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Single-Cell and Spatial Analysis of Emergent Organoid Platforms

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Cancer Systems and Integrative Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2660))

Abstract

Organoids have emerged as a promising advancement of the two-dimensional (2D) culture systems to improve studies in organogenesis, drug discovery, precision medicine, and regenerative medicine applications. Organoids can self-organize as three-dimensional (3D) tissues derived from stem cells and patient tissues to resemble organs. This chapter presents growth strategies, molecular screening methods, and emerging issues of the organoid platforms. Single-cell and spatial analysis resolve organoid heterogeneity to obtain information about the structural and molecular cellular states. Culture media diversity and varying lab-to-lab practices have resulted in organoid-to-organoid variability in morphology and cell compositions. An essential resource is an organoid atlas that can catalog protocols and standardize data analysis for different organoid types. Molecular profiling of individual cells in organoids and data organization of the organoid landscape will impact biomedical applications from basic science to translational use.

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References

  1. Clevers H (2016) Modeling development and disease with organoids. Cell 165(7):1586–1597

    Article  CAS  PubMed  Google Scholar 

  2. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6(3):331–343

    Article  CAS  PubMed  Google Scholar 

  3. Muthuswamy SK (2017) Bringing together the organoid field: from early beginnings to the road ahead. Development 144:963–967

    Article  CAS  PubMed  Google Scholar 

  4. Rossi G, Manfrin A, Lutolf MP (2018) Progress and potential in organoid research. Nat Rev Genet 19(11):671–687

    Google Scholar 

  5. Petersen OW, Rønnov-Jessen L, Howlett AR, Bissell MJ (1992) Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A 89(19):9064–9068

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Eiraku M, Takata N, Ishibashi H et al (2011) Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472:51–56

    Article  CAS  PubMed  Google Scholar 

  7. Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265

    Article  CAS  PubMed  Google Scholar 

  8. Betge J, Rindtorff N, Sauer J et al (2019) Multiparametric phenotyping of compound effects on patient derived organoids. bioRxiv. https://doi.org/10.1101/660993

  9. Driehuis E, Kretzschmar K, Clevers H (2020) Establishment of patient-derived cancer organoids for drug-screening applications. Nat Protoc 15(10):3380–3409

    Article  CAS  PubMed  Google Scholar 

  10. Ooft SN, Weeber F, Dijkstra KK et al (2019) Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients. Sci Transl Med 11. https://doi.org/10.1126/scitranslmed.aay2574

  11. Chen J, Lau BT, Andor N et al (2019) Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model. Sci Rep 9(1):4536

    Article  PubMed  PubMed Central  Google Scholar 

  12. Cattaneo CM, Dijkstra KK, Fanchi LF et al (2020) Tumor organoid-T-cell coculture systems. Nat Protoc 15(1):15–39

    Article  CAS  PubMed  Google Scholar 

  13. Nozaki K, Mochizuki W, Matsumoto Y et al (2016) Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes. J Gastroenterol 51(3):206–213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kim J, Koo BK, Knoblich JA (2020) Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol 21:571–584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Urbischek M, Rannikmae H, Foets T et al (2019) Organoid culture media formulated with growth factors of defined cellular activity. Sci Rep 9:6193

    Article  PubMed  PubMed Central  Google Scholar 

  16. Karzbrun E, Reiner O (2019) Brain organoids-A bottom-up approach for studying human neurodevelopment. Bioengineering (Basel) 6(1):9

    Article  CAS  PubMed  Google Scholar 

  17. Qin X, Sufi J, Vlckova P et al (2020) Cell-type-specific signaling networks in heterocellular organoids. Nat Methods 17(3):335–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lancaster MA, Huch M (2019) Disease modelling in human organoids. Dis Model Mech 12(7):dmm039347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Camp JG, Treutlein B (2017) Human organomics: a fresh approach to understanding human development using single-cell transcriptomics. Development 144:1584–1587

    Google Scholar 

  20. Fiorini E, Veghini L, Corbo V (2020) Modeling cell communication in cancer with organoids: making the complex simple. Front Cell Dev Biol 8. https://doi.org/10.3389/fcell.2020.00166

  21. Huch M, Koo BK (2015) Modeling mouse and human development using organoid cultures. Development 142(18):3113–3125

    Article  CAS  PubMed  Google Scholar 

  22. de Souza N (2018) Organoids. Nat Methods 15:23

    Article  Google Scholar 

  23. Drost J, Clevers H (2017) Translational applications of adult stem cell-derived organoids. Development 144(6):968–975

    Article  CAS  PubMed  Google Scholar 

  24. Fujii M, Sato T (2021) Somatic cell-derived organoids as prototypes of human epithelial tissues and diseases. Nat Mater 20:156–169

    Article  CAS  PubMed  Google Scholar 

  25. Qian X, Nguyen HN, Song MM et al (2016) Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165(5):1238–1254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chugh RM, Bhanja P, Norris A, Saha S (2021) Experimental models to study COVID-19 effect in stem cells. Cell 10:91. https://doi.org/10.3390/cells10010091

    Article  CAS  Google Scholar 

  27. Quiroz EJ, Ryan AL (Firth) (2019) In: Stem Cell-Based Ther Lung Dis (Eds: JK Burgess, IH Heijink), Springer International Publishing, Cham, pp. 153–178

    Google Scholar 

  28. Longmire TA, Ikonomou L, Hawkins F et al (2012) Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10(4):398–411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Liang P, Lan F, Lee AS et al (2013) Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity. Circulation 127(16):1677–1691

    Article  CAS  PubMed  Google Scholar 

  30. Bartfeld S, Clevers H (2017) Stem cell-derived organoids and their application for medical research and patient treatment. J Mol Med (Berl) 95(7):729–738

    Article  CAS  PubMed  Google Scholar 

  31. Naglea PW, Plukkerc JTM, Muijsb CT, van Luijkb P, Coppes RP (2018) Patient-derived tumor organoids for prediction of cancer treatment response. Semin Cancer Biol 53:258–264

    Article  Google Scholar 

  32. Jabs J, Zickgraf FM, Park J et al (2017) Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations. Mol Syst Biol 13(11):955

    Article  PubMed  PubMed Central  Google Scholar 

  33. Costales-Carrera A, Fernández-Barral A, Bustamante-Madrid P et al (2020) Comparative study of organoids from patient-derived normal and tumor colon and rectal tissue. Cancers (Basel) 12(8):2302

    Article  CAS  PubMed  Google Scholar 

  34. Driehuis E, Spelier S, Beltrán Hernández I et al (2019) Patient-derived head and neck cancer organoids recapitulate EGFR expression levels of respective tissues and are responsive to EGFR-targeted photodynamic therapy. J Clin Med 8:1880

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Eiraku M, Watanabe K, Matsuo-Takasaki M et al (2008) Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3(5):519–532

    Article  CAS  PubMed  Google Scholar 

  36. Nakano T, Ando S, Takata N et al (2012) Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10(6):771–785

    Article  CAS  PubMed  Google Scholar 

  37. Sinagoga KL, Schumacher M, Rockich BE et al (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516(7531):400–404

    Article  PubMed  PubMed Central  Google Scholar 

  38. Wong AP, Bear CE, Chin S et al (2012) Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol 30(9):876–882

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Takebe T, Sekine K, Enomura M et al (2013) Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499(7459):481–484

    Article  CAS  PubMed  Google Scholar 

  40. Takasato M, Er PX, Becroft M et al (2014) Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 16(1):118–126

    Article  CAS  PubMed  Google Scholar 

  41. Takasato M, Er PX, Chiu HS et al (2015) Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526(7574):564–568

    Article  CAS  PubMed  Google Scholar 

  42. Maimets M, Rocchi C, Bron R et al (2016) Long-term in vitro expansion of salivary gland stem cells driven by Wnt signals. Stem Cell Reports 6(1):150–162

    Article  CAS  PubMed  Google Scholar 

  43. Octavian SA, Pei WY (2018) Organoids as reliable breast cancer study models: an update. Int J Oncol Res 1. https://doi.org/10.23937/ijor-2017/1710008

  44. Gleave AM, Ci X, Lin D, Wang Y (2020) A synopsis of prostate organoid methodologies, applications, and limitations. Prostate 80(6):518–526

    Article  PubMed  Google Scholar 

  45. Wallach TE, Bayrer JR (2017) Intestinal organoids: new frontiers in the study of intestinal disease and physiology. J Pediatr Gastroenterol Nutr 64(2):180–185

    Article  PubMed  PubMed Central  Google Scholar 

  46. Giese C, Lubitz A, Demmler CD et al (2010) Immunological substance testing on human lymphatic micro-organoids in vitro. J Biotechnol 148(1):38–45

    Article  CAS  PubMed  Google Scholar 

  47. Lancaster MA, Renner M, Martin CA et al (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467):373–379

    Article  CAS  PubMed  Google Scholar 

  48. Dedhia PH, Bertaux-Skeirik N, Zavros Y et al (2016) Organoid models of human gastrointestinal development and disease. Gastroenterology 150(5):1098–1112

    Article  PubMed  Google Scholar 

  49. Duval K, Grover H, Han LH et al (2017) Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda) 32(4):266–277

    CAS  PubMed  Google Scholar 

  50. Yin X, Mead BE, Safaee H et al (2016) Engineering stem cell organoids. Cell Stem Cell 18(1):25–38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Shah SB, Singh A (2017) Cellular self-assembly and biomaterials-based organoid models of development and diseases. Acta Biomater 53:29–45

    Article  CAS  PubMed  Google Scholar 

  52. Pham MT, Pollock KM, Rose MD et al (2018) Generation of human vascularized brain organoids. Neuroreport 29(7):588–593

    Article  PubMed  PubMed Central  Google Scholar 

  53. Bagley JA, Reumann D, Bian S, Lévi-Strauss J, Knoblich JA (2017) Fused cerebral organoids model interactions between brain regions. Nat Methods 14(7):743–751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Wang Y, Wang L, Zhu Y, Qin J (2018) Human brain organoid-on-a-chip to model prenatal nicotine exposure. Lab Chip 18:851

    Article  CAS  PubMed  Google Scholar 

  55. Czerniecki SM, Cruz NM, Harder JL et al (2018) High-throughput screening enhances kidney organoid differentiation from human pluripotent stem cells and enables automated multidimensional phenotyping. Cell Stem Cell 22(6):929–940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lancaster MA, Corsini NS, Wolfinger S et al (2017) Guided self-organization and cortical plate formation in human brain organoids. Nat Biotechnol 35(7):659–666

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Broutier L, Andersson-Rolf A, Hindley CJ et al (2016) Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11(9):1724–1743

    Article  CAS  PubMed  Google Scholar 

  58. Broutier L, Mastrogiovanni G, Verstegen MM et al (2017) Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med 23(12):1424–1435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Low JH, Li P, Chew EGY et al (2019) Generation of human PSC-derived kidney organoids with patterned nephron segments and a de novo vascular network. Cell Stem Cell 25(3):373–387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Morizane R, Lam AQ, Freedman BS et al (2015) Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat Biotechnol 33(11):1193–1200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. van den Berg CW, Ritsma L, Avramut MC et al (2018) Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo. Stem Cell Reports 10(3):751–765

    Article  PubMed  PubMed Central  Google Scholar 

  62. Asai A, Aihara E, Watson C et al (2017) Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells. Development 144(6):1056–1064

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Rennert K, Steinborn S, Gröger M et al (2015) A microfluidically perfused three dimensional human liver model. Biomaterials 71:119–131

    Article  CAS  PubMed  Google Scholar 

  64. Leite SB, Roosens T, El Taghdouini A et al (2016) Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro. Biomaterials 78:1–10

    Article  CAS  PubMed  Google Scholar 

  65. Koehler K, Hashino E (2014) 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat Protoc 9:1229–1244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. DeJonge RE, Liu XP, Deig CR (2016) Modulation of Wnt signaling enhances inner ear organoid development in 3D culture. PLOS ONE 11(9):e0162508

    Article  PubMed  PubMed Central  Google Scholar 

  67. Koehler KR, Nie J, Longworth-Mills E et al (2017) Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nat Biotechnol 35(6):583–589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Liu XP, Koehler K, Mikosz A et al (2016) Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells. Nat Commun 7:11508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Jeong M, O’Reilly M, Kirkwood NK et al (2018) Generating inner ear organoids containing putative cochlear hair cells from human pluripotent stem cells. Cell Death Dis 9:1

    Article  Google Scholar 

  70. Seino T, Kawasaki S, Shimokawa M et al (2018) Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression. Cell Stem Cell 22(3):454–467

    Article  CAS  PubMed  Google Scholar 

  71. Boj SF, Hwang CI, Baker LA et al (2015) Organoid models of human and mouse ductal pancreatic cancer. Cell 160:324–338

    Article  CAS  PubMed  Google Scholar 

  72. Huang L, Holtzinger A, Jagan I et al (2015) Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids. Nat Med 21(11):1364–1371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Greggio C, De Franceschi F, Figueiredo-Larsen M et al (2013) Artificial three-dimensional niches deconstruct pancreas development in vitro. Development 140(21):4452–4462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Chen HY, Kaya KD, Dong L, Swaroop A (2016) Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation. Mol Vis 22:1077–1094

    CAS  PubMed  PubMed Central  Google Scholar 

  75. DiStefano T, Chen HY, Panebianco C et al (2018) Accelerated and improved differentiation of retinal organoids from pluripotent stem cells in rotating-wall vessel bioreactors. Stem Cell Reports 10(1):300–313

    Article  CAS  PubMed  Google Scholar 

  76. Völkner M, Zschätzsch M, Rostovskaya M et al (2016) Retinal organoids from pluripotent stem cells efficiently recapitulate retinogenesis. Stem Cell Reports 6(4):525–538

    Article  PubMed  PubMed Central  Google Scholar 

  77. Deng WL, Gao ML, Lei XL et al (2018) Gene correction reverses ciliopathy and photoreceptor loss in iPSC-derived retinal organoids from retinitis pigmentosa patients. Stem Cell Reports 10(4):1267–1281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Reichman S, Slembrouck A, Gagliardi G et al (2017) Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in xeno-free and feeder-free conditions. Stem Cells 35(5):1176–1188

    Article  CAS  PubMed  Google Scholar 

  79. Gracz AD, Ramalingam S, Magness ST (2010) Sox9 expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro. Am J Physiol Gastrointest Liver Physiol 298(5):G590–G600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Petersen N, Reimann F, Bartfeld S et al (2014) Generation of L-cells in mouse and human small intestine organoids. Diabetes 63:410–420

    Article  CAS  PubMed  Google Scholar 

  81. Dekkers JF, Wiegerinck CL, de Jonge HR et al (2013) A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 19(7):939–945

    Article  CAS  PubMed  Google Scholar 

  82. Wilson SS, Tocchi A, Holly MK, Parks WC, Smith JG (2015) A small intestinal organoid model of non-invasive enteric pathogen-epithelial cell interactions. Mucosal Immunol 8(2):352–361

    Article  CAS  PubMed  Google Scholar 

  83. Tsuruta T, Saito S, Osaki Y et al (2016) Organoids as an ex vivo model for studying the serotonin system in the murine small intestine and colon epithelium. Biochem Biophys Res Commun 474(1):161–167

    Article  CAS  PubMed  Google Scholar 

  84. Sachs N, de Ligt J, Kopper O et al (2018) A living biobank of breast cancer organoids captures disease heterogeneity. Cell 172(1–2):373–386

    Article  CAS  PubMed  Google Scholar 

  85. Srivastava V, Huycke TR, Phong KT, Gartner ZJ (2020) Organoid models for mammary gland dynamics and breast cancer. Curr Opin Cell Biol 66:51–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Chandhoke AS, Chanda A, Karve K et al (2017) The PIAS3-Smurf2 sumoylation pathway suppresses breast cancer organoid invasiveness. Oncotarget 8(13):21001–21014

    Article  PubMed  PubMed Central  Google Scholar 

  87. Walsh AJ, Cook RS, Sanders ME et al (2014) Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer. Cancer Res 74(18):5184–5194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Goldhammer N, Kim J, Timmermans-Wielenga V, Petersen OW (2019) Characterization of organoid cultured human breast cancer. Breast Cancer Res 21(1):141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Gao D, Vela I, Sboner A et al (2014) Organoid cultures derived from patients with advanced prostate cancer. Cell 159(1):176–187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Karthaus WR, Iaquinta PJ, Drost J et al (2014) Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159(1):163–175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Chua CW, Shibata M, Lei M et al (2014) Single luminal epithelial progenitors can generate prostate organoids in culture. Nat Cell Biol 16(10):951–954

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Calderon-Gierszal EL, Prins GS (2015) Directed differentiation of human embryonic stem cells into prostate organoids in vitro and its perturbation by low-dose bisphenol A exposure. PLoS One 10(7):e0133238

    Article  PubMed  PubMed Central  Google Scholar 

  93. Richards Z, McCray T, Marsili J et al (2019) Prostate stroma increases the viability and maintains the branching phenotype of human prostate organoids. iScience 12:304–317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Saito Y, Onishi N, Takami H et al (2018) Development of a functional thyroid model based on an organoid culture system. Biochem Biophy Res Comm 497:783–789

    Article  CAS  Google Scholar 

  95. Bartfeld S, Clevers H (2015) Organoids as model for infectious diseases: culture of human and murine stomach organoids and microinjection of Helicobacter Pylori. J Vis Exp 105:53359

    Google Scholar 

  96. Yan HHN, Siu HC, Law S et al (2018) A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening. Cell Stem Cell 23(6):882–897

    Article  CAS  PubMed  Google Scholar 

  97. McCracken KW, Catá EM, Crawford CM et al (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516(7531):400–404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Bouchi R, Foo KS, Hua H et al (2014) FOXO1 inhibition yields functional insulin-producing cells in human gut organoid cultures. Nat Commun 5:4242

    Article  CAS  PubMed  Google Scholar 

  99. Steele NG, Chakrabarti J, Wang J et al (2019) An organoid-based preclinical model of human gastric cancer. Cell Mol Gastroenterol Hepatol 7(1):161–184

    Article  PubMed  Google Scholar 

  100. Sato T, Stange DE, Ferrante M et al (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141(5):1762–1772

    Article  CAS  PubMed  Google Scholar 

  101. Lukovac S, Roeselers G (2015) In Impact food bioact health vitro ex vivo models (Eds: K Verhoeckx P, Cotter I et al), Springer International Publishing, Cham, pp. 245–253

    Google Scholar 

  102. Spence JR, Mayhew CN, Rankin SA et al (2011) Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470(7332):105–109

    Article  PubMed  Google Scholar 

  103. DeWard AD, Cramer J, Lagasse E (2014) Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population. Cell Rep 9(2):701–711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Trisno SL, Philo KED, McCracken KW et al (2018) Esophageal organoids from human pluripotent stem cells delineate Sox2 functions during esophageal specification. Cell Stem Cell 23(4):501–515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Dye BR, Hill DR, Ferguson MAH et al (2015) In vitro generation of human pluripotent stem cell derived lung organoids. elife 4:e05098

    Article  PubMed  PubMed Central  Google Scholar 

  106. Zhang Y, Yang Y, Jiang M et al (2018) 3D modeling of esophageal development using human PSC-derived basal progenitors reveals a critical role for notch signaling. Cell Stem Cell 23(4):516–529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Shacham-Silverberg V, Wells JM (2020) In Methods Cell Biol (Ed JR Spence), Academic Press, pp. 1–22

    Google Scholar 

  108. Miller AJ, Dye BR, Ferrer-Torres D et al (2019) Generation of lung organoids from human pluripotent stem cells in vitro. Nat Protoc 14(2):518–540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Gjorevski N, Sachs N, Manfrin A et al (2016) Designer matrices for intestinal stem cell and organoid culture. Nature 539(7630):560–564

    Article  CAS  PubMed  Google Scholar 

  110. Chen YW, Huang SX, de Carvalho ALRT et al (2017) A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol 19(5):542–549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Kim M, Mun H, Sung CO et al (2019) Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening. Nat Commun 10(1):3991

    Article  PubMed  PubMed Central  Google Scholar 

  112. Lehmann R, Lee CM, Shugart EC et al (2019) Human organoids: a new dimension in cell biology. Mol Biol Cell 30(10):1129–1137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Angus HCK, Butt AG, Schultz M, Kemp RA (2020) Intestinal organoids as a tool for inflammatory bowel disease research. Front Med 6. https://doi.org/10.3389/fmed.2019.00334

  114. Takebe T, Wells JM, Helmrath MA, Zorn AM (2018) Organoid center strategies for accelerating clinical translation. Cell Stem Cell 22(6):806–809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Ma R, Morshed SA, Latif R, Davies TF (2015) Thyroid cell differentiation from murine induced pluripotent stem cells. Front Endocrinol 6. https://doi.org/10.3389/fendo.2015.00056

  116. Mullenders J, de Jongh E, Brousali A et al (2019) Mouse and human urothelial cancer organoids: a tool for bladder cancer research. Proc Natl Acad Sci USA 116(10):4567–4574

    Google Scholar 

  117. Lõhmussaar K, Kopper O, Korving J et al (2020) Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids. Nat Commun 11:2660

    Article  PubMed  PubMed Central  Google Scholar 

  118. Nanki Y, Chiyoda T, Hirasawa A et al (2020) Patient-derived ovarian cancer organoids capture the genomic profiles of primary tumours applicable for drug sensitivity and resistance testing. Sci Rep 10(1):12581

    Google Scholar 

  119. Maenhoudt N, Defraye C, Boretto M et al (2020) Developing organoids from ovarian cancer as experimental and preclinical models. Stem Cell Reports 14(4):717–729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Rothschild D, Srinivasan T, Aponte-Santiago L et al (2016) The ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids. J Vis Exp. https://doi.org/10.3791/54033

  121. Lee SH, Hu W, Matulay JT et al (2018) Tumor evolution and drug response in patient-derived organoid models of bladder cancer. Cell 173(2):515–528

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Eldred KC, Hadyniak S E, Hussey KA et al (2018) Thyroid hormone signaling specifies cone subtypes in human retinal organoids. bioRxiv. https://doi.org/10.1101/359950

  123. Drost J, Karthaus WR, Gao D et al (2016) Organoid culture systems for prostate epithelial and cancer tissue. Nat Protoc 11(2):347–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Yakoub AM (2019) Cerebral organoids exhibit mature neurons and astrocytes and recapitulate electrophysiological activity of the human brain. Neural Regen Res 14(5):757–761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Rosenbluth JM, Schackmann RCJ, Gray GK et al (2020) Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages. Nat Commun 11(1):1711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Roccio M, Edge ASB (2019) Inner ear organoids: new tools to understand neurosensory cell development, degeneration and regeneration. Development 146(17):dev177188

    Article  PubMed  PubMed Central  Google Scholar 

  127. Subramanian A, Sidhom EH, Emani M et al (2019) Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation. Nat Commun 10:5462

    Article  PubMed  PubMed Central  Google Scholar 

  128. Yoshida S, Miwa H, Kawachi T et al (2020) Generation of intestinal organoids derived from human pluripotent stem cells for drug testing. Sci Rep 10(1):5989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Akbari S, Arslan N, Senturk S, Erdal E (2019) Next-generation liver medicine using organoid models. Front Cell Dev Biol 7. https://doi.org/10.3389/fcell.2019.00345

  130. Barkauskas CE, Chung MI, Fioret B et al (2017) Lung organoids: current uses and future promise. Development 144(6):986–997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Whelan KA, Muir AB, Nakagawa H (2018) Esophageal 3D culture systems as modeling tools in esophageal epithelial pathobiology and personalized medicine. Cell Mol Gastroenterol Hepatol 5(4):461–478

    Article  PubMed  PubMed Central  Google Scholar 

  132. Cowan CS, Renner M, De Gennaro M et al (2020) Cell types of the human retina and its organoids at single-cell resolution. Cell 182(6):1623–1640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Hohwieler M, Illing A, Hermann PC et al (2017) Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut 66(3):473–486

    Article  CAS  PubMed  Google Scholar 

  134. Moreira L, Bakir B, Chatterji P et al (2017) Pancreas 3D organoids: current and future aspects as a research platform for personalized medicine in pancreatic cancer. Cell Mol Gastroenterol Hepatol 5(3):289–298

    Article  PubMed  PubMed Central  Google Scholar 

  135. Elbadawy M, Abugomaa A, Yamawaki H et al (2020) Development of prostate cancer organoid culture models in basic medicine and translational research. Cancers 12:777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. McCray T, Richards Z, Marsili J et al (2019) Handling and assessment of human primary prostate organoid culture. Vis Exp JoVE. https://doi.org/10.3791/59051

  137. Seidlitz T, Merker SR, Rothe A et al (2019) Human gastric cancer modelling using organoids. Gut 68(2):207–217

    Article  CAS  PubMed  Google Scholar 

  138. Kurmann AA, Serra M, Hawkins F et al (2015) Regeneration of thyroid function by transplantation of differentiated pluripotent stem cells. Cell Stem Cell 17(5):527–542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Dekkers JF, Alieva M, Wellens LM et al (2019) High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc 14(6):1756–1771

    Article  CAS  PubMed  Google Scholar 

  140. Glaser AK, Reder NP, Chen Y et al (2019) Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues. Nat Commun 10(1):2781

    Article  PubMed  PubMed Central  Google Scholar 

  141. Brazovskaja A, Treutlein B, Camp JG (2019) High-throughput single-cell transcriptomics on organoids. Curr Opin Biotechnol 55:167–171

    Article  CAS  PubMed  Google Scholar 

  142. Zanotelli VR, Leutenegger M, Lun XK et al (2020) A quantitative analysis of the interplay of environment, neighborhood, and cell state in 3D spheroids. Mol Syst Biol 16(12):e9798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Tanaka Y, Cakir B, Xiang Y, Sullivan GJ, Park IH (2020) Synthetic analyses of single-cell transcriptomes from multiple brain organoids and fetal brain. Cell Rep 30(6):1682–1689

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Andilla J, Jorand R, Olarte O et al (2017) Imaging tissue-mimic with light sheet microscopy: a comparative guideline. Sci Rep 7:44939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Yang B, Chen X, Wang Y et al (2019) Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution. Nat Methods 16(6):501–504

    Google Scholar 

  146. Hof L, Moreth T, Koch M et al (2020) Long-term live imaging of epithelial organoids and corresponding multiscale analysis reveal high heterogeneity and identifies core regulatory principles. bioRxiv 2020.07.12.199463

    Google Scholar 

  147. Bolhaqueiro ACF, van Jaarsveld RH, Ponsioen B et al (2018) In Methods Cell Biol (Eds: H Maiato, M Schuh), Academic Press, pp. 91–106

    Google Scholar 

  148. Kim S, Choung S, Sun RX et al (2020) Comparison of cell and organoid-level analysis of patient-derived 3D organoids to evaluate tumor cell growth dynamics and drug response. SLAS Discov Adv Sci Drug Discov 25:744

    Article  CAS  Google Scholar 

  149. Kanton S, Boyle MJ, He Z et al (2019) Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574(7778):418–422

    Article  CAS  PubMed  Google Scholar 

  150. Smits LM, Magni S, Kinugawa K et al (2020) Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids. Cell Tissue Res 382(3):463–476

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Zhou J, Li C, Sachs N et al (2018) Differentiated human airway organoids to assess infectivity of emerging influenza virus. Proc Natl Acad Sci U S A 115:6822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Buzzelli JN, Ouaret D, Brown G et al (2018) Colorectal cancer liver metastases organoids retain characteristics of original tumor and acquire chemotherapy resistance. Stem Cell Res 27:109–120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Velasco S, Kedaigle AJ, Simmons SK et al (2019) Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 570:523–527

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Alladin A, Chaible L, Reither S et al (2019) Tracking the cells of tumor origin in breast organoids by light sheet microscopy. bioRxiv. https://doi.org/10.1101/617837

  155. Fujii E, Yamazaki M, Kawai S et al (2018) A simple method for histopathological evaluation of organoids. J Toxicol Pathol 31(1):81–85

    Article  CAS  PubMed  Google Scholar 

  156. Xie Y, Park ES, Xiang D, Li Z (2018) Long-term organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube. Stem Cell Res 32:51–60

    Article  CAS  PubMed  Google Scholar 

  157. Pastuła A, Middelhoff M, Brandtner A et al (2016) Three-dimensional gastrointestinal organoid culture in combination with nerves or fibroblasts: a method to characterize the gastrointestinal stem cell niche. Stem Cells Int:3710836

    Google Scholar 

  158. Lu W, Rettenmeier E, Paszek M et al (2017) Crypt organoid culture as an in vitro model in drug metabolism and cytotoxicity studies. Drug Metab Dispos 45(7):748–754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. Yakoub AM, Sadek M (2018) Development and characterization of human cerebral organoids: an optimized protocol. Cell Transplant 27(3):393–406

    Article  PubMed  PubMed Central  Google Scholar 

  160. Xia C, Fana J, Emanuel G et al (2019) Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression. Proc Natl Acad Sci 116:19490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Shah S, Lubeck E, Zhou W, Cai L (2016) In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus. Neuron 92(2):342–357

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Mayr U, Serra D, Liberali P (2019) Exploring single cells in space and time during tissue development, homeostasis and regeneration. Development 146(12):dev176727

    Article  CAS  PubMed  Google Scholar 

  163. Schürch CM, Bhate SS, Barlow GL et al (2020) Coordinated cellular neighborhoods orchestrate antitumoral immunity at the colorectal cancer invasive front. Cell 182(5):1341–1359

    Article  PubMed  PubMed Central  Google Scholar 

  164. Lin JR, Fallahi-Sichani M, Chen JY, Sorger PK (2016) Cyclic Immunofluorescence (CycIF), A highly multiplexed method for single-cell imaging. Curr Protoc Chem Biol 8(4):251–264

    Article  PubMed  PubMed Central  Google Scholar 

  165. Lin JR, Izar B, Wang C et al (2018) Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. elife 7:e31657

    Article  PubMed  PubMed Central  Google Scholar 

  166. Dueñas ME, Essner JJ, Lee YJ (2017) 3D MALDI Mass spectrometry imaging of a single cell: spatial mapping of lipids in the embryonic development of zebrafish. Sci Rep 7:14946

    Article  PubMed  PubMed Central  Google Scholar 

  167. Fouquet T, Mertz G, Desbenoit N et al (2014) TOF-SIMS/MALDI-TOF combination for the molecular weight depth profiling of polymeric bilayer. Mater Lett 128:23–26

    Article  CAS  Google Scholar 

  168. Kaitin KI (2010) Deconstructing the drug development process: the new face of innovation. Clin Pharmacol Ther 87(3):356–361

    Article  CAS  PubMed  Google Scholar 

  169. Brehm-Stecher BF (2014) In Encycl Food Microbiol Second Ed. (Eds: CA Batt, ML Tortorello), Academic Press, Oxford, pp. 943–953.

    Google Scholar 

  170. Allam M, Cai S, Coskun AF (2020) Multiplex bioimaging of single-cell spatial profiles for precision cancer diagnostics and therapeutics. NPJ Precis Oncol 4:11

    Article  PubMed  PubMed Central  Google Scholar 

  171. Saglam-Metiner P, Gulce-Iz S, Biray-Avci C (2019) Bioengineering-inspired three-dimensional culture systems: organoids to create tumor microenvironment. Gene 686:203–212

    Article  CAS  PubMed  Google Scholar 

  172. Yu F, Hunziker W, Choudhury D (2019) Engineering microfluidic organoid-on-a-chip platforms. Micromachines 10:165

    Article  PubMed  PubMed Central  Google Scholar 

  173. Wong CH, Siah KW, Lo AW (2019) Estimation of clinical trial success rates and related parameters. Biostatistics 20(2):273–286

    Article  PubMed  Google Scholar 

  174. Zhou Y, Othus M, Araki D et al (2016) Pre- and post-transplant quantification of measurable (‘minimal’) residual disease via multiparameter flow cytometry in adult acute myeloid leukemia. Leukemia 30(7):1456–1464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Garcez PP, Loiola EC, Madeiro da Costa R et al (2016) Zika virus impairs growth in human neurospheres and brain organoids. Science 352(6287):816–818

    Article  CAS  PubMed  Google Scholar 

  176. Quadrato G, Nguyen T, Macosko EZ et al (2017) Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545(7652):48–53

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  177. Öhlund D, Handly-Santana A, Biffi G et al (2017) Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J Exp Med 214(3):579–596

    Article  PubMed  PubMed Central  Google Scholar 

  178. Bartucci M, Ferrari AC, Kim IY et al (2016) Personalized medicine approaches in prostate cancer employing patient derived 3D organoids and humanized mice. Front Cell Dev Biol 4. https://doi.org/10.3389/fcell.2016.00064

  179. Al-Lamki RS, Bradley JR, Pober JS (2017) Human organ culture: updating the approach to bridge the gap from in vitro to in vivo in inflammation, cancer, and stem cell biology. Front Med 4. https://doi.org/10.3389/fmed.2017.00148

  180. Willemse J, Lieshout R, van der Laan LJW, Verstegen MMA (2017) From organoids to organs: bioengineering liver grafts from hepatic stem cells and matrix. Best Pract Res Clin Gastroenterol 31(2):151–159

    Article  PubMed  Google Scholar 

  181. Herron LA, Hansen CS, Abaci HE (2019) Engineering tissue-specific blood vessels. Bioeng Transl Med 4:e10139. https://doi.org/10.1002/btm2.10139

    Article  PubMed  PubMed Central  Google Scholar 

  182. Kolesky DB, Truby RL, Gladman AS et al (2014) 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv Mater 26(19):3124–3130

    Article  CAS  PubMed  Google Scholar 

  183. Zhang B, Montgomery M, Chamberlain MD et al (2016) Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis. Nat Mater 15(6):669–678

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  184. Akhtar A (2015) The flaws and human harms of animal experimentation. Camb Q Healthc Ethics 24(4):407–419

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

A.F.C. holds a Career Award at the Scientific Interface from Burroughs Wellcome Fund and a Bernie-Marcus Early-Career Professorship. A. F. C. was supported by start-up funds from the Georgia Institute of Technology and Emory University. A.K., S.C., and M.A. contributed equally to work and are equal first co-authors on this study. Figures were created with BioRender.com.

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Kumar, A. et al. (2023). Single-Cell and Spatial Analysis of Emergent Organoid Platforms. In: Kasid, U.N., Clarke, R. (eds) Cancer Systems and Integrative Biology. Methods in Molecular Biology, vol 2660. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3163-8_22

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