2020 A Brief History of Organoids

1 A brief history of organoids
2
3
4 Claudia Corrò1,*, Laura Novellasdemunt1,*, Vivian S.W. Li1
5
6
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7 Stem Cell and Cancer Biology Lab, The Francis Crick Institute, 1 Midland Road,
8 London NW1 1AT, UK
9 *These authors contributed equally to this work
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13 Correspondence should be addressed to Vivian Li: Vivian.Li@crick.ac.uk
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15 Keywords: Organoids, Embryonic stem cells, Adult stem cells, Pluripotent stem cells,
16 Disease modelling, Biobanking, Drug screening, Regenerative medicine.
17
Downloaded from journals.physiology.org/journal/ajpcell at Uppsala Universitetsbibliotek (130.238.007.040) on May 28, 2020.

18 Abstract
19 In vitro cell cultures are crucial research tools for modelling human development and
20 diseases. While the conventional monolayer cell cultures have been widely used in the past,
21 the lack of tissue architecture and complexity of such model fails to inform the true biological
22 processes in vivo. Recent advances in the organoid technology have revolutionized the in
23 vitro culture tools for biomedical research by creating powerful three-dimensional (3D)
24 models to recapitulate the cellular heterogeneity, structure and functions of the primary
25 tissues. Such organoid technology enables researchers to recreate human organs and
26 diseases in a dish, thus holds great promises for many translational applications such as
27 regenerative medicine, drug discovery and precision medicine. In this review, we provide an
28 overview of the organoid history and development. We discuss the strengths and limitations
29 of organoids, as well as their potential applications in the laboratory and the clinic.
30
31 Introduction
32 The modern term organoid refers to cells growing in defined 3D environment in vitro
33 to form mini-clusters of cells that self-organize and differentiate into functional cell types,
34 recapitulating the structure and function of an organ in vivo (hence also called “mini-
35 organs”). Organoids can be derived from either embryonic stem cells (ESCs), induced
36 pluripotent stem cells (iPSCs), neonatal or adult stem cells (ASCs)1, 2 through a process
37 similar to the way in which the organ acquires its distinctive organization. Self-organization
38 within the organoid occurs through spatially restricted lineage commitment and cell sorting,
39 which requires activation of various signaling pathways mediated by intrinsic cellular
40 components or extrinsic environment such as extracellular matrix (ECM) and media.
41 ASC-derived organoids are generated directly from postnatal or adult tissues, either
42 from single ASC or ASC-containing tissue units. This is supported by a cocktail of growth
43 factors in the culture media that recapitulate signaling control under normal tissue
44 homeostasis. Besides normal tissues, ASC-derived organoids can also be established from
45 patient-specific material for disease modeling and precision medicine (see Organoid
46 Applications section below). On the other hand, ESC/iPSC-derived organoids involve
47 stepwise differentiation protocols using various growth-factors or inhibitors that resemble the
48 developmental cues during gastrulation and organogenesis. The pluripotent property of
49 ESCs and iPSCs enables the generation of organoids from all three germ layers. This is
50 particularly useful for studies of early-stage embryonic development where primary human
51 material is limited. In this review, we will discuss the history and development of 3D organoid
52 culture, and provide the most recent update on organoid research that covers whole range
53 of systems. We will explore various applications of organoid technology in biomedicine and
54 discuss its promises and challenges. Finally, we will evaluate the pros and cons of 3D
55 organoid technology compared to other conventional models.
56
57 3D culture models: from cell aggregates to organoids
58
59 The 3D culture system is established by suspension culture to avoid direct physical
60 contact to the plastic dish. This can be achieved using scaffold or scaffold-free techniques.
61 Scaffolds are biological or synthetic hydrogels that resemble the natural ECM. The most
62 commonly used one is Matrigel, which is a heterogeneous and gelatinous protein mixture
63 secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells3. It comprises mainly
64 adhesive proteins such as collagen, entactin, laminin and heparin sulphate proteoglycans
65 which resemble the extracellular environment to provide structural support and ECM signals
66 to the cells. For scaffold-free techniques, cells are cultured in droplets of a defined culture
67 medium hanging from a plate by gravity and surface tension4. Alternatively, the 3D structure
68 of the organoids can also be established via “air-liquid-interface”. In this case, cells are
69 cultured on a basal layer of fibroblasts or Matrigel that are initially submerged in medium,
70 which gradually evaporates and exposes the upper cell layers to the air to allow polarization
71 and differentiation5, 6.

72 Back in 1907, Henry Van Peters Wilson described the first attempt of in vitro
73 organism regeneration, where he demonstrated that dissociated sponge cells can self-
74 organize to regenerate a whole organism7. Few decades later, several groups performed
75 dissociation-reaggregation experiments to generate different types of organs from
76 dissociated amphibian pronephros8 and chick embryos9. In 1964, Malcolm Steinberg
77 introduced the differential adhesion hypothesis, proposing that cell sorting and
78 rearrangement can be explained by thermodynamics mediated by differential surface
79 adhesion10. Stem cell research began to thrive when pluripotent stem cells (PSCs) were first
80 isolated and established from mouse embryos in 198111, 12. But it was not until 1998 that
81 scientists were able to isolate and culture embryonic stem cells derived from human
82 blastocysts for the first time13. Later on, iPSCs were subsequently established by
83 reprogramming of mouse and human fibroblasts, which have brought significant impact to
84 stem cell and organoid research14-16.
85 In 1987, scientists began to improve cell culture conditions by simulating the in vivo
86 microenvironment. Bissell and colleagues demonstrated that breast epithelia can form 3D
87 ducts and lumen when grown on EHS ECM extract, where they appeared to be able to
88 synthetize and secrete milk protein as opposed to two-dimensional (2D) culture17. Similarly,
89 alveolar type II epithelial cells were able to maintain their differentiation in the presence of
90 ECM matrix18, highlighting the importance of cell-matrix interactions in tissue maintenance
91 and differentiation. Organoid research began to shift from 2D to 3D when Sasai and
92 colleagues were able to generate cerebral cortex tissue from ESCs using 3D aggregation
93 culture method19. In 2009, a landmark study from Hans Clevers laboratory showed that
94 single leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5)-expressing adult
95 intestinal stem cells can form 3D intestinal organoids in Matrigel that self-organize and
96 differentiate into crypt-villus structures in the absence of a mesenchymal niche20. This was
97 the first report on establishing 3D organoid culture derived from single ASC, which set the
98 scene for many subsequent organoid works in other systems, including mesendoderm (e.g.
99 stomach, liver, pancreas, lung and kidney) and neuroectoderm (brain and retina) using
100 either ASCs or PSCs (Figure 1). Below, we will provide the most recent updates on organoid
101 technology in various systems.
102
103 Progress in organoid research
104
105 Gastrointestinal organoids
106 The gastrointestinal (GI) tract arises from the endoderm during development, which
107 forms a tube that can be divided in three different regions: the foregut, the midgut and the
108 hindgut21. The foregut gives rise to the oral cavity, pharynx, respiratory tract, pancreas,
109 stomach and the liver; the midgut gives rise to the small intestine and the ascending colon;
110 and the hindgut gives rise to the remaining colon and the rectum. Understanding the
111 molecular mechanism and signaling regulation underlying the GI tract development and
112 homeostasis is crucial for establishment and maintenance of ASC/PSC-derived organoids
113 from these regions.
114 Intestinal organoids. In adult intestine, Wnt and Egf are known to play key roles for
115 stem cell maintenance in the crypt, while Bmp drives differentiation in the villi22, 23. In 2009,
116 Hans Clevers laboratory described the first establishment of long-term 3D culture of intestine
117 organoids from single Lgr5+ stem cells20. These organoids were grown in Matrigel in the
118 presence of Wnt agonist R-spondin, Egf and Bmp inhibitor Noggin to form crypt-villus
119 structures and were able to differentiate into all intestinal cell types, recapitulating the
120 organization and function of the small bowel in vivo. Similar protocols for long-term culture of
121 human colon, adenoma and adenocarcinoma were subsequently established24, 25.
122 Importantly, transplantation of these intestinal organoids in mice showed long-term
123 engraftment into the damaged colonic epithelium in vivo, highlighting the regenerative
124 potential of these 3D organoids26. Building on these adult-derived organoid cultures, a
125 modified protocol of human PSC-derived intestinal organoids was further established27. In
126 particular, human PSCs were first treated with activin A to drive mesendodermal identity,

127 followed by Wnt3a and Fgf4 to promote hindgut specification. The hindgut spheroids were
128 subsequently cultured in Matrigel following the adult-derived organoid protocol to promote
129 maturation. The major difference of PSC-derived intestinal organoids from adult-derived
130 ones is the presence of surrounding mesenchymal cells in the culture, which allows
131 formation of both epithelium and mesenchyme supported by mouse vasculature upon
132 engraftment in vivo28.
133 Gastric organoids. Stomach and intestinal epithelia share many molecular and
134 physiological similarities, including the presence of proliferating Lgr5+ stem cells at the base
135 of the glands/crypts. With minor modification of the intestinal culture system, gastric
136 organoids were established from adult mouse pyloric Lgr5+ stem cells or Troy+ chief cells in
137 corpus gland with addition of Wnt3a and Fgf1029, 30. A similar method was adopted for the
138 establishment of long-term culture of human adult gastric organoids31. Subsequently, human
139 PSC-derived gastric organoids were generated by adding Wnt3a, Fgf4, Noggin and retinoic
140 acid (RA) to drive posterior foregut fate followed by 3D culture in Matrigel for maturation32.
141 These PSC-derived organoids are believed to adopt predominantly pyloric lineage.
142 Tongue and salivary gland organoids. Apart from intestine and stomach,
143 organoids derived from tongue in the upper GI tract have also been explored. The initial
144 approach was to derive lingual organoids from Bmi-expressing stem cells from adult tongue
145 epithelium, which formed stratified squamous epithelia without salivary acinar cells or taste
146 bud cells33. Later on, taste bud organoids were established using LGR5+, LGR6+ or CD44+
147 stem/progenitor cells derived from taste buds in circumvallate papilla tissue with taste
148 receptor expression34, 35. Moreover, long-term expansion of mouse salivary gland organoids
149 driven by Wnt signals has also been reported36. More recently, it has been shown that
150 transcription factors Sox9 and Foxc1 can drive differentiation of mouse ESC-derived oral
151 ectoderm to salivary gland organoids, which can mature to functional salary gland following
152 orthotopic transplantation37.
153
154 Liver and pancreatic organoids
155 The liver derives mainly from the foregut endoderm epithelium during development
156 that gives rise to the hepatic bud structure, which generates hepatoblasts and subsequently
157 hepatocytes and biliary epithelium38. An early study showed that dissociated chick
158 embryonic hepatic tissue can reaggregate and form secretory units with functional bile
159 ducts9. Adult liver and pancreas are slow cycling under homeostasis. It has been shown that
160 cycling Lgr5+ cells were found near the bile ducts after damage in mice39. These cells were
161 able to generate organoids (budding cysts) when grown in 3D culture conditions with
162 Matrigel and can be differentiated to form mature, functional hepatocytes39. These liver
163 organoids consist mostly of progenitor cells expressing bile duct and hepatocyte markers,
164 but can differentiate into functional hepatocytes when transplanted into a mouse model of
165 liver disease39. In a follow-up study, long-term expansion of adult bile duct-derived bipotent
166 progenitors was established from human liver 40. In 2018, two studies further reported the
167 successful long-term expansion of human and mouse hepatocyte as 3D organoid culture
168 with high engraftment efficiency41, 42. An alternative method has also been described to
169 generate vascularized human liver from human iPSCs43. This protocol involves
170 differentiation of human PSCs into hepatic endodermal cells in 2D together with human
171 mesenchymal stem cells and human endothelial cells. When grown in Matrigel, these cells
172 spontaneously form vascularized 3D aggregates that can further engraft in vivo to form
173 functional liver with vascular network.
174 Pancreatic organoids can also be generated by plating mouse embryonic pancreatic
175 progenitor cells in Matrigel44. Similarly, mouse and human pancreatic organoids were
176 subsequently established from adult pancreas, which can further differentiate to ductal and
177 endocrine lineages after transplantation45, 46.
178
179 Brain organoids
180 Vertebrate central nervous system is derived from the neuroectoderm during
181 development47. The human brain is a highly complex system which can be broadly divided

182 into three regions: forebrain, midbrain and hindbrain that are composed primarily of neurons
183 and glia cells. Previous dissociation-reaggregation experiments using chick neural
184 progenitors derived from early developing brain formed clusters of neuroepithelial cells in a
185 radial manner around a lumen similar to the neural tube, suggesting a self-organizing
186 capacity of these brain cells48. Similarly, neural progenitor cells (NPCs) also have the ability
187 to aggregate and form neurospheres in suspension culture with the capacity to differentiate
188 into neurons and astrocytes49. Neural aggregates can also be generated from PSC-derived
189 embryoid body (EB)50. More recently, neural rosettes were further established from PSCs,
190 which contained NPCs surrounding a central lumen resembling the neural tube51.
191 Remarkably, they can be further specified into various mature cell types with characteristics
192 of different brain regions52-57. However, these models are still largely based on 2D culture or
193 simple aggregates, which lack the complexity for the study of brain development and
194 function.
195 Yoshiki Sasai laboratory has pioneered in developing 3D culture of different brain
196 regions from mouse or human PSCs to recapitulate the complex brain tissue organization.
197 They first generated forebrain tissues by plating mouse58 or human59 EBs in 2D. When
198 transferred to 3D aggregation culture, these neuroepithelium formed more complex
199 structures recapitulating the dorsal forebrain19. This 3D protocol was further optimized later
200 on to allow self-organization of neuronal layers similar to early cortical development that can
201 be cultured up to 112 days60. Different brain regional identities can also be developed from
202 ESCs by manipulating growth factors such as Hedgehog, Fgf, Bmp and Wnt61-63.
203 In 2013, the laboratory of Jürgen Knoblich further established the 3D cerebral
204 organoids that contain different brain regions within single organoids64. This is an improved
205 method from Sasai’s one by embedding EBs in Matrigel, which allows polarization and
206 outgrowth of large neuroepithelial buds. These mini-brains can further grow up to a few
207 millimeters when transferred to spinning bioreactor, and develop into different brain regions,
208 including retina, dorsal cortex, ventral forebrain, midbrain-hindbrain boundary, choroid
209 plexus, and hippocampus. Subsequent studies further generated other organoid protocols to
210 model specific brain regions, such as midbrain-specific organoids65, hippocampal
211 organoids66 and cerebellar organoids67. Using 3D printing technology, a miniaturized
212 spinning bioreactor was further generated to allow cost-effective generation of forebrain-
213 specific organoids from human iPSCs68.
214
215 Retinal organoids
216 The neuroectoderm-derived retina originates from optic vesicle during development,
217 where the front of the vesicle invaginates to form two adjacent epithelial layers: the outer
218 retinal pigmented epithelium and the inner neural retina69. Reaggregation experiments in
219 chick retina showed self-organization of retina in vitro70, 71. These reaggregates can further
220 organize into a correctly laminated structure when cultured in the presence of Wnt2b72. Later
221 on, 3D culture of mouse EB aggregates further allowed the establishment of optic cup
222 organoids resembling early retina with retinal stratification and apical-basal polarity73. Optic
223 cup organoids can also be generated from human PSCs74. These human retinal organoids
224 are larger than mouse organoids and have the capacity to grow into multi-layered tissue
225 containing both rods and cones.
226
227 Kidney organoids
228 The kidney arises from the intermediate mesoderm through Wnt and Fgf signaling
229 which develops into the ureteric bud and the metanephric mesenchyme to form early renal
230 tubes75. Similar to other tissues, dissociation-reaggregation experiments in chick and mouse
231 embryonic kidney demonstrated the ability to self-organize and form organotypic renal
232 structures9, 76. In 2013, ureteric bud organoids were established from human PSCs that were
233 first cultured in Bmp4 and Fgf2 for mesodermal specification, followed by exposure to RA,
234 Activin A and Bmp2 to generate ureteric bud-committed renal progenitors77. These human
235 progenitor cells were further co-cultured with disaggregated mouse embryonic kidney cells
236 to self-organize and form 3D ureteric bud structures. In addition, metanephric mesenchyme

237 identity can also be generated from mouse EB and human PSCs by sequential exposure to
238 Activin, Bmp4 and the Wnt activator CHIR99021 followed by RA and Fgf978. Co-culture of
239 the metanephric mesenchyme with spinal cord tissue form 3D structures with organized
240 nephric tubules and glomeruli. Similarly, hESCs can also be differentiated to ureteric and
241 metanephric progenitors through primitive streak and intermediate mesoderm, which further
242 form 3D structures similar to ureteric epithelium and proximal tubules when co-cultured with
243 dissociated mouse embryonic kidney79. In 2015, a simplified and improved protocol was
244 established by direct differentiation of human PSCs to complex multicellular kidney
245 organoids that contain nephrons associated with a collecting duct network surrounded by
246 endothelial cells and renal interstitium80. More recently, long-term culture of kidney tubular
247 organoids was further established from adult human or mouse kidney tissues or from human
248 urine, which form proximal and distal nephron segments81.
249
250 Other organoid types
251 Organoids can be generated from a broad range of tissues in addition to the ones
252 mentioned above. For example, Jamieson et al have recently established mammary
253 organoids from single adult mammary epithelial cells containing both polarized secretory
254 epithelium surrounded by myoepithelial cells82. Prostate organoids can also be derived
255 from adult mouse and human prostate epithelia to form both luminal and basal cells83, 84.
256 Thyroid organoids were generated by transient expression of the transcription factors
257 NKX2-1 and PAX8 to direct mouse ESC differentiation into thyroid follicular cells and form
258 3D follicular structures when treated with thyrotropin85. Cardiovascular organoids can be
259 generated from EBs by modulating substrate stiffness86. Lung organoids can be generated
260 by co-culturing adult bronchioalveolar stem cells and lung endothelial cells in Matrigel87.
261 Similarly, human airway organoids were established from broncho-alveolar resections which
262 comprise basal cells, functional ciliated cells, mucus-producing cells, and CC10-secreting
263 club cells88. Stable fallopian tube organoids were also established from human fallopian
264 tubes containing both ciliated and secretory cells89. In addition, pituitary organoids have
265 also been generated from EBs when grown under ectoderm-promoting conditions, which
266 can further mature and synthesize pituitary hormones90. A similar protocol has been used to
267 generate inner ear organoids from EBs, which consist of functional inner ear sensory
268 epithelia with stereocilia and kinocilia91.
269 Besides modelling individual organs, organoids have also been recently used to
270 explore early mammalian embryonic development. Embryonic organoids or gastruloids
271 were established by 3D aggregation of mouse ESCs in suspension that developed into
272 embryo-like structures with polarized gene expression in the absence of external asymmetry
273 clues92. These embryonic organoids self-organize and exhibit behaviors reminiscent of
274 mammalian gastrulation, giving rise to cell types that correspond to the three germ layers
275 with axial organization in a time-scale similar to mouse embryos. Comparison of mouse
276 gastruloids and embryos further reveal somitogenesis dynamics, highlighting the power of
277 these gastruloids as a model for exploring early embryonic development in vitro93. It will be
278 important to further develop equivalent gastruloid system in primates to model human
279 embryo development in vitro.
280 In fact, organoid technology has also been extended to other animal models in
281 addition to mouse and human. Methods of generating intestinal, mammary, keratinocyte,
282 and liver organoids have been reported in different species such as bovine, porcine, ovine,
283 chicken, feline, and canine94, 95. A recent study has further reported the generation of snake
284 venom gland organoids that express high levels of toxin transcripts, which can potentially be
285 used for toxicology studies96.
286 Despite the diversity of organoid systems and their corresponding culture protocols,
287 there are some core growth factors and chemical modulators shared between systems. In
288 particular, vast majority of the ASC-derived organoids are cultured in Matrigel suspension
289 that require serum-free basal media supplemented with Wnt agonists and/or ligands (R-
290 spondin, Wnt3a), Egf and BMP inhibitor (Noggin). Depending on the signaling or hormonal
291 requirements of their tissues of origin, additional growth factors or inhibitors (such as FGF in

292 liver39-41, gastric29-31 and pancreatic45, 46 organoids, gastrin in gastric organoids29-31 and
293 dihydrotestosterone in prostate organoids83, 84) are added to the cultures. On the other hand,
294 stepwise differentiation protocols are required for ESCs/PSCs-derived organoids. Notably,
295 activin A is required to drive differentiation of ESCs/PSCs to definitive endoderm, whereas
296 Fgf and Wnt can promote neuromesoderm differentiation97, 98. In essence, the similarities
297 and differences of the culture protocols between systems reflect the growth signal
298 requirement during development and tissue homeostasis.
299
300
301 Organoid applications
302
303 Organoids are becoming one of the mainstream cell culture tools in many biomedical
304 studies. The wide range of tissue types, the long-term expansion capacity and the
305 physiological 3D architecture of organoids make them a powerful new technology for many
306 biological and clinical applications. Notably, organoids have been widely used for
307 development and disease modelling, precision medicine, toxicology studies and
308 regenerative medicine (Figure 2). Below, we focus on the applications of organoids in
309 disease modelling, biobanking, precision medicine and regenerative medicine.
310
311 Disease modelling
312 Genetic diseases
313 Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations
314 in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In
315 2013, Dekkers and colleagues generated the first human CF-patient derived intestinal
316 organoids carrying F508del CFTR mutation to recapitulate the disease in vitro99. They
317 developed a swelling assay where healthy organoids respond to Forskolin treatment by
318 rapid swelling while such effect is strongly reduced in CF-organoids. This organoid-swelling
319 assay has proven to be very reliable to predict responders to CFTR modulators, and has
320 become the first organoid-based personalized medicine application for CF patients in the
321 Netherlands100. Interestingly, gene editing by CRISPR-mediated homologous recombination
322 in primary patient-derived organoids (PDOs) can repair the CFTR mutation and function,
323 implying the potential application of such gene correction approach to single-gene hereditary
324 defects101. Hereditary multiple intestinal atresia (HMIA) is another autosomal recessive
325 disorder characterized by bowel obstructions. Pathogenic mutations in the tetratricopeptide
326 repeat domain 7A (TTC7A) have been identified102. Patient-derived intestinal organoids
327 showed activation of RhoA kinase pathway and apicobasal polarity inversion, which could be
328 restored by adding RhoA kinase inhibitor (Y-27632)103. Similarly, liver organoids derived
329 from patients with α1-antitrypsin (A1AT) deficiency and Alagille syndrome can also
330 recapitulate the in vivo pathology, where accumulation of misfolded precipitates of A1AT
331 protein in hepatocytes and biliary defects were observed respectively40.
332 Cerebral organoids have been used to model human microcephaly, a genetic
333 disease caused by a mutation in CDK5RAP2, where organoids generated from patient-
334 derived iPSCs were smaller with reduced progenitor regions64. Forebrain organoids have
335 been used to model a genetic condition that causes lissencephaly (smooth brain) which
336 showed defects in progenitors and Wnt signaling104, 105. Brain organoids could also be
337 relevant models for neurodegenerative diseases such as Alzheimer’s disease (AD), the most
338 common type of dementia characterized by extracellular deposition of misfolded amyloid-β
339 (Aβ) containing plaques and intracellular neurofibrillary tangles (NFTs)106, 107. Raja and
340 collaborators have developed a scaffold-free culture method to generate iPSCs-derived
341 brain organoids from patients with familial AD, which could reproduce several AD
342 pathologies like Aβ aggregation, hyperphosphorylated tau protein and abnormalities of
343 endosomes108. Treatment of these patient organoids with β- and γ- secretase inhibitors can
344 significantly reduce the Aβ and tau pathology, demonstrating the potential of using human
345 brain organoids for drug discovery in AD108. More recently, mini-brains have further been
346 used to model Parkinson´s disease (PD)109. These organoids were generated from midbrain

347 floor plate NPCs containing midbrain dopaminergic neurons (mDANs) that resemble key
348 features of the human midbrain to produce and secrete dopamine. PD PDOs carrying
349 LRRK2-G2019S mutation recapitulated main features of the disease with decreased number
350 and complexity of mDANs. In parallel, Kim and colleagues have generated isogenic midbrain
351 hiPSCs-derived organoids by introducing heterozygous LRKK2-G2019S point mutation
352 using CRISRP/Cas9 system to model PD110. Transcriptome analysis of control versus
353 mutant organoids identified thioredoxin-interacting protein as the key factor to mediate the
354 LRRK2-G2019S pathological phenotype110.
355 In addition, iPSC-derived retinal organoids carrying a mutation in CEP290 have been
356 used to model Leber congenital amaurosis, a ciliopathy that leads to inherited blindness. By
357 restoring the expression of full-length CEP290, cilia length and protein trafficking in cilium
358 were restored111. Human PSC-derived lung bud organoids have also been used to model
359 intractable pulmonary fibrosis by introducing mutation in HPS1, leading to accumulation of
360 ECM and mesenchymal cells reminiscent of the features of fibrotic lung disease112.
361 Together, these results highlight the advantage of 3D organoid-based culture system for
362 studying genetic diseases.
363
364 Infectious diseases
365 The 3D organoid technology offers excellent models for the study of host-pathogen
366 interaction in different human infectious diseases involving viruses, bacteria, and protozoan
367 parasites. For instance, cerebral organoids have been recently adopted to study the
368 mechanisms of microcephaly caused by Zika virus infection that showed overall smaller
369 sizes of infected organoids compared to controls, which is consistent with the pathology
370 observed in patients68, 113, 114. Treatment strategies have further been explored in these Zika-
371 infected organoids to prevent the effects of Zika virus infection on neural progenitors115, 116.
372 Intestinal organoids also present valuable models to study a number of infectious diseases.
373 For instance, by using human primary intestinal organoids, scientists suggested that human
374 intestinal tract may serve as an alternative infection route for Middle East Respiratory
375 Syndrome Coronavirus (MERS-CoV), which has caused a major human respiratory infection
376 outbreak in 2012117. Human enteroids (organoids derived from small intestine) have also
377 been used to study norovirus, where Nitazoxanide treatment showed great inhibition of
378 norovirus replication through activation of cellular antiviral response, indicating the
379 therapeutic potential118. Other viral infection studies using organoid systems include
380 rotavirus and enteric adenovirus using intestinal organoids, herpes simplex virus 1 and
381 cytomegalovirus in cerebral organoids, and BK virus infection in human kidney organoids95.
382 Organoids are also increasingly popular for modelling parasitic infections. In 2018,
383 Hans Clevers and colleagues showed that microinjection of Cryptosporidium parvum into
384 human intestinal and lung organoids allows the parasites to propagate within the organoids
385 and complete its complex life cycle, which was not possible previously in conventional 2D
386 culture systems119. Similarly, Toxoplasma gondii has been shown to successfully infect and
387 propagate in bovine and porcine small intestinal organoids120.
388 3D organoid constructs have also been employed to investigate the relationship
389 between infectious pathogens and corresponding cancers. For instance, epidemiological
390 association between Helicobacter pylori and stomach cancers has been investigated
391 through co-culture of the pathogen with gastric organoids31. Similarly, fallopian tube
392 organoids were used to model the long-term impact of Chlamydia trachomatis infections in
393 the human epithelium that may contribute to the development of ovarian cancer121. Other
394 use of intestinal organoids to model bacterial pathogenesis include Escherichia coli, Vibrio
395 cholerae, Clostridium difficile, and Shigella95. Very recently, primary human intestinal
396 organoids have been used to study the genotoxic pks+ E.coli carrying the colibactin-
397 producing pks pathogenicity island122. Long-term exposure of the pks+ E. coli induces a
398 distinct mutational signature that is absent from organoids exposed to the isogenic pks-
399 mutant bacteria. Importantly, the same mutational signature is detected in a subset of
400 colorectal cancer (CRC), implying that exposure to pks+ E. coli may be the direct cause of
401 the mutational signature.

402
403 Cancers
404 For many years, immortalized human cancer-derived cell lines were the fundamental
405 in vitro models for cancer studies. Patient-derived xenografts (PDXs) have subsequently
406 been developed to better model tumor tissue architecture and heterogeneity in vivo. Despite
407 being physiological, PDXs are very costly and time-consuming. The emergence of organoid
408 technology in recent years has opened up an unprecedented approach to model human
409 cancers in vitro. Organoids derived from different mouse or human tumors have now been
410 widely adopted for the study of different types of cancer. CRC organoids were first
411 established from different anatomical sites and displayed distinctive sensitivities to Wnt3a
412 and R-spondin24. Human liver cancer organoids were derived from patients by extensive
413 refinement of medium conditions to expand the three common subtypes: hepatocellular
414 carcinoma, cholangiocarcinoma, and combined hepatocellular-cholangiocarcinoma123. Long-
415 term maintenance and enrichment of pancreatic ductal adenocarcinoma (PDAC) organoids
416 have also been established from mouse and human primary tissues that retain the
417 histoarchitecture and phenotypic heterogeneity of the primary tumors46, 124, 125. In addition,
418 primary breast cancer organoids have been reported to faithfully recapitulate the
419 corresponding parent tumors in morphology, histopathology, hormone receptor status and
420 mutational landscape126. Organoids of other cancer types have also been subsequently
421 established, including gastric127-130, prostate131, ovarian132, brain133, bladder134, kidney135,
422 lung88 and oesophageal cancers136.
423 Alternatively, human cancer can also be engineered by introducing pathological
424 mutations to wild-type organoids using gene editing tools such as gene transfer, CRISPR-
425 Cas9 or RNA interference methods. For instance, colorectal adenoma-carcinoma sequence
426 can be recreated by introducing driver mutations (APC, KRAS, TP53, SMAD4 and PIK3CA)
427 to healthy wild-type organoids and form invasive carcinoma after transplantation137, 138.
428 Further interrogation of different APC truncating mutations in intestinal organoids revealed
429 the critical regulatory region for pathological Wnt activation in CRCs139. Similarly, Seino and
430 colleagues modelled PDAC organoids by engineering driver genes KRAS, CDKN2A,
431 SMAD4, and TP53 via CRISPR-targeting, which revealed an unexpected Wnt niche
432 adaptive response mediated by TP53 mutations124.
433 Unlike 2D cancer cell lines, cancer-derived organoids often retain their tumor
434 heterogeneity and are thus ideal for study of tumor evolution. By comparing organoids
435 derived from primary colorectal tumors and metastatic lesions isolated from the same
436 patients, Fujii and colleagues revealed that these tumors shared the same common origin
437 and driver mutations, implying that the driver mutations precede metastatic dissemination140.
438 Later on, Roerink and colleagues generated clonal organoids derived from multiple single
439 cells from three CRCs as well as from adjacent normal intestinal crypts to study intra-tumor
440 diversification141. Global mutational landscape was used to construct phylogenetic trees,
441 which showed extensive mutational diversification in CRC cells, and that most mutations
442 were acquired during the final dominant clonal expansion of the cancer. Taken together,
443 these 3D organoids present revolutionary in vitro tools for disease modelling, phylogenetic
444 and drug discovery studies.
445
446 Biobanking and precision medicine
447 The long-term expansion capacity of organoids has opened possibilities for
448 biobanking of disease-derived organoids. These biobanks represent valuable resources for
449 clinical applications such as omics analysis for cancer stratification and drug screening for
450 precision medicine. In the past few years, extensive efforts have been made to establish
451 living organoid biobanks derived from many different tumor types, including colorectal140, 142,
452 gastric143, liver123, 144, pancreatic145, breast146, prostate147, lung88, 148, glioblastoma149 and
453 bladder134 cancer. Large-scale genomic and functional analysis from various studies have
454 shown that tumor-derived organoids can faithfully recapitulate the phenotypic and genomic
455 features of the primary tumors both in vitro and in vivo after transplantation46, 126, 131, 142, 150.
456 Importantly, the tumor heterogeneity and clonal dynamics were preserved after serial

457 passaging of PDOs, indicating that these ‘mini-tumors’ are genetically stable with enormous
458 clinical applicability135. With increasing interest of the use of organoids for disease modelling,
459 biobanking can soon be extended beyond cancer, such as intestinal and lung organoids for
460 cystic fibrosis patients and liver organoids for patients with various metabolic diseases.
461 The PDOs also provide unique opportunities to precision medicine through drug
462 screening and drug safety test. Failure of many drug development at clinical trials could
463 partly be attributed to the inadequate evaluation of the drug toxicity at preclinical trial stage.
464 The emerging 3D organoid technology with the ability to grow matched normal and tumor
465 PDOs enables proper assessment of drug toxicity and the possibility to determine the
466 optimal and effective doses that would kill tumor cells with minimal damage to normal tissue.
467 For instance, liver and kidney organoids would be excellent platforms to evaluate potential
468 drug-related hepatic and nephron-toxicity.
469 Another important clinical application of PDOs is to screen for drug responders. In a
470 recent study, a living PDO biobank has been established from patients with metastatic,
471 colorectal and gastroesophageal cancer with the aim to screen for a library of 55 drugs
472 either in phase 1 to 3 clinical trials or in clinical practice such as EGFR, BRAF and
473 PI3K/mTOR inhibitors151. The results showed that PDOs can faithfully recapitulate drug
474 responses and predict clinical outcome in patients. Another study has generated over 100
475 primary and metastatic breast cancer PDOs for high-throughput screening of drugs targeting
476 HER signaling that showed high correlation with clinical drug responses126. Similarly,
477 Broutier and colleagues performed a compound screening on PDOs from hepatocellular
478 carcinoma and identified ERK signaling as a potential therapeutic target for primary liver
479 cancer123. In addition, PDOs have also been used to screen for CFTR modulators100, drug
480 combination strategies152, chemotherapy and radiotherapy responses153, 154. These findings
481 provide supportive evidence that PDOs are powerful unprecedented tools for disease
482 modelling and drug screening, paving the way towards precision medicine.
483
484 Regenerative medicine
485 Currently, organ replacement therapy of diseased or damaged tissues relies largely
486 on allogeneic transplantation. However, the shortage of matched donor tissues and
487 complications of life-long immunosuppression represent some of the major challenges of
488 organ transplantation. The recent organoid technology with high expansion capacity and
489 genetically stable property suggests that PDOs could potentially be explored as alternative
490 treatment strategies to organ transplantation. Following the first establishment of mouse
491 intestinal organoids, Watanabe and collaborators have demonstrated that mouse colonic
492 organoids could indeed be expanded and engrafted into damaged mouse colon and formed
493 functional crypt units155. Similar results were observed using fetal progenitor-derived small
494 intestinal organoids156. Human PSC-derived intestinal organoids have also been
495 subsequently transplanted to mice under kidney capsule and showed crypt-villus structure
496 with permeability and peptide uptake functions, highlighting the translational potential for
497 treatment of short bowel syndrome and other gastrointestinal diseases28. Organoids can
498 also be combined with synthetic or biological (decellularized) scaffolds to engineer intestinal
499 grafts in vitro157-159.
500 Besides intestinal organoids, mouse adult liver organoids have also been shown to
501 rescue liver failure and prolong the survival rate after transplantation into
502 fumarylacetoacetate hydrolase mutant mice, a mouse model for tyrosinemia type I liver
503 disease, or chemically damaged liver39, 41. Similarly, PSC-derived liver organoids were able
504 to rescue acute liver failure and restore the hepatic functions160. Treatment of common bile
505 duct disorders has further been explored by engineering extrahepatic biliary tree using
506 extrahepatic cholangiocyte organoids161. The resulting engineered ducts could reconstruct
507 the gallbladder wall and repair the biliary epithelium following transplantation.
508 In addition, organoids could potentially be combined with gene correction as an
509 alternative approach to treat single-gene hereditary degenerative diseases. For instance, as
510 proof of concept, gene correction of CFTR mutation in PDOs using CRISPR/Cas9 gene
511 editing could repair the CFTR function162. It will be important to explore the therapeutic

512 potential of other single-gene-associated degenerative diseases, such as PD mediated by
513 LRRK2-G2019S mutation, using gene corrected-PDOs.
514 While the potential of organoid applications in precision medicine and regenerative
515 medicine is promising and exciting, it is important to address the safety, ethical and legal
516 concerns before moving to the clinic. One of the major concerns is the informed consent and
517 ownership of the PDOs and the associated commercial interests. It will be important to
518 define how and to what extend organoids are related to donors, and the subsequent
519 governance of any organoid-associated data, such as all the omics data generated from
520 PDOs. When considering the application of organoids in regenerative medicine, it is
521 particularly important to address all the safety and ethical concerns before applying to
522 patients. For instance, a global regulatory consensus of stem cell products and therapies
523 may be needed to resolve the discrepancies of the medical regulations between countries.
524 Open dialogues between scientists, policy-makers and the public are also needed to decide
525 to what extend these technologies should be used in the clinic.
526
527 Strengths and limitations of 3D organoid cultures over conventional models
528
529 Conventional 2D cell or tissue cultures have long been used to model human
530 development and diseases. Despite being widely adopted in many biomedical studies, 2D
531 cell lines are generally considered as non-physiological as they are mostly immortalized and
532 lack tissue architecture and complexity. On the other hand, genetically engineered mouse
533 models (GEMMs) and PDXs are considered to be improved in vivo alternatives to model
534 biological processes of diseases. Although GEMMs are the current workhorse in
535 developmental and cancer research, the production of GEMMs (from design to generation
536 and breeding) often takes years to establish. In addition, GEMMs cannot 100% recapitulate
537 human conditions (e.g. microbiome and diversity), genetics and/or physiology, which may
538 impact their predictive power in assessing clinical outcome. PDXs are another step forward
539 to model human cancer by xenotransplantation of patient material into immunodeficient
540 mice, but the establishment of PDXs is inefficient and time consuming. The newly emerged
541 ex vivo PDOs offer superior alternatives to cell lines, GEMMs and PDXs for disease
542 modelling. Below we discuss the advantages and limitations of the 3D PDOs as compared to
543 other disease models (Table 1).
544 Generation of PDOs are relatively easy once the culture condition is optimized, and
545 can be derived from limited primary tissue materials such as needle biopsies, urine81 or
546 bronchial lavage material88. On the contrary, derivation of cell lines from primary tissues is
547 often inefficient and involves extensive adaptation to the 2D culture conditions, resulting in
548 substantial genetic changes. Compared to immortalized cell lines, organoids are considered
549 superior in recapitulating the 3D architecture, heterogeneity and cell functions of the primary
550 tissues, hence are more physiologically relevant for modelling human diseases and
551 predicting drug response. Other models such as PDXs and GEMMs can better recapitulate
552 human diseases in vivo, yet they are very costly, labor and time-consuming and, therefore,
553 not suitable for high-throughput screening.
554 Although organoid technology bridges the gap between cell lines and in vivo models,
555 there are still limitations of the current system. Despites being heterogeneous, most PDOs
556 lack surrounding stromal cells in the culture, which fail to reconstitute the tumor
557 microenvironment (TME). The TME includes not only the surrounding fibroblasts and
558 endothelial cells, but also immune cells and ECM. Lack of TME in PDOs may perhaps
559 compromise the application to predict clinical outcome. For instance, the response rate to
560 immunotherapy (such as checkpoint blockade) varies among tumor types despite being
561 promising in the clinic. A potential in vitro screening platform will be important to predict the
562 immunotherapy drug response for personalized medicine. However, most PDOs from solid
563 tumors lack TME, and are thus not suitable for such screening. A recent study reported the
564 generation of PDOs from different cancer types using air-liquid interface method that retains
565 fibroblasts and immune cells in the culture, which could potentially be used for personalized
566 immunotherapy testing163. However, the fibroblasts and immune cells of these PDOs

567 progressively decline over a 1- to 2-month period, indicating that they can only be used for
568 short-term disease modelling. Additionally, organoids generated from chordoma patients
569 have also been shown to contain both PD-L1 positive tumor cells and PD-1/CD8-positive
570 lymphocytes, and displayed marked response to nivolumab treatment164. On the other hand,
571 co-cultures of PDOs and peripheral blood lymphocytes have also been explored to assess
572 the efficiency of T cell-mediated killing of matched tumor organoids, while the co-culture
573 efficiency beyond three days has not been tested165. These studies show that, with further
574 optimization, PDOs may have the potential for immuno-oncology investigations.
575 Most organoids are suspended in Matrigel and cultured in media saturated with
576 growth factors. The presence of Matrigel could affect functional/biochemical assays and
577 complicate the cell harvesting and passaging as compared to 2D cell line culture. Also, the
578 enriched growth factors surrounding the organoids may compromise the natural morphogen
579 gradients of the tissues. Spinning bioreactors optimized for brain organoid culture may
580 resolve some of these issues. However, Bhaduri and colleagues have recently shown that
581 cortical organoids ectopically activate cellular stress pathways that impair cell-type
582 specification, thus do not recapitulate distinct cellular subtype identities and appropriate
583 progenitor maturation166. The data suggest that the fidelity of these mini-brain organoids
584 requires further evaluation.
585 Apart from the limitations described above, there are still some practicality issues
586 that need to be addressed before large-scale roll-out to the clinic. For example, the high
587 reagent cost for PDO production makes it unlikely to be affordable by patients or the health
588 care system. Notably, scaling up of PDOs is not as easy as it is in cell lines due to the
589 complex 3D culture system. Finally, developing consistent and standardized drug screening
590 strategies and readout is critical to reliably predict the patient treatment outcome in the clinic.
591
592 Perspectives
593
594 Since the report of the first long-term expansion of ASC-derived organoids in 200920,
595 it has become clear that organoid technology has unique and powerful properties to
596 revolutionize the conventional in vitro research tools for modelling human development and
597 diseases. In particular, organoid studies have bridged the longstanding gaps in
598 developmental biology and precision medicine. The 3D architecture and heterogenous
599 properties of organoids enable us to study cell lineage specifications with spatial and
600 temporal information. The ESC/iPSC-derived organoids have opened up the possibilities for
601 gastrulation studies and regeneration of patient-derived organs, which were largely limited
602 by the use of disorganized EB previously. Importantly, the establishment of PDOs from
603 various disease models has further bridged the studies between basic research and
604 precision medicine by providing more efficient, physiological and reliable models as
605 compared to PDXs and 2D cell lines. Increasing evidence suggests that PDOs functionally
606 recapitulate primary human cancers, which present valuable translational tools for disease
607 modelling, biobanking, drug discovery and precision medicine. However, there is still room
608 for improvement in the current organoid culture. More effort will be needed to standardize
609 the culture protocol and to monitor the tumor heterogeneity after prolonged culture, which
610 can directly affect the drug screening results. It will also be important to develop an improved
611 long-term expansion protocol including the surrounding TME to better recapitulate the
612 primary tumors. In addition, organoids can also be combined with other recent
613 bioengineering tools such as organ-on-a-chip for microfluidic studies167. For instance,
614 microfluidic devices have been used to investigate the behavior of immune cells towards
615 tumor cells168. Several studies have further developed multi-organoid approaches to model
616 the kinetics of metastasis and drug responses169, 170. Further research on the combination of
617 organoid and engineering technologies will open up exciting avenues for the next generation
618 organoid platforms to model more complex human physiology and pathology, as well as to
619 exploit their potential in regenerative medicine.
620
621 Acknowledgements

622 We thank Joe Brock from the Research Illustration & Graphics team for the contributions to
623 the figures. This review is a snapshot of 3D organoid technologies at the current status, and
624 we apologize to many colleagues whose work could not be cited here due to space
625 limitation.
626
627 Grants
628 The authors’ research is supported by the European Union’s Horizon 2020 research and
629 innovation programme (668294) and the Francis Crick Institute, which receives its core
630 funding from Cancer Research UK (FC001105), the UK Medical Research Council
631 (FC001105) and the Wellcome Trust (FC001105).
632
633
634 Disclosures
635 No conflicts of interest, financial or otherwise, are declared by the authors.
636
637 Author contributions
638 L.N., C.C., and V.S.W.L. drafted manuscript; L.N. and C.C. prepared figures; L.N., C.C., and
639 V.S.W.L. edited and revised manuscript; V.S.L. approved final version of manuscript.
640
641
642
643 Table 1: Comparison of different in vitro and in vivo disease models.
644
Characteristics 2D cell lines 3D PDOs PDXs GEMMs
Establishment
Inefficient Easy Inefficient N/A
efficiency
Maintenance time Low Moderate Moderate to high High
Reproducibility High Medium Medium High
Cost Low Moderate to high High High

Self-organize in Conserved.
Tissue 2D constrains Conserved but
3D resembling in Recapitulate
organization morphogenesis murine specific
vivo architecture patient’s tissue
Heterogeneity Homogenous Heterogeneous Heterogeneous Heterogeneous
Conserved and Not always

Cell function Limited Moderate relevant to relevant to
human biology human biology
Preserved except
Stromal
Absent Mostly absent immune cell Preserved
microenvironment
populations
Easy. But could Easy after tissue Easy after tissue
Functional be complicated sampling; sampling;
Easy
analysis by the presence complex in vivo complex in vivo
of matrix analysis analysis
Mediocre
Possible but
Disease modelling Poor Good Good challenging for
some human
diseases

Limited to Limited to
Scalability Easy diffusion of engraftment N/A
nutrients efficiency
High throughput More difficult but
Easy Difficult Difficult
assay possible
Not always
Not very More relevant to More relevant to
Drug screening relevant to
physiological the patient the patient
human diseases
Personalized Not always
Not possible Possible Possible
medicine possible
645
646
647
648
649 Figure Legends
650
651 Figure 1: Timeline for the development of organoid cultures. A summary of key
652 landmark studies and breakthroughs leading to the establishment of various organoid
653 technologies.
654
655 Figure 2: Diverse applications of organoid technology. Schematic diagram summarising
656 various applications of organoids in many areas including developmental biology, disease
657 modelling, precision medicine, regenerative medicine, toxicology, drug discovery studies,
658 host-microbiome interactions, gene editing, multi-OMICs and phylogenetic studies.
659
660

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1291

Gastric Retinal
Breast epithelia organoids organoids
organize into 3D from mouse
ducts and ductules 3D cerebral Cerebral
Dissociation- cortex tissue ES cells. Snake
in ECM extract. Gut organoids organoids
reaggregation from ESCs from hPSCs. venom
experiments with Differentiation of from hPSCs gland
Differentiation of alveolar type II iPSCs from and primary kidney, liver,
dissociated amphibian Pancreatic organoids
pronephros embryoid bodies epithelial cells mouse adult stem
in vitro in ECM extract fibroblasts cells organoids
1907
1944
1960
1961
1981
1987
1998
2006
2008
2009
2010
2012
2013
2014
2015
2020
2011
Dissociation- Embryonic Gut Mammary
Sponge cells Pluripotent Retinal Prostate,
reaggregation stem cell line organoids lung gland,
self-organize stem cells organoids
experiments with established from from adult organoids. fallopian
to regenate a established from from
several organs from human intestinal tube,
whole organism mouse embryos hPSCs Gastruloids
embryonic chick blastocysts stem cells hippocampal
organoids

Precision Regenerative
Medicine medicine
Disease
modelling Toxicology
Development Drug discovery
Phylogenetic Host-microbe
studies interactions
OMICs Gene editing

AJP review- Abbreviations
2D Two-dimensional
3D Three-dimensional
A1AT α1-antitrypsin
AD Alzheimer’s disease
ASC Adult Stem Cell
Aβ Amyloid-β
BMP Bone Morphogenetic Protein
CF Cystic Fibrosis
CFTR Cystic Fibrosis Conductance Regulator
CRC colorectal cancer
EB Embryoid body
ECM Extracellular Matrix
EGF Epithelial Growth Factor
EHS Engelbreth-Holm-Swarm
ESC Embryonic Stem Cell
FGF Fibroblast Growth Factor
GEMM Genetically Engineered Mouse Model
GI Gastrointestinal
GIP Gastric inhibitory polypeptide
HMIA Hereditary Multiple Intestinal Atresia
iPSC Induced Pluripotent Stem Cell
LGR5 Leucine-rich repeat containing G protein-coupled Receptor 5
LGR6 Leucine-rich repeat containing G protein-coupled Receptor 6
mDAN Midbrain Dopaminergic Neuron
MERS-CoV Middle East Respiratory Syndrome Coronavirus
NFT Neurofibrillary tangles
NPC Neural Progenitor Cell
PD Parkinson’s disease
PDAC Pancreatic Ductal Adenocarcinoma
PDO Patient-Derived Organoid
PDX Patient-Derived Xenograft
PSC Pluripotent Stem Cell
RA Retinoic Acid
TME Tumor Microenvironment
TTC7A Tetratricopeptide Repeat Domain 7A
WNT Wingless-related integration site
Y-27632 Rho Kinase Inhibitor

2020 A Brief History of Organoids

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1 A brief history of organoids

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Maintenance time Low Moderate Moderate to high High

Reproducibility High Medium Medium High

Cost Low Moderate to high High High

Heterogeneity Homogenous Heterogeneous Heterogeneous Heterogeneous

Conserved and Not always

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Development Drug discovery

OMICs Gene editing

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