differential expression analysis ( extended data fig. 8e – h ). taken altogether, these data provide a framework for protocol assessment and dataset incorporation into an integrated organoid cell atlas. we next sought to use the heoca as a cohort to assess organoid perturbations. we conducted two perturbation experiments aimed at modeling response to viral infection ( interferon ( ifn ) α, ifnβ and ifnɣ ) and acute pathogenic inflammation ( tnf, oncostatin m ( osm ), ifnɣ, stem cell factor ( scf ), il - 6, il - 17a and il - 18 ) ( fig. 6a ). we treated asc - derived ileum organoids with these cytokines for 24 h and performed scrna - seq on control ( 4, 191 cells ) and treated samples from the same batch ( viral response, 3, 305 cells; inflammation, 2, 158 cells ). heoca projection and annotation revealed diverse cell types including stem cells, enterocytes, goblet cells and enteroendocrine cells ( fig. 6b, c ). distance - to - atlas analysis revealed that, compared with the control sample, both viral response and inflammation samples had higher distances in all cell types ( fig. 6d, e ). differential expression analysis between perturbation samples and the paired nearest neighbor cells in the heoca cohort revealed 618 genes specific to viral response ( for example, isg15, oas1 - 3 ), 259 specific to inflammation ( lcn2, il32, tnfaip2 ), 717 shared ( stat1, wars1 ) and 996 genes upregulated in the atlas ( fig. 6f, g ). gene ontology ( go ) enrichment analysis showed that viral response - specific upregulated genes were enriched in functions related to the defense response to viruses, response to type i ifn and ifnβ, and regulation of autophagy. inflammation - specific differentially expressed genes ( degs ) were associated with the inflammatory responses and cellular responses to chemok ines. genes commonly upregulated in both viral response and inflammation samples were involved in regulating epithelial cell proliferation, chromosome organization, epithelial cell migration, intracellular signal transduction and response to cytokines. by contrast, genes with higher expression in the atlas cohort were enriched in atp biosynthetic processes, messenger rna processing and cellular respiration ( fig. 6h ). we found that degs identified from comparison with the heoca cohort were similar to the set identified through comparison with the isogenic control ( extended data fig. 9a, b ). to assess the biological relevance of the identified states, we compared transcriptomes with counterpart epithelium in an atlas of inflammatory bowel disease ( ibd ) patient samples. interestingly, we found that the perturbation - induced degs were also differentially expressed between healthy individuals and patients with ibd ( fig. 6i, j ). this finding confirms that these perturbations generate organoid cell states not prevalent in the atlas, that the integrated atlas can be used as a diverse cohort for perturbation assessment and that these perturbation states have relevance to primary counterparts. we next assessed the utility of the integrated atlas to understand organoid models of disease. through comparison with the heoca we assess cell proportion, identify disease-associated states and perform differential expression analysis against the atlas data ( fig. 5a ). we first explored colorectal cancer ( crc ) using a dataset compose d of crc organoids from a patient resection and normal organoids from adjacent healthy tissue ( fig. 7a ).