Dawn of the organoid era Prospects & Overviews Dawn of the organoid era 3D tissue and organ cultures revolutionize the study of development, disease, and regeneration Ninouk Akkerman and Libert H K De[.]
Prospects & Overviews 3D tissue and organ cultures revolutionize the study of development, disease, and regeneration Ninouk Akkerman and Libert H.K Defize Novel and updated approaches of culturing cells in 3D are rapidly advancing our understanding of development, health, and disease As tissues have been found to behave more realistically in 3D than in 2D cultures, organoid technology in combination with recent advances in the isolation and generation of stem cells, has rapidly become a promising concept in developmental and regenerative research The development of all kinds of tissues can now be studied ‘‘in a dish,’’ allowing more detailed observations of stem cell maintenance, morphogens, and differentiation This review explores how organoids have revolutionized academic research over the last decades, and how they may continue to so It also addresses remaining hurdles in 3D cell culturing, and how they may be overcome Keywords: 3D tissue culturing; cancer; development; organoids; stem cells; tissue regeneration Introduction It has been a long-standing aim of biologists to unravel the processes governing the formation of functional tissues and organs However, the complexity of this process has made it extremely difficult to develop reliable in vitro model systems DOI 10.1002/bies.201600244 University College Utrecht, Utrecht, The Netherlands *Corresponding author: Libert H.K Defize E-mail: l.h.k.defize@uu.nl Abbreviations: ESC, embryonic stem cells; iPSC, induced pluripotent stem cell to study it At best, a couple of different cell types residing in the tissue of interest could be grown in a flat layer in a dish, doing little to recapitulate the real-life structure and functionality However, a modern method of culturing is rapidly augmenting the possibilities in developmental and regenerative research: organoid technology, by which stem cells can be grown in three dimensions, generating rudimentary organ-like structures Organoids can be generated in a number of ways, which have the common property that cells are not restricted and molded to a flat surface, such as a petri dish This allows them to engage in cell-cell and cell-matrix interactions, which are lacking in a 2D environment This cross-talk appears to be essential for mimicking in vivo situations, as without cell-cell and cell-matrix interactions cells may lose their distinctive phenotype and react differently to cues from the outside [1–3] Organoids, thus, allow the spatial organization of heterogeneous tissue-specific cells in a manner that closely resembles real-life composition and functionality Initially, growing 3D tissues was limited to transformed cell lines [2], but due to pioneering work on stem cells [4, 5] it is currently revolutionizing our understanding of development and regeneration, as shown by the generation of cerebral [4, 6], intestinal [5, 7], gastric [8, 9], kidney [10–12], retina [13, 14], pituitary gland [15], inner ear [16], liver [17, 18], pancreas [19], breast [20], heart [21], lung [22], and embryonic organoids (gastruloids) [23–27] Although still small in size (about mm in diameter, at max), organoids have great promise for developmental and regenerative research, being relatively stable model systems of animal and human organs and tissues that are amenable to long-term cultivation and manipulation [25, 27, 28] The fact that they recapitulate native human tissue so well makes them attractive substitutes for animal models; these, although similar to humans to a certain extent, remain fundamentally different in many aspects [6, 29] Moreover, access to laboratory animals is limited for ethical reasons, which impedes research in many instances Now that stem cells can be coaxed to produce realistic mini-organs, a lack of available animals may no longer yield an impasse Furthermore, it appears we are making the first crucial steps towards Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited www.bioessays-journal.com 1600244 (1 of 10) Review essays Dawn of the organoid era Prospects & Overviews Review essays N Akkerman and L.H.K Defize Figure General method of growing organoids from stem cells with different origins growing human tissues and organs, which may in time alleviate the pressing shortage of donors This review will describe recent developments in organoid technology and will critically assess their impact on our understanding of developmental and regenerative biology, with special emphasis on the remaining hurdles, while offering putative solutions As such, it offers a broad overview of the field For more in-depth analyzes of topic covered, the reader is referred to a number of excellent reviews that have recently appeared [23, 30–44] Generating organoids Unlike other organisms, the early development of humans cannot be studied in vitro, hence organoids offer a great opportunity to investigate uniquely human properties This has encouraged researchers to embrace organoid technology, fine-tuning it for many types of cells and tissues Organoids have been generated from adult stem cells (ASCs) [8, 9, 18, 45], embryonic stem cells (ESCs) [11, 46, 47] as well as induced pluripotent stem cells (iPSCs) [10, 46] (Fig and “Box 1”) The latter two require a more extensive procedure of coaxing, as they are pluripotent For instance, to generate intestinal stem cells from ESCs or iPSCs, the cells are first treated with activin A to obtain definitive endoderm, after which specific signaling molecules are employed to steer the cells to a front- mid- or hindgut fate [7] A clear advantage of using these stem cells as a source, is that they can be used to generate a wider variety of tissues and may be easier to obtain (the latter is true particularly for iPSCs) Two main approaches are employed for the generation of 3D structures: scaffold and scaffold-free techniques [21, 48, 49] The former utilizes natural or synthetic hydrogels to recreate the extracellular matrix, the most common of which is Matrigel (see “Box 1”), and the cultures may be maintained in a bioreactor that spins to provide a uniform environment and facilitate 1600244 (2 of 10) nutrient exchange In the latter, cells are cultured in drops of a defined culture medium that facilitates growth, either hanging from a plate sustained by gravity and surface tension [50, 51], or on low adhesion plates [23, 50, 52] Another method is to culture on cells on inserts that are submerged in medium, which gradually evaporates, termed “air-liquid -interface” [23, 53, 54] “Box 1” This way, the culture is partly exposed to air, which may allow for more polarized or heterogeneous structures to form [23, 51, 54, 55] The culture medium can be supplied with specific growth factors and other signaling molecules, depending on the type of starting material and the tissue that is aimed to be generated It is of vital importance that the agents as well as nutrients and oxygen can diffuse throughout the structure, which puts constraints on the size of the organoids that can be grown One of the greatest puzzles currently being solved is how to equip organoids with their own vascular system, either by means of seeding endothelial precursors or some form of synthetic microchannels [28, 42] Organoids as models of development Among the earliest and most diverse tissues to be grown in organoid-context are those comprising the digestive tract Cells of the small intestine grow in crypts and villi, with the stem cells responsible for renewal of the intestinal walls’ lining residing near the bottom of the crypts [56] Up until recently, it was impossible to model the intestinal morphology and physiology in vitro, but in 2009 Clevers et al succeeded in doing so They suspended single mice intestinal stem cells in laminin-rich Matrigel and supplied the cultures with epidermal growth factor (EGF), R-spondin (a Wnt agonist) and Noggin, which allowed them to develop into multiple crypts and villus-like domains, containing all differentiated cell types present in the mouse small intestine Impressively, the organoids could be passaged and maintained for over months without losing their characteristics, strongly resembling newly isolated crypts [5] Spence et al continued this research and generated intestinal organoids from human ESCs and iPSCs These model systems proved their usefulness by allowing the researchers to discover the signaling molecules required for hindgut specification (Fgf4 and Wnt3) and morphogenesis (Fgf4 only) [7] Barker et al created gastric organoids and performed lineage tracing to identify the stem cells (Lgr5 ỵ ) maintaining the gastric epithelium [9] and in a similar manner Stange et al found a marker (Troy þ ) for chief cells capable of regenerating gastric units upon tissue damage [8] Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc Prospects & Overviews N Akkerman and L.H.K Defize Box Review essays Key terms Organoid culture Three-dimensional organ-like structure, which selforganizes in vitro from stem cells or biopsies, yielding organ-specific cell types, that can demonstrate organspecific functions [42] Adult stem cells Undifferentiated (multipotent) cells found throughout the adult body, which are capable of self-renewal and generating progeny of the cell types that are found in the organ of origin [110] Embryonic stem cells Pluripotent stem cells, i.e cells that can generate all three germ layers, which are derived from the inner cell mass of a blastocyst [111, 112] Human embryonic stem cells are obtained from the surplus of embryos that is generated in in vitro fertilization procedures, with consent of the donors [113] Induced pluripotent stem cells Differentiated cells that are “reprogrammed” to pluripotency by reactivating specific pluripotencyassociated genes, most commonly Oct 3/4, Sox2, Klf4, and Myc [114] Matrigel1 Heterogeneous and gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells [115] Resembles the extracellular environment in many tissues Main constituents: adhesive proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans Growth factors such as TGFb and EGF are present to prevent differentiation and stimulate proliferation A growth factor-reduced version is available as well [116] Air-liquid interface Culture method in which cells are seeded on a basal layer containing fibroblasts or Matrigel, which provide adhesion to the stem cells that are seeded on top The cells are submerged in medium, which evaporates over time, exposing the upper cell layers to the air and allowing gradual differentiation [23, 53, 54] Figure Cerebral organoid showing distinct populations of neural stem cells (Sox2 in red) in the ventricular zone, and neurons in the outer layer of the cortex (Tuj1 in green) DAPI stains the nuclei (blue) Image courtesy of Madeline Lancaster produces the neural tube and ventricles [57, 58] Morphogen gradients of Sonic hedgehog (Shh)-Wnt/Bmp and retinoic acid (Ra)-Fgf determine the ventral-dorsal and rostral-caudal axes, respectively [59, 60] Neurogenesis occurs in an inside-out manner, with the early formed neurons residing in the center and later neurons migrating through the early structures to generate for instance the optic tectum and layers of the cerebral cortex [60] These properties are well recapitulated by the organoids generated from embryoid bodies, 3D aggregates of pluripotent stem cells [6] When embedded in Matrigel, these structures develop into buds of neuroepithelium which expand to form several brain regions and show the characteristic layering of the cortex, without the need of additional growth factors [4, 6] (Fig 2) Dorsal neural brain tissue appears to be the “default” fate of undifferentiated neuroectoderm [61–64], but applying the appropriate growth factors at the correct time allows the development of specific brain structures, such as cerebellar and hypothalamic tissues [42] Kidney organoids recapitulate the kidney’s complexity in detail Brain organoids mimic cortical architecture Being arguably the most complex and enigmatic organs of the human body, brain structures have featured many a time as the subject of organoid genesis Animal models have provided a fundamental understanding of brain development, yet the most intricate details cannot be easily extrapolated to humans, a gap in understanding that organoids may help to fill The central nervous system (CNS) arises from the neural ectoderm, which through rigorous folding and fusing Kidney organoids have also been successfully generated [10, 11, 46, 65] In vivo, the kidney develops from the intermediate mesoderm (IM) through Fgf and Wnt signaling, forming the ureteric bud and the metanephric mesenchyme The final result is a complex organ with nephrons containing intricate systems of glomeruli and tubules As the kidney has a poor self-regenerative capacity, the source for modeling kidney development are generally ESCs or iPSCs Takasato et al were among the first to generate renal tissue in 3D [11], Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc 1600244 (3 of 10) Review essays N Akkerman and L.H.K Defize Prospects & Overviews which they derived from human ESCs, guiding them through primitive streak formation by creating a gradient of activin A and Bmp4 and supplementing a canonical Wnt agonist Exposure to Fgf9 gave the cells an intermediate mesoderm identity, and the cells subsequently differentiated into ureteric bud and metanephric mesenchyme Importantly, when hESC cultures were allowed to grow in 3D after IM induction, they developed more advanced structures, reminiscent of early nephrons [11] Xia et al took a different approach, developing their own protocol of growth factor exposure to prime human iPSCs and ESCs to become ureteric bud progenitor-like cells Dissociation of the cells and mixing with murine embryonic kidney cells resulted in reaggregation and the formation of chimeric ureteric bud structures, while allowing nephrogenesis to occur as normal [46] Taguchi et al used lineage tracing to define the conditions for the development of the metanephric mesenchyme, which generates most of the kidney components They used their findings to develop a robust protocol for generating metanephric mesenchyme from iPSCs, which in 3D develop into nephric tubules and glomeruli Upon transplantation in immunodeficient mice, these were successfully vascularized by the host [10] Recently, Takasato et al published a refinement of their differentiation protocol, more precisely balancing the patterning along the anterior-posterior axis with small molecules This way, they generated renal organoids containing nephrons with distal and proximal tubules, loops of Henle, and glomeruli equipped with podocytes Impressively, these organoids even showed indications of vascularization, and all anticipated kidney components were present [65] Gastruloids shed light on mammalian embryonic development Mammalian embryonic development has been particularly hard to study, as the major part of it takes place within the uterine wall of the mother Although genetic research has identified mutations that disrupt normal development and connect these to the molecular processes at work, the precise patterning process remains incompletely understood [66, 67] Embryonic organoids, or Gastruloids, have been generated to shed light on this matter [24–26, 68] Introduced by Van Den Brink et al., these clusters of about 300 mouse ESCs recapitulate early embryonic development, including symmetry-breaking, germ layer specification and the formation of the body axes [25, 26, 68] Turner et al employed mice Gastruloids to study the initial symmetrybreaking of an embryo, which establishes its anterior– posterior axis [24] In vivo, this process depends on interacting Nodal and Wnt (originating from the epiblast) and Bmp (from the extraembryonic ectoderm or trophoectoderm) signaling [69–71] Gastruloids, however, not contain extraembryonic tissue and hence lack Bmp signaling, yet they show axial organization It appears that the embryonic tissue of the Gastruloids is intrinsically capable of patterning, independent of extraembryonic tissue Turner et al suggest that the Bmp from the trophoectoderm does not initiate, but rather biases and 1600244 (4 of 10) stabilizes the intrinsic capacity of the epiblast to break the embryonic symmetry [25] Since mice Gastruloids develop in a time-scale similar to mouse embryos and are highly reproducible, they provide an excellent model system for studying early developmental processes [25] Organoids help define morphogen gradients, aiding the improvement of protocols for organoid generation Organoid models have great potential to help uncover developmental processes, as stem cells can be studied stepwise while they differentiate, form niches and give rise to the various cell types of the tissue [5, 6, 8–10, 18, 19, 22, 24, 25, 47, 56, 68, 72–74] Several methods can be employed to gain insight into mechanisms involved in tissue development and regeneration (Fig 3A) One of the first things to be considered is whether or not to supply growth factors, and if so, at what time and concentration For instance, by minimizing exogenous growth factors, Lancaster et al generated heterogeneous cerebral organoids, which allows the conserved developmental pathways of the stem cells to be studied [6, 75] Here, the manner in which morphogen gradients arise spontaneously could be studied On the other hand, by mimicking endogenous levels of Shh or Bmp4/Wnt3, for example, tissue with specific identities can be generated, such as ventral forebrain or cerebellar tissue [76, 77] Systematically varying the levels and timing of application of these growth factors can help to deduce their functions in developmental patterning Lineage tracing is an invaluable technique in this respect, as visualization of stem cells and their progeny helps to discover the patterns in which they develop and replenish the tissue This has been successfully applied to study lung [22, 72], liver [18], stomach [8, 9], small intestine [5, 56, 73], colon [56], kidney [10, 47], pancreas [19, 74], and brain tissue [6] and will undoubtedly continue to contribute to key insights in the mechanisms of tissue development and regeneration Organoids as models of disease Human diseases represent another subject of research that can benefit from 3D modeling with organoids By providing us with a more representative model system, the use of organoids may in time reduce the amount of animals currently needed for research and drug screening [42] Thus, patient-derived iPSCs can be employed to obtain a model of the disorder or disease of interest [6, 78], allowing for highly specific (developmental) disease modeling, while, importantly, not all genetic and epigenetic characteristics of the particular disease are required to be known in advance in order to create realistic model systems Especially in the fields of neurodevelopmental and psychiatric disorders the use of organoids has started to flourish, understandably so, as these diseases are commonly multifactorial, and particularly difficult to capture in animal models [6, 78] For instance, cerebral organoids derived from iPSCs from a microcephaly patient have helped to identify a loss-of-function mutation that results in premature neural differentiation [6], an effect that could be Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc Prospects & Overviews N Akkerman and L.H.K Defize Review essays Figure Applications of organoids A: Types of applications in developmental research, including “default” development (in minimal media), the study of morphogens, stem cell identification, and tissue replenishment B: Applications in the study of developmental disease, including the effects of infection, hereditary diseases, and mutations C: Applications in regenerative therapy, including tissue replenishment in case of damage or disease ZIKV, Zika virus; iPSCs, induced pluripotent stem cells; ASCs, adult stem cells; PKD, polycystic kidney disease; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, caspase 9; CFTR, cystic fibrosis transmembrane conductance regulator rescued by reintroducing the protein encoded for by the defective gene In a similar vein, organoids generated from a patient with autism spectrum disorder (ASD) have allowed researchers to pinpoint a transcription factor (FOXG1), which when overexpressed causes an increase in GABAergic inhibitory neurons [78] Finally, brain organoids have been used to study the effects of Zika virus infection [79], which resulted in a 40% growth reduction of the neural tissues These studies strongly indicate that organoids can enhance our understanding of not only the developmental abnormalities themselves, but also on their possible treatment, even in cases of polygenic disorders If specific mutations are hypothesized to be responsible for a certain disorder, the role of these mutations can also be studied in organoid models, through loss- and gain-offunction mutations CRISPR-Cas9 [80, 81] is a particularly helpful tool to create gain- and loss-of-function mutations, and short hairpin RNA (shRNA) [82] or RNA interference [83] may be used for modifications on a post-translational level For instance, in a polycystic kidney disease (PKD) model, loss-of-function of PKD1 or PKD2 caused cyst formation in the organoids, confirming their importance in cystogenesis in PKD [47] Furthermore, specific mutagenesis in organoids has already been applied to investigate the molecular basis of congenital anomalies of the intestine [7] and lung [22] Finally, organoids contribute to our understanding of cancer [39, 84–90] Neoplastic cells grown in 3D represent a type of culture midway between the 2D petri dish and the xenograft, but are more physiologically relevant than cell lines and are more accessible for high-throughput screening than xenografts [40, 84, 85] Cancer organoids are not only generated from stem cells, but also from tumor biopsies, directly, by embedding these into Matrigel [84, 85] Additional heterogeneity can be achieved by co-culturing with healthy cells which reside adjacent to the in vivo tumor [85] Excitingly, it was recently reported that DMSO-frozen tumor tissue is a reliable substitute for freshly isolated biopsies, yielding viable organoids with similar morphology, metabolism, coenzyme environment, and drug responsivity as organoids from fresh biopsies [86] This expands the availability and applicability of tumor organoids in research, as fresh tumors are not always easy to come by Currently, the model systems are intensively put to use in uncovering the progression and invasion of various tumor tissues, including prostate [87, 88], colorectal [84, 89], pancreatic cancer [90] and glioblastomas [39] Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc 1600244 (5 of 10) N Akkerman and L.H.K Defize Prospects & Overviews Review essays Therapeutics Besides aiding in the understanding of the molecular mechanisms of tumorigenesis and metastasis, organoids are also being proposed for therapeutic screening For this purpose, Van de Wetering et al are developing a “living organoid biobank,” containing patient-derived model systems representing a wide repertoire of colorectal cancer subtypes Already, they have generated twenty colorectal carcinoma organoids, each from different patients The ultimate goal is to enhance the efficiency of high throughput drug screening and allow the design of personalized treatment [84] Moreover, organoids could advance regenerative medicine, as transplants with a certain tissue integrity may be integrated into patients’ own tissue more successfully than single stem cells [10, 42, 91] In mice, this possibility has already been explored for liver [17], pancreatic, and kidney buds [92], which upon transplantation mature into wellvascularized structures that function in a tissue-specific manner In the future, this may even allow autologous transplantation with organoids, limiting tissue rejection Even “corrected” organoids could be considered for transplantation, such as intestinal organoids of cystic fibrosis patients in which the cystic fibrosis transmembrane receptor (CFTR) mutations have been repaired [93] As the shortage of donor organs is immense and only ever-increasing [94], these types of novel tissue regeneration techniques are highly anticipated (Fig 3C) Problems and prospects 3D tissue culturing offers a wide range of utilities in cell biological research Organoid technology can complement and validate animal models, as it can identify factors that are specific to human beings or instead conserved across species The optimal growing conditions for a variety of tissues have already been described, providing the possibility to generate stable model systems that are amenable to extended cultivation and manipulation Yet, several challenges regarding the size and maturation of the model systems need to be resolved in order to bring this technology to its full potential Sufficient nutrients and efficient supply are essential for organoid growth and sustenance The major difficulty that is being dealt with at the moment is the matter of how to optimize organoid growth conditions The size of the cultures is limited by the maximum distance of diffusion of nutrients and particularly oxygen Mathematical models suggest that for cerebral organoids the maximum diameter is 1.4 mm, and indeed larger sizes are accompanied by cell death in the center regions of the organoids [6, 95, 96] One method to increase oxygen availability within the organoids is to use experimental conditions with higher oxygen levels [4], but this can cause dangerous situations if not controlled carefully [97] Some researchers use spinning bioreactors to facilitate higher extents of diffusion, but in order to grow markedly larger structures, vascular networks 1600244 (6 of 10) would be needed It is not yet possible for organoids to develop their own vascular network, but seeding endothelial cells along with the stem cells has yielded some promising results (neoangiogenesis) [17, 31, 92] Another option would be to equip the cultures with synthetic vessels or scaffolds, as is done in microfluidics and bioprinting Although this technique allows the perfusion of larger structures, adapting it to the shape, function and physiological needs of specialized tissues represents another issue [28] However, vascularization seems to be less of a challenge in transplantation, as multiple studies have shown that transplants are effectively innervated by the host vascular network [17, 92] The medium contributes to (the lack of) organoid differentiation A related matter is one of tissue maturation: Although a high level of tissue heterogeneity can be achieved, it is not always the case that all cell types of the tissue of interest can be reproduced in the organoid Moreover, though the cultures can be grown and maintained for several months, it is unclear to what extent they can mature in vitro This puts limits on their value for developmental research, as organoids may recapitulate the first months of development, but not the stages beyond These issues may be related to the most common environment that is used for organoids to grow: Matrigel This gelatinous protein mixture is rich in growth factors that stimulate proliferation and inhibit differentiation These properties have great advantages for the continued growth and expansion of organoids, but if one has the goal to study more differentiated tissues, it could be wise to consider alternatives [98] It is remarkable that there are, in fact, no particularly advanced commercial substitutes available Obtaining ECM material from tissue-specific environments is possible, but the composition will unavoidably vary from batch to batch, which is undesirable with regard to reproducibility Again, synthetic materials may be a solution [28, 99] Hydrogels equipped with synthetic or natural polymers for cell adhesion and properties such as porosity and stiffness are being developed for this purpose [100–102] Although the complexity of these engineered ECMs is still low in comparison to Matrigel, interesting advancements have been made that even surpass the utility of this conventional substrate [31] For instance, a hydrogel that softens due to light exposure allows tuning the mechanical properties of the gel, even locally, to generate gradients of stiffness [103] This could be used to mimic mechanical stimulation in organoids, and may facilitate the growth of morphological features that are still missing, such as the villi in intestinal organoids [28, 98] Growth factors supplied more precisely may yield more reproducible and realistic organoids Another technical factor that could improve the validity of organoids is a more precise way of growth factor delivery, creating gradients that can guide development in a manner that is true to nature In vivo, development of complex body Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc Prospects & Overviews N Akkerman and L.H.K Defize Cell-cell and cell-matrix interactions in organoids add to their complexity Figure Intestinal organoids demonstrate Wnt gradients arising from the bottom of the crypt-like compartments, which gradually diminish towards the inner mass of the organoid Confocal immunofluorescent microscopy picture, Wnt3 labeled green, and EPCAM membrane staining in blue Image courtesy of Henner Farin structures is governed by morphogen gradients For instance, the dorsal-ventral axis of the neural tube is patterned by opposing gradients of TGF-b family proteins and Shh Depending on the relative concentration of these morphogens, different sets of genes are expressed along the axis, with the most ventral part expressing the transcription factors Nkx6.1 and Nkx2.2, the most dorsal part expressing Pax6, and the intermediate region producing Olig2 and Pax6 These initial differences set in motion further accumulation of distinctions in gene expression, specifying the ventral neurons, motor neurons and interneurons of V1 and V2 [104] In generating organoids, it is attempted to mimic the endogenous patterning by supplying growth factors to the medium in varying concentrations and times Although this has yielded organoids that in certain cases contain all expected cell types, a full-sized organ is still far away This has been suggested to be due to the lack of body axes to pattern the tissue [96], which are relevant for the spatial distribution of morphogens and thus for the correct positioning of tissues within the developing organism As a result, organoids generally consist of randomly positioned tissues [96] The exception to this rule are Gastruloids, which develop all three embryonic axes and hence develop in a predictable and reproducible way [25, 68] The lack of morphogen gradients and body axes to pattern organoids thus appears to prevent them from developing in a reproducible manner This constraint may be overcome if growth factors are not only supplied in a time- and concentration-, but also location-specific manner Currently, in most laboratories growth factors are added to the medium that surrounds the organoid, but this does not result in realistic, polarized patterns of morphogen gradients The use of bioreactors may even distribute them more evenly across the culture Interestingly, Gastruloids, even when stimulated homogeneously with specific factors, still appropriately form the embryonic axes, which makes them unique with respect to other organoids Once the issue of adequate nutrient delivery is solved, formation of more complete organ-like structures may be achieved by more precise localized administration of growth factors (for example via morphogen-secreting beads) A continued issue in organoid technology is the trade-off between generating as natural as possible, self-organizing structures, and maintaining control over their composition As the complexity of these culture systems is much higher than in simple petri dishes, keeping track of, and control over the constituents of the organoids is difficult To a certain extent, the supplementation of growth factors allows us to specify the fate of the tissue, however, cell-cell and cell-matrix interaction are also crucial in differentiation Interactions between adhesive epithelial and more loose mesenchymal cells, as well as transitions between the two, provide the basis for the formation of the kidney and brain, for instance Extracellular matrix components are indispensable in this context, as they mediate cell adhesion However, they are more than just anchors for cells: They can serve as binding sites for growth factors (proteoglycans) and guides for cell migration (fibronectin, laminin) [104] In organoids, this cross-talk between cells and the matrix occurs as well, for instance via integrin receptors that bind to Matrigel components [13] A permissive 3D environment such as Matrigel serves as a starting point for cell-cell and cell-matrix interactions to ensue This way, it is possible that patterns and gradients of morphogens arise in organoids, just like in embryos, which allows the developmental blueprint to be studied in vitro In accordance with this, Wnt gradients arise in Gastruloids in patterns reminiscent of those in embryos [25, 26] Furthermore, neuroepithelial 3D cultures can spontaneously produce opposing gradients of COUP-TF1 (posterior identity) and Sp8 (anterior identity), which can be modulated by administering Fgf8 [108] In vivo, this patterns the anteroposterior axis of the brain [109] In intestinal organoids, Wnt gradients arise from the crypt-like structures, where the stem cell reside, to the more differentiated cells at the villi-like compartments [73] (Fig 4) These results show that realistic cell-cell and cellmatrix interactions can and appear in organoids, which further strengthens their reputation as being truthful model systems Ironically, this key strength of organoids as a model system, cell-autonomous self-organization, is also one that impedes the reproducibility of the studies in which they are employed Even within a highly systematic experiment, but particularly between preparations, the morphology and composition of organoids may differ because of deviations in nutrient distribution [96] By including appropriate control cultures, preferably within the same medium, major differences in morphology and cellular composition can be ruled Bioessays 39: 1600244, ß 2017 The Authors BioEssays Published by WILEY Periodicals, Inc 1600244 (7 of 10) Review essays to approximate the axes that develop in vivo Again, advancements in hydrogels may facilitate improvements in the years to come; light- or bioresponsive gels may allow more control over the graded release or activation of growth factors within the organoid culture [99, 105–107] Review essays N Akkerman and L.H.K Defize Prospects & Overviews out In order to allow replication and expansion of published results, it is crucial that optimized protocols for generating tissue-specific organoids are available Indeed, protocols for various tissues such as cerebral cortex [96], kidney [46], prostate [87], liver [17], kidney and intestine [92] have already been published, and more will undoubtedly follow Conclusion and outlook Recognition of the importance of environmental factors has revolutionized our view of development, structure, and functionality Organoids provide evidence that the genome is not the sole determinant of an organism’s final characteristics Rather, the development of organs, tissues, and cells depend on many more factors, of which the environment is a major one [23] 3D tissue culturing allows us to examine the “default” development of tissues, morphogens, stem cells and regeneration in novel, exciting ways In the future, larger and more complex organoids may be produced, as the methods for nutrient supply improve Furthermore, the development of synthetic growth media may facilitate a more reliable administration of growth factors and increase the reproducibility of organoid generation In any case, the fact that organoids have already proven incredibly helpful in discovering developmental and disease-related pathways bears great promise for the years to come Acknowledgments The authors would like to thank Madeline Lancaster for providing Fig and Henner Farin for providing 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