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Modeling human development in 3D culture
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ScienceDirect Modeling human development in 3D culture Marius Ader and Elly M Tanaka Recently human embryonic stem cell research has taken on a new dimension — the third dimension. Capitalizing on increasing knowledge on directing pluripotent cells along different lineages, combined with ECM supported threedimensional culture conditions, it has become possible to generate highly organized tissues of the central nervous system, gut, liver and kidney. Each system has been used to study different aspects of organogenesis and function including physical forces underlying optic cup morphogenesis, the function of disease related genes in progenitor cell control, as well as interaction of the generated tissues with host tissue upon transplantation. Pluripotent stem cell derived organoids represent powerful systems for the study of how cells selforganize to generate tissues with a given shape, pattern and form. Addresses DFG-Center for Regenerative Therapies Dresden (CRTD), Technische Universita¨t Dresden, Fetscherstr. 105, 01307 Dresden, Germany
objects, important aspects influencing organogenesis are missing in conventional 2D cultures and thus such approaches cannot fully recapitulate in vivo development and function. Indeed, pioneering work initiated more than 30 years ago started to use collagen gels and extra cellular matrix (ECM) matrices to provide 3D scaffolds for epithelial cells of diverse origins (e.g. mammary gland/breast cancer; liver, skin/melanoma; smooth muscle; adipocytes; prostate/malignant prostate), that allowed the formation of so-called 3D spheroids. Importantly, such matrices not only provided structural support but influenced cell–cell interactions via transmembrane receptors leading to changes in cyto-skeleton organization, chromatin modulation, and gene expression [1].
Introduction
In parallel, during the last three decades stem cell research has blossomed and has had a major impact on most fields of biological research including developmental-, regenerative-, or cellular studies. Specifically, the isolation and in vitro propagation of pluripotent embryonic stem cells (ESCs), first reported from mouse [2] and later also from human [3] blastocysts, tremendously changed the field of developmental biology by providing practically an unlimited source of cells with the potential to differentiate along the three main lineages of the body. With the advent of induced pluripotent stem cells (iPSCs) generated from somatic cells by introduction of pluripotency genes [4,5,6] a further major step for the generation of unlimited material, that can be isolated from a variety of humans and patients for studying human development and disease, are now available. Finally, defined procedures to control the differentiation of these cells along each of the three major lineages to generate cell types in two dimensional culture has been a major step forward in controlling the enormous potential of the system.
The development of organs and tissues from the fertilized oocyte represents a highly complex but well organized system. Studies using animal models, cell- and organotypic cultures have significantly increased our knowledge about fundamental mechanisms for organogenesis. However, continuous and detailed analysis of human development over time with its network of cell– cell interactions, cell fate decisions and differentiation steps besides inducing chemical-, genetic- and environmental factors is challenging due to limitations in human tissue availability. Thus, cell culture methodologies using in vitro expandable cell lines are widely used to study cellular and molecular events in lineage fate decision, differentiation and maintenance of human cells. However, as organs and tissues represent 3D
Previously, three-dimensional differentiation of PSCs was performed under relatively uncontrolled conditions to generate embryoid bodies in which cell aggregates formed tissues of several different germlayers within one aggregate. Such structures may be considered akin to teratomas that are formed when PSCs are injected subcutaneously into rodent models. This impressive ability of PSCs to self-organize into three-dimensional tissues has recently been harnessed for the formation of defined human tissues and organoids by combining 3D culture techniques with directed PSC specification protocols. Considering the possibilities to rapidly generate reporter cell lines in PSCs, as well as the ability to generate PSCs from individuals with defined genetic disorders, these
Corresponding authors: Ader, Marius ([email protected]) and Tanaka, Elly M ([email protected])
Current Opinion in Cell Biology 2014, 31:23–28 This review comes from a themed issue on Cell cycle, differentiation and disease Edited by Stefano Piccolo and Eduard Batlle
http://dx.doi.org/10.1016/j.ceb.2014.06.013 0955-0674/# 2014 Elsevier Ltd. All rights reserved.
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Current Opinion in Cell Biology 2014, 31:23–28
24 Cell cycle, differentiation and disease
methods will revolutionize not only regenerative medicine approaches for which they are often envisioned, but will open up new horizons for studying the cell biology of human organogenesis in structures including the brain, gut, kidney and liver. Here we describe some of the major recent developments in human 3D organoid formation, including the strategies that have led to reconstitution of complex tissue architecture, as well as the application of these organoids to address previously inaccessible questions on cell differentiation and tissue morphogenesis.
Neuroectoderm: cortex and retina The central nervous system (CNS) and in particular the brain, is one of the most accessible fate choices of ESCs. Recently the vast experience in directing pluripotent cells toward neural fates in two dimensions has been combined with three-dimensional culture in matrigel to produce various defined subregions of the developing mouse and human CNS including the cortex, retina, subpallium and adeno-hypophysis. Such organoids have been used to study the physical forces underlying retinal cup formation, species-specific dimensionality of eye tissue, as well as factors underlying progenitor cell control in the cortex.
Cortical organoids reproduce progenitor subtypes and layered structure The cortex represents by far the biggest structure (approx. 75%) within the human brain. Its complex architecture is tightly regulated during development with the formation of distinct progenitor layers that eventually build up the six-layered adult cortex. Directed differentiation of human PSCs in two dimensional culture toward cortical neuron fate revealed a ‘default’ pathway of neural differentiation when PSCs are cultured under minimal conditions in the absence of exogenous morphogens and supplementation either with the bone morphogenetic protein (BMP) inhibitor Noggin [7,8] or with retinoids in combination with SMAD inhibitors [9]. Under these conditions, cortical pyramidal neuron specification followed the temporal and sequential order seen in vivo. By adapting culture principles established first with mouse ESCs the Sasai group pioneered work also for early human cortical development in vitro [10]. Rapid aggregation of a defined number of PSCs in single wells of low attachment dishes was followed by suspension cultivation in medium containing ECM components (matrigel), yielding three dimensional neural epithelia with apical-basal polarity that further subdivided into the characteristic ventricular (VZ) and subventricular (SVZ) progenitor cell zones. Such progenitors generated cortical neurons in an appropriate temporal order with so called Cajal-Retzius-type neurons produced first forming the marginal zone basally, followed by cortical neurons constituting the cortical plate, a layer located between the SVZ and the marginal zone. Indeed, the generated Current Opinion in Cell Biology 2014, 31:23–28
neurons within these 3D cortical structures showed signs of maturation including fast-wave Ca2+ oscillations and formation of synapses [10,11]. Maturation of the organoids to fetal stages equivalent to the beginning of the second trimester, was achieved by culturing under increased O2 and nutrient supplementation yielding a defined outer subventricular progenitor zone characteristic of human brains, but formation of cortical layers I–VI and mature pyramidal neurons have not yet been achieved [12,13]. However, disease modeling of early onset brain conditions was possible using iPSCs derived from a patient with microcephaly which generated organoids harboring reduced neuroepithelial progenitor zones with a larger proportion of differentiation, suggesting that premature differentiation causes the microcephaly phenotype, a conclusion that was supported via overexpression and knockdown of the diseasecausing gene, CDK5RAP2 [13]. Interestingly, the large cerebral organoids also included other brain regions including forebrain, hindbrain, dorsal cortex, prefrontal cortex, hippocampus, choroid plexus occipital lobe and retina, leading to the term ‘mini-brains’ [13]. Previously alternate media conditions were used to produce different CNS tissue regions from mouse ESCs including subpallial patterning [14] and adeno-hypophysis [15], that might also be soon recapitulated from human PSC sources. These exciting results provide access to a broad diversity of human CNS structures for functional and morphological studies.
Eyecup morphogenesis in retinal organoids An important highlight in self-organizing CNS tissue is the retina and fundamental insights into retinal morphogenesis have been attained through ESC-derived organoids. Slight modifications to the cortical induction protocol, particularly the use of matrigel early during controlled aggregate formation, combined with Wnt inhibition for rostralization and addition of Hh agonist, yielded retinal organoids [16]. Such retinal epithelia expressed characteristic eyefield transcription factors like Rx and Pax6, and remarkably evaginated from the aggregates and formed optic vesicle–like structures. The retinal tissue then spontaneously invaginated forming a two-walled optic cup-like tissue with a distal neural retina portion and a proximal monolayer of retinal pigment epithelial cells. A long held concept in developmental biology was that signaling from the overlying lens epithelium directs optic cup invagination. In the first work using mouse ESCs to generate retinal organoids, Eiraku et al. [17] provided new insights into optic cup morphogehesis, as they showed that the organoids form optic cups in the apparent absence of lens epithelium. Additionally, the authors defined four morphological phases of optic cup morphogenesis, and using a combination of myosin light chain localization, pharmacological www.sciencedirect.com
Human pluripotent stem cell-derived tissue culture Ader and Tanaka 25
disruption of myosin activity, and laser ablation, the authors defined stage and location specific modulation of actin-based contraction that contributes to cup formation. Amazingly when human organoids were formed, the size of the optic cup was larger than in the mouse cultures, scaling with the sizes in the respective embryos, indicating that tissue scaling is an intrinsic property of mouse versus human eyefield cells [16,17].
non-retinal regions secrete factors that inhibit maturation. In the detached optic cups early born cone photoreceptors were detected apically, but they were also found inappropriately in an intermediate-deep layer. Further cultivation for 100+ days lead to rod photoreceptor differentiation and a few immature horizontal cells [16]. However only very few late-born retinal neurons like bipolar cells, the main interneurons of the retina that connect photoreceptors and RGCs, were detected. In line with these observations no plexiform layers, that is, the synaptic zones of the retina, were formed. Thus, whether correct neural circuits can be generated in PSC-derived retinal tissue in vitro remains to be elucidated and might require pro-longed culturing times (Figure 1).
Further maturation of retinal organoids depended on separating the optic cup-like part of the tissue from the rest of the aggregate leading to stratified retinal tissue in a temporal sequence resembling in vivo development [16]. It is not yet clear if this step is necessary to provide increased space for maturation, or whether the surrounding
Figure 1
embryoid body
intermediate mesoderm
nascent mesoderm
epiblast
AcvinA FGF2
BMP4
BMP4
3D primive streak BMP4/AcnA or Wnt agonist monolayer
FGF9
FGF9 Wnt agonist (low)
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Wnt agonist (high)
RA/Wnt agonist (mid) intermediate mesoderm
FGF9/RA/ BMP7 or FGF9
metanephric mesoderm
metanephric mesenchyme co-culture spinal cord 3D
nephric tubule
glomeruli re-aggregaon
dissociaon 3D
early nephron
ureteric bud
mesoderm MZ CP
neuroectodermal sphere
embryoid body
spinning bioreactor RA
3D Matrigel hPSC
neuroectoderm
suspension
3D
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strafied cortex
cerebral organoid
Wnt agonist opc vesicle excision
cyst formaon
opc cup
renal cyst dissociaon 3D Matrigel
definive AcvinA endoderm
SP IZ OSVZ ISVZ VZ
RPE epithelium
hindgut spheroid FGF4/Wnt3a
monolayer
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R-Spondin1/Noggin/EGF 3D Matrigel
3D MSCs Hepac cells
re-aggregaon
gut organoid
liver bud
dissociaon 3D Matrigel
HUVECs
Current Opinion in Cell Biology
Schematic for the formation of 3D tissue structures from human PSCs in vitro. Three-dimensional PSC-derived tissues/organoids of ectodermal (CNS, cortex, retina), endodermal (gut, liver), and mesodermal (kidney) origin have been generated. Main factors for the stepwise lineage specification and time points for the initiation of 3D culturing are shown. Organogenesis in the neuroectodermal lineage generating cortical tissue, cerebral organoids and optic cups follows a continuous 3D culture system with slight changes in media composition facilitating regionalization (for further details see [12,13,16]). BMP: bone morphogenetic protein; CP: cortical plate; EGF: epidermal growth factor; FGF: fibroblast growth factor; hPSC: human pluripotent stem cell; HUVEC: human umbilical vein endothelial cell; ISVZ: inner subventricular zone; IZ: intermediate zone; MSC: mesenchymal stem cell; MZ: marginal zone; OSVZ: outer subventricular zone; RA: retinoic acid; RPE: retinal pigment epithelium; SP: subplate. www.sciencedirect.com
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These observations point out that the retina forming organoids deriving from ESC aggregation, while impressive in morphogenesis are still heterogeneous in composition and differentiation to mature phenotypes is lengthy, which would limit the usefulness of cultures for biochemical approaches. Zhu et al. [18] used a different approach to initiate three dimensional neuroepithelium formation from human ESCs. Applying principles developed for three dimensional kidney cyst formation, the authors embedded smaller clusters of human ESCs directly in matrigel, which yielded neuroepithelial cysts with well-defined, single lumens [18]. In the absence of any growth factor, this condition led to >95% of cysts acquiring eyefield identity within five days. To show the potential of eyefield cysts to form the two major cell layers of the eye, Zhu and colleagues plated the eyefield epithelium onto transwell filters and obtained uniform differentiation to retinal pigment epithelium (RPE) in response to ActivinA within 30 days, and in the absence of growth factors, differentiation into neural retina within 60 days. These results raise the possibility to co-culture RPE and neural retina in opposed layers under controlled conditions to study the crucial cellular interactions that take place between RPE and photoreceptors. In addition, the development of eyefield epithelium and RPE was highly accelerated compared to normal human development [18], and an interesting issue is how timing of cell maturation may be accelerated under specific culturing conditions.
Definitive endoderm: gut and liver Whereas 3D neuroectodermal tissues emerge upon continuous suspension culture, endoderm organoids are initiated by a 2D step prior to 3D culture. Previously, the Clevers lab [19,20] pioneered intestinal 3D organoid formation from Lgr5-expressing intestinal stem cells that grew to elaborate crypt-villus structures and all major celltypes of the gut in matrigel plus EGF, Wnt agonist (R-spondin-1) and Noggin (BMP inhibitor). Interestingly, their findings demonstrated that intestinal stem cells receive niche support by their own progeny (i.e. the Paneth cells) [19,20]. Indeed, in addition to in vivo systems the use of such 3D intestinal organoid cultures represents an effective experimental tool to decipher important compensatory mechanisms for intestinal formation and homeostasis and might set further light on the controversial role of Paneth cells within the intestinal stem cell niche [20–22]. Furthermore, intestinal organoid technology might provide a potential platform for personalized medicine approaches as demonstrated by the forscolin-induced organoid swelling assay that allows analysis of individual drug responses in cystic fibrosis [23]. Spence et al. on the other hand induced PSC monolayers to form definitive endoderm using ActivinA followed by 3D hindgut spheroid formation in the presence of FGF-4 and Wnt3a [24]. These PSC-derived spheroids were further cultured in matrigel supported Current Opinion in Cell Biology 2014, 31:23–28
medium as described by Sato et al. [19] to induce intestinal organoids with crypt-like progenitor niches and villus-like domains including Lgr5 stem cells, enterocytes, goblet cells, Paneth cells and enteroendocrine cells. Interestingly, the PSC-derived intestinal organoids also developed a mesenchymal layer — probably generated from the 2% of mesoderm that remained in the population following initial ActivinA treatment — and intestinal subepithelial myofibroblasts beside smooth muscle and fibroblasts at later stages. Thus, the parallel development of intestinal epithelia and mesenchyme suggests a highly coordinated interaction of several germ layers in organoid morphogenesis [24]. The authors used this system to investigate loss-of-function mutations in neurogenin 3 (NEURG3) that underlies malabsorptive diarrhea presumably due to lack of intestinal enteroendocrine cells [25]. Indeed adenoviral mediated overexpression of NEURG3 in hESC-derived intestinal organoids resulted in an approximately fivefold increase of enteroendocrine cells in comparison to control organoids, and shRNA mediated knock-down led to a 90% reduction in enteroendocrine cells [24].
Reconstitution of vascularized liver from hybrid cultures Remarkably, the previous examples of organoids attained sizeable dimensions in the absence of any vascularization, which may explain some of the cell death or deficits in cell differentiation, since blood vessels provide important nutrients and are themselves considered an important part of stem cell niches. Takebe and colleagues [26] have cleverly reconstituted 3D hepatic tissue by combining PSC-derived endoderm with somatically derived endothelial and mesenchymal cells. By inducing definitive endoderm using ActivinA followed by FGF2/BMP4 signaling, PSCs efficiently differentiate toward hepatic cells in 2D culture systems. However, as a complex and highly vascularized organ, the development of liver tissue requires the orchestration of interactions between endodermal epithelial-progenitors, endothelial-progenitors and mesenchymal progenitors. Therefore, Takebe et al. [26] co-cultured human iPSC-derived hepatic cells, human umbilical vein endothelial cells and mesenchymal stem cells in matrigel to generate 3D liver buds that showed high similarity at the gene expression level to primary fetal liver and improved maturation compared to human iPSC-derived hepatic cells cultured without stroma. Liver maturation and functionality requires connection to the blood system. Therefore human iPSC-derived liver buds were transplanted into a cranial window mouse model to follow the fate of transplanted donor cells over time. The liver buds connected to the host vasculature within 48 hours developing a vascular network similar in density and morphology to adult liver. The organoids functioned to produce serum albumin and human-specific metabolites such that when these www.sciencedirect.com
Human pluripotent stem cell-derived tissue culture Ader and Tanaka 27
organoids were transplanted into a gancyclovir-induced TK-NOG mouse liver failure model, increased survival rates were observed [26]. Interestingly, the connection between host and donor vessels was necessary for transplantation success, as human iPSC-derived hepatic cells without endothelial cells failed to vascularize and engraft [26]. These results pave the way for reconstitution of complex, multi-tissue organs, and provide some insight into the cell compositions required to obtain interaction between donor and host tissue.
Mesoderm: kidney 3D cultures of primary metanephric mesenchyme is a well established technique to generate kidney tissue in vitro [27]. Co-culture of embryonic metanephric nephron progenitors with Wnt-providing embryonic tissues, specifically spinal cord or Wnt-4 expressing cells, at the air-fluid interface on polycarbonate filters leads to mesenchyme-to-epithelial transition and formation of glomeruli and renal tubes [27]. Therefore, the challenge in starting from ES cells laid in directing human PSCs toward metanephric mesenchyme, but diagnostic markers to identify such early progenitors had been missing. Taguchi et al. [28] worked backward using colony forming assays of dissociated mouse embryonic tissue starting with late metanephric mesenchyme as identified by reliable metanephric mesenchyme markers [28]. This was followed by identification of conditions that could induce earlier progenitors to turn on these markers yielding a defined, stepwise sequence of kidney induction that was then used to differentiate mouse ESCs and human iPSCs into metanephric mesenchyme progenitors, that then reconstituted 3D kidney structures upon cocultivation with embryonic spinal cord. In future, further maturation of these nephron components by co-culturing with ureteric bud-derived structures will be necessary to confer physiological kidney functions. Interestingly, synchronous induction of ureteric bud and metanephric mesenchyme cells from human ESCs was recently achieved [29]. Re-aggregation after dissociation of the initially monolayer cultures upon ureteric bud and metanephric mesenchyme commitment allowed the formation of small, self-organized kidney organoids. However, further improvements in regard to nephron progenitor maintenance over time and 3D culture conditions will be necessary for the generation of more mature and larger kidney structures.
indeed impressive and point to the enormous potential of such systems to understand the mechanistic basis of human organogenesis. Not really addressed yet in most studies is how the ECM modulates tissue formation over time, as well as the role of factors expressed by the developing organoid itself to feedback and promote its own organization. The CNS organoids develop directly in three dimentional conditions to elaborate complex tissue architecture, something that would be difficult to achieve from co-culturing later stage cell types. In this regard, the early epithelialization observed in CNS organoid formation is likely crucial to the progression to neuroepithelium and further differentiation. How larger organoids subsection themselves into recognizable subregions of the brain is a fascinating dimension of the remarkable fluidity and adaptability of embryonic patterning systems. The ability of tissues to achieve coherent order and pattern from seemingly random cell aggregates will be a fascinating aspect of dynamic tissue organization for future study.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG) FZT 111, Center for Regenerative Therapies Dresden, Cluster of Excellence. And grants from the European Research Council (ERC) and DFG Staatsministerium fu¨r Wissenschaft und Kunst (SMWK) to MA and EMT. EMT is also a Max Planck Society Fellow.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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Summary/conclusion The combination of 3D culture methodologies, including the supply of ECM matrices, re-aggregation techniques, together with inductive factors identified from developmental studies to direct human PSCs along a given lineage represents a highly efficient means to recapitulate early human development in vitro. The generation of such organoids described in this review, and the first studies of disease states or tissue morphogenesis, are www.sciencedirect.com
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Current Opinion in Cell Biology 2014, 31:23–28
19. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ et al.: Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009, 459:262-265. 20. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H: Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 2011, 469:415-418. 21. Durand A, Donahue B, Peignon G, Letourneur F, Cagnard N, Slomianny C, Perret C, Shroyer NF, Romagnolo B: Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1). Proc Natl Acad Sci U S A 2012, 109:8965-8970. 22. Farin HF, Van Es JH, Clevers H: Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology 2012, 143:1518-1529 e1517. 23. Dekkers JF, Wiegerinck CL, de Jonge HR, Bronsveld I, Janssens HM, de Winter-de Groot KM, Brandsma AM, de Jong NW, Bijvelds MJ, Scholte BJ et al.: A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 2013, 19:939-945. 24. Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM et al.: Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 2011, 470:105-109. First report demonstrating the formation of three-dimensional human PSC-derived intestinal tissue in vitro that resembled the architecture and cellular composition of human fetal intestine. 25. Wang J, Cortina G, Wu SV, Tran R, Cho JH, Tsai MJ, Bailey TJ, Jamrich M, Ament ME, Treem WR et al.: Mutant neurogenin-3 in congenital malabsorptive diarrhea. N Engl J Med 2006, 355:270-280. 26. Takebe T, Zhang RR, Koike H, Kimura M, Yoshizawa E, Enomura M, Koike N, Sekine K, Taniguchi H: Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 2014, 9:396-409. By re-aggregation of human iPSC-derived hepatic cells with human MSCs and HUVECs Takebe et al. demonstrated the formation of vascularized liver buds in culture that following transplantation into pre-clinical mouse models functionally engrafted and connected to the host blood system. 27. Kispert A, Vainio S, McMahon AP: Wnt-4 is a mesenchymal signal for epithelial transformation of metanephric mesenchyme in the developing kidney. Development 1998, 125:4225-4234. 28. Taguchi A, Kaku Y, Ohmori T, Sharmin S, Ogawa M, Sasaki H, Nishinakamura R: Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 2014, 14:53-67. In this report Taguchi et al. identified the developmental origin and propagation of distinct metanephric progenitors by studying kidney formation in embryonic mice. Adapting this knowledge to PSC specification allowed the reconstruction of three-dimensional kidney tissue in vitro. 29. Takasato M, Er PX, Becroft M, Vanslambrouck JM, Stanley EG, Elefanty AG, Little MH: Directing human embryonic stem cell differentiation towards a renal lineage generates a selforganizing kidney. Nat Cell Biol 2014, 16:118-126. Here, the authors provide evidence for the coordinated induction of ureteric and metanephric progenitor lineages from human ESCs leading to the self-organized formation of three-dimensional kidney tissue.
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ScienceDirect Modeling human development in 3D culture Marius Ader and Elly M Tanaka Recently human embryonic stem cell research has taken on a new dimension — the third dimension. Capitalizing on increasing knowledge on directing pluripotent cells along different lineages, combined with ECM supported threedimensional culture conditions, it has become possible to generate highly organized tissues of the central nervous system, gut, liver and kidney. Each system has been used to study different aspects of organogenesis and function including physical forces underlying optic cup morphogenesis, the function of disease related genes in progenitor cell control, as well as interaction of the generated tissues with host tissue upon transplantation. Pluripotent stem cell derived organoids represent powerful systems for the study of how cells selforganize to generate tissues with a given shape, pattern and form. Addresses DFG-Center for Regenerative Therapies Dresden (CRTD), Technische Universita¨t Dresden, Fetscherstr. 105, 01307 Dresden, Germany
objects, important aspects influencing organogenesis are missing in conventional 2D cultures and thus such approaches cannot fully recapitulate in vivo development and function. Indeed, pioneering work initiated more than 30 years ago started to use collagen gels and extra cellular matrix (ECM) matrices to provide 3D scaffolds for epithelial cells of diverse origins (e.g. mammary gland/breast cancer; liver, skin/melanoma; smooth muscle; adipocytes; prostate/malignant prostate), that allowed the formation of so-called 3D spheroids. Importantly, such matrices not only provided structural support but influenced cell–cell interactions via transmembrane receptors leading to changes in cyto-skeleton organization, chromatin modulation, and gene expression [1].
Introduction
In parallel, during the last three decades stem cell research has blossomed and has had a major impact on most fields of biological research including developmental-, regenerative-, or cellular studies. Specifically, the isolation and in vitro propagation of pluripotent embryonic stem cells (ESCs), first reported from mouse [2] and later also from human [3] blastocysts, tremendously changed the field of developmental biology by providing practically an unlimited source of cells with the potential to differentiate along the three main lineages of the body. With the advent of induced pluripotent stem cells (iPSCs) generated from somatic cells by introduction of pluripotency genes [4,5,6] a further major step for the generation of unlimited material, that can be isolated from a variety of humans and patients for studying human development and disease, are now available. Finally, defined procedures to control the differentiation of these cells along each of the three major lineages to generate cell types in two dimensional culture has been a major step forward in controlling the enormous potential of the system.
The development of organs and tissues from the fertilized oocyte represents a highly complex but well organized system. Studies using animal models, cell- and organotypic cultures have significantly increased our knowledge about fundamental mechanisms for organogenesis. However, continuous and detailed analysis of human development over time with its network of cell– cell interactions, cell fate decisions and differentiation steps besides inducing chemical-, genetic- and environmental factors is challenging due to limitations in human tissue availability. Thus, cell culture methodologies using in vitro expandable cell lines are widely used to study cellular and molecular events in lineage fate decision, differentiation and maintenance of human cells. However, as organs and tissues represent 3D
Previously, three-dimensional differentiation of PSCs was performed under relatively uncontrolled conditions to generate embryoid bodies in which cell aggregates formed tissues of several different germlayers within one aggregate. Such structures may be considered akin to teratomas that are formed when PSCs are injected subcutaneously into rodent models. This impressive ability of PSCs to self-organize into three-dimensional tissues has recently been harnessed for the formation of defined human tissues and organoids by combining 3D culture techniques with directed PSC specification protocols. Considering the possibilities to rapidly generate reporter cell lines in PSCs, as well as the ability to generate PSCs from individuals with defined genetic disorders, these
Corresponding authors: Ader, Marius ([email protected]) and Tanaka, Elly M ([email protected])
Current Opinion in Cell Biology 2014, 31:23–28 This review comes from a themed issue on Cell cycle, differentiation and disease Edited by Stefano Piccolo and Eduard Batlle
http://dx.doi.org/10.1016/j.ceb.2014.06.013 0955-0674/# 2014 Elsevier Ltd. All rights reserved.
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Current Opinion in Cell Biology 2014, 31:23–28
24 Cell cycle, differentiation and disease
methods will revolutionize not only regenerative medicine approaches for which they are often envisioned, but will open up new horizons for studying the cell biology of human organogenesis in structures including the brain, gut, kidney and liver. Here we describe some of the major recent developments in human 3D organoid formation, including the strategies that have led to reconstitution of complex tissue architecture, as well as the application of these organoids to address previously inaccessible questions on cell differentiation and tissue morphogenesis.
Neuroectoderm: cortex and retina The central nervous system (CNS) and in particular the brain, is one of the most accessible fate choices of ESCs. Recently the vast experience in directing pluripotent cells toward neural fates in two dimensions has been combined with three-dimensional culture in matrigel to produce various defined subregions of the developing mouse and human CNS including the cortex, retina, subpallium and adeno-hypophysis. Such organoids have been used to study the physical forces underlying retinal cup formation, species-specific dimensionality of eye tissue, as well as factors underlying progenitor cell control in the cortex.
Cortical organoids reproduce progenitor subtypes and layered structure The cortex represents by far the biggest structure (approx. 75%) within the human brain. Its complex architecture is tightly regulated during development with the formation of distinct progenitor layers that eventually build up the six-layered adult cortex. Directed differentiation of human PSCs in two dimensional culture toward cortical neuron fate revealed a ‘default’ pathway of neural differentiation when PSCs are cultured under minimal conditions in the absence of exogenous morphogens and supplementation either with the bone morphogenetic protein (BMP) inhibitor Noggin [7,8] or with retinoids in combination with SMAD inhibitors [9]. Under these conditions, cortical pyramidal neuron specification followed the temporal and sequential order seen in vivo. By adapting culture principles established first with mouse ESCs the Sasai group pioneered work also for early human cortical development in vitro [10]. Rapid aggregation of a defined number of PSCs in single wells of low attachment dishes was followed by suspension cultivation in medium containing ECM components (matrigel), yielding three dimensional neural epithelia with apical-basal polarity that further subdivided into the characteristic ventricular (VZ) and subventricular (SVZ) progenitor cell zones. Such progenitors generated cortical neurons in an appropriate temporal order with so called Cajal-Retzius-type neurons produced first forming the marginal zone basally, followed by cortical neurons constituting the cortical plate, a layer located between the SVZ and the marginal zone. Indeed, the generated Current Opinion in Cell Biology 2014, 31:23–28
neurons within these 3D cortical structures showed signs of maturation including fast-wave Ca2+ oscillations and formation of synapses [10,11]. Maturation of the organoids to fetal stages equivalent to the beginning of the second trimester, was achieved by culturing under increased O2 and nutrient supplementation yielding a defined outer subventricular progenitor zone characteristic of human brains, but formation of cortical layers I–VI and mature pyramidal neurons have not yet been achieved [12,13]. However, disease modeling of early onset brain conditions was possible using iPSCs derived from a patient with microcephaly which generated organoids harboring reduced neuroepithelial progenitor zones with a larger proportion of differentiation, suggesting that premature differentiation causes the microcephaly phenotype, a conclusion that was supported via overexpression and knockdown of the diseasecausing gene, CDK5RAP2 [13]. Interestingly, the large cerebral organoids also included other brain regions including forebrain, hindbrain, dorsal cortex, prefrontal cortex, hippocampus, choroid plexus occipital lobe and retina, leading to the term ‘mini-brains’ [13]. Previously alternate media conditions were used to produce different CNS tissue regions from mouse ESCs including subpallial patterning [14] and adeno-hypophysis [15], that might also be soon recapitulated from human PSC sources. These exciting results provide access to a broad diversity of human CNS structures for functional and morphological studies.
Eyecup morphogenesis in retinal organoids An important highlight in self-organizing CNS tissue is the retina and fundamental insights into retinal morphogenesis have been attained through ESC-derived organoids. Slight modifications to the cortical induction protocol, particularly the use of matrigel early during controlled aggregate formation, combined with Wnt inhibition for rostralization and addition of Hh agonist, yielded retinal organoids [16]. Such retinal epithelia expressed characteristic eyefield transcription factors like Rx and Pax6, and remarkably evaginated from the aggregates and formed optic vesicle–like structures. The retinal tissue then spontaneously invaginated forming a two-walled optic cup-like tissue with a distal neural retina portion and a proximal monolayer of retinal pigment epithelial cells. A long held concept in developmental biology was that signaling from the overlying lens epithelium directs optic cup invagination. In the first work using mouse ESCs to generate retinal organoids, Eiraku et al. [17] provided new insights into optic cup morphogehesis, as they showed that the organoids form optic cups in the apparent absence of lens epithelium. Additionally, the authors defined four morphological phases of optic cup morphogenesis, and using a combination of myosin light chain localization, pharmacological www.sciencedirect.com
Human pluripotent stem cell-derived tissue culture Ader and Tanaka 25
disruption of myosin activity, and laser ablation, the authors defined stage and location specific modulation of actin-based contraction that contributes to cup formation. Amazingly when human organoids were formed, the size of the optic cup was larger than in the mouse cultures, scaling with the sizes in the respective embryos, indicating that tissue scaling is an intrinsic property of mouse versus human eyefield cells [16,17].
non-retinal regions secrete factors that inhibit maturation. In the detached optic cups early born cone photoreceptors were detected apically, but they were also found inappropriately in an intermediate-deep layer. Further cultivation for 100+ days lead to rod photoreceptor differentiation and a few immature horizontal cells [16]. However only very few late-born retinal neurons like bipolar cells, the main interneurons of the retina that connect photoreceptors and RGCs, were detected. In line with these observations no plexiform layers, that is, the synaptic zones of the retina, were formed. Thus, whether correct neural circuits can be generated in PSC-derived retinal tissue in vitro remains to be elucidated and might require pro-longed culturing times (Figure 1).
Further maturation of retinal organoids depended on separating the optic cup-like part of the tissue from the rest of the aggregate leading to stratified retinal tissue in a temporal sequence resembling in vivo development [16]. It is not yet clear if this step is necessary to provide increased space for maturation, or whether the surrounding
Figure 1
embryoid body
intermediate mesoderm
nascent mesoderm
epiblast
AcvinA FGF2
BMP4
BMP4
3D primive streak BMP4/AcnA or Wnt agonist monolayer
FGF9
FGF9 Wnt agonist (low)
Acvin/BMP4
Wnt agonist (high)
RA/Wnt agonist (mid) intermediate mesoderm
FGF9/RA/ BMP7 or FGF9
metanephric mesoderm
metanephric mesenchyme co-culture spinal cord 3D
nephric tubule
glomeruli re-aggregaon
dissociaon 3D
early nephron
ureteric bud
mesoderm MZ CP
neuroectodermal sphere
embryoid body
spinning bioreactor RA
3D Matrigel hPSC
neuroectoderm
suspension
3D
3D Matrigel
strafied cortex
cerebral organoid
Wnt agonist opc vesicle excision
cyst formaon
opc cup
renal cyst dissociaon 3D Matrigel
definive AcvinA endoderm
SP IZ OSVZ ISVZ VZ
RPE epithelium
hindgut spheroid FGF4/Wnt3a
monolayer
FGF2/BMP4
R-Spondin1/Noggin/EGF 3D Matrigel
3D MSCs Hepac cells
re-aggregaon
gut organoid
liver bud
dissociaon 3D Matrigel
HUVECs
Current Opinion in Cell Biology
Schematic for the formation of 3D tissue structures from human PSCs in vitro. Three-dimensional PSC-derived tissues/organoids of ectodermal (CNS, cortex, retina), endodermal (gut, liver), and mesodermal (kidney) origin have been generated. Main factors for the stepwise lineage specification and time points for the initiation of 3D culturing are shown. Organogenesis in the neuroectodermal lineage generating cortical tissue, cerebral organoids and optic cups follows a continuous 3D culture system with slight changes in media composition facilitating regionalization (for further details see [12,13,16]). BMP: bone morphogenetic protein; CP: cortical plate; EGF: epidermal growth factor; FGF: fibroblast growth factor; hPSC: human pluripotent stem cell; HUVEC: human umbilical vein endothelial cell; ISVZ: inner subventricular zone; IZ: intermediate zone; MSC: mesenchymal stem cell; MZ: marginal zone; OSVZ: outer subventricular zone; RA: retinoic acid; RPE: retinal pigment epithelium; SP: subplate. www.sciencedirect.com
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26 Cell cycle, differentiation and disease
These observations point out that the retina forming organoids deriving from ESC aggregation, while impressive in morphogenesis are still heterogeneous in composition and differentiation to mature phenotypes is lengthy, which would limit the usefulness of cultures for biochemical approaches. Zhu et al. [18] used a different approach to initiate three dimensional neuroepithelium formation from human ESCs. Applying principles developed for three dimensional kidney cyst formation, the authors embedded smaller clusters of human ESCs directly in matrigel, which yielded neuroepithelial cysts with well-defined, single lumens [18]. In the absence of any growth factor, this condition led to >95% of cysts acquiring eyefield identity within five days. To show the potential of eyefield cysts to form the two major cell layers of the eye, Zhu and colleagues plated the eyefield epithelium onto transwell filters and obtained uniform differentiation to retinal pigment epithelium (RPE) in response to ActivinA within 30 days, and in the absence of growth factors, differentiation into neural retina within 60 days. These results raise the possibility to co-culture RPE and neural retina in opposed layers under controlled conditions to study the crucial cellular interactions that take place between RPE and photoreceptors. In addition, the development of eyefield epithelium and RPE was highly accelerated compared to normal human development [18], and an interesting issue is how timing of cell maturation may be accelerated under specific culturing conditions.
Definitive endoderm: gut and liver Whereas 3D neuroectodermal tissues emerge upon continuous suspension culture, endoderm organoids are initiated by a 2D step prior to 3D culture. Previously, the Clevers lab [19,20] pioneered intestinal 3D organoid formation from Lgr5-expressing intestinal stem cells that grew to elaborate crypt-villus structures and all major celltypes of the gut in matrigel plus EGF, Wnt agonist (R-spondin-1) and Noggin (BMP inhibitor). Interestingly, their findings demonstrated that intestinal stem cells receive niche support by their own progeny (i.e. the Paneth cells) [19,20]. Indeed, in addition to in vivo systems the use of such 3D intestinal organoid cultures represents an effective experimental tool to decipher important compensatory mechanisms for intestinal formation and homeostasis and might set further light on the controversial role of Paneth cells within the intestinal stem cell niche [20–22]. Furthermore, intestinal organoid technology might provide a potential platform for personalized medicine approaches as demonstrated by the forscolin-induced organoid swelling assay that allows analysis of individual drug responses in cystic fibrosis [23]. Spence et al. on the other hand induced PSC monolayers to form definitive endoderm using ActivinA followed by 3D hindgut spheroid formation in the presence of FGF-4 and Wnt3a [24]. These PSC-derived spheroids were further cultured in matrigel supported Current Opinion in Cell Biology 2014, 31:23–28
medium as described by Sato et al. [19] to induce intestinal organoids with crypt-like progenitor niches and villus-like domains including Lgr5 stem cells, enterocytes, goblet cells, Paneth cells and enteroendocrine cells. Interestingly, the PSC-derived intestinal organoids also developed a mesenchymal layer — probably generated from the 2% of mesoderm that remained in the population following initial ActivinA treatment — and intestinal subepithelial myofibroblasts beside smooth muscle and fibroblasts at later stages. Thus, the parallel development of intestinal epithelia and mesenchyme suggests a highly coordinated interaction of several germ layers in organoid morphogenesis [24]. The authors used this system to investigate loss-of-function mutations in neurogenin 3 (NEURG3) that underlies malabsorptive diarrhea presumably due to lack of intestinal enteroendocrine cells [25]. Indeed adenoviral mediated overexpression of NEURG3 in hESC-derived intestinal organoids resulted in an approximately fivefold increase of enteroendocrine cells in comparison to control organoids, and shRNA mediated knock-down led to a 90% reduction in enteroendocrine cells [24].
Reconstitution of vascularized liver from hybrid cultures Remarkably, the previous examples of organoids attained sizeable dimensions in the absence of any vascularization, which may explain some of the cell death or deficits in cell differentiation, since blood vessels provide important nutrients and are themselves considered an important part of stem cell niches. Takebe and colleagues [26] have cleverly reconstituted 3D hepatic tissue by combining PSC-derived endoderm with somatically derived endothelial and mesenchymal cells. By inducing definitive endoderm using ActivinA followed by FGF2/BMP4 signaling, PSCs efficiently differentiate toward hepatic cells in 2D culture systems. However, as a complex and highly vascularized organ, the development of liver tissue requires the orchestration of interactions between endodermal epithelial-progenitors, endothelial-progenitors and mesenchymal progenitors. Therefore, Takebe et al. [26] co-cultured human iPSC-derived hepatic cells, human umbilical vein endothelial cells and mesenchymal stem cells in matrigel to generate 3D liver buds that showed high similarity at the gene expression level to primary fetal liver and improved maturation compared to human iPSC-derived hepatic cells cultured without stroma. Liver maturation and functionality requires connection to the blood system. Therefore human iPSC-derived liver buds were transplanted into a cranial window mouse model to follow the fate of transplanted donor cells over time. The liver buds connected to the host vasculature within 48 hours developing a vascular network similar in density and morphology to adult liver. The organoids functioned to produce serum albumin and human-specific metabolites such that when these www.sciencedirect.com
Human pluripotent stem cell-derived tissue culture Ader and Tanaka 27
organoids were transplanted into a gancyclovir-induced TK-NOG mouse liver failure model, increased survival rates were observed [26]. Interestingly, the connection between host and donor vessels was necessary for transplantation success, as human iPSC-derived hepatic cells without endothelial cells failed to vascularize and engraft [26]. These results pave the way for reconstitution of complex, multi-tissue organs, and provide some insight into the cell compositions required to obtain interaction between donor and host tissue.
Mesoderm: kidney 3D cultures of primary metanephric mesenchyme is a well established technique to generate kidney tissue in vitro [27]. Co-culture of embryonic metanephric nephron progenitors with Wnt-providing embryonic tissues, specifically spinal cord or Wnt-4 expressing cells, at the air-fluid interface on polycarbonate filters leads to mesenchyme-to-epithelial transition and formation of glomeruli and renal tubes [27]. Therefore, the challenge in starting from ES cells laid in directing human PSCs toward metanephric mesenchyme, but diagnostic markers to identify such early progenitors had been missing. Taguchi et al. [28] worked backward using colony forming assays of dissociated mouse embryonic tissue starting with late metanephric mesenchyme as identified by reliable metanephric mesenchyme markers [28]. This was followed by identification of conditions that could induce earlier progenitors to turn on these markers yielding a defined, stepwise sequence of kidney induction that was then used to differentiate mouse ESCs and human iPSCs into metanephric mesenchyme progenitors, that then reconstituted 3D kidney structures upon cocultivation with embryonic spinal cord. In future, further maturation of these nephron components by co-culturing with ureteric bud-derived structures will be necessary to confer physiological kidney functions. Interestingly, synchronous induction of ureteric bud and metanephric mesenchyme cells from human ESCs was recently achieved [29]. Re-aggregation after dissociation of the initially monolayer cultures upon ureteric bud and metanephric mesenchyme commitment allowed the formation of small, self-organized kidney organoids. However, further improvements in regard to nephron progenitor maintenance over time and 3D culture conditions will be necessary for the generation of more mature and larger kidney structures.
indeed impressive and point to the enormous potential of such systems to understand the mechanistic basis of human organogenesis. Not really addressed yet in most studies is how the ECM modulates tissue formation over time, as well as the role of factors expressed by the developing organoid itself to feedback and promote its own organization. The CNS organoids develop directly in three dimentional conditions to elaborate complex tissue architecture, something that would be difficult to achieve from co-culturing later stage cell types. In this regard, the early epithelialization observed in CNS organoid formation is likely crucial to the progression to neuroepithelium and further differentiation. How larger organoids subsection themselves into recognizable subregions of the brain is a fascinating dimension of the remarkable fluidity and adaptability of embryonic patterning systems. The ability of tissues to achieve coherent order and pattern from seemingly random cell aggregates will be a fascinating aspect of dynamic tissue organization for future study.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG) FZT 111, Center for Regenerative Therapies Dresden, Cluster of Excellence. And grants from the European Research Council (ERC) and DFG Staatsministerium fu¨r Wissenschaft und Kunst (SMWK) to MA and EMT. EMT is also a Max Planck Society Fellow.
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