1、ABDispatchDate:16.09.2020 ProofNo:804,p.1123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263Review ARticlehttps:/doi.org/10.1038/s41563-020-00804-41Pluripotency for Organ Regeneration,Institute for Bioengineering of Catalonia,the Barc
2、elona Institute of Technology,Barcelona,Spain.2University of Barcelona,Barcelona,Spain.3Department of Biological Engineering and Department of Mechanical Engineering,Massachusetts Institute of Technology,Cambridge,MA,USA.4Department of Anatomy and Embryology,Leiden University Medical Center,Leiden,t
3、he Netherlands.5MRC Laboratory of Molecular Biology,Cambridge Biomedical Campus,Cambridge,UK.6Department of Biological Engineering,Massachusetts Institute of Technology,Cambridge,MA,USA.7Synthetic Biology Center,Massachusetts Institute of Technology,Cambridge,MA,USA.8Centro de Investigacin Biomdica
4、en Red en Bioingeniera,Biomateriales y Nanomedicina,Barcelona,Spain.9Instituci Catalana de Recerca i Estudis Avanats,Barcelona,Spain.10Institute for Bioengineering of Catalonia,the Barcelona Institute of Technology,Barcelona,Spain.11Unitat de Biofsica i Bioenginyeria,Universitat de Barcelona,Barcelo
5、na,Spain.12Department of Bioethics,School of Medicine,Case Western Reserve University,Cleveland,OH,USA.13Center for Bioethics,Harvard Medical School,Boston,MA,USA.14These authors contributed equally:Elena Garreta,Roger D.Kamm.e-mail:nmontserratibecbarcelona.euOver the past two decades,stem cell rese
6、arch has advanced our understanding of key aspects of organogenesis through the exploitation of the self-organizing properties of adult stem cells(ASCs)and pluripotent stem cells(PSCs).Such progresses have led to the development of cell culture procedures to generate micro-and miniorgan-like structu
7、res on demand,the so-called organoids.In parallel,the emergence of the bioengineering field is leading to technological advances that allow proper instructive environments(physical and chemical),boosting cellular responses towards the formation of organ-specific multicellular structures in these min
8、iorgan-like structures.Current methods rely on tra-ditional three-dimensional(3D)culture techniques that exploit cell-autonomous self-organization of human PSCs(hPSCs)(Fig.1).Nevertheless,hPSC-derived organoids still exhibit several short-comings.These include the lack of reproducibility;lack of spe
9、cific-ity with regard to cell-type(s)composition;uncontrolled size;shape heterogeneity;absence of proper vascular,immune and innervation components and organ-specific morphological features;and lack of functionality.Therefore,major goals of organoid technology are now focused on improving organoids
10、cellular and morphological complexity(for example,via the induction of properly organized regional identities in brain organoids,or providing a developing branching collecting-duct system in kidney organoids),providing perfusable vascular networks(to facilitate organoid differentiation and lifespan,
11、but also organoid-to-organoid connection for studying complex interactions between different tissue types),and enhanc-ing organoid maturation in order to achieve relevant tissue-specific functionalities.Understanding and integrating self-formation capacities and programmability of hPSCs with bioengi
12、neering Q1Q2design may increase control of self-organization and functionality of hPSC-derived organoids.This knowledge will help the community to generate higher-grade organoids(in terms of cellular composi-tion,architecture,function and reproducibility)for developmental biology,drug screening,dise
13、ase modelling and personalized/precise medicine applications,and in the future to derive clinically relevant tissue-like structures for regenerative medicine applications(Fig.1).In this Review,we first look back on the historical origin of organoid technology and how early developments in 3D cell cu
14、l-ture systems exploiting the self-organization ability of hPSCs have enabled generation of these powerful platforms.We will collectively examine how to apply current knowledge in organoid mechanics and transcriptomics to further control the arrangement function and composition of hPSC-derived organ
15、oids.Then,we foresee the immediate impact of engineering approaches(that is,biomimetic hydrogels,3D bioprinting and microtechnologies)to overcome current hPSC-derived organoids challenges in the upcoming years.Finally,we will discuss on how ethicists,engineers and stem cell biologists will need to c
16、ollaborate on engineering ethics and how this joint effort will benefit the success of the entire hPSC-derived organoid field.Engineering hPSC-derived organoidsInsert some text here.Evolution of 3D cultures in the organoids history.Since the 1900s,developmental biologists have selected different mod
17、el organisms to study the morphogenetic processes occurring dur-ing tissue and organ development,including sponges,amphibians,Q3Q4Q5Q6Rethinking organoid technology through bioengineeringElena Garreta1,2,14,Roger D.Kamm 3,14,Susana M.Chuva de Sousa Lopes 4,Madeline A.Lancaster5,Ron Weiss 6,7,Xavier
18、Trepat8,9,10,11,Insoo Hyun12,13 and Nuria Montserrat 1,8,9In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells(hPSCs).An important step in this direction has also been the derivation of organoids.This te
19、chnol-ogy relies on traditional three-dimensional culture techniques that exploit cell-autonomous self-organization responses of hPSCs with minimal control over the external inputs supplied to the system.The convergence of stem cell biology and bio-engineering offers the possibility to provide these
20、 stimuli in a controlled fashion,resulting in the development of naturally inspired approaches to overcome major limitations of this nascent technology.Based on the current developments,we empha-size the achievements and ongoing challenges of bringing together hPSC organoid differentiation,bioengine
21、ering and eth-ics.This Review underlines the need for providing engineering solutions to gain control of self-organization and functionality of hPSC-derived organoids.We expect that this knowledge will guide the community to generate higher-grade hPSC-derived organoids for further applications in de
22、velopmental biology,drug screening,disease modelling and personalized medicine.NATuRE MATERIALS| ProofNo:804,p.2646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129Review ARticleNature Mater
23、ialschick and mice.All these different systems have contributed sub-stantially to address key long-standing questions in this field.Groundbreaking observations performed in these organisms dur-ing the first decades of the last century highlighted the intrinsic capacities of tissues to follow predete
24、rmined developmental and functional programmes and how these dictated the mutual and exclusive relations between cells during reaggregation(Box1).By the 1940s,different researchers demonstrated that growing tissue removed from the avian limb rudiment would rearrange and pat-tern invitro and in ovo1,
25、2.In addition,disaggregated and reaggre-gated chick embryonic kidneys became proper kidney epithelial tubules surrounded by mesenchyme-derived stroma in culture3.These experiments pinpointed that cell reaggregation could result in self-reassembly of tissue-like structures,whereby cells organized aut
26、onomously into pre-patterned structures.Paul Weiss and A.Cecil Taylor showed that when chick embryonic cells from differ-ent organs at advanced stages of development were reaggregated and grafted into a highly vascularized neutral environment,as the chick chorioallantoic membrane(CAM),the resulting
27、cell masses formed well-organized organs ex vivo4.Interestingly,in that study the authors highlighted the phenomenon of self-organization as the major cause driving morphogenesis instead of external inductions,which was the major trend explaining development at that time4.Q7Q8By contrast,experiments
28、 by Clifford Grobstein demonstrated that when an inducing source,such as spinal cord,was reaggregated with the kidney metanephric mesenchyme,nephron-like structures were able to develop invitro5.As the knowledge of 3D cultures progressed,intense research was devoted to explore how the extracellular
29、matrix(ECM)dic-tates morphogenesis and function in a wide range of cell cultures6.Later,Hans Clevers and colleagues applied this knowledge to cul-ture single-cell suspensions of Lgr5+mouse intestinal stem cells embedded in Matrigel under specific culture conditions succeeding in the generation of in
30、testinal organoids with a crypt-villus architec-ture7.This seminal study and others facilitated the development of ASC-derived organoids,such as stomach,pancreas,colon,prostate and liver.Progress in ASC-derived organoids and major advances in this field have recently been addressed in several excell
31、ent reviews8,9.Since the isolation of human embryonic stem cells(ESCs)in 199810 and the reprogramming of human somatic cells into induced pluripotent stem cells(iPSCs)11,both sources have become instru-mental in recapitulating the fundamental principles of tissue differ-entiation and morphogenesis.B
32、ecause PSCs represent the starting point of differentiation(pluripotency stage),they offer a model of organ ontogeny and a minimal system for discerning in a systematic manner the relative contribution of different cellular components to Establishment of hESCs and hiPSCs 3D cortical structures(Eirak
33、u,2008)Differentiation methodologies to generate hPSC-derived organoids 2D micropatterns to guide hPSC self-organization Intestinal organoids(Spence,2011)Gastric organoids(McCracken,2014)Optic cup organoids(Nakano,2012)Brain organoids(Lancaster,2013)Kidney organoids(Xia,2013;Takasato,2015)Lung organ
34、oids(Dye,2015)Boold vessel organoids(Wimmer,2019)Generation of self-organized cardiac micro-tissues(Ma,2015)Generation self-organizing germ layers,so called gastruloids(Martyn,2018)Combination with a microfluidic system to provide controlled morphogen gradients for hPSC differentiation(Manfrin,2019)
35、Biomimetic materials to control 3D organoid differentiation Microfluidic devices to mimic tissuetissue interactions Unveiling organoid mechanics for disease mode3D bioprinting for improving tissue architecture Brain organoid formation in micro-structured scaffolds(Lancaster,2017)Use of synthetic PEG
36、-based hydrogels for intestinal organoids derivation(Cruz-Acua,2017)Compliant hydrogels accelerate the formation of kidney organoids(Garreta,2019)A physiological model of the human motor unit for disease modelling(Osaki,2018)Multi-layer human retina-ona-chip platform for drug screening(Loskill,2019)
37、Mechanics of self-organizing optic cups(Okuda,2018)An on-chip approach for model ling the physics of brain folding(Karzbrun,2018)Fabrication of a perfusable vascularized cardiac tissue(Skylar-Scott,2019)Current organoid challenges:Increase organoid lifespan Provide proper vascularization Achieve rel
38、evant tissufic architecture and functionality Bioengineering ethics Informed consent requirements for procuring human cells Development of ethical guidelines for organoid researchNovel ethical issues include:Biobanking and research sharing of organoids Potential role of organoids in personalized med
39、icine Impact of organoids on animal research and clinical trials1998200620082010s201520172018201920203D culture systems hPSCs Cell embedding in matrigel 3D suspension culture Cell pellet culture in transwell system Engineering solutionsEmerging technologiesMicropatterningtechniques Biomimeticmateria
40、ls 3D bioprintingFluidic devices/microfluidicsSingle-cell RNA sequencing Probing organoids mechanics Fig.1|Advances in engineering hPSC-derived organoids.Timeline of milestones for the generation of organoids from hPSCs.To date,several engineering solutions controlling self-organization,differentiat
41、ion and tissue boundary conditions have explored micropatterning and microfluidics to improve organoid outcomes.Other approaches aiming to gain control on cell-to-cell and cell-to-ECM are considered when fabricating new materials emulating biochemical or biophysical properties of native tissues.Thes
42、e biomimetic materials can be further exploited for 3D bioprinting creating better tissue architectures.The application of these engineering approaches together with emergent technologies from the fields of transcriptomics and mechanics are expected to provide a better control of hPSC organoid gener
43、ation.At the same time,the ethical dimension in this field compromises policies for patient consent,biobanking or animal use.All these considerations call for responsibility in communicating results to the public and the need to discuss these topics between ethicists,engineers and stem cell biologis
44、ts.NATuRE MATERIALS| ProofNo:804,p.3130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187188189190191192193194195Review ARticleNature Materialscomplex morphogenetic processes.Prof
45、iting from extensive knowl-edge accumulated from mouse development and mouse PSCs,research studies employ(extrapolated)growth signalling molecules to instruct hPSC differentiation in two-dimensional(2D)condi-tions or generating hPSC aggregate-like structures named embry-oid bodies(EBs).This knowledg
46、e was key for obtaining for the first time self-patterned stratified cortical tissue after plating EBs gener-ated in serum-free medium on a coated surface12.Later Sasai and colleagues maintained EBs as floating neuroepithelial cysts with minimal exogenously provided signals(Matrigel)13,which further
47、 self-organized into optic cup organoids containing spatially sepa-rated domains of neural retina and retinal pigmented epithelium.Building on this,further studies using spontaneous differentiation generated brain organoids with a wide variety of regional identi-ties14,15,while more directed approac
48、hes with small molecules could generate specific brain regions16,17.A common feature of the various methods developed for hPSC-derived neural organoids is that the factors that are applied to the cells attempt to reproduce the invivo signalling networks and associated timing to which the rudimentary
49、 organ is exposed during development.Similarly,other hPSC-derived organoids seek to mimic these developmental events to generate tissues that mimic the invivo counterparts.This has now been quite successful for kidney18,intestine19,lung20 and inner ear21,to name a few.Understanding self-organization
50、 and symmetry breaking.For an aggregate of cells(PSCs or committed organ progenitors)to evolve from a simple spheroid to an organoid with complex tissue architec-ture,symmetry breaking must occur.While the precise mechanisms that underlie this process are in most cases still unclear,in general it in