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Organoid Research Techniques

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Organoid Research Techniques 2ïäÞëìæá5âðâÞïàå7âàåëæîòâð 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. μP Figure 1. Light microscope visualization of a mouse intestinal organoid. 6RXUFH67(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&DVPLJKWSURYHYLWDOWRPRUHWKRURXJKO\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 WKHVHRUJDQRLGV6DWRÊVWHDPUHSRUWHGÌ: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-
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