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Cover Image: STEMCELL Technologies
Copyright © 2019 Wiley Periodicals, Inc. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means without the prior permission in writing from the copyright holder. 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
CONTENTS 3 INTRODUCTION 6 HISTORIC MILESTONES 12 IN PRACTICE 25 PROBLEMS & SOLUTIONS 27 WHAT’S NEXT 29 REFERENCES 31 FURTHER READING 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð INTRODUCTION
Ì, KHDUG DERXW RUJDQRLGV DQG WKRXJKW WKH\ PLJKW EH WKH SHUIHFW PRGHO WR VWXG\ DOO WKH SURFHVVHV WKDW ,ÊP LQWHUHVWHG LQÍ VD\V 7DPDUD =LHWHN 3ULQFLSDO ,QYHVWLJDWRU LQ WKH 1XWULWLRQ3K\VLRORJ\/DERUDWRU\DWWKH7HFKQLFDO8QLYHUVLW\ RI 0XQLFK %HIRUH XVLQJ RUJDQRLGV =LHWHNÊV JURXS ZRUNHG Eoth witK DQ LQ YLYRÈPLFHÈDQG LQ YLWURÈFHOO OLQHVÈ V\VWHP WR DQVZHU WKHLU UHVHDUFK TXHVWLRQV EXW WKH\ ZHUH RQ WKH KXQW IRU D V\VWHP WKDW FRPELQHG WKH DGYDQWDJHV RI ERWK 8VLQJ SULPDU\ FHOO FXOWXUHV ZDV DQ LPSURYHPHQW EXWEHFDXVHWKHFHOOVFRXOGQÊWEHSDVVDJHGLWZDVQRWDORQJ WHUP V\VWHP 6R =LHWHN XVHG RUJDQRLG PRGHO V\VWHPV WR EHWWHU XQGHUVWDQG LQWHVWLQDO QXWULHQW WUDQVSRUW DQG VHQVLQJ.
In recent years, our cumulative understanding of organ physiology, development, and maintenance has resulted in the creation of three- GLPHQVLRQDO ' RUJDQRLGVÈFXOWXUHG'FHOOVWUXFWXUHVWKDWPRGHO features of organ function, composition, and development. Given that the RUJDQVRIRXUERG\RFFXS\'VSDFHRUJDQRLGVEHWWHUUHSUHVHQWWKHSK\VL- ological system than their two-dimensional culture counterparts for the purpose of organ studies as they recapitulate the signaling and morphologi- cal cues that can occur within the human body. Scientists have thus turned to using organoids to study normal and pathological conditions, and as a method to test potential therapeutics for human disease. 7KHPHWKRGVIRUFXOWXULQJRUJDQRLGVDUHWLVVXHGHSHQGHQWEXWVRPH RYHUDOOSULQFLSOHVDSSO\$VFLHQWLVWHPEHGVWKHVWDUWLQJPDWHULDOÈVXFKDV SURJHQLWRUFHOOVGHULYHGIURPSOXULSRWHQWVWHPFHOOV 36&V RUDGXOWVWHP FHOOV $6&V KDUYHVWHGIURPWLVVXHVDPSOHVÈLQDQH[WUDFHOOXODUPDWUL[ 7KHFHOOVDUHPDLQWDLQHGLQFXOWXUHPHGLDFRQWDLQLQJWKHQXWULHQWVDQG growth factors necessary to mimic the LQYLYR cellular environment. Under WKHVHFRQGLWLRQVWKHVWDUWLQJFHOOVH[SDQGDQGVHOIRUJDQL]HWREXLOGD 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
'VWUXFWXUHÈWKHRUJDQRLGVÈZKLFKFDQEHPDLQWDLQHGIRUORQJSHULRGVRI WLPH)RUVRPHHSLWKHOLDORUJDQRLGV HJLQWHVWLQDORUJDQRLGV WKHFXOWXUHV can be maintained indefinitely through routine passaging (i.e., breaking WKHRUJDQRLGVLQWRVPDOOVHFWLRQVDQGUHVHHGLQJWKHPLQQHZFXOWXUHV 'HYHORSLQJFXOWXUHFRQGLWLRQVIRURUJDQRLGVLVQRWDWULYLDOPDWWHU Certain conditions must be met for the organ-resident stem cells to be maintained and differentiated into the appropriate complement of cells. 7KLVIXQGDPHQWDOSURSHUW\RIRUJDQRLGJHQHUDWLRQPDNHVWKHV\VWHPLGHDO for understanding organ development and the biology of the adult- or tissue- resident stem cells that maintain a given tissue throughout life. Organoids also lend themselves to many other applications, such as cell biology, GUXJGHYHORSPHQWDQGWKHXQGHUVWDQGLQJRIGLVHDVH7KHLUZLGHUDQJHRI applications, coupled with their potential to significantly reduce the use of DQLPDOPRGHOVZKLOHDOORZLQJIRUIDFLOHH[SHULPHQWDWLRQRQKXPDQFHOOV makes organoids valuable model systems. Consequently, organoid model systems are seeing rapid adoption in laboratories world-wide.
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Figure 1. Light microscope visualization of a mouse intestinal organoid. 6RXUFH 67(0&(//7HFKQRORJLHV 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
Organoids can be classified in a variety of ways, depending on the starting cells used, the tissue, and whether cells are from healthy or diseased VRXUFHV7RGDWHRUJDQRLGVKDYHEHHQVXFFHVVIXOO\JHQHUDWHGIURPDZLGH variety of tissues, including brain, breast, intestine, kidney, liver, lung, optic cup, pancreas, and prostate. Additionally, organoids can be generated from tumor tissue, resulting in organoid cultures that closely model the LQYLYRWXPRUFKDUDFWHULVWLFVDQGJHQHWLFKHWHURJHQHLW\ÈVRPHWKLQJWKDW KDVQRWEHHQSRVVLEOHLQ'FXOWXUHV Organoids do represent a significant leap in understanding stem cell biology and organ development, but combinatorial and/or complemen- WDU\WHFKQLTXHVFRXOGGULYHHYHQPRUHDSSOLFDWLRQV7KHUHIRUHFRPELQLQJ organoid generation with evolving techniques, such as the gene-editing WHFKQLTXH&5,635&DV PLJKWSURYHYLWDOWRPRUHWKRURXJKO\LQYHVWLJDW- ing biological and medical questions. 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð HISTORIC MILESTONES
7KHPDMRUDFKLHYHPHQWVLQUHVHDUFKRQDQGZLWKRUJDQRLGVFDQEH H[SORUHGLQWKLVFKURQRORJLFDOOLVWRINH\SXEOLFDWLRQV • 2RWDQL$/L;6DQJLRUJL(HWDO 6XVWDLQHGLQYLWUR intestinal epithelial culture within a Wnt-dependent stem cell niche. 1DW0HG. Ç ˜In search of a culture system that mimics the growth and differen- WLDWLRQRIWKHLQWHVWLQH&DOYLQ.XRÈ0DXUHHQ/\OHV'Ê$PEURJLR 3URIHVVRURI0HGLFLQHDW6WDQIRUG8QLYHUVLW\ÈDQGKLVFROOHDJXHV GHYHORSHGDPHWKRGIRUFUHDWLQJLQWHVWLQDORUJDQRLGV7KH\DOVR showed that an antagonist of the Wnt growth factor inhibited growth of the organoids, and that other treatments could be used to trigger the differentiation of specific types of cells, such as goblet and enteroendo- crine cells. • 6DWR79ULHV5*6QLSSHUW+-HWDO 6LQJOH/JUVWHPFHOOV build crypt-villus structures LQYLWUR without a mesenchymal niche. 1DW Ç ˜New cells in the intestinal wall arise in the crypts, which are the GLSVEHWZHHQYLOOL7RVKLUR6DWRRIWKH.HLR8QLYHUVLW\6FKRRORI 0HGLFLQHÈWKHQZRUNLQJLQWKHODERI+DQV&OHYHUVDWWKH8QLYHUVLW\ 0HGLFDO&HQWHU8WUHFKWDQG8WUHFKW8QLYHUVLW\ÈDQGKLVFROOHDJXHV developed a system in which crypt cells could be turned into orga- noids. In particular, the scientists started these organoids with crypt FHOOVH[SUHVVLQJ/JU, which triggers cycling in these cells. • 6DWR76WDQJH'()HUUDQWH0HWDO /RQJWHUPH[SDQVLRQRI epithelial organoids from human colon, adenoma, adenocarcinoma, DQG%DUUHWWÊVHSLWKHOLXP*DVWURHQWHURORJ\ Ç ˜Continuing the work with intestinal organoids created from crypt- derived stem cells, Sato and his colleagues developed methods to grow 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
healthy and disease-specific organoids from mouse colon and human VPDOOLQWHVWLQHDQGFRORQ$IWHURSWLPL]LQJPHWKRGVIRUFXOWXULQJ WKHVHRUJDQRLGV6DWRÊVWHDPUHSRUWHG Ì:HGHYHORSHGDWHFKQRORJ\ that can be used to study infected, inflammatory, or neoplastic tissues IURPWKHKXPDQJDVWURLQWHVWLQDOWUDFWÍ7KHUHVHDUFKHUVDGGHGWKDW WKH\IRXQGQRÌLQKHUHQWUHVWULFWLRQLQWKHUHSOLFDWLYHSRWHQWLDORI adult stem cells . . . H[YLYR.” • 6SHQFH-50D\KHZ&15DQNLQ6$ 'LUHFWHGGLIIHUHQWLD- tion of human pluripotent stem cells into intestinal tissue LQYLWUR. 1DW. Ç ˜8VLQJKXPDQ36&V-DVRQ6SHQFHÈWKHQDW&LQFLQQDWL&KLOGUHQÊV+RV- SLWDO0HGLFDO&HQWHUDQGQRZDVVRFLDWHSURIHVVRURILQWHUQDOPHGLFLQH DWWKH8QLYHUVLW\RI0LFKLJDQ0HGLFDO6FKRROÈDQGKLVFROOHDJXHV GHYHORSHGZKDWWKH\GHVFULEHGDVÌDUREXVWDQGHIILFLHQWSURFHVVWR direct the differentiation of human PSCs into intestinal tissue LQYLWUR using a temporal series of growth factor manipulations to mimic HPEU\RQLFLQWHVWLQDOGHYHORSPHQWÍ7KLVLQYROYHGHQGRGHUPIRUPD- tion and patterning, hindgut specification and morphogenesis, plus a culture system for intestinal growth, morphogenesis, and cytodif- IHUHQWLDWLRQÌ7KHUHVXOWLQJWKUHHGLPHQVLRQDOLQWHVWLQDOÉRUJDQRLGVÊ FRQVLVWHGRIDSRODUL]HGFROXPQDUHSLWKHOLXPWKDWZDVSDWWHUQHGLQWR YLOOXVOLNHVWUXFWXUHVDQGFU\SWOLNHSUROLIHUDWLYH]RQHVWKDWH[SUHVVHG LQWHVWLQDOVWHPFHOOPDUNHUVÍ6SHQFHÊVWHDPQRWHG • .DGRVKLPD76DNDJXFKL+1DNDQR7HWDO 6HOIRUJDQL]DWLRQ RID[LDOSRODULW\LQVLGHRXWOD\HUSDWWHUQDQGVSHFLHVVSHFLILFSURJHQL- WRUG\QDPLFVLQKXPDQ(6FHOOGHULYHGQHRFRUWH[3URF1DWO$FDG 6FL86$ Ç ˜Working with cortical neuroepithelium derived from human HPEU\RQLFVWHPFHOOV7DLVXNH.DGRVKLPDÈWKHQDWWKH5,.(1 &HQWHUIRU'HYHORSPHQWDO%LRORJ\DQGQRZDW$VXELR3KDUPDÈDQG his collaborators studied neocorticogenesis in neural organoids. 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
Ì6HOIRUJDQL]HGFRUWLFDOWLVVXHVSRQWDQHRXVO\IRUPVDSRODULW\DORQJ WKHGRUVRFDXGDOYHQWURURVWUDOD[LVDQGXQGHUJRHVUHJLRQVSHFLILF rolling morphogenesis that generates a semispherical structure,” the UHVHDUFKHUVZURWHÌ7KHQHXURHSLWKHOLXPVHOIIRUPVDPXOWLOD\HUHG VWUXFWXUHLQFOXGLQJWKUHHQHXURQDO]RQHV VXESODWHFRUWLFDOSODWH DQG&DMDO5HW]LXVFHOO]RQHV DQGWKUHHSURJHQLWRU]RQHV YHQWULFXODU VXEYHQWULFXODUDQGLQWHUPHGLDWH LQWKHVDPHDSLFDOEDVDORUGHUDV VHHQLQWKHKXPDQIHWDOFRUWH[LQWKHHDUO\VHFRQGWULPHVWHUÍ%DVHGRQ WKHVHDQGRWKHUIHDWXUHVWKHVFLHQWLVWVFRQFOXGHGWKDWÌKXPDQQHRFRU- ticogenesis involves intrinsic programs that enable the emergence of FRPSOH[QHRFRUWLFDOIHDWXUHVÍ • /DQFDVWHU0$5HQQHU00DUWLQ&$HWDO &HUHEUDO organoids model human brain development and microcephaly. 1DW. Ç ˜Ì7KHFRPSOH[LW\RIWKHKXPDQEUDLQKDVPDGHLWGLIILFXOWWRVWXG\ many brain disorders in model organisms, highlighting the need for an LQYLWURPRGHORIKXPDQEUDLQGHYHORSPHQWÍZURWH0DGHOLQH /DQFDVWHUÈWKHQDWWKH,QVWLWXWHRI0ROHFXODU%LRWHFKQRORJ\RIWKH Austrian Academy of Science and now a principal investigator at the 0HGLFDO5HVHDUFK&RXQFLO 05& /DERUDWRU\RI0ROHFXODU%LRORJ\È and her coworkers. Cerebral organoids were generated using human- derived PSCs in healthy controls as well as microcephalic patients. ,QDGGLWLRQWRIRUPLQJUHJLRQVOLNHWKHFHUHEUDOFRUWH[WKHVHRUJD- QRLGVÌUHFDSLWXODWHIHDWXUHVRIKXPDQFRUWLFDOGHYHORSPHQWQDPHO\ FKDUDFWHULVWLFSURJHQLWRU]RQHRUJDQL]DWLRQZLWKDEXQGDQWRXWHU UDGLDOJOLDOVWHPFHOOVÍ/DQFDVWHUÊVWHDPSRLQWHGRXW • %DJOH\-$5HXPDQQ'%LDQ6HWDO )XVHGFHUHEUDORUJD- noids model interactions between brain regions. 1DW0HWKRGV Ç ˜7REXLOGDQHXUDORUJDQRLGWKDWDOORZVPRUHDGYDQFHGVWXGLHVRIWKH EUDLQWKH'VWUXFWXUHQHHGVVRPHUHSUHVHQWDWLRQRIEUDLQUHJLRQV 7RPRYHWRZDUGVXFKDQHXUDORUJDQRLG-RVKXD%DJOH\ÈDSRVWGRFWRUDO 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
UHVHDUFKHULQ-XHUJHQ.QREOLFKÊVODEDWWKH,QVWLWXWHRI0ROHFXODU%LR- WHFKQRORJ\RIWKH$XVWULDQ$FDGHP\RI6FLHQFHLQ9LHQQD$XVWULDÈ and his colleagues used small molecule patterning to create dorsal- and YHQWUDOEUDLQRUJDQRLGVIURPKXPDQLQGXFHG36&V L36&V DQGIXVHG WKHP7KHVHVWUXFWXUHVH[KLELWHGVRPHRIWKHGHYHORSPHQWDOSURFHVVHV found in human embryonic brains, including γ-aminobutyric acid *$%$ SURGXFLQJLQWHUQHXURQVGHYHORSHGLQWKHYHQWUDOSRUWLRQRI WKHEUDLQDQGPLJUDWLQJWRWKHGRUVDOUHJLRQ5HVHDUFKHUVVKRZHGWKH PLJUDWLQJFHOOVZHUHQHXURQVDVLQGLFDWHGE\WKHH[SUHVVLRQRI+X&' DQGPDWXUHE\WKHH[SUHVVLRQRI0$3%DVHGRQRWKHUPROHFXODU PDUNHUVWKHVFLHQWLVWVUHSRUWHG Ì2XUUHVXOWVLQGLFDWHWKDWRUJDQRLG fusions contain diverse interneuron subtypes originating from the PDMRUYHQWUDOIRUHEUDLQVXEUHJLRQVÍ%\SURGXFLQJRUJDQRLGVWKDW mimic other brain regions and fusing them, this methodology can be used to study a wide range of neuronal circuits. • %LUH\)$QGHUVHQ-0DNLQVRQ&'HWDO $VVHPEO\RIIXQF- WLRQDOO\LQWHJUDWHGKXPDQIRUHEUDLQVSKHURLGV1DW Ç ˜7KHGRUVDOIRUHEUDLQDOVRNQRZQDVWKHSDOOLXPFRQWDLQVH[FLWDWRU\ QHXURQVWKDWUHOHDVHWKHQHXURWUDQVPLWWHUJOXWDPDWH7KHYHQWUDO forebrain, or subpallium, contains inhibitory neurons that release *$%$7RVWXG\WKHGHYHORSPHQWRIQHXUDOFLUFXLWVLQWKHFRUWH[ )LNUL%LUH\ÈDSRVWGRFWRUDOUHVHDUFKHULQ6HUJLX3DșFDÊVODEDW6WDQIRUG 8QLYHUVLW\ÈDQGKLVFROOHDJXHVFUHDWHGKXPDQFRUWLFDO GRUVDO VSKHURLGV K&6 DQGKXPDQVXESDOOLXPVSKHURLGV K66 IURPK36&V Placing one of each kind of spheroid in a tube, they combined in three GD\V8VLQJJUHHQIOXRUHVFHQWSURWHLQ *)3 WKHVFLHQWLVWVVKRZHG FHOOVPRYLQJIURPWKHK66WRWKHK&67KHWHDPXVHGWKLVPRGHOWR VWXG\7LPRWK\V\QGURPH 76 LQZKLFKGHIHFWVLQFRUWLFDOQHXURQ migration result in various neural deficits, including autism spec- WUXPGLVRUGHUV7KHVFLHQWLVWVPDGHK&6DQGK66IURPSDWLHQWVZLWK 76&RPSDUHGWRK&6K66VWUXFWXUHVIURPFRQWUROVXEMHFWVQHXURQV PLJUDWHGPRUHVORZO\LQWKHVWUXFWXUHVIURPSHRSOHZLWK766WXGLHV LQURGHQWPRGHOVKDYHVXJJHVWHGWKDWWKHPLJUDWLRQRI/W\SHFDOFLXP 10 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
FKDQQHOV /7&&V SOD\VRPHSDUWLQ76%LUH\ÊVWHDPVKRZHGÌWKDWWKH PLJUDWLRQGHIHFWLQLQWHUQHXURQVFDUU\LQJWKH76JDLQRIIXQFWLRQ PXWDWLRQFDQEHUHVWRUHGE\UHGXFLQJWKHDFWLYLW\RI/7&&VÍ7KH FRUWLFDOH[FLWDWRU\LQKLELWRU\EDODQFHLQSDWLHQWGHULYHGPRGHOVRI DXWLVPFDQQRZEHVWXGLHGPHFKDQLVWLFDOO\LQYLWUR7KLVZRUNGHP- onstrated how anatomical and molecular aspects of neurodevelop- PHQWHYHQLQWKHFRUWH[FDQEHH[SORUHGZLWKRUJDQRLGV • )UHHGPDQ%6%URRNV&5/DP$4HWDO 0RGHOOLQJNLGQH\ GLVHDVHZLWK&5,635PXWDQWNLGQH\RUJDQRLGVGHULYHGIURPKXPDQ pluripotent epiblast spheroids. 1DW&RPPXQ ˜%HQMDPLQ)UHHGPDQÈWKHQDW+DUYDUG0HGLFDO6FKRRODQGQRZDW WKH8QLYHUVLW\RI:DVKLQJWRQ6FKRRORI0HGLFLQHÈDQGKLVFRDXWKRUV ZDQWHGWRNQRZLIKXPDQ36& K36& GHULYHGNLGQH\FHOOVFRXOG ÌUHFRQVWLWXWHWLVVXHVSHFLILFSKHQRW\SHVÍ7RILQGRXWWKHUHVHDUFKHUV PDGH'FXOWXUHVRIHSLEODVWVWDJHK36&VDQGVKRZHGWKDWLQKLELW- LQJDQHQ]\PHÈJO\FRJHQV\QWKDVHNLQDVHEHWD *6.β ÈFDXVHG WKHVSKHURLGVWRGLIIHUHQWLDWHÌLQWRVHJPHQWHGQHSKURQOLNHNLGQH\ RUJDQRLGVFRQWDLQLQJFHOOSRSXODWLRQVZLWKFKDUDFWHULVWLFVRISUR[L- mal tubules, podocytes and endothelium.” In addition, these scien- tists showed that gene manipulation could be used to create models RIKHDOWKFRQGLWLRQV)RUH[DPSOHWKHDXWKRUVUHSRUWHG Ì.QRFNRXW of the polycystic kidney disease genes 3.' or 3.' induces cyst formation from kidney tubules.” • 7DNDVDWR0(U3;&KLX+6HWDO .LGQH\RUJDQRLGVIURP human iPS cells contain multiple lineages and model human nephro- genesis. 1DW Ç ˜7KHNLGQH\SRUWUD\VWKHGLYHUVLW\RIFHOOW\SHVWKDWFDQEHUHTXLUHG to build an organoid that reliably replicates the actual structure. Ì5HJHQHUDWLQJWKHNLGQH\UHTXLUHVWKHLQGXFWLRQRIWKHPRUHWKDQ GLVWLQFWFHOOW\SHVUHTXLUHGIRUH[FUHWLRQDQGWKHUHJXODWLRQRIS+DQG HOHFWURO\WHDQGIOXLGEDODQFHÍZURWH0LQRUX7DNDVDWRÈWKHQDWWKH 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
8QLYHUVLW\RI4XHHQVODQGDQGQRZDW5,.(1&HQWHUIRU%LRV\VWHPV '\QDPLFV5HVHDUFKÈDQGDWHDPRIVFLHQWLVWV7KHVHFHOOVEXLOGVSHFLILF parts of the kidney, such as the collecting duct and nephrons. In this DUWLFOH7DNDVDWRDQGKLVFROOHDJXHVGHVFULEHGDWHFKQLTXHWRJHQHUDWH kidney organoids that included these structures. As the researcher ZURWH Ì:LWKLQWKHVHRUJDQRLGVLQGLYLGXDOQHSKURQVVHJPHQWLQWR GLVWDODQGSUR[LPDOWXEXOHVHDUO\ORRSVRI+HQOHDQGJORPHUXOLFRQ- taining podocytes elaborating foot processes and undergoing vascular- L]DWLRQÍ7KHUHVHDUFKHUFRQFOXGHGWKDWWKHVHNLGQH\RUJDQRLGVFRXOG EHXVHGIRUIXWXUHDSSOLFDWLRQVLQFOXGLQJQHSKURWR[LFLW\VFUHHQLQJ disease modelling and as a source of cells for therapy.” 12 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð IN PRACTICE
2UJDQRLG7\SHVDQG&XOWXUH&RQGLWLRQV 7KHUHDUHWZRSULPDU\FODVVLILFDWLRQVRIRUJDQRLGV\VWHPV RUJDQRLGV cultured from PSCs and organoids cultured from adult stem or progenitor cells. PSC-derived organoids are generated by differentiation of PSCs to lineage-specific progenitor cells that form the desired organoid. Organoid formation is then achieved by culturing these progenitors in conditions that mimic the LQYLYR developmental conditions of that organ. For brain organoids, by omitting or adding patterning factors, the organoid can HLWKHUEHDOORZHGWRVHOIRUJDQL]HLQWRDQRUJDQRLGZLWKVHYHUDOGLIIHUHQW EUDLQUHJLRQV HJFHUHEUDORUJDQRLGV RUFDQEHGLUHFWHGWRIRUPDQRUJD- QRLGZLWKVSHFLILFEUDLQUHJLRQV HJIRUHEUDLQRUPLGEUDLQRUJDQRLGV 7KHDGYDQWDJHRI36&GHULYHGRUJDQRLGVLVWKHLUHDV\WRDFFHVVVWDUWLQJ material, because previously-established or patient-specific PSC lines can EHXVHG7KHVHRUJDQRLGVWHQGWRPRGHOWKHGHYHORSLQJUDWKHUWKDQWKH adult organ. ASC-derived organoids are cultured from tissue-specific stem or progenitor cells that are responsible for maintenance of a given organ LQYLYR7KHRUJDQRLGVJURZQIURPWKHVHSURJHQLWRUVWHQGWRPRUHFORVHO\ model adult tissue and have the advantage that they can recapitulate both congenital and non-congenital disease states, including modeling epige- netic and tumor signatures. Both classes of organoids are cultured in specific cell-culture media ZLWKLQDQH[WUDFHOOXODUPDWUL[WKDWLVUHTXLUHGWRVXSSRUWWKH'VWUXFWXUH 7KHFHOOFXOWXUHPHGLXPXVHGGHSHQGVRQWKHW\SHDQGWLVVXHRIWKHRUJD- noid, and in general mimics the signaling environment the cultured cells ZRXOGH[SHULHQFHLQYLYR)RU36&GHULYHGFHUHEUDORUJDQRLGVIRUH[DPSOH the culture conditions mimic the signaling present in the developing brain, allowing the neural progenitors present in early stages of the organoid to GLIIHUHQWLDWHDQGVHOIRUJDQL]HLQWRWKHODPLQDUVWUXFWXUHRIWKHFHUHEUXP In similar fashion, the culture conditions for ASC-derived intestinal orga- noids mimic the signaling present at the intestinal crypt base, the stem cell 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
QLFKHRILQWHVWLQDOVWHPFHOOV7KLVDOORZVIRUVHOIUHQHZDORIWKHVWHPFHOO population as well as differentiation of a subset of this population to form the cellular complement of the intestine. $GXOW6WHP&HOOâ'HULYHG2UJDQRLGV Sato HWDO GHYHORSHGDPHWKRGIRUFXOWXULQJ$6&GHULYHGLQWHV- WLQDORUJDQRLGVIURPLVRODWHGLQWHVWLQDOFU\SWV7KLVNLFNHGRIIDGHFDGHRI LQQRYDWLRQLQRUJDQRW\SLF'FHOOFXOWXUHWHFKQLTXHV7KHFXOWXUHPRGHO RIWKHVHLQWHVWLQDORUJDQRLGVH[SORLWVWKHELRORJ\RIWKHLQWHVWLQDOVWHP cells, which LQYLYR actively divide throughout life to replenish their own SRSXODWLRQDQGWXUQRYHUWKHHQWLUHLQWHVWLQDOHSLWKHOLXPHYHU\ÇGD\V 7KHHQYLURQPHQWDOFRQGLWLRQVWKDWHQDEOHWKHVHUHSOHQLVKLQJSURSHUWLHVLQ YLYRDUHUHFDSLWXODWHGLQWKHRUJDQRLGFXOWXUHV\VWHP7KLVDOORZVWKHVDPH cells to create a faithful intestinal model LQYLWUR. Although not all tissues have ASCs with the same regenerative capacity during homeostasis, many organs contain a population of cells that carry the ability to regenerate WLVVXHXQGHUVSHFLILFFRQGLWLRQV7KLVKDVHQDEOHGGHYHORSPHQWRIRUJDQRLG culture conditions for a wide variety of tissues, including liver, pancreas, prostate, breast, and airway. Generating organoids from ASCs has several advantages. First, because the cultures are generated from phenotypically adult cells, the resultant FXOWXUHVDOVRWHQGWRH[KLELWDQDGXOWSKHQRW\SH7KHSURWRFROVDOVRWHQGWR be shorter than those needed to generate PSC-derived organoids, because WKHVWDUWLQJPDWHULDOLVDOUHDG\DWLVVXHVSHFLILFSURJHQLWRU7KHWHFKQLTXHV are comparatively easy to learn and adopt, allowing labs to add organoid H[SHULPHQWVWRWKHLUDOUHDG\HVWDEOLVKHGODERUDWRU\UHSHUWRLUH2QFHHVWDE- lished, many ASC-derived organoid cultures can be maintained long-term through routine passaging and can be cryopreserved; however, the origi- nal starting material requires patient biopsy and associated consent that can significantly limit access for many researchers, especially industrial laboratories. While there is substantial overlap in the applications of ASC- and PSC-derived organoids, ASC-derived organoids are uniquely suited to some 14 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
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Figure 2. Adult stem cell-derived organoids: (a) Human colonic organoids; (b) mouse prostate organoids; (c) pancreatic exocrine organoids; and (d) hepatic progenitor organoids. 6RXUFH 67(0&(//7HFKQRORJLHV 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
VSHFLILFDSSOLFDWLRQV)RUH[DPSOH$6&GHULYHGRUJDQRLGVFDQEHXVHGWR model non-genetic pathologies and cancers, both of which are challenging for a PSC-derived system. Indeed, tumor organoids derived from ASCs are rapidly gaining recognition for their utility in patient-specific tumor mod- HOV7KHFDQFHUFKDUDFWHULVWLFVJHQHWLFKHWHURJHQHLW\DQGGUXJUHVSRQVH profile are maintained with high fidelity in the organoids, allowing for precision-medicine applications. 7KHVRXUFHRIWKHDGXOWVWHPFHOOVLVFULWLFDOWRWKHVXFFHVVIXOJHQHUDWLRQ DQGXVHRIRUJDQRLGV5HVHDUFKHUVRIWHQXVHPRXVHRUJDQRLGVWRFRPSDUH results from LQYLYR and LQYLWUR studies. Organoids made from human stem cells, however, allow unique uses, such as studying therapeutic impact on DSDWLHQWÊVRZQWXPRU SUHFLVLRQPHGLFLQH RUWKHEDVLFELRORJ\RIKXPDQ stem cells. As with any emerging technology, organoids must be validated as DSSURSULDWHPRGHOV\VWHPVIRUDQ\JLYHQDSSOLFDWLRQ$VDQH[DPSOH=LHWHN HWDO FUHDWHGLQWHVWLQDORUJDQRLGVIURPZLOGW\SHPLFHDQGRQHVODFN- LQJVSHFLILFQXWULHQWWUDQVSRUWHUV7KHUHVXOWVVKRZHGWKDWWKHVWUXFWXUHV VHUYHDVH[FHOOHQWPRGHOVIRUUHVHDUFKEHFDXVHWKH\PDLQWDLQWKHSKHQR- W\SLFDQGIXQFWLRQDOIHDWXUHVRIPRXVHLQWHVWLQHV/LNHZLVH$6&GHULYHG RUJDQRLGVDUHEHLQJH[SORUHGDQGDGRSWHGIRUDZLGHUDQJHRIDSSOLFDWLRQV ranging from basic, academic research to drug development. $GXOW6WHP&HOO&DVH6WXGLHV 7KHHSLWKHOLXPLQWKHPXFRVDOVXUIDFHVRIWKHLQWHVWLQHVLVUHSODFHG HYHU\ÇGD\V6WHPFHOOVLQWKHFU\SWVDUHWKHVRXUFHRIWKLVVHOIUHQHZDO and these cells can be stimulated to differentiate into any of the cell types WKDWPDNHXSWKHOLQLQJRIWKHLQWHVWLQH7RH[SORUHWKHPROHFXODUGHWDLOVRI those mechanisms, Sehgal HWDO XVHG*HQWOH&HOO'LVVRFLDWLRQ5HDJHQW to dissociate crypts from mouse small intestine, and then cultured the FU\SWVZLWK,QWHVWL&XOWģ2UJDQRLG*URZWK0HGLXP 0RXVH WRPDNHHQWHU- RLGV6FLHQWLVWVDOUHDG\NQHZWKDWFRORQ\VWLPXODWLQJIDFWRU &6) SOD\VD role in maintaining gut epithelium, but the mechanism was unknown. 16 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
CSF1 drives the differentiation of macrophages from monocytes. 7KURXJKPDFURSKDJHDEODWLRQDQG&6)5EORFNDGHVWXGLHVWKHVFLHQWLVWV VKRZHGWKDW&6)GHSHQGHQWPDFURSKDJHVÌLQIOXHQFHLQWHVWLQDOHSLWKHOLDO differentiation and homeostasis” by aiding in the differentiation of intesti- nal epithelial Paneth cells, which release antimicrobials that block bacterial infection in the crypts and prevent bacteria from crossing the intestinal
Day 1 Day 3 Day 5 Control
50 μm Anti-CSF1R CSF1-Fc
Figure 3. No obvious morphological changes distinguish mouse intestinal organoids treated with anti-CSF1R or CSF1. 6RXUFH 6HKJDO$'RQDOGVRQ'63ULGDQV&HWDO 7KHUROHRI&6)5GHSHQGHQWPDFURSKDJHVLQFRQWURORIWKH intestinal stem cell niche. 1DWXUH&RPPXQ 'RL V 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
HSLWKHOLXP7KLVZRUN\LHOGVWZRJHQHUDOFRQFOXVLRQV WKHXWLOLW\RI organoids allowed quick assessment of the role of CSF1 in a physiologi- cally relevant LQYLWURFHOOFXOWXUHPRGHO DQG WKLVZRUNSURYLGHGIXUWKHU XQGHUVWDQGLQJRI&6)5GHSHQGHQWFU\SWDVVRFLDWHGPDFURSKDJHVWKDWDUH ÌFRQVWLWXWLYHO\UHTXLUHGWRPDLQWDLQWKHLQWHVWLQDOVWHPFHOOQLFKHLQWKH small intestine.” $QRWKHUH[DPSOHRIWKHXWLOLW\RIRUJDQRLGVFDQEHIRXQGLQVWXGLHVRI the liver. Broutier HWDO GHVFULEHGJHQHUDWLQJWXPRURUJDQRLGVIURP liver tumor tissue from patients who had not been treated for primary liver FDQFHU 3/& :LWKWKLVWHFKQLTXHWKHUHVHDUFKHUVJHQHUDWHGRUJDQRLGV IURPWKHWKUHHPRVWFRPPRQVXEW\SHVRI3/& KHSDWRFHOOXODUFDUFLQRPD +&& FKRODQJLRFDUFLQRPD && DQGFRPELQHG+&&&& &+& WXPRUV Several comparisons confirmed that the organoids mimic the original WXPRU+LVWRORJLFDOO\WKHWXPRURLGVPDLQWDLQHGSDWLHQWVSHFLILFKHWHURJH- QRXVPRUSKRORJLFDOIHDWXUHVRIHDFKWXPRUVXEW\SH)RULQVWDQFH+&&DQG &+&WXPRURLGVLQFOXGHGFRPSDFWVWUXFWXUHVDQG&&WXPRURLGVLQFOXGHG LUUHJXODUF\VWVWUXFWXUHVÈDOOGLIIHUHQWWKDQWKHRUGHUHGF\VWOLNHVWUXFWXUHV in healthy liver organoids. Based on genome-wide transcriptomic analysis 51$VHTXHQFLQJRU51$VHT DQGFRPSDULVRQWRWKHNQRZQJHQHH[SUHVVLRQ SDWWHUQVIRUWKHVXEW\SHVRI3/&%URXWLHUHWDOUHSRUWHG Ì*HQHH[SUHVVLRQ correlation analysis indicated that each tumoroid line correlated to its cor- responding tissue-of-origin, but not with the other subtypes.” Broutier HWDO. used these organoids to identify an inhibitor of the H[WUDFHOOXODUVLJQDOUHJXODWHGNLQDVH (5. SDWKZD\WKDWPLJKWOHDGWR D3/&WUHDWPHQW$VWKHUHVHDUFKHUVFRQFOXGHGWKLVZRUNGHPRQVWUDWHV ÌWKHZLGHUDQJLQJELRPHGLFDOXWLOLWLHVRI3/&GHULYHGRUJDQRLGPRGHOVLQ furthering the understanding of liver cancer biology and in developing SHUVRQDOL]HGPHGLFLQHDSSURDFKHVIRUWKHGLVHDVHÍ 3OXULSRWHQW6WHP&HOO'HULYHG2UJDQRLGV $VQRWHGDERYHRUJDQRLGVFDQDOVREHFUHDWHGIURP36&V+HUHWKH starting cells can be established PSC cell lines or patient-specific induced 36&V L36&V 7KHFDSDFLW\RI36&VWRGLIIHUHQWLDWHLQWRDQ\FHOOW\SHRIWKH 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
(a) AFP+ AFP+/– HepPar1+/– HepPar1+ KRT19+ KRT19+ +/– +/– EpCAM EpCAM– +/– SALL4 Loss of biliary SALL4 + Well-diff. Mucins markers Poorly diff. HCC + Biliary markers + Classical HCC AFP Stem cell HepPar1+ HCC CHC – CHC KRT19 AFP+ HCC-1 – +/– CHC-1 EpCAM HepPar1 SALL4– KRT19+ – EpCAM+ HCC-3 AFP – SALL4+ CC-3 CC-1 HepPar1 CHC-2 + DIFFERENTIATION CC-2 KRT19 EpCAM+ Poorly diff. – CC Well-diff. SALL4 Loss of hepatocyte CC markers
(b) HCC-1_T HCC-3_T CHC-1_T CHC-2_T CC-1_T CC-2_T HepPar1 EpCAM
(c) HCC-1_O HCC-3_O CHC-1_O CHC-2_O CC-1_O CC-2_O AFP EpCAM
Figure 4. Human PLC tumoroids maintain the expression patterns of the original tissue in long-term culture. (a) HCC and CC subtypes of PLC arise from distinct pathways. (b) Immunohistochemistry assays for the hepatocyte/HCC (HepPar1) and ductal/CC (EpCAM) markers on subtypes of PLC tissue. (Scale bar, 125 μm; dashed red square indicates focal staining.) (c) On tumoroids cultured for at least three months, immunofluorescence analysis shows presence of the HCC marker AFP (red) and the ductal/CC marker EpCAM (green). (Nuclei were counterstained with Hoechst33342 (blue); Scale bar, 30 μm.) 6RXUFH %URXWLHU/0DVWURJLRYDQQL*9HUVWHJHQ00$HWDO +XPDQSULPDU\OLYHUFDQFHUGHULYHGRUJDQRLG cultures for disease modeling and drug screening. 1DW0HG ÇGRL QP body, combined with their ability to proliferate indefinitely, make PSCs a convenient starting material for organoid culture. PSC-derived organoid culture systems have been developed to model tissues derived from all three germ layers: endoderm, ectoderm, and 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
PHVRGHUP7KHVHV\VWHPVPLPLFWKHELRORJ\RIWKHWLVVXHVWKH\PRGHO IRU H[DPSOHFHUHEUDORUJDQRLGVUHFDSLWXODWHWKHGHYHORSPHQWDOSURFHVVHVDQG RUJDQL]DWLRQRIWKHGHYHORSLQJKXPDQEUDLQ6XFKRUJDQRLGVFDQEHJHQHU- DWHGDQGPDLQWDLQHGZLWKWKH67(0GLIIģ&HUHEUDO2UJDQRLG.LW/LNHWKH cerebral tissue these organoids model, the cells within the organoids can be maintained for weeks and months, if they have access to the right nutri- HQWVEXWFDQQRWEHXVHGWRLQLWLDWHQHZRUJDQRLGFXOWXUHV7KLVLVVLPLODU to the LQYLYR brain that contains proliferative progenitors during develop- PHQWDQGORVHVWKHYDVWPDMRULW\RILWVDELOLW\WRJHQHUDWHQHZQHXURQVLQ DGXOWKRRG7KLVFRQWUDVWVZLWKWKHHSLWKHOLDOFRPSRQHQWRIVRPHRUJDQV such as the intestine, which maintain an active adult stem cell population WRFRQVWDQWO\UHSOHQLVKWKHRUJDQÊVFHOOV36&VFDQEHGLUHFWHGWRWKHVHOLQH- ages and generate organs that share many of the growth properties of ASC- GHULYHGRUJDQRLGV,QWHVWLQDO36&GHULYHGRUJDQRLGVIRUH[DPSOHFDQEH generated and maintained long-term through routine passaging with the 67(0GLIIģ,QWHVWLQDO2UJDQRLG.LW Generating organoids from PSCs comes with advantages. It is relatively easy to access starting material to generate organoids, compared with REWDLQLQJSDWLHQWELRSVLHV0DNLQJSDWLHQWVSHFLILFL36&VDOVRDOORZVWKH generation of organoids with a specific genetic code. With human cerebral organoids, starting with hPSCs enables studies not possible with any other model system, because ethical limitations severely constrain access to primary neural tissue. Building organoids from PSCs also allows for co- differentiation of multiple lineages within one culture, thus providing the opportunity to create organoids that mimic the various tissues found in an actual organ. 'HVSLWHWKHDGYDQWDJHVRIPDNLQJRUJDQRLGVIURP36&VWKLVDSSURDFK also raises some challenges. For one, the scientist needs to be proficient in PDLQWDLQLQJ36&VWRHQVXUHKLJKTXDOLW\VWDUWLQJFHOOV7KHSURWRFROVIRU differentiating the cells to organoids can also be quite lengthy, requiring PXOWLSOHVWHSVWKDWPLPLFWKHGHYHORSPHQWDOWUDMHFWRU\RIFHOOVLQDGHYHO- oping body. 20 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
(a) (b)
Figure 5. Pluripotent stem cell-derived organoids: (a) Human intestinal organoids; and (b) human cerebral organoids. 6RXUFH 67(0&(//7HFKQRORJLHV Still, organoids created from PSCs fit many applications. For intestinal organoids, using hPSCs as the starting material provides an opportunity to study developmental stages of the intestine, such as a more fetal-like organ in early passages. Genetic manipulation also provides the opportunity for other applications of PSC-derived organoids, and the cells can be geneti- FDOO\PDQLSXODWHGE\DGGLQJ'1$WKURXJKWUDQVIHFWLRQRUHOHFWURSRUDWLRQ $OVRD&5,635&DV WHFKQLTXHFDQEHXVHGWRHGLWJHQHVZLWKLQRUJDQRLG V\VWHPV7KLVPHWKRGLVIDVWHUWKDQPDNLQJNQRFNRXWPLFH7KHVHRUJD- noid systems can also be used in combination with other technologies. For LQVWDQFHLQIRUPDWLRQIURPVWXGLHVRINQRFNRXWPLFHFDQEHUHH[DPLQHG LQRUJDQRLGVZKHUHPHFKDQLVPVFDQEHH[SORUHGLQDQHQYLURQPHQWWKDW better reflects physiological conditions. 3OXULSRWHQW6WHP&HOO&DVH6WXGLHV Neural organoids derived from PSCs can be used in many ways, from VWXG\LQJQRUPDOQHXUDOGHYHORSPHQWDQGSURFHVVHVWRDQDO\]LQJEUDLQ based diseases. Scientists did not know, however, if neural organoids could EHXVHGWRVWXG\WKHGHYHORSPHQWDOJXLGDQFHRID[RQVLQYROYHGLQQHXUDO FLUFXLWV7RILQGRXW*LDQGRPHQLFRHWDO JHQHUDWHGQHXUDORUJDQRLGV IURPKXPDQHPEU\RQLFVWHPFHOOVFXOWXUHGZLWKWKH67(0GLIIģ&HUHEUDO 2UJDQRLG.LW0DWXUHQHXUDORUJDQRLGVZHUHFRPELQHGZLWKGLVVHFWLRQV 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
of mouse spinal cord, including some nearby muscle tissue, and sliced LQμPVHFWLRQVWRIRUPDLUOLTXLGLQWHUIDFHFHUHEUDORUJDQRLGV $/, &2V 7KHDSSOLFDWLRQRISUHDQGSRVWV\QDSWLFPDUNHUVVKRZHGV\QDSVHV on the dendrites of mature neurons in the organoids, and Giandomenico HWDO. recorded spontaneous electrical activity using BrainPhys™ Neuronal 0HGLXPLQWKHQHXURQV0RUHRYHULQMHFWLQJSRVLWLYHFXUUHQWYLDZKROH cell patch clamping triggered action potentials, demonstrating functional maturity of the organoids. 7KH$/,&2VDOVRVKRZHGPDQ\IHDWXUHVRIDQDFWXDOEUDLQ%\DSSO\LQJ 51$VHTIRUFHOOW\SHVDQGFOXVWHULQJWKHUHVXOWVEDVHGRQH[SUHVVLRQZLWK principal components analysis, Giandomenico HWDOIRXQGÌDZHOOGHILQHG DVVRFLDWLRQEHWZHHQFHOOW\SHVDQGWKHLUH[SHFWHGIXQFWLRQVXJJHVWHGE\WKH PROHFXODUSURILOHVLQHDFKFOXVWHUÍ0RUHRYHUZLWKWKHHDVHRIOLYHLPDJLQJ in the sliced culture, Giandomenico HWDOWUDFNHGD[RQJXLGDQFHLQ*)3 ODEHOOHGQHXURQV7KHQHXURQVLQWKH$/,&2VFRXOGHYHQGULYHFRQWUDFWLRQ in the muscle tissue near the cord. Interestingly, contractions could be LQKLELWHGSKDUPDFRORJLFDOO\RUE\OHVLRQRIWKHD[RQDOWUDFW7KLVPRGHO system will enable detailed mechanistic studies of the regenerative capacity of the human nervous system. 2YHUDOOWKLVZRUNVKRZVWKHEHQHILWVRIWKH$/,&2DSSURDFKLQFOXG- LQJORQJWHUPYLDELOLW\7KH$/,&2VUHPDLQHGYLDEOHIRUXSWRILYH months, the longest time tested. Giandomenico HWDODOVRQRWHG Ì7KHVH H[SHULPHQWVDUHWKHILUVWWRRXUNQRZOHGJHWRVKRZDIXQFWLRQDORXWSXW from a neural organoid.” 7KHXVHVRI36&GHULYHGRUJDQRLGVZLOOH[SDQGDVVFLHQWLVWVFRPELQH systems to make even more accurate models of human organs. In one approach, Spence HWDO GHYHORSHGDPHWKRGIRUPDNLQJKXPDQLQWHV- tinal organoids. Starting with human embryonic stem cells and iPSCs, this SURWRFROZDVODWHUOLFHQVHGWRVHUYHDVWKHEDVLVIRUWKH67(0GLIIģ,QWHVWLQDO 2UJDQRLG.LW7KLVPHWKRG6SHQFHHWDOH[SODLQHGLQYROYHVDÌWHPSRUDO series of growth factor manipulations” to induce definitive endoderm formation, posterior endoderm patterning, hindgut specification and PRUSKRJHQHVLVDQGDÌSURLQWHVWLQDOFXOWXUHV\VWHPWRSURPRWHLQWHVWLQDO 22 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
STEM121 MAP2 Mouse tissue
ALI-CO
Figure 6. Brightfield image (left), shows axon tracks (arrow) in combined human ALI-CO and mouse spinal cord, plus associated tissue. Staining (right) with human-specific STEM121 (pink) shows that the tracks originate in the ALI-CO; MAP2 (green) indicates mouse neurons in the spinal cord and human neurons in the ALI-CO. 6RXUFH *LDQGRPHQLFR6/0LHUDX6%*LEERQV*0HWDO &HUHEUDORUJDQRLGVDWWKHDLUOLTXLGLQWHUIDFHJHQHUDWH diverse nerve tracts with functional output. 1DW1HXURVFL Ç GRL V
JURZWKPRUSKRJHQHVLVDQGF\WRGLIIHUHQWLDWLRQÍ7KLVLVWKHILUVWGHP- onstration of a robust and efficient method of directed differentiation LQ YLWURIURPKXPDQ36&VWRZDUGD'DUFKLWHFWXUHDQGFHOOXODUFRPSRVLWLRQ ÌUHPDUNDEO\VLPLODUWRWKHIHWDOLQWHVWLQHÍ Workman HWDO WRRNWKLVWHFKQLTXHIXUWKHUWRPDNHKXPDQ LQWHVWLQDORUJDQRLGV +,2V ZKLFKWKH\FRPELQHGZLWKQHXUDOFUHVWFHOOV GHULYHGIURPKXPDQ36&V K36&V 7KLVUHVXOWHGLQLQWHVWLQDORUJDQRLGV ZLWKDIXQFWLRQDOHQWHULFQHUYRXVV\VWHP (16 7KH(16FRQWUROVWKH JDVWURLQWHVWLQDOWUDFWÊVPRELOLW\DQGSHUPHDELOLW\,QWKH+,2(16RUJD- noids, Workman HWDOXVHG51$VHTWRVKRZWUDQVFULSWLRQDOFKDQJHVLQ WKH+,2(16YHUVXV+,2RUJDQRLGV,QDGGLWLRQWKHUHVHDUFKHUVVKRZHG QHXURQDODFWLYLW\LQWKH(16FHOOV(16GULYHQPXVFOHFRQWUDFWLRQVDQG intestinal-like motility. $VWKHVFLHQWLVWVSRLQWHGRXW Ì3HUWXUEDWLRQVLQ(16GHYHORSPHQWRU IXQFWLRQDUHFRPPRQ\HWWKHUHLVQRKXPDQPRGHOIRUVWXG\LQJ(16 intestinal biology and disease.” Workman HWDOH[SORUHGWKHSRVVLELOLW\ RIXVLQJ+,2(16RUJDQRLGVWRPRGHO+LUVFKVSUXQJÊVGLVHDVHZKLFKLV 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
(a) d2 d3 d4 d9 d13
d0 d1 14-day organoid 28-day organoid (b) (d) Ki67 / Nuc / CDX2
(c) (e) SOX9 / CDX2 / KLF5
e12.5 mouse e16.5 mouse (f) (g) SOX9 / CDX2 / KLF5
SOX9 LGR5 ASCL2 (h) (i) (j) H9 organoids
Figure 7. Human embryonic stem cells and induced pluripotent stem cells can be directed to create intestinal organoids. (a) In less than two weeks, they form highly convoluted epithelial structures surrounded by mesenchyme. (b–e) Expression of intestinal transcription factors (KLF5, CDX2, SOX9) and cell proliferation on serial sections of organoids after 14 and 28 days resemble mouse fetal intestinal development (f, g). (h, i, j) Whole mount of 56-day-old organoids show epithelial expression of Sox9 (h) and some “crypt-like” expression of the stem cell markers Lgr5 (i) and Ascl2 (j). (Insets show sense controls for each probe.) 6RXUFH 6SHQFH-50D\KHZ&15DQNLQ6$HWDO 'LUHFWHGGLIIHUHQWLDWLRQRIKXPDQSOXULSRWHQWVWHPFHOOVLQWR intestinal tissue in vitro. 1DWXUH Ç GRL QDWXUH 24 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
DFRQJHQLWDOFRQVWLSDWLRQFDXVHGE\DGHYHORSPHQWDOGHIHFWLQWKH(16 9DULRXVJHQHVKDYHEHHQLPSOLFDWHGLQWKLVGLVHDVHDQGRQHLVWKHPXWDWHG SDLUHGOLNHKRPHRER[% 3+2;% ZKLFKFDQFDXVHWKHFRPSOHWHODFN RIQHXURQVLQWKHLQWHVWLQHVRIKXPDQVDQGPLFHÌ'HVSLWHWKHREYLRXVSKH- notype, the molecular pathways that are affected by 3+2;% mutations KDYHQRWEHHQLGHQWLILHGLQKXPDQVVRZHXVHG+,2(16DVDPRGHOV\VWHP WRVWXG\WKLVIRUPRI+LUVFKVSUXQJÊVGLVHDVHÍ:RUNPDQHWDOH[SODLQHG Workman HWDO. generated hPSCs with various 3+2;% mutations DQGXVHGWKHPWRJHQHUDWH+,2(16RUJDQRLGV7KH\FRQFOXGHGWKDWÌWKLV human-PSC-derived intestinal model seems well suited for mechanistically VWXG\LQJJHQHWLFIRUPVRI+LUVFKVSUXQJÊVGLVHDVHLQKXPDQVÍ
No NCC (HIO only) PHOX2B+/+ PHOX2B+/Y14X PHOX2BY14X/Y14X Bright-field 3/3 3/3 2/3 1/3 DAPI / DES TUBB3/ DAPI / DES S100B/
Figure 8. HIOs+ENS generated with 3+2;% neural crest cells. Bright-field images (top) show harvested organoids after seven weeks of post-transplantation growth with ENS carrying one of three 3+2;% mutations. (Fractions indicate the number of organoids that grew and contained intestinal epithelium; Scale bars, 2 mm.) TUBB3 (middle) indicates the development of neurons in the organoids, and S100β (bottom) shows the development of glia. (Scale bars, 100 μm.) 6RXUFH :RUNPDQ0-0DKH007ULVQR6HWDO (QJLQHHUHGKXPDQSOXULSRWHQWVWHPFHOOGHULYHGLQWHVWLQDO tissues with a functional enteric nervous system. 1DWXUH0HGLFLQH Ç GRL QP 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð PROBLEMS & SOLUTIONS
6WDQGDUGL]LQJFXOWXUHFRQGLWLRQVLVWKHNH\FKDOOHQJHWRXVLQJRUJD- noids in research. Some methods are inherently prone to variability and PDNHJHQHUDWLRQRIUHSURGXFLEOHGDWDDFKDOOHQJH7KHVHSUREOHPVXVX- DOO\DULVHIURPWKHPHGLXPXVHGLQFXOWXUHRIWHQLQFOXGLQJYHU\FRPSOH[ components that are labor-intensive to prepare and prone to variable performance. Published media also vary in composition, which makes VWDQGDUGL]DWLRQQHDUO\LPSRVVLEOH7KHLQFRQVLVWHQF\PDNHVFROODERUDWLRQ and reproducibility across labs challenging. Using a complete commercial medium can be an effective option to PDQDJHODEUHVRXUFHVZKLOHPDNLQJLWHDVLHUWRFRPSDUHGDWDDFURVVH[SHUL- PHQWVXVHUVDQGFROODERUDWRUV7RHVWDEOLVKDQGPDLQWDLQPRXVHLQWHVWLQDO RUJDQRLGVIURPPRXVHLQWHVWLQHFHOOVIRUH[DPSOH,QWHVWL&XOWģ2UJDQRLG *URZWK0HGLXP 0RXVH LVDVHUXPIUHHRSWLRQ7KLVPHGLXPDOVRFUHDWHV PRXVHLQWHVWLQDORUJDQRLGVWKDWLQFOXGHDOOWKHH[SHFWHGW\SHVRIFHOOVLQ the adult intestinal epithelium. Similarly, IntestiCult™ Organoid Growth 0HGLXP +XPDQ FDQEHXVHGWRHVWDEOLVKDQGPDLQWDLQKXPDQLQWHVWLQDO RUJDQRLGV2WKHUSURGXFWVDUHDYDLODEOHIURP67(0&(//7HFKQRORJLHVWR support culture of ASC-derived organoids from a variety of tissues, includ- ing liver and pancreas. In addition to differing media compositions, variations on differentia- tion protocols for stem cells can produce significantly different results, PDNLQJLWH[WUHPHO\GLIILFXOWRULPSRVVLEOHWRFRPSDUHGDWDDFURVVODEV using different directed differentiation protocols. 7KHH[WUDFHOOXODUPDWUL[WKDWVXSSRUWVRUJDQRLGJURZWKFDQDOVRFRQ- WULEXWHWRYDULDEOHUHVXOWV0DQ\UHVHDUFKHUVXVHXQGHILQHGPRXVHWXPRU GHULYHGH[WUDFHOOXODUPDWUL[ZLWKLQKHUHQWO\KLJKORWWRORWYDULDELOLW\ 7RGHYHORSVWDQGDUGL]HGSURWRFROVDQGFRQVLVWHQWUHVXOWVWKHPDWUL[LGH- ally should be defined or at least screened to ensure compatibility with the required cell culture conditions. 26 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
7KHSK\VLFDOFKDUDFWHULVWLFVRIRUJDQRLGVPDNHDQDO\VLVFKDOOHQJLQJ 7KHRUJDQRLGVKDSHÈIURPDVLPSOHVSKHUHWRPRUHFRPSOH[VWUXFWXUHVÈ affects analysis. At times, the same kind of organoid can vary in shape. 7KHVHFKDUDFWHULVWLFVFDQPDNHTXDQWLWDWLYHFKDUDFWHUL]DWLRQGLIILFXOW especially since an organoid is thick enough that a typical compound microscope cannot image the entire structure. As a result, scientists often consider sectioning an organoid for analysis or using a more complicated form of deep-tissue imaging, which is still unlikely to provide a complete SLFWXUHRIDQRUJDQRLGÊVLQWHUQDOVWUXFWXUH1RQHWKHOHVVPHWKRGVRIPXOWL photon microscopy, such as two- and three-photon imaging, let scientists H[SORUHPRUHGHHSO\LQWRRUJDQRLGVDQGYLHZWKHHQWLUHVWUXFWXUHLQ some cases. Some of the challenges in organoid research could be addressed WKURXJKFROODERUDWLRQVDQGVWDQGDUGL]DWLRQRISURWRFROVDFURVVODEV(YHQ VWDQGDUGL]LQJWKHWHUPLQRORJ\XVHGLQWKHILHOGZRXOGKHOS)RULQVWDQFH VRPHSXEOLFDWLRQVVWLOOXVHWKHZRUGÌRUJDQRLGÍWRGHVFULEHZKDWDUHFRP- monly understood to be spheroids, or tissue-specific terms (e.g., enteroids or EURQFKRVSKHUH WKDWDUHQRWDOZD\VXVHGFRQVLVWHQWO\ 'HYHORSLQJJUHDWHUFRQVLVWHQF\LQRUJDQRLGFXOWXUHVLVNH\7KHPRUH that scientists develop and use consistent protocols and materials that can be more completely defined, the more data can be reproduced, compared, and the results applied in healthcare applications. 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð WHAT’S NEXT
Future benefits from organoids will arise not only from where they are DSSOLHGEXWKRZWKH\DUHXVHG2QHH[DPSOHLVPRQROD\HUV2UJDQRLGVFDQ be broken up and used to seed cultures made of a single layer, providing the FHOOXODUKHWHURJHQHLW\RIDQRUJDQRLGZLWKWKHFRQYHQLHQFHRI'FXOWXUH YDQGHU+HH IRXQGWKDWDPRQROD\HUPDGHIURPRUJDQRLGVSURYLGHG the ability to measure nutrient transport, barrier function, and interac- tions with gut bacteria. Other structural adaptations include organoids being incorporated in organs-on-a-chip, which are devices that include a system of small chan- nels that are used to provide nutrients to the cells inside. Boston-based (PXODWHQRWHVWKDWVXFKV\VWHPVFDQEHXVHGWRVWXG\ELRORJ\LPSURYH KXPDQKHDOWKDQGGHYHORSQHZSHUVRQDOKHDOWKDSSOLFDWLRQV7KLVFRP- pany already offers lung, liver, and intestine chips. In the Netherlands, 0,0(7$6GHYHORSHGLWV2UJDQR3ODWHģZKLFKWKHFRPSDQ\GHVFULEHVDV ÌDPLFURIOXLGLF'FHOOFXOWXUHSODWHVXSSRUWLQJXSWR WLVVXHPRGHOVRQ DVLQJOHSODWHÍ0,0(7$6KDVXVHGWKHLUPLFURIOXLGLFV\VWHPWRJHQHUDWH models including perfused-gut epithelium tubules, various human neu- ronal and human liver models, and a model of the human kidney. By using cells grown in organoid cultures to populate the organ-on-a-chip system, UHVHDUFKHUVDGGDOHYHORIELRORJLFDOUHOHYDQFHDQGFRPSOH[LW\QRWDFKLHY- able with either system alone. 2QHRIWKHJUHDWVWHSVIRUZDUGIRURUJDQRLGVZLOOEHUHFRJQL]LQJWKH true utility and limitations of the system, especially in clinical applica- WLRQV)RUH[DPSOH%HUNHUVHWDO FUHDWHGRUJDQRLGVIURPUHFWDOWLVVXH WDNHQIURPSDWLHQWVZLWKF\VWLFILEURVLV &) $FRPSRXQGFDOOHGIRUVNROLQ causes swelling in organoids created from healthy rectal tissue, but not IURP&)SDWLHQWV'UXJGULYHQLPSURYHPHQWVLQ&)75DFWLYLW\WULJJHUHG IRUVNROLQLQGXFHGVZHOOLQJ ),6 LQWKH&)GHULYHGRUJDQRLGV WKHVDPH effect is produced in patients. Berkers HWDOFRQFOXGHG Ì,QYLWUR drug efficacy measurements by FIS in rectal organoids of individuals with CF correlate 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð with the most important LQYLYRUHVSRQVHLQGLFDWRUVRI&)75PRGXODWRUVÍ 'HVSLWHWKLVZRUNDQGRWKHUVZRUNLQJVHSDUDWHO\WRDFFRPSOLVKVLPLODU goals, more collaboration could be instrumental to see near-term impact on patient care. Another potential clinical application is cellular therapy via the func- WLRQDORUJDQRLGWUDQVSODQWDWLRQ&RUWH]HWDO WUDQVSODQWHGKXPDQ intestinal organoids into the mesentery of immunosuppressed mice, after ZKLFKWKHRUJDQRLGVVXUYLYHGDQGJUHZ9DULRXVVWXGLHVDUHDOUHDG\XQGHU- ZD\WRH[SORUHWKHSRVVLELOLW\RIWUDQVODWLQJUHVXOWVJHQHUDWHGIURPSURRI RIFRQFHSWVWXGLHVWRFOLQLFDOSUDFWLFH 7DNHEHHWDO 7KHZRUNLQRUJDQRLGVZLOOFRQWLQXHWRH[SDQGEDVHGRQQHZDQGPRUH consistent methodologies. In addition, combining the results from other technologies with those from organoids will also provide finer-grained understanding of biological systems and open new pathways to disease treatments. 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð REFERENCES
%HUNHUV*YDQ0RXULN39RQN$0HWDO 5HFWDORUJDQRLGV HQDEOHSHUVRQDOL]HGWUHDWPHQWRIF\VWLFILEURVLV&HOO5HSRUWV ÇGRL MFHOUHS %HUVKWH\Q01RZDNRZVNL7-3ROOHQ$$HWDO +XPDQL36& derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. &HOO6WHP&HOO, Ç GRL MVWHP %URXWLHU/0DVWURJLRYDQQL*9HUVWHJHQ00$HWDO +XPDQ primary liver cancer-derived organoid cultures for disease mod- eling and drug screening. 1DWXUH0HGLFLQH ÇGRL QP &RUWH]$53ROLQJ+0%URZQ1(HWDO 7UDQVSODQWDWLRQRI human intestinal organoids into the mouse mesentery: A more physiologic and anatomic engraftment site. 6XUJHU\ ÇGRL MVXUJ 'MRPHKUL6,%XUPDQ%*RQ]DOH]0(HWDO $UHSURGXFLEOH VFDIIROGIUHH'RUJDQRLGPRGHOWRVWXG\QHRSODVWLFSURJUHVVLRQLQ breast cancer. -RXUQDORI&HOO&RPPXQLFDWLRQDQG6LJQDOLQJ ÇGRL V *DEULHO(5DPDQL$.DURZ8HWDO 5HFHQW=LNDYLUXVLVR- lates induce premature differentiation of neural progenitors in human brain organoids. &HOO6WHP&HOO ÇGRL M VWHP *LDQGRPHQLFR6/0LHUDX6%*LEERQV*0HWDO &HUHEUDO organoids at the air-liquid interface generate diverse nerve tracts with functional output. 1DW1HXURVFL Ç GRL V 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
0F&UDFNHQ.:+RZHOO-&:HOOV-0HWDO *HQHUDWLQJ human intestinal tissue from pluripotent stem cells LQYLWUR. 1DWXUH 3URWRFROV Ç GRL QSURW 0LOOHU$-+LOO'51DJ\06HWDO ,QYLWUR induction and LQYLYR engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells. 6WHP&HOO5HSRUWV Ç GRL MVWHPFU 6HKJDO$'RQDOGVRQ'63ULGDQV&HWDO 7KHUROHRI &6)5GHSHQGHQWPDFURSKDJHVLQFRQWURORIWKHLQWHVWLQDO stem cell niche. 1DWXUH&RPPXQLFDWLRQV 'RL V 6SHQFH-50D\KHZ&15DQNLQ6$HWDO 'LUHFWHGGLIIHUHQ- tiation of human pluripotent stem cells into intestinal tissue in vitro. 1DWXUH Ç GRL QDWXUH 7DNHEH7:HOOV-0+HOPUDWK0$DQG=RUQ$0 2UJDQRLG center strategies for accelerating clinical translation. &HOO6WHP&HOO, Ç GRL MVWHP 9ODFKRJLDQQLV*+HGD\DW69DWVLRX$HWDO 3DWLHQWGHULYHG organoids model treatment response of metastatic gastrointestinal cancers. 6FLHQFH Ç GRL VFLHQFHDDR :RUNPDQ0-0DKH007ULVQR6HWDO (QJLQHHUHGKXPDQ pluripotent-stem cell-derived intestinal tissues with a functional enteric nervous system. 1DWXUH0HGLFLQH Ç GRL QP =LHWHN75DWK(+DOOHU+HWDO ,QWHVWLQDORUJDQRLGVIRUDVVHVV- ing nutrient transport, sensing and incretin secretion. 6FLHQWLILF 5HSRUWV GRL VUHS 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð FURTHER READING
$EHUOH05%XUNKDUW5$7LULDF+HWDO 3DWLHQWGHULYHGRUJD- QRLGPRGHOVKHOSGHILQHSHUVRQDOL]HGPDQDJHPHQWRIJDVWURLQWHV- tinal cancer. %ULWLVK-RXUQDORI6XUJHU\ HÇHGRL EMV 2UJDQRLGVFDQEHXVHGWRVFUHHQIRUVDIHUPRUHHIIHFWLYHFDQFHUGUXJV &KHQ+,6RQJ+0LQJ*/ $SSOLFDWLRQVRIKXPDQEUDLQ organoids to clinical problems. 'HYHORSPHQWDO'\QDPLFV ÇGRL GYG\ 1HXUDORUJDQRLGVRIIHUPDQ\SRVVLELOLWLHVLQWUDQVODWLRQDOPHGLFLQH *KDHPL59&R,/0F)HH0&HWDO %UDLQRUJDQRLGV $QHZ transformative investigational tool for neuroscience research. $GYDQFHG%LRV\VWHPV GRLRUJDGEL 1HXUDORUJDQRLGVFUHDWHQHZPHWKRGVRIVWXG\LQJEUDLQGHYHORSPHQW DQGQHXURGHJHQHUDWLRQ +\QGV5(%RQIDQWL3-DQHV60 5HJHQHUDWLQJKXPDQ epithelia with cultured stem cells: feeder cells, organoids and beyond. (0%20ROHFXODU0HGLFLQH ÇGRL HPPP 'HVFULEHVGHULYLQJRUJDQRLGVIURPYDULRXVHSLWKHOLDOW\SHVDQGKRZ WKHVHPHWKRGVPLJKWEHXVHGWRFUHDWHVWHPFHOOWKHUDSLHV .DXVKLN*3RQQXVDP\03%DWUD6. &RQFLVHUHYLHZ &XUUHQW status of three-dimensional organoids as preclinical models. 6WHP&HOOV ÇGRL VWHP 'LVFXVVHVWKHEHQHILWVRIXVLQJRUJDQRLGVLQSUHFOLQLFDOUHVHDUFK .DU]EUXQ(7VKXYD5<5HLQHU2 $QRQFKLSPHWKRGIRU long-term growth and real-time imaging of brain organoids. &XUUHQW3URWRFROVLQ&HOO%LRORJ\ HGRL FSFE %ULDQRUJDQRLGVRQPLFURIDEULFDWHGGHYLFHVDOORZORQJWHUPVWXGLHV 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð
0DMROR)0DULQRZLF'50RXUD$HWDO 8VHRILQGXFHG SOXULSRWHQWVWHPFHOOV L36&V DQGFHUHEUDORUJDQRLGVLQPRGHOLQJ the congenital infection and neuropathogenesis induced by =LNDYLUXV-RXUQDORI0HGLFDO9LURORJ\ ÇGRL MPY 6XPPDUL]HVVWXGLHVRIXVLQJL36&VDQGRUJDQRLGVWRGHVFULEHDQGWUHDW WKH=LNDYLUXV 0LXUD66X]XNL$ %ULHIVXPPDU\RIWKHFXUUHQWSURWRFROVIRU generating intestinal organoids. 'HYHORSPHQW*URZWK 'LIIHUHQ WLDWLRQ Ç GRL GJG 'HVFULEHVWKHYDULRXVIRUPVRILQWHVWLQDORUJDQRLGVDQGKRZWKH\FDQEH PDGHDQGXVHG 0LWWDO5:RR):&DVWUR&6HWDO 2UJDQRQFKLSPRGHOV Implications in drug discovery and clinical applications. -RXUQDORI &HOOXODU3K\VLRORJ\ ÇGRL MFS ([SORUHVFRPELQDWLRQVRI'RUJDQRLGVPLFURIDEULFDWLRQDQGELRSULQW LQJWRVWXG\RUJDQV Organoid research: https://www.stemcell.com/technical-resources/ area-of-interest/organoid-research.html $FROOHFWLRQRILQIRUPDWLRQRQYDULRXVW\SHVRIRUJDQRLGV 5DQMDQ9'4LX/7DQ(.HWDO 0RGHOOLQJ$O]KHLPHUÊV disease: Insights from LQYLYR to LQYLWUR three-dimensional culture platforms. -RXUQDORI7LVVXH(QJLQHHULQJDQG5HJHQHUDWLYH0HGLFLQH, Ç GRL WHUP 'HVFULEHVWKHXVHRIQHXUDORUJDQRLGVDQGWLVVXHHQJLQHHULQJWRXQGHU VWDQG$O]KHLPHUÊV '&HOO&XOWXUHDQG$QDO\VLV (YROXWLRQDQG$SSOLFD- tions.: https://www.essentialknowledgebriefings.com/ GRZQORDGVGBFHOOFXOWXUHBDQGBDQDO\VLV 6XUYH\VWKHKLVWRU\PHWKRGVDQGDSSOLFDWLRQVRI'FHOOFXOWXUH WILEY END USER LICENSE AGREEMENT Go to www.wiley.com/go/eula to access Wiley’s ebook EULA.