resultantesetnod32id

REVIEWOpenAccessGenerationofpancreaticβcellsfortreatmentofdiabetes:advancesandchallengesHussainMd.
Shahjalal1,2,AhmedAbdalDayem1,KyungMinLim1,Tak-ilJeon1andSsang-GooCho1*AbstractHumanembryonicstemcells(hESC)andinducedpluripotentstemcells(hiPSC)areconsideredattractivesourcesofpancreaticβcellsandisletorganoids.
Recently,severalreportspresentedthathESC/iPSC-derivedcellsenrichedwithspecifictranscriptionfactorscanformglucose-responsiveinsulin-secretingcellsinvitroandtransplantationofthesecellsameliorateshyperglycemiaindiabeticmice.
However,theglucose-stimulatedinsulin-secretingcapacityofthesecellsislowerthanthatofendogenousislets,suggestingtheneedtoimproveinductionprocedures.
OneofthecriticalproblemsfacinginvivomaturationofhESC/iPSC-derivedcellsistheirlowsurvivalrateaftertransplantation,althoughthisrateincreaseswhentheimplantedpancreaticcellsareencapsulatedtoavoidtheimmuneresponse.
SeveralgroupshavealsoreportedonthegenerationofhESC/iPSC-derivedislet-likeorganoids,butdevelopmentoftechniquesforcompleteisletstructureswiththeeventualgenerationofvascularizedconstructsremainsamajorchallengetotheirapplicationinregenerativetherapies.
ManyissuesalsoneedtobeaddressedbeforethesuccessfulclinicalapplicationofhESC/iPSC-derivedcellsorisletorganoids.
Inthisreview,wesummarizeadvancesinthegenerationofhESC/iPSC-derivedpancreaticβcellsorisletorganoidsanddiscussthelimitationsandchallengesfortheirsuccessfultherapeuticapplicationindiabetes.
Keywords:Embryonicstemcells(ESC),Inducedpluripotentstemcells(iPSC),Differentiation,Pancreaticβcell,Isletorganoids,Transplantation,βCellmaturationBackgroundDiabetesmellitusisalife-threateningdisease,anditsprevalenceisincreasingworldwide.
Theavailabletreat-mentcanneithercurenorcompletelycontrolthecom-plicationsofthisdisorder,whichresultsinsubstantiallossesoflifeinalmostallcountriesintheworld.
Fur-thermore,currentlife-longtreatmentstrategiesimposelargesocialandeconomicburdensonafamily.
Forthelastfewdecades,humanbeingshavebeentryingtode-velopatreatmentstrategythatcaneffectivelycontrolthisdisorderandsavelives.
Despitetremendousefforts,however,humanshavebeenfarfromsuccessinfindinganeffectivetreatmentstrategyfordiabetes.
Type1diabetesresultsfromanabsolutedeficiencyofinsulinduetoTcell-mediatedautoimmunedestructionofpancreasβcells[1].
Thecurrenttreatmentfortype1diabetesissolelydependentontheadministrationofex-ogenousinsulin.
Althoughthisapproachmanagesthedisease,unwantedrisksandlong-termcomplicationspersistbecauseoftheinabilitytotightlymaintainglucoselevelswithinanormalphysiologicalrange.
Complicationsincludelife-threateningepisodesofhypoglycemia,aswellaslong-termcomplicationsthatincludemicro-andmacro-angiopathyleadingtocardio-vascularpathologies,kidneyfailure,andneuropathy.
Thus,thereisaneedfornewtreatmentsthatprovidesuperiorcontrolofbloodglucosetominimizethesecomplications[2].
Oneexistingapproachtotreatingdia-betesistransplantationofpurifiedhumancadavericis-letsintotheportalveintoreplacethedestroyedβcellsofthepatients.
Thisproceduretypicallyresultsinbetterglycemiccontrol,canrenderpatientsinsulinindepend-entforprolongedperiodsoftime,andimprovesoverall*Correspondence:ssangoo@konkuk.
ac.
kr;ssangoo33@gmail.
comHussainMd.
ShahjalalandAhmedAbdalDayemcontributedequallytothiswork.
1DepartmentofStemCell&RegenerativeBiotechnologyandIDASI(IncurableDiseaseAnimalmodel&StemcellInstitute),KonkukUniversity,120Neungdong-ro,Gwangjin-gu,Seoul05029,SouthKoreaFulllistofauthorinformationisavailableattheendofthearticleTheAuthor(s).
2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Shahjalaletal.
StemCellResearch&Therapy(2018)9:355https://doi.
org/10.
1186/s13287-018-1099-3qualityoflife[3,4].
Althoughpromising,becauseofdif-ficultiessuchasthescarcityofcadavericdonorscom-paredtothelargenumberofdiabeticpatients,lowyieldoftransplantableisletsfromcadavericpancreases,andnecessityforchronicimmunosuppressiontopreventre-jectionoftheallograft[5,6],analternativesourceofsurrogatecellsisneeded.
Moreover,thenumberoffunc-tionalβcellsthatcanbeextractedfromasinglecadav-ericpancreasisoftennotenoughtorestoreeuglycemiainasinglediabeticpatient[7].
Thisalsoillustratestheneedforalternativesourcesofβcellstotreatthein-creasingnumberofdiabeticpatients.
Humanpluripotentstemcells(hPSCs),includinghu-manembryonicstemcells(hESC)andinducedpluripo-tentstemcells(hiPSC),areconsideredveryattractivealternativesourcesofsurrogateβcellsbecauseoftheirabilitytodifferentiateintoallmajorsomaticcelllineages[8,9].
Todate,themostsuccessinproducingpancreaticβ-likecellsfromhPSCshascomefromapproachesthatmimicnormalpancreasdevelopment.
Manyresearchgroupshavefollowedthisapproach,whichinvolvesex-posingthecellstovariousgrowthfactorsandsignalingmoleculesatspecificdosesandinaparticularsequencetosuccessfullydifferentiatethecellsintopancreaticendodermorendocrinecells(ECs)[2,10–29].
However,inmanystudies,alargenumberofpolyhormonalinsulin-expressingcellshavebeenobservedinculturethatresembletransientECsseeninmid-gestationhu-manfetalpancreases[10,11,15–17,30–32].
Thesepoly-hormonalcellslackexpressionofkeyβcelltranscriptionfactorsanddonotsecreteinsulininvitroinresponsetoglucosechallenge—thehallmarkfunctionofbonafideβcells[10,32–34](Fig.
1a).
Concurrently,inseveralotherstudies,analternativestrategyhasbeenadoptedinwhichglucose-responsiveinsulin-secretingcellscanbegeneratedfollowingtransplantationofhESC/iPSC-derivedpancreaticprogenitorcellsintoectopicsitesinimmuno-deficientortype1diabeticmice[12,14,18–21,26].
Inre-cipientmice,theresultingcellscanproducehumaninsulintoreversediabetes[18,20,21](Fig.
1b).
Inrecentyears,optimizeddifferentiationprotocolshavebeensuc-cessfullydevelopedtogenerateglucose-responsiveinsulin-secretingcellsinvitrofromhESC/iPSC,whichex-pressmatureβcellmarkers,andtransplantationoftheseabcFig.
1Differentiation,maturation,andfunctionofpancreaticβcellsderivedfromhESC/iPSC.
Insulin-positivepolyhormonalcellsmostlyformedinmanyinvitrocellcultureprotocolswhichshowlimitedornoGSIS(a).
Alternatively,EPcellswereformedfromhESC/iPSCinamonolayerand/orrotatingsuspensionculture,andtransplantationofthesecellsgeneratedislet-likeECsthatexhibitedGSISandcouldreversehyperglycemia(b).
Recently,pancreaticβ-likecellsexpressingmatureβcellmarkersandexhibitingGSISinvitroweregeneratedineitherlowadhesioncultureorrotatingsuspensionculture;aftertransplantation,thesecellsunderwentfurthermaturation,secretedinsulininresponsetoglucose,andamelioratedhyperglycemiaindiabeticmice(c).
GSIS,glucose-stimulatedinsulinsecretion;AFP,hepaticprogenitorcellsexpressingAFP;CDX2,intestinalprogenitorcellsexpressingCDX2;PP,pancreaticprogenitor;EP,endocrineprecursor;INS,β-likecellsexpressinginsulin;GCG,αcellsexpressingglucagon;SST,δcellsexpressingsomatostatinShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page2of19cellshasbeenshowntoamelioratehyperglycemiaindia-beticmice[2,22,23,29](Fig.
1c).
Theβ-likecellsgener-atedshowgeneexpression,ultrastructuralcharacteristics,andglucoseresponsivenessbothinvitroandinvivo,whichcloselyresemblingthefeaturesofβcellsfoundinpancreaticislets[2,22,23].
Inthesemultistageprotocols,thefinalcellpopulationhasabout30–60%β-likecells,andthemajorityoftheremainingcellsarerelativelyuncharacterizedcellsthatcanbeundifferentiatedprogeni-torsorothertypesofunwantedcells.
Thus,improvingef-ficiency,intermsofthepercentageofdifferentiatedcellsthatbecomeβcells,remainsanimportantchallenge.
Althoughtremendoussuccesshasbeenachievedinthelastfewyears,lowsurvivalratesofhESC/iPSC-der-ivedpancreaticcellsaftertransplantationintoectopicsitesinrecipientsremainacriticalproblem[20,25,35].
Therefore,anefficientculturesystemthatcanbeusedtogeneratefunctionalandterminallydifferentiatedβcells,alongwithaneffectivetransplantationtechnique,isneededforclinicalapplicationofhESC/iPSC-derivedβcellsfordiabetestreatment.
However,phase1/2clin-icaltrialsfortheapplicationofhESC-derivedpancreaticprogenitorsintype1diabetespatientshavealreadybegun[36].
Inthisreview,wesummarizeadvancesinthedifferentiationofhESC/iPSC-derivedcellsintopan-creaticβcellsandislet-likeorganoidsanddiscussthelimitationsandchallengesfortheirsuccessfulgenerationandtherapeuticapplicationintype1diabetes.
PluripotentstemcellsandtheirreprogrammingESCsshowunlimitedreplicativepropertiesandthepo-tentialtodifferentiateintoanyadultcelltype[37–39].
iPSCs,establishedfromsomaticcellsofmouseandhu-man[40–42],havethesameabilitytoexpandanddiffer-entiateasESCs.
Therefore,bothESCsandiPSCshavegreatpotentialforuseincelltherapies.
However,theuseofiPSCshasfewerethicalcomplicationsthanESCsthatarederivedfromtheinnercellmassoflivingembryos.
iPSCsarederivedfromvarioussomaticcellsafterexpos-uretoacombinationoftranscriptionfactorssuchasOct3/4,Sox2,Klf4,andc-Myc[40,41].
iPSCgenerationiscarriedoutviaviral-basedandnon-viral-basedmethodsassummarizedinFig.
2.
Thesemethodsarevariedintheirefficiencies,transductionperiod,genomeFig.
2GenerationofiPSCsfromvarioussomaticcells.
iPSCgenerationcarriedoutviaviral-basedandnon-viral-basedmethodsaresummarizedShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page3of19integration,andcost[43–48].
Therefore,selectionofthereprogrammingmethoddeterminesthefurtherapplica-tionoftheproducediPSCinregenerativemedicine.
Generally,viral-basedmethodsleadtogenomeintegra-tionandareoflowsafety,albeit,thehighefficiency.
MostiPSCsaremadeusingretrovirusvectors,whichin-tegratereprogrammingfactorsintohostgenomes.
Retrovirusvectorscanspontaneouslyinfectvariouscelltypesandinserttheircodinggenesintohostgenomesusingreversetranscriptase,whichallowscontinuoustransgeneexpressionduringreprogramming.
RetroviraltransgeneexpressioncontinuesuntilthecellsbecomeiPSCs,andthen,theretroviralpromoterisinactivated,possiblybecauseofepigeneticmodificationssuchashis-tonemethylation[49].
Thisguidedreprogrammingandautomaticsilencingmechanismisconsideredveryim-portantforiPSCinductionfromsomaticcells.
Recently,severalvirus-freetechniqueshavebeendevelopedfortheproductionoffootprint-freeiPSCs;theirefficientculturetechniqueshavealsobeenestablished[50–57].
ESCsvs.
iPSCs:similaritiesanddifferencesSimilartoESCs,iPSCshaveacharacteristicmorphology,abilitytogenerateembryoidbodiesandteratomas,andunlimitedproliferationcapabilityinvitro,whiletheymaintaintheirpluripotencybyexpressingpluripotencygenes.
However,severalstudieshaverevealedsomedif-ferencesbetweenhESCsandhiPSCsintermsofgeneex-pressionprofiles[58],epigeneticmodificationssuchasDNAmethylation[59],geneticstability[60],imprintedgeneexpressionstability[61],differentiationpotentials[62,63],anddiseasemodeling[64].
iPSCshavesome"memory"oftheirsomaticoriginandthereforearenotidenticaltoESCs.
ThememoryofiPSCmayaffecttheirsafety[65].
However,thereisnosufficientevidenceyettodeterminewhetheriPSCmemorycanbefatalincelltherapies.
DifferentiationofhESC/iPSCintopancreaticβcellsInsulin-producingcellswithpancreaticβcellcharacter-isticswerefirstsuccessfullyderivedinembryoidbodiesfromspontaneousdifferentiationofhESC[66].
Sincethen,numerousmethodstogeneratepancreaticendo-dermorβ-likecellsfromhESC/iPSChavebeenreported[2,10–29].
Thesestudiesdemonstratedthegenerationofinsulin-positivecells,aswellasglucagon-andsomatostatin-positivecells(Table1).
However,theper-centagesofinsulin-positivecellsobtainedinculturevaryamongtheprotocols.
Thekeystagesofembryonicpancreasdevelopmentin-cludedevelopmentofthedefinitiveendoderm(DE),primitiveguttube(PG),pancreaticprogenitor(PP),endo-crineprogenitor(EP),andhormone-expressingECs.
Basedoninformationaboutembryonicpancreasdevelop-ment,eachdifferentiationprotocolhasbeendesignedtousevariouscytokinesorsignalingmodulatorsatspecificdosesandinparticularsequencestoactivateorinhibitkeysignalingpathways,includingnodal/activin,Wnt,PI3K,fibroblastgrowthfactors(FGF),bonemorphogeneticpro-tein(BMP),retinoicacid,hedgehog,proteinkinaseC,notch,epidermalgrowthfactor,andtransforminggrowthfactor-β(TGF-β).
Othergrowthfactorssuchasinsulin-likegrowthfactors1and2,hepatocytegrowthfac-tor(HGF),andglucagon-likepeptide-1(GLP-1)orexendin-4(apeptideanalogofGLP-1)havealsobeenusedtofacilitatedifferentiationofpancreatichormone-ex-pressingcells[10,11,13,15,16,24].
Inaddition,variousclassesofsmallmoleculeshavebeenreportedtobeeffect-ivefordifferentiationofhESC/iPSCintoinsulin-produ-cingcells.
Nicotinamide,apoly(ADP-ribose)synthetaseinhibitor,isusedinsomeprotocolstoimprovetheyieldofpancreaticECs[11,13,16,17,24].
Further,forskolin(anactivatorofadenylylcyclase)anddexamethasone(asyn-theticadrenocorticalsteroid)havebeenshowntoenhancecellularmaturation,andtheseagentscanbecombinedwithothersmallmoleculestoobtainsynergisticeffects[17].
Thyroidhormonepromotespostnatalβcelldevelop-mentandglucose-responsiveinsulinsecretioninratsthroughthetranscriptionfactorMAFA[67].
Thisinsighthasincreasedtheuseofthyroidhormoneinrecentproto-colstoimproveglucoseresponsivenessofhESC/iPSC-der-ivedβcells[22,23,25,27,28,68].
Severalreportshaverecognizedpancreaticprogenitorsco-expressingPancre-aticandDuodenalHomeobox1(PDX1)andNK6homeo-box1(NKX6.
1)asindispensableprecursorsofmaturepancreaticβcells[18,21,26].
Differentiationintopancre-aticprogenitorsco-expressingPDX1andNKX6.
1canbeenhancedinvitrobyeitherdissociatingdenselyformedendodermalcellsandre-platingthesecellsatalowdensityfollowedbyexposuretoalongerperiodofretinoidandFGF10signaling[68]orcultureswithhigh-densityag-gregates[26]orrotatingasuspensioncultureaftertheadditionoffactorssuchasALK5i(aTGF-βtypeIreceptorkinaseinhibitorII),TBP(aPKCactivator),and/orLDN(aBMPinhibitor)[18,21,23].
Theuseofepidermalgrowthfactor(EGF)andnicotinamideinthepancreaticprogenitorspecificationstagecanalsosignifi-cantlyenhancepancreaticprogenitorco-expressingPDX1andNKX6.
1[69].
MaturationofhESC/iPSC-derivedβcellsThematurationofpancreaticβ-likecellsobtainedbydifferentiationfromhESC/iPSCinvitroremainscontro-versial.
Intheearlystudies,eitherMatrigelorlow-densitymouseembryonicfibroblast(MEF)wasusedasa2DcultureplatformonwhichhESC/iPSCwereseeded[10,11,15–17,30–32].
TheseprotocolsShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page4of19Table1Generationofinsulin-positiveβ-likecellsfromhESC/iPSC,theirmaturation,andfunctionsinvitroandinvivoReferences[Celllinesused]DifferentiationconditionCelltypesinducedPercentinsulin+cellsGSISRecipients/transplantationsiteAmeliorationofhyperglycemiaD'Amouretal.
[10]CyT203[hESCline]Onlow-densityMEFsINS+,GCG+,SST+,PPY+,GHRL+7.
3%(3–12%)Non.
a.
n.
d.
Jiangetal.
[11]H1,H9[hESCline]OnMatrigelINS+,GCG+,SST+[n.
t:PPY+,GHRL+]>15%Yes(invitro)BALB/cnudemice/kidneycapsuleYesShimetal.
[12]Miz-hES4,Miz-hES6[hESCline]SuspensioncultureINS+,GCG+,SST+n.
d.
n.
d.
BALB/cnudemice/kidneycapsuleYesEshpeteretal.
[13]H1[hESCline]OnMatrigelINS+,GCG+,SST+5.
3%Yes(invivo)DiabeticC57BL6Rag1/1mice/kidneycapsuleNoKroonetal.
[14]CyT49,CyT203[hESCline]Onlow-densityMEFsINS+,GCG+,SST+,PPY+,GHRL+n.
d.
Yes(invivo)SCID-beigemice/epididymalfatpadYesChenetal.
[15]HUES-2,HUES-4,HUES-8,HUES-9[hESCline]Onlow-densityMEFsINS+,GCG+,SST+,[n.
t:PPY+,GHRL+]0.
8±0.
4%Low(invitro)CD1nudemice/kidneycapsulen.
d.
Zhangetal.
[16]H1,H9[hESCline];C1,C2,C5[hiPSCline]OnMatrigelINS+,SST+25%Yes(invitro)n.
a.
n.
d.
Kunisadaetal.
[17]253G1,201B7,297A1,297F1,and297L1[hiPSCline]Onlow-densityMEFsINS+,GCG+,SST+,GHRL+11.
8%(8.
0–16.
9%)Non.
a.
n.
d.
Rezaniaetal.
[18]H1,ESI-49[hESCline]Matrigel/suspensionculture(stirred)INS+,GCG+,SST+,PPY+~10%Yes(invivo)SCID-beigeandSTZ-diabeticmice/kidneycapsuleYesSchulzetal.
[19]CyT49[hESCline]Suspension(aggregates)INS+,GCG+,SST+,[n.
t:PPY+,GHRL+]n.
d.
Yes(invivo)SCID-beigemice/epididymalfatpadYesRezaniaetal.
[21]H1[hESCline]Matrigel/suspensionculture(stirred)INS+,GCG+,SST+,PPY+,GHRL+~55–60%(post-transplant)Yes(invivo)SCID-beigeandSTZ-diabeticmice/subcutaneouswithencapsulationdeviceYesBruinetal.
[20]H1[hESCline]Matrigel/suspensionculture(stirred)INS+,GCG+,SST+n.
d.
Yes(invivo)SCID-beigeandSTZ-diabeticmice/kidneycapsuleandsubcutaneouswithencapsulationdeviceYesShahjalaletal.
[24]Toe,201B7[hiPSC];KhES-3[hESCline]OnSynthemax(xeno-free)INS+,GCG+,SST+5–8%Yes(invitro)n.
a.
n.
d.
Rezaniaetal.
[22]H1[hESCline];anepisomalhiPSCPlanarculture/air-liquidinterfacecultureINS+,GCG+,SST+~50%Yes(invitroandinvivo)Non-diabeticandSTZ-diabeticmice/kidneycapsuleYesPagliucaetal.
[23]HUES8[hESC];hiPSC-1,hiPSC-2[hiPSCline]SuspensioncultureonastirplateINS+,GCG+,SST+>30%Yes(invitroandinvivo)SCID-beigeanddiabeticmice(NRG-Akita)/kidneycapsuleYesShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page5of19Table1Generationofinsulin-positiveβ-likecellsfromhESC/iPSC,theirmaturation,andfunctionsinvitroandinvivo(Continued)References[Celllinesused]DifferentiationconditionCelltypesinducedPercentinsulin+cellsGSISRecipients/transplantationsiteAmeliorationofhyperglycemiaRussetal.
[2]MEL1INSGFP/W[hESCline]Low-adherenceplatesINS+,GCG+,SST+~60%Yes(invitroandinvivo)STZ-diabeticNODmice/kidneycapsuleYesAgulnicetal.
[25]CyT49[hESCline]SuspensioncultureINS+,GCG+,SST+40–50%Yes(invivo)SCID-beigemice/subcutaneouswithencapsulationdevicen.
d.
Toyodaetal.
[26]KhES-3[hESCline];585A1,604B1,692D2,648B1and409B2[hiPSCline]OnMatrigel/lowadhesionplateINS+,GCG+,SST+n.
d.
Yes(invivo)NOD–SCIDmice/kidneysubcapsulen.
d.
Millmanetal.
[27]ND-1andND-2[NDhiPSCline];T1DhiPSClineOnMatrigel/SpinnerflasksonastirplateINS+,GCG+[n.
t.
.
:SST+]24–27%Yes(invitroandinvivo)ND-SCIDmiceandalloxan-induceddiabeticmice/kidneycapsuleYesManzaretal.
[28]T1DhiPSClineMatrigel/3DcultureINS+,GCG+,SST+~56%Low(invitro)ImmunodeficientSTZ-diabeticmice/shoulderregionYesYabeetal.
[29]TkDN4-M,253G1,454E2[hiPSCline]OnMatrigel/aggregateonultra-lowadhesionplateINS+,GCG+,SST+30–33.
6%Yes(invitro)STZ-diabeticNOD-SCIDmice/kidneycapsulesYesPercentagesofinsulin-positivecells,glucose-stimulatedinsulinsecretion(GSIS),andinvivofunctionsreportedfromvariousstudiesaresummarizedMEFs,mouseembryonicfibroblasts;INS+,insulin-positivecells;GCG+,glucagon-positivecells;SST+,somatostatin-positivecells;PPY+,pancreaticpolypeptide-positivecells;GHRL+,ghrelin-positivecells;n.
t.
,nottested;n.
a.
,notapplied;n.
d.
,notdeterminedShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page6of19efficientlyestablishedPDX1+progenitorsbyusingretin-oicacidincombinationwithinhibitorsofBMPandhedgehogsignalingpathways,whilesimultaneouslyadd-ingeitherFGF10orFGF7.
Theβ-likecellsgeneratedinsuchmonolayerculturewerelargelypolyhormonalinsulin-expressingcells(Fig.
1a).
Polyhormonalcellslackexpressionofkeyβcelltranscriptionfactorsandexhibitlimitedglucose-stimulatedinsulinsecretion(GSIS)invitro[10,32–34].
Formationofnon-functionalpolyhor-monalcellsisconsideredthelimitationoftheseproto-cols.
Whetherthecultureplatformortheinappropriatecombinationsofgrowthfactorsintheculturemediapromotesuchcellsarenotclearlyknown.
Varyingde-greesofinvitroGSISfromhESC/iPSC-derivedinsulin-positivecellshavebeenreportedbyseveralstud-ies,includinganapproximately1.
7-foldincreaseob-servedbyChenetal.
[15],a2-foldincreasenotedbyJiangetal.
[11]andZhangetal.
[16],andapparentlynoGSISreportedbyD'Amouretal.
[10]andKunisadaetal.
[17](Fig.
1a)(Table1).
Thesedifferencesandlowlevelsofsecretedinsulincouldbeduetothegenerationofvaryingnumbersofpolyhormonalcellsinculture.
Thepolyhormonalcellsmayresembletheimmatureβcellsobservedinmid-gestationhumanfetalpancreases[70,71].
Theroleandfateofpolyhormonalcellsduringhu-manfetaldevelopmentarepoorlyunderstood;however,immunohistochemicalcharacterizationindicatesthatthesecellspossessanαcelltranscriptionfactorprofile[72].
Severalreportshavedescribedtheformationofglucagon-expressingαcellsinvivofollowingtransplant-ationofhESC-derivedpolyhormonalcells[21,33,73](Fig.
1a),anddynamicchromatinremodelingwasre-portedtooccurduringthistransitionintomaturedcelltypes[73,74].
StudiesofBruinetal.
[32]revealedsev-eralkeyfeaturesofpolyhormonalinsulin-positivecellsthatdifferfromthoseofmaturepancreaticβcells,in-cludingdefectsinglucosetransporterexpression,KATPchannelfunction,andprohormoneprocessingenzymes.
Thesedeficienciesmustbeaddressedwithfurtherproto-colmodificationstogeneratehESC/iPSC-derivedpan-creaticβcellsthatshowGSISinvitro.
AlthoughseveralofthesereportsdescribedthedetectionofGSISinvitro,noneofthereportedcellswerecapableofefficientlyre-storingeuglycemiainaninvivodiabeticanimalmodel.
Toovercomethislimitation,analternativestrategytoobtainglucose-responsiveinsulin-producingcellshasbeenestablishedinseveralstudies[12,14,18–21,26](Fig.
1b).
MostofthesestudiesusedMatrigelasthe2DplatformforESC/iPSCmonolayerculture,followedbysuspensionculturewithorw/ostirringusinglowadhe-sionplate.
Continuousstirringpromotescell-cellandcell-matrixinteractionswithintheculture.
TheresultantEPcellswerethentransplantedintorecipientmiceforfurtherdifferentiationinvivo.
ThesestudiesdemonstratedthathESC/iPSC-derivedpancreaticprogenitorcellswhentransplantedintoectopicsitesinimmunodeficientortype1diabetesmice;theyunderwentfurtherdifferentiationandmaturationintoglucose-responsiveinsulin-secretingcells,whichcouldreversediabetesinrecipientmice[18,20,21](Fig.
1b)(Table1),suggestingthatpancreaticpre-cursorsorimmatureislet-likecellsobtainedinvitrocouldmatureinvivo.
Thisalsoindicatesthatsomeinvivofac-torsarestillmissingininvitrogrowthfactorcocktails.
Therefore,growthfactorsandsignalingmoleculesin-volvedinpancreasdevelopmentneedtobebetterscreenedtodetecttheirpotentialabilitiestocausehESC/iPSCtodifferentiateintomaturepancreaticβcellsinvitro.
Inrecentyears,tremendoussuccesshasbeenachievedinestablishingdifferentiationprotocolsthatcangenerateglucose-responsiveinsulin-secretingβcellsfromhESC/iPSCinvitroexpressingmatureβcellmarkers,andameli-oratinghyperglycemiaindiabeticmicefollowingtrans-plantationofthesecells[2,22,23,29](Fig.
1c)(Table1).
Inthesestudies,hESCs/iPSCswereculturedeitherinlowad-hesionplateorinthree-dimensional(3D)suspensioncul-turewithcontrolledstirring,whichpromotecell-cellandcell-matrixinteractionsresultingintheformationofcellag-gregates.
Sequentialandtime-dependentuseofsignalingmoleculesinsuchculturesystemsconcurrentlyguideshESC/iPSC-derivedcellstoappropriatelydifferentiateto-wardspancreaticβcellswithbetterphenotypesinvitro.
Aftertransplantationintorecipientmice,cellsresidingintheaggregatesunderwentfurtherdifferentiationandmatur-ationintomatureβcells.
Thesesimplifieddifferentiationconditionsenabletheefficientgenerationofhumanpancre-aticandmorerestrictedendocrineprogenitorpopulationsfrompluripotentstemcellswithoutunwantedformationofpolyhormonalcells.
Furthermore,theinducedβcellsinthisprotocolshowbothinvitroandinvivogeneexpressionpatterns,ultrastructuralcharacteristics,andglucoserespon-sivenesstoinsulinsecretionthatcloselyresemblethoseofβcellsfrompancreaticislets[2,22,23].
Moreover,com-paredtopreviouslyreportedimplantationsofhESC/iPSC--derivedpancreaticprogenitors,forwhichittook3–4monthsafterimplantationforcellstomature,recentad-vancesinthegenerationofβcellsinvitrosubstantiallyshortenthewaitingtimetotherapeuticeffectsafterim-plantation.
Inastudy,Rezaniaetal.
[22]optimizedtheirpreviousdifferentiationprotocolbyaddingfactorssuchasvitaminC,proteinkinaseCactivators,transforminggrowthfactor-βreceptorinhibitors,andthyroidhormonestogen-erateinsulin-producingcellsataninductionrateofap-proximately50%.
Furthermore,theyidentifiedR428,aselectivesmall-moleculeinhibitoroftyrosinekinaserecep-torAXL,asacrucialfactorforthematurationofβcellsinvitro.
Pagliucaetal.
[23]alsooptimizedadifferentiationmethodtogenerateβcellsfromhESC/iPSCinvitroatanShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page7of19inductionefficiencyof>30%.
Inthisstudy,ascalablesuspension-basedculturesystemthatadoptedfromSchulzetal.
[19]wasusedtogeneratemorethan108hPSCsforfurtherdifferentiation.
Thisprotocoltakes4–5weeksandinvolvesauniquecombinationofsequentialculturestepsusingfactorsthataffectsignalinginnumerouspathways,includingsignalingbyWnt,activin,hedgehog,EGF,TGFβ,thyroidhormone,andretinoicacid,aswellasγ-secretaseinhibition.
Later,in2015,Russetal.
[2]showedthattheuseofBMPinhibitorstospecifypancreaticcellspromotestheprecociousinductionofendocrinedifferentiationinPDX1+pancreaticprogenitors,whichultimatelyresultsinthefor-mationofnon-functionalpolyhormonalcells.
Therefore,intheirculturesystem,thecommonlyusedBMPinhibitorswereomittedduringpancreaticspecification,whichpreventprecociousendocrineformation,whiletreatmentwithret-inoicacidfollowedbycombinedEGF/keratinocytegrowthfactor(KGF)efficientlygeneratesbothPDX1+andsubse-quentPDX1+/NKX6.
1+pancreaticprogenitorpopulations,respectively.
TheprecisetemporalactivationofendocrinedifferentiationinPDX1+/NKX6.
1+progenitorsfinallypro-ducesglucose-responsiveβ-likecellsinvitroataninduc-tionefficiencyof~60%.
Thus,thisprotocolisconsideredtobemorecloselyresembleskeyaspectsofearlyhumanpancreasdevelopmentand,assuch,representsanimprove-mentoverpreviousprotocols.
Alltheseobservationssug-gestthatinsulin-producingcellssuitablefordiabetescelltherapiescanbeproducedfromhESC/iPSCinvitro.
How-ever,theformationofhESC/iPSC-inducedβcellsinvitrodependsonmultiplefactors,suchastheuseofplatforms/materials,applicationofsuspensionculture,useofalargenumberofgrowthfactorsandtheircombinations,andthetimingofrotatingthecultures.
Inthesemultistageproto-cols,althoughthefinalcellpopulationhasonlyabout30–60%β-likecells,themajorityoftheremainingcellpopula-tioncomprisedrelativelyuncharacterizedcellsthatmaybeundifferentiatedprogenitorsorothertypesofunwantedcells.
Thus,improvingdifferentiationefficiencytogeneratehigherpercentagesofβcellsinvitroremainsanimportantchallenge.
AsimilariPSC-derivedβcellgenerationprotocolhasbeenreportedforpatientswithtype1diabetes(T1D)[27,28].
Millmanetal.
[27]reportedthattheinducedcellsexpressβcellmarkers,respondtoglucosebothinvitroandinvivo,preventalloxan-induceddiabetesinmice,andrespondtoseveralcategoriesofantidiabeticdrugs.
NomajordifferenceswereobservedinT1Dstemcell-derivedβcellscomparedtostemcell-inducedβcellsderivedfromnon-diabeticpatients.
Furthermore,T1DiPSC-derivedβcellsrespondedtodifferentformsofβcellstressinaninvitrodiseasemodel.
Manzaretal.
[28]generatedglucose-responsiveinsulin-producingcellsvia3Dculture.
Inthisstudy,T1DiPSCswereini-tiallyresistanttodifferentiation,buttransientdemethylationtreatmentsignificantlyenhancedtheyieldofinsulin-producingcells.
Thecellsrespondedtohigh-glucosestimulationbysecretinginsulininvitro.
Theshape,size,andnumberoftheirgranuleswereiden-ticaltothosefoundincadavericβcells.
Whentheseinsulin-producingcellsweretransplantedintoimmuno-deficientmicethathaddevelopedstreptozotocin(STZ)-induceddiabetes,hyperglycemiadecreaseddra-matically,sothatthemicebecomenormoglycemic.
Thus,T1DiPSC-derivedβcellsareasuitablecandidateforuseasanautologouscellsourceforthetreatmentofdiabetes.
However,amoreefficientculturesystemisre-quiredtogeneratefunctionalandterminallydifferenti-atedβcellsforfutureresearchandclinicalapplications.
IsletorganoidgenerationfordiabetestreatmentPancreaticisletsarecomposedofECs,includinginsulin-producingβcells,glucagon-producingαcells,somatostatin-producingδcells,pancreaticpeptide-pro-ducing(PP)cells,andghrelin-producingεcells[75–77].
Afterfunctionalmaturation,theseECsintheisletshelptoregulatebloodglucoselevels.
ReciprocalinteractionsamongECsintheisletsarecriticalforregulationofin-sulinsecretioninresponsetoglucose[78–80].
Thus,pancreaticisletstructuresofferaneffectivemeansofphysiologicallyregulatinginsulinsecretioninpatientswithdiabetesmellitus.
Organoidsareagroupofprimarycells,ESCs,oriPSCsgrowninvitrothatowetheirself-renewalcapacitiesandabilitytodifferentiateinto3Dstructuresthatassumeasimilarorganizationandfunctionalityasanorgan.
Thegenerationofisletorganoidscontainingmatureβcellswithfullfunctionalityisyettobedemonstrated.
How-ever,generatingsuchfunctionalorganoidswouldbevaluableinperformingpathologystudiesofdiabetesde-velopment,treatment,anddrugscreening[81,82].
Inthelastdecade,severalresearchgroupshavereportedonthegenerationofhESC/iPSC-derivedislet-likeclus-ters/aggregates,aswellasislet-likeorganoids[81–87].
Intheearlystudies,onlythefeasibilityofgeneratingislet-likeclustersoraggregatesfromhESC/iPSCwasstudied.
However,inrecentyears,considerablesuccesshasbeenachievedingeneratingislet-likeorganoids(Fig.
3).
Islet-likeorganoidsdevelopedfromhPSCbyKimetal.
[86]showedglucoseresponsivenessinvitro,aswellasinvivo.
Inthatstudy,ECsexpressingpancre-aticendocrinehormoneswerefirstgeneratedfromhESCandiPSCusingastep-wiseprotocol,andECclusters(ECCs)werethenformedspontaneouslyin1dayfromthedissociatedECsinanoptimized3Dculture.
ThesizesofthehESC-derivedECCswereapproximately50–150μmindiameter,whichissimilartothesizesofhu-manpancreaticislets.
TheECCscomprisedseveralpan-creaticECtypes,exceptforαcells,andthusshowedShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page8of19thathESC-derivedECCsare,tosomeextent,analogoustohumanpancreaticisletsintermsofsizeandcellcom-position.
Levelsofβcell-associatedgenetranscriptionandGSISwerefoundtobehigherinECCsthaninECs.
Inaddition,intracellularCa2+influxoscillatedinECCsduringglucosestimulation,andSTZ-treateddiabeticmicetransplantedwithECCsbecamenormoglycemicwithin3daysaftertransplantationandsurvivedforap-proximately2months.
Thisstudy,therefore,supportedtheideathatfunctionalislet-likeorganoidscanbegener-atedfromhPSCs,whichcouldserveasanalternativesourceoftherapeuticcellsforthetreatmentofdiabetes.
AnotherstudybyWangetal.
[81]demonstratedthede-velopmentofisletorganoidsfromhESCin3Dbiomim-eticscaffoldsusingseveralgrowthfactors,whichpromotepancreaticECdifferentiation.
Theorganoidsformedinthisstudyconsistedofpancreaticα,β,δ,andPPcells,and,importantly,mostinsulin-secretingcellsgenerateddidnotco-expressglucagon,somatostatin,orPP.
Matureβcellmarkergeneswereexpressed,andinsulin-secretorygranules,whichareindicationsofβcellmaturity,weredetectedinthese3D-inducedcellclus-ters.
The3D-inducedorganoidcellsweresensitivetoglucoselevels;exposingthecellstoahighconcentrationofglucoseinducedasharpincreaseininsulinsecretion.
However,theseisletorganoidswerenottransplantedintoanimalmodelstoconfirmtheirbiologicalfunction.
Theconventional2Dcultureofstemcellsoverseveralpassagesinfluencecellphenotypeandfunction[88].
Cellsareonlypartiallypolarizedonaflatsubstrate.
Incontrast,3Dcellcultureonaspecializedmatrixpreventscellsfromattachingtothebottomoftheplatebymain-tainingthecellsinsuspensionorembeddingtheminthematrixinwhichcell-cellandcell-matrixinteractionsaremaintained,therebypromisesphenotypicmainten-anceandself-assemblyoffunctionaltissue-likepolarizedstructures[89–91].
3Dplatformsrecapitulatemechan-icalandbiochemicalstimuliaspresentinnativetissueandthusdictatepolarization[92].
However,suchstruc-turesoftendonotentirelymatchmulticellularorganizationseeninnativetissue.
Extracellularmatrix(ECM)isacriticalregulatorofcellularprocesseswhichservediversefunctionssuchassequesteringsignalingmoleculesandtransmittingligand-specificcuesviacellreceptorsandisamenabletosynthesis,degradation,andreassemblyovertime[93].
Cellsreadilyshapeandre-modeltheirextracellularenvironmentbyenzymaticallydegradingandresynthesizingtheECM.
Intissues,ECMnotonlyactsasanimmobilizationplatformforahigherorderofself-assemblybutalsofacilitatestherelayofabFig.
3Schematicoffabricationprocessesforislet-likeorganoids,adoptedandmodifiedfromCandielloetal.
[82]andTakahashietal.
[87].
aGenerationofhESC-derivedisletspheroidsandislet-likeorganoidsonAmikagelhydrogelplatform.
Pre-differentiatedhESC-derivedpancreaticprogenitorcells(hESC-PPs)on2DMatrigelwereharvestedandthenseededontotheAmikagelhydrogelplatformtoeitherformhomogenousisletspheroidsorbecombinedwithendothelialcells(HUVEC)toformendothelializedheterogeneousislet-likeorganoids.
Severalotherscaffold-basedstrategieshavealsobeenappliedtogeneratehESC-derivedislet-likeorganoidssuchascollagen-Matrigelscaffolds.
bGenerationofvascularizedislet-likeorganoidsinself-condensationculture.
Inthisprocess,isolatedadultmouse/humanisletsorhiPSC-derivedpancreatictissueswereco-culturedwithendothelialcells(HUVECs)andhumanmesenchymalstemcells(hMSCs).
Inthebeginning,thecellswerescatteredthroughouttheculturewell,andthen,theybeganmovingtowardsthecenterofthewelltoformcondensedtissue.
EachcondensedtissuecontainedpancreaticisletswithendothelialcellssurroundingthemShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page9of19biochemicalandmechanicalcuesinabufferedandhy-dratedenvironment[93].
ECMinteractionshavebeenshowntoimproveβcellproliferation[94],insulinsecre-tion[95,96],andisletdevelopment[96–98].
Collagenhasbeenusedwidelyfor3Dstemcellcultures[99].
Acollagenscaffoldconstitutesasoftandflexiblefibrousnetworkthatmaintainscellmorphologyandallowscellstofreelyreachout,migrate,andform3Dstructures.
However,collagenaloneisinsufficienttoprovidemul-tiplecuesandsophisticatedgeometryandcompositionthatexistinanativeextracellularmatrix.
Inpreviousstudies,thecombinationofcollagenwithMatrigel,acomplexheterogeneousmixtureofbasementmembraneproteinssuchaslaminin,collagenIV,fibronectin,hep-arinsulfateproteoglycans,andentactin,hasbeenusedastheunderlyingmaterialtoreconstructcardiacmuscleanduterinetissuesinvitro[100,101].
Inastudy,Wangetal.
[81]usedafour-stagedifferentiationstrategytodifferentiatehESCintopancreaticendodermandtoma-turethesecellsintoisletorganoidswithincollagen-Matrigelscaffolds.
Theirresultsshowedthataugmenta-tionofcollagenscaffoldswithMatrigelcreatesbetter3DnichesforisletorganoiddevelopmentfromhESC.
Al-thoughneitherMatrigelnorrattailcollagenIisaUSFoodandDrugAdministration-approvedmaterialforclinicalapplications,thestudyonlydemonstratedthefeasibilityofgeneratingisletorganoidsfromhESC.
How-ever,thecellclusterscanbepurifiedbyenzymaticallydigestingthescaffolds.
AnotheralternativeistouseaporcinedecellularizedECMtoconstructscaffolds.
Ashortageofdonorisletsiscurrentlylimitingthewide-spreadimplementationofislettransplantationtotreatdia-betes[102].
Inresponsetotheseneeds,recentstudieshavefocusedprimarilyonderivingisletβcellsfromhESC/iPSCasanalternativetodonorislets[2,22,23,27–29].
Thenon-endocrinecomponentsofisletsalsoplayacriticalroleintheirfunction.
Previously,heterogeneouspancreaticorganoidshavebeengeneratedbyaggregatingadultmouseβcellswithendothelialandmesenchymalcells[103].
Theseorganoidswerefoundtosuccessfullyintegratewithhostvasculatureandnormalizebloodglucoseindiabeticmice.
Systematicgenerationofheterogeneousorganoidsinvitrorequiresanorgan-specificcellsourceanda3Dcultureplat-formtoinduceself-organization,lineagespecification,func-tionalmaturationoforgan-specificcells,andintegrationofsupportingcellpopulations[104,105].
Scaffold-basedstrat-egieshaveprimarilyreliedonlaminin-richMatrigel,aswellasothernaturalorsyntheticbiomaterials,whichtypicallyconfinedcellswithinthe3Dscaffold.
Recently,isletorga-noidshavebeengeneratedfromhPSCsbyaggregatingthesecellsintohomogenous3Dislet-likespheroidsusing2Dnon-adherentculture[86]orbyembeddingtheminacollagen-Matrigelmatrix[81].
Effectivebioengineeredplat-formsthatwillsupportspecificorganoidproduction,finecontrolovertheorganoidsizeandcellularcomposition,scalingupofproduction,andeaseoforganoidrecoveryafterculturearestillneeded.
Hydrogelshavebeenafavoredchoiceofmaterialsintissueengineeringapplicationsduetotheirabilitytomimicthearchitectureandmechanicsofpli-ablecellularmicroenvironment[106,107].
Tissue-likeflu-idity,faciletransportofsolublenutrients,easeoffabrication,andintegrationwithbiologicalinterfacesaresomeofthemainadvantagesofahydrogelsystem.
Inare-centstudy,isletorganoidsofaprecisesizeandcellularhet-erogeneitywereengineeredfromhESC-derivedpancreaticisletcellsutilizinganovelhydrogelplatform,Amikagel[82](Fig.
3).
TheAmikagel-basedplatformwasshowntofacili-tatecontrolledandspontaneous,ratherthanforced,aggre-gationofhESC-derivedpancreaticprogenitorcells(hESC-PPs)intorobusthomogeneousspheroids.
Thefor-mationofAmikagel-inducedhESC-PPspheroidsenhancedpancreaticislet-specificPDX1andNKX6.
1geneandpro-teinexpression,whilealsoincreasingthepercentageofcellsco-expressingboth.
Amikagelalsoenabledco-aggregationofhESC-PPwithsupportingendothelialcells,resultinginself-organizedmulticellularpancreaticorganoidsthatwereclosertoisletphysiologyintermsoftheirheterogeneitythanhPSC-PPhomogenousspheroids.
.
TheseAmikagel-in-ducedhESC-PPspheroidsandheterogeneousorganoidsspontaneouslydifferentiatedintomatureβ-likecellsthatshowexpressionoftheβcell-specificINS1gene,aswellasC-peptideprotein,andproduceinsulininresponsetoinvitroglucosechallenge.
Aftermaturation,theAmikagel-in-ducedheterogeneousorganoidsalsoshowasignificantlydevelopedextracellularmatrixsupportsystem.
Therefore,theAmikagelplatformcouldbeidealforengineeringmulti-cellular3DisletorganoidsfromhPSCs.
Thepurposeforin-ducingandmaintaining3D-aggregatedorganoidsistoenhancetissue-ororgan-specificfunctionsbyreproducingcells'nativeenvironments.
Commonlyusedtechniquesforengineeringisletspheroidsneitherhaveprecisecontrolovertheultimatesizeofanaggregate,nordotheysupportcellinclusion,whichisthenextstepneededtogenerateisletorganoidswithmulticellularcomplexityandeventualgenerationofvascularizedconstructs.
TheAmikagelplat-formisparticularlysuitableinthiscontext.
Adevelopedextracellularmatrixbaseisimportantforisletstructure,cellhealth,andoverallfunction,whilealsobeingaprerequisiteforadevelopedvascularsystem[108].
Integrationofendothelialcellswouldbeanim-portantinitialsteptowardsvasculardevelopment[109].
Inthecontextofregenerativemedicine,tissuesurvivalandneovesselorganizationofhESC-derivedcellsmaybedependentonendothelialinclusionandmesenchymalsupplementation[110].
In2018,acomplexorganoiden-gineeringmethodtogeneratepancreaticisletswasre-ported[87](Fig.
3).
Usingthisprotocol,pancreaticislet-likeorganoidswithvascularnetworkswereformedShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page10of19byco-culturingeitherisolatedadultmouse/humanislettissuesorhiPSC-derivedpancreatictissueswithvascularendothelialcells(HUVECs)andhumanmesenchymalstemcells(hMSCs).
Pancreaticislet-likeorganoidsweregeneratedbyself-condensationafterseedingcellsontotheMatrigelbed.
Transplantationofthesevascularizedislet-likeorganoidsintothekidneysubcapsuleoffulmin-anttype1diabeticmicesignificantlyimprovedthesur-vivalofthediabeticmiceandeffectivelynormalizedbloodglucosecomparedtoconventionalislettransplant-ation.
Thisapproach,therefore,offersapromisingalter-nativetotherapeuticislettransplantation.
Thefunctionalityofisletorganoidsgeneratedsofarremainspartial.
Someofthelimitationsoftheseorganoidsarethattheyoftenlackcelltypesneededforcompleteisletfunctionsandthegenerationofbloodvesselsandnerves.
Toapplyisletorganoidstothetreatmentofdia-betes,morecompleteisletstructuresmustbepreparedforbetterfunctionindiabeticrecipients.
Despitethepromiseofemergingisletorganoid-basedapproaches,developingvascularnetworksremainsamajorchallengetotheirapplicationinregenerativetherapies.
ESC/iPSC-derivedβcellsfordiabetestreatment:limitationsandchallengesTherearestillmanypointstoaddressandproblemstoovercomebeforehESC/iPSC-derivedcellscanbeclinic-allyappliedindiabeticpatients,includingthefollowing:(1)Safetyissues:Sofar,mostpatient-specificiPSCshavebeenestablishedwithretrovirusvectors.
TheseiPSCshavenumeroustransgeneintegrationsintheirgenomes,andtheseintegrationsmaycauseleakyexpressionthatcaninterruptthefunctionofendogenoustranscriptionfactornetworksandleadtodifferentiationfailure.
An-otherimportantproblemoftransgeneintegrationistumorigenicriskaftertransplantation.
Inparticular,c-Myc,oneofthereprogrammingfactors,isawell-knownoncogene,anditsreactivationcangiverisetotransgene-derivedtumorsinchimericmice[111].
TomakesafeiPSCs,oneimportantapproachmaybeelim-inatingthec-Myctransgeneinthereprogrammingcock-tail.
HumanandmouseiPSCscanbeestablishedfromfibroblastswithonlyOct3/4,Sox2,andKlf4,butboththeefficiencyofiPSCgenerationandthequalityofthesecellsaresignificantlyreduced[112].
Chimericmicepro-ducedwithc-Myc-freeiPSCsdidnotshowenhancedtumorformationincomparisonwithcontrolmice.
How-ever,retroviralinsertionsinthegenomeitselfmaydis-turbendogenousgenestructureandincreasetheriskoftumors[113].
ToincreasethesafetyofhiPSC-basedcelltherapies,itisnecessarytogeneratehiPSCswithoutvec-torintegrationandcontinuousc-MYCexpression.
ThegenerationofhiPSCswithtransientexpressionfromnon-integratingvectors[52,56,114]mayaddresstheseconcerns.
Todate,variousintegration-freetechniqueshavebeenreported,includingtransientexpressionofre-programmingfactorsusingadenovirus[115]orSendaivirusvectors[116],thepiggyBacsystem[51],episomalvectors[52,56],aminicirclevector[53],anddirectde-liveryofprotein[50]orsyntheticRNA[54].
However,theiriPSCinductionefficienciesarelowerthanthosewithretrovirusvectors,possiblybecauseoflowtrans-ductionefficiencyandunstableexpression[117].
(2)Variationindifferentiationefficiencies:DifferentiationpropensitiesarereportedtovaryamonghESClines[118].
Dependingonthecelloriginorderivationproced-ure,someiPSClinesalsodemonstratevaryingdegreesofdifferentiationefficiency,resistancetodifferentiation,ortumorigenicity[65,84].
Abnormalitiesinkaryotypeandvariationsinthetechniquesusedtoobtainormain-tainiPSClinesandepigeneticdifferencesamongthemhavealsobeenconsideredvitalfactorsthatalterdiffer-entiationpotential.
Epigeneticvariationsaremorepro-nouncediniPSCthanESC.
Thus,selectionofgoodiPSClineswithlowbatch-to-batchvariationindifferen-tiationefficiencyisessentialtodifferentiatingthesecellsintotargetlineagespriortouseinspecificcelltherapies[118].
Inaddition,differentiatedcellspreparedfrompatient-specificiPSCcanincreasethesuccessrateofcelltherapiesinthefuture.
(3)Formationofpolyhormonalcells:Polyhormonalinsulin-expressingcellsarefre-quentlyformedfromhESC/iPSC-derivedcellsinvitro,asdiscussedabove.
AvailablereportssuggestthatoncehESC/iPSC-derivedcellsbecomepolyhormonal,theycannotbedifferentiatedintomatureβcells[33].
There-fore,itisnecessarytofindouttherightcombinationoffactorstoreducetheformationofpolyhormonalcellsinculture,aswellastoinducethesecellsintomatureβcellsiftheyaregenerated.
(4)Xenogeneiccontamina-tionsandunknowneffects:Althoughmostrecentdiffer-entiationprotocolshavebeendevelopedonfeeder-freeculturesystems,manyprotocolsstillcallforavarietyofundefinedanimal-derivedproductsthatmayhaveun-knowneffectsoncellcharacteristicsanddifferentiationability.
Thepotentialconsequencesoftransplantinghu-mancellsexposedtoanimal-derivedproductsintopa-tientscouldincludeincreasedriskofgraftrejection,immunoreactions,microbialinfectious,prions,andyetunidentifiedzoonoses[119–121].
Toreducetheeffectsofxenogeneiccontamination,Micallefetal.
[122]usedxeno-freemedia;however,theyusedMEFforpassaging.
Inanotherstudy,Schulzetal.
[19]expandedhESCinxeno-freemediawithoutfeedercells,buttheyusedfetalbovineserumduringdifferentiation.
Later,Shahjalaletal.
[24]expandedanddifferentiatedhESCandiPSCinasyntheticscaffoldundercompletelyxeno-freeconditionsusingrecombinantand/orhumanizedcomponentsandsuccessfullygeneratedinsulin-expressingcellsinvitro.
Shahjalaletal.
StemCellResearch&Therapy(2018)9:355Page11of19ThesestudiesindicatethefeasibilityofgeneratinghESC/iPSC-derivedpancreaticβcellsunderxeno-freecondi-tions.
Forsuccessfulclinicalapplications,hESC/iPSCshouldbeprepared,maintained,anddifferentiatedinxeno-freeculturesystems.
Humanizedand/orrecombin-antfactors,chemicallydefinedsupplements,andsyn-theticscaffoldscanbeappliedinvitrotoaddressthisissue.
(5)Lackofmaturation:Todate,insulin-expressingcellsgeneratedfromhESC/iPSCinvitrolacktheproper-tiesofmaturepancreaticβcells.
Transplantationofim-maturehumanislet-likecellstoimmunodeficientmiceenablesfurthermaturationofisletcells.
Thisisevi-dencedbyhumanC-peptidesecretioninglucosetoler-ancetestsandbymorphologicalandultrastructuralstudies[2,22,23,123].
Itisimportanttounderstandwhatfactorsintheinvivomilieuarecriticaltofunc-tionalmaturation.
Thesecouldberelatedtolocalsignalsprovidedbytheinvivonicheatthetransplantsite.
Re-cently,ithasbeenshownthatmaturationoccursfasterandmoreefficientlyinfemalerecipientmice,pointingtothepotentialroleofestrogenreceptorsignalinginmaturation[124].
Geneprofilingstudiessuggestthatthecreationofmatureβcells,inwhichinsulinsecretionistightlycoupledtoglucoseconcentrations,requiresthecoordinatedupregulationofcertaingenesandrepressionofothers[125].
Arecentreportshowedthatligand-dependenttranscriptionfactorestrogen-relatedreceptor-γ(ERRγ)isadriveroftheoxidativemetabolicgenenetworkinmatureβcellsandthatitspostnatalin-ductionorchestratesthemetabolicmaturationofβcells[123].
Thisreportalsoindicatedthatβcell-specificERRγ-deficientmiceareglucoseintolerantandfailtoappropriatelysecreteinsulininresponsetoglucosechal-lenge.
ERRγexpressionduringpostnatalβcellmatur-ationdrivesatranscriptionalprogramthatpromotesthemitochondrialoxidativemetabolismnecessaryforGSIS.
Assuch,promotingERRγexpressionandactivityduringthelatestageofhiPSCdifferentiationinvitroresultsinglucose-responsiveβcellsthatarecapableofrestoringbloodglucoseintype1diabeticmice.
Thus,futurestud-iestoimprovefunctionalmaturationofinsulin-expressingcellsshouldfocusonsignalingpath-waysthatregulatethematurationofpancreaticβcells.
(6)Lowsurvivalrateandimmunogenicity:Becauseofthelackofasuitabletransplantationtechnique,there-coveryofwell-demarcatedgraftsaftertransplantationintoectopicsitesinexperimentalanimalsandexamin-ationofglucoseresponsivenessofhESC/iPSC-derivedpancreaticcellsinvivoarestilldifficult.
Thiscouldbeduetoimmunerejectioninthehostanimals.
Atpresent,twodifferenttransplantationstrategiesareapplied:oneisdirectimplantationofhESC/iPSC-derivedpancreaticcellsintoectopicsites,andotheroneisimplantationofadevicecontaininginducedpancreaticcells(Fig.
4).
Inthefirstmethod,pretreatmenttoinduceangiogenesisatimplantationsitesisusedtopromoteengraftmentandlong-termsurvivaloftheimplantedcells.
Onerecentstudyshowedthatanyloncatheterembeddedintothesubcutaneoustissuesofhostmicefor1monthbeforecellimplantationgeneratedavascularizedspace[126].
Theformationofvascularnetworksattheimplantationsiteandtheimplantationofpancreaticcellsafterinflam-matoryreactionshavediminisheddevelopealessin-tolerantenvironmentfortheimplantedcells.
Inthesecondmethod,pancreaticcellsareencapsulatedinadevicemadeofbiocompatiblematerialthatincludessemipermeablemembranes.
Oxygenandnutrientscanpassthroughthemembranestopromotecellsurvival,differentiation,andmaturation,whereasimmunemole-culesandcellscannot.
Severalstudieshaveshownthat,whenimplantedsubcutaneouslyintohostmice,hESC/iPSC-derivedpancreaticcellsencapsulatedbythesesemipermeablemembranedevicescanfurtherdifferenti-ateintomatureinsulin-secretingcellsandsurvivefromhostimmuneresponses,primarilybyTcells[20,21,25,35].
Inaddition,becauseofvasculogenesisaroundthesedevices,thedifferentiatedβcellscansecreteinsulininresponsetochangesinglucoseconcentrations.
ArecentstudyalsodemonstratedimplantationofhESC-derivedβ-likecellsencapsulatedwithanalginatederivative.
Thisdevicemitigatesforeignbodyresponsesandimplantsfi-brosis,andinducesglycemiccorrectionwithoutim-munosuppressioninimmune-competentmice[127].
Thesedevice-basedimplantationmethodscanreduceoreliminatetheneedforimmunosuppressiveagents.
Fur-thermore,thesemethodsmayhavetheadvantageofallowingremovaloftheimplantedcellswiththedevicewhenadverseeventssuchastumorigenesisordysfunc-tionoccur.
However,itispresentlyunclearwhetherpro-tectionfromsolubleantibodiesdirectedagainstdifferentiatedβcellswillbeasignificantproblem[128].
Althoughinsulin-expressingcellmaturationcanoccurwithorwithouttheimplantedcell/devicecombination,thesurvivalrateoftheimplantedcellsinhostsisstillrelativelylow.
Thus,asuitabletransplantationtechnique,alongwithaneffectivecombinationofinducers,isre-quiredtoovercomethistransplantationchallenge.
Des-piteseverallimitations,phase1/2clinicaltrialshavealreadystartedforthetreatmentoftype1diabetespa-tientsusingasemipermeablemembranecapsuledevicethatcarrieshESC-derivedpancreaticprogenitorsco-expressingPDX1andNKX6.
1[36].
Thistrialhasattractedattentionworldwide,asitrepresentsanim-portantfirststepforthedevelopmentofnewstemcelltherapiesfordiabetes.
Todate,noclinicaltrialswithhiPSC-derivedpancreaticcellshavebeencarriedout.
However,thepotentialadvantagesofhiPSCoverhESCmaymakesuchtherapiesavailableinthefuture.
(7)IsletShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page12of19βcellheterogeneity:βcellsinhumanpancreaticisletshavebeenthoughttobeahomogenouscellpopulation.
Despitethisprevailingparadigm,therehavealsobeenreportsofβcellheterogeneityinhumanislets[129,130].
Recently,astudybyDorrelletal.
hasidentifiedfourantigenicallydistinctsubtypesofhumanβcells,whicharedistinguishedbydifferentialexpressionofST8SIA1andCD9[131].
Theseβcellsubpopulationsarealwayspresentinnormaladultisletsandhavedi-versegeneexpressionprofilesanddistinctbasalandglucose-stimulatedinsulinsecretion.
Dissimilarbasalandglucose-stimulatedinsulinsecretioncharacteristicsindicatethattheβcellsubtypesarefunctionallydistinct.
Dorrelletal.
intheirstudyisolatedlivepancreaticβcellsfromhumanisletsamplesbyFACSandco-labeledthemwithantibodiesrecognizingST8SIA1andCD9andfi-nallyidentifiedfourantigenicallydistinctβcellsubpopu-lations.
TheyalsoaccessedtheexpressionofST8SIA1andCD9insectionsofhumanpancreasandfurtherconfirmedtheexistenceoffourdistinctβcellsubpopulationsinhumanislets.
TranscriptomeanalysesbyRNAsequencingoftheβcellsubsetshaveshownthatmostofthedifferentiallyexpressedgenesareofun-knownfunctioninβcells,butsomehavebeenclearlyassociatedwithinsulinsecretionorareknowntobedys-regulatedintype2diabetesmellitus.
Dorrelletal.
havealsoobservedthatthefrequenciesofβcellsubtypesarealteredinthemajorityofindividualswithtype2dia-betes.
Thus,theβcellsubpopulationsmayhaverele-vancetodiabetesandthisissueneedstobeaddressedbyextensiveresearch.
AlmostallpreviousstudieshaveattemptedtogeneratematureβcellsfromhumanESCs/iPSCs.
Recently,researchersaremainlyfocusingoneffi-cienttechniquestoenhancetheyieldofmatureβcellsandtheirsuccessfulsurvivalpost-transplantationtore-tainnormoglycemiaindiabetes.
However,βcellhetero-geneityisanemergingissue.
Thedistinctpropertiesofhumanβcellsubtypesfoundinhumanisletslikelyhaveanimportantimpactonmetabolicregulationandhu-mandiseaseprocesses.
Thus,futurestudiesshouldfocusabFig.
4MethodsforimplantinghESC/iPSC-derivedpancreaticcells.
Twodifferentmethodshavebeenapplied.
Inonemethod,hESC/iPSC-derivedpancreaticcellsareimplanteddirectlyintotransplantationsitesorintopre-developedvascularizedsitesofdiabeticand/ornon-diabeticSCIDmice(a).
Inanothermethod,hESC/iPSC-derivedpancreaticcellsencapsulatedinimmunoprotectivesemipermeabledevicesareimplantedintoectopicsitesofSCID-Beigeand/ordiabeticmice(b).
Oxygen,nutrients,insulin,andglucosecanpassthroughthemembranesofthedevicestopromotethesurvival,differentiation,maturation,andglucose-responsiveinsulinsecretionoftheencapsulatedpancreaticcellsfollowingimplantationintothehostmice.
Inaddition,vasculogenesisoccursaroundthedevices,supportingsecretionofinsulinfromthedifferentiatedβcellsinresponsetochangesinglucoseconcentrations.
Incontrast,immunecellsormoleculessuchasantibodiesandcomplementscannotpassthroughthemembranes,preventingimmunerejectionorautoimmuneresponsesagainstthecellsShahjalaletal.
StemCellResearch&Therapy(2018)9:355Page13of19Table2StandardcriteriafordesigningandperformingfuturepreclinicalstudiesinvitroandinvivoPreclinicaltoolProsCons(challenges)Possibleimprovements-NODmouseisidealforstudyingtype1diabetesandthecharacterizationoftheimmunopathologyofthedisease.
-BioBreedingratisasuitablemodelforunderstandingthegeneticsoftype1diabetes[132]andstudyingofneuropathy-associateddiabetes[133].
-Diabetesinductioninthemurinemodelispossibleviatheexposuretocertainchemicals,namelystreptozotocinandalloxan,andthusoffersausefultoolfortestingthepotentialsoftherapeuticagentsortransplantedcellstoreduceglucoselevel.
-SomediseasesusceptibilitylociinNODmousehavenomarkedimpactinhumandisease.
-VariousdrugsandantibodytherapyshowedanexcellenteffectinNODmicebutnoeffectintheclinicaltrials[134].
-InductionofdiabetesinNODmiceiscorrelatedwithmicrobialinfections.
-Inductionofdiabetesusingchemicalsintheanimalmodelcouldshowtoxiceffectstotheotherorgans,suchasthekidney,liver,brain,intestine,andreproductiveorgans.
-Applyinghumanizedmousemodelhavingthecomponentsofthehumanimmunesystem.
-Takingintoaccountthegender-dependentdiabetespathogenicityinanimalmodels.
-Settingupnewanimalmodelsthatrecapitulatediabetespathogenesisinhuman.
-MaintainingNODmiceunderspecificpathogen-freeenvironmentduringdiabetesexperimentation.
-Consideringtoxicactionsinanimalmodelsduringthechemicalinductionofdiabetesinvivo.
PreviousreportsshowedtheoccurrenceoflymphopeniaandhighproductionofTregulatorycells[135].
-Forstudyingtype2diabetes,theoccurrenceandthecauseofobesityshouldbeconsidered.
-Studyingdiabetescomplications(neuropathy)needtoavoidselectingneuropathy-resistantmousesuchasC57BL/6strain[136].
Stemcellquality-PSCscouldobviatethehurdlesofisletapplicationsuchaslackofdonorsandweaksecretionofinsulinpost-implantation.
-ApplicationofPSCsallowstheunderstandingofpatient-specificdiseasepathogenicityandalsothedevelopmentofpotentialtherapeutics.
-GenerationofiPSCsusingintegrativeorviral-basedmethodshinderstheirclinicalapplicationindiabetestherapy.
-PSCculturesusingundefinedorxenogeneicconditionsproducecellshavingunusualcharacteristicsandpoorphenotypes,andthuscannotbeappliedintheclinic.
-Usingnon-integrativeandsafemethodsforthegenerationofiPSCs.
-DevelopingaccurateassaysforevaluatingthequalityofiPSCs,suchaskaryotyping,analysisofthepluripotencymarkers,andthedifferentiationcapacity.
-Developingefficientmethodsforheterogeneityandteratomaassays.
-Microbiologicalassaysforthedetectionofcellcontamination,suchasmycoplasmatest.
-Usingdefinedandxeno-freecultureconditions.
Organoid/spheroidculture-Organoid/spheroidcultureallowsadetailedunderstandingofdiabetespathogenicity,molecularmechanisms,anddiseasemodelandprovidesausefultoolfordrugscreening.
-Fororganoidculture,Matrigel,collagen-Matrigel,orhydrogelsaremainlyusedasaplatform.
-Applicationofanimal-derivedECMsuchasMatrigelhampersthefurtherapplicationofgeneratedorganoidsintheclinic.
-Organoidcultureiscostlyandlaboriousforthelarge-scaleproduction.
-Designingsuitablesafexenogeneicfreescaffolds(physicalcues)withgrowthfactors(biochemicalcues)forthegenerationofstemcellniche.
-Discoveringacost-effectiveagentsandprotocolsforefficientorganoidcultureatthelargescale.
-Developingefficientassaysfortheevaluationofthegeneratedorganoids/spheroidspriortotheirapplicationfordiseasemodelingordrugscreening.
DifferentiationmethodsVariousdifferentiationprotocolsaredevelopedforthegenerationofinsulin-producingβ-likecellsfromPSCsineithermonolayeror3Dcultureusingacocktailofvariouschemicals,growthfactors,inhibitors,andcytokinesinordertoemulatetheinvivosystem.
-Differentiationprotocolsdependonagentsofhighcosts.
-Manyofthedevelopedprotocolsarenotreproducible.
-Themolecularmechanismsofmostofthechemicalsusedineachstepofthedifferentiationmethodremainunrevealed.
-CharacterizingthereproducibilityofthecurrentβcelldifferentiationprotocolsfromPSCs.
-Settinguphighlyefficientprotocolsforthegenerationofmatureβcellsandtheirtransplantation.
-Cultureconditionssuchasculturemedia,celldensity,ECM,cell-cell,andcell-ECMinteractionshaveanimpactonPSCdifferentiation[68,137–139]andthusshouldbeoptimized.
-Characterizingthemolecularmechanismsofthefactorsusedinthecurrentdifferentiationprotocols.
Shahjalaletal.
StemCellResearch&Therapy(2018)9:355Page14of19Table2Standardcriteriafordesigningandperformingfuturepreclinicalstudiesinvitroandinvivo(Continued)PreclinicaltoolProsCons(challenges)PossibleimprovementsTransplantationdevices-Encapsulationdevicesusedforcelltransplantation,suchassemipermeablecapsuleormembrane,possessvariousfunctions[140]:Avoidingtheundesirablehostimmunereactionsagainstthetransplantedcells.
Protectingthepatientfromtumorigenicactionofstemcells.
Avoidingthelossofviabilityofthetransplantedcells.
Maintainingstableinsulinsecretion-Theencapsulationdevicesneedtheapplicationofimmunemodulatingagents.
-Encapsulationdevicesmayprovokethepatient'simmunesystemandultimatelyleadtocelldeath.
-Applyingsuitableagentswithimmunemodulatingfunctions,summarizedpreviously[140],whichprotectthetransplantedstemcellsfromrejection.
-Designinganefficientencapsulationdevicewiththefollowingfeatures:Allowingenoughbloodsupplytotheencapsulatedcells.
Havingbiocompatibility.
Avoidingthestimulationofhostimmunereactions.
Permittingtheefficienttransferofthesecretedinsulintothecirculation.
Shahjalaletal.
StemCellResearch&Therapy(2018)9:355Page15of19notonlyonthegenerationofmatureβcellsbutalsoontheformationofsubpopulationsofβcellsfromhumanESCs/iPSCsandrevealtheirfunctionalpropertiestopo-tentiallyapplyindiabetes.
ConclusionsTheabilitytoobtainalargenumberofmatureinsulin-producingcellsfromhESC/iPSCcouldprovideun-limitedsuppliesofsurrogateβcellstoreplacedamagedcellsinpatientswithdiabetes.
Insulin-expressingpancreaticcellsgeneratedfromhESC/iPSCtodateappearsimilartoneonatalcells.
Generatingmatureinsulin-expressingcellswiththesameGSIScapabilityasendogenousβcellsandtheirsurvivalfollowingtransplantationintoectopicsitesinexperimentalhostanimalsarechallengesforfuturere-search.
Theprocessesofβcelldifferentiationandisletorga-noidformationarecontrolledbyacomplexnetworkthatdependsontranscriptionalregulationofgenesinvolvedinpancreasdevelopment,acertaintypeandnumberofdiffer-entiationfactors,andspecifictypesandconditionsofstemcellculture.
Althoughmanyfactorsareimportantforthesuccessfulgenerationandtransplantationofinsulin-secret-ingβcellsorislet-likeorganoids,asuitablecombinationoffactorsandconditionsstandsoutasoneofthemostcriticalfactors.
Thus,moreworkisneededtoincreasematurityandpost-transplantsurvivalofhESC/iPSC-derivedinsulin-producingcellsorislet-likeorganoidsthoseresem-bleendogenouspancreaticβcellsorislets,respectively.
Toachievethisgoal,chemicalscreeningofvariousregulatoryfactorsandsmallmoleculesinlatestagesofdifferentiationinvitromaybeagoodoption.
AlistofstandardcriteriafordesigningandperformingfuturepreclinicalstudiesforhPSC-derivedβcells/isletorganoidsinvitroandinvivoisgiveninTable2.
Althoughanumberofconsiderationscur-rentlylimittheuseofhESC/iPSC-derivedcellsincellre-placementtherapies,priorityshouldbegiventotheissuesdiscussedabove.
AbbreviationsBMP:Bonemorphogeneticprotein;DE:Definitiveendoderm;EC:Hormone-expressingendocrinecells;ECCs:Endocrinecellclusters;ECM:Extracellularmatrix;EGF:Epidermalgrowthfactor;EP:Endocrineprogenitor;ERRγ:Estrogen-relatedreceptor-γ;ESC:Embryonicstemcells;FGF:Fibroblastgrowthfactors;GCG+:Glucagon-positivecells;GHRL+:Ghrelin-positivecells;GLP-1:Glucagon-likepeptide-1;GSIS:Glucose-stimulatedinsulinsecretion;HGF:Hepatocytegrowthfactor;HUVEC:Humanumbilicalveinendothelialcell;INS+:Insulin-positivecells;iPSC:Inducedpluripotentstemcells;KGF:Keratinocytegrowthfactor;MEFs:Mouseembryonicfibroblasts;MSCs:Humanmesenchymalstemcells;ND:Non-diabetic;NKX6.
1:NK6homeobox1;NOD:Non-obesediabetes;PDX1:Pancreas/duodenumhomeobox1;PG:Primitiveguttube;PI3K:Phosphatidylinositol-3-kinase;PP:Pancreaticprogenitor;PPY+:Pancreaticpolypeptide-positivecells;PSC:Pluripotentstemcell;SCID:Severecombinedimmunodeficiency;SST+:Somatostatin-positivecells;STZ:Streptozotocin;T1D:Type1diabetes;TGF-β:Transforminggrowthfactor-β;Wnt:WntsignalingAcknowledgementsTheauthorsthankGarrySweeneyforcarefulproofreadingofthetext.
FundingThisworkwassupportedbygrantsfromtheNationalResearchFoundation(NRF)fundedbytheKoreangovernment(2017M3A9C6029562and2015R1A5A1009701).
AvailabilityofdataandmaterialsDatasharingisnotapplicabletothisarticleasnodatasetsweregeneratedoranalyzedduringthecurrentstudy.
Authors'contributionsHMScontributedtotheconception,draft,andmodificationofthemanuscript.
AADcontributedtothedraftandmodificationofthemanuscript.
KMNandTJcontributedtothecollectionandsortingofthedata.
SGCcontributedtothetop-layerdesignandcriticalreviewofthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
EthicsapprovalandconsenttoparticipateNotapplicable.
ConsentforpublicationNotapplicable.
CompetinginterestsTheauthorsdeclarethattherearenocompetinginterests.
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
Authordetails1DepartmentofStemCell&RegenerativeBiotechnologyandIDASI(IncurableDiseaseAnimalmodel&StemcellInstitute),KonkukUniversity,120Neungdong-ro,Gwangjin-gu,Seoul05029,SouthKorea.
2DepartmentofBiochemistryandMolecularBiology,JahangirnagarUniversity,Savar,Dhaka1342,Bangladesh.
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