BACKGROUND: Loss of intestine from infection,ischemia,or injury can lead to the inability to absorb enough nutrients to sustain life,a condition called short bowel syndrome(SBS).Chronic medical management and surgical interventions provide little success in increasing intestinal function.Given the high rates of morbidity and mortality in SBS patients,development of patient-specific intestinal tissue for transplantation is essential.We have generated human intestinal organoid-derived tissue-engineered small intestine(HIO-TESI)from embryonic stem cells.However,HIO-TESI fails to develop an enteric nervous system(ENS).The purpose of our study is to establish an ENS in HIO-TESI derived exclusively from human pluripotent stem cells(hPSCs).METHODS: H9 hESCs or WTC hiPSCs were maintained in mTeSR.H9 cells were temporally exposed to Activin A,CHIR99021,and FGF4 as previously described to generate HIOs.Enteric neural crest(ENC)progenitor cells were derived from hPSCs exposed to LDN193189,SB431542,CHIR99021 and Retinoic Acid as previously described.HIOs and unsorted ENC progenitor cells were seeded onto biodegradable scaffolds,wrapped in the omentum of adult NSG mice,and allowed to mature for 3 months.ENS-HIO-TESI was immunostained for neuronal,glial,and smooth muscle cell markers: EDNRB,RET,TrkC,Tuj1,GFAP,s100b,ChAT,Calbindin,Calretinin and SMA.RESULTS: H&E analysis of ENS-HIO-TESI demonstrates mature villus formation with the presence of underlying smooth muscle and ganglia within the submucosal and muscular layers.Excitatory neurons(Tuj1/ChAT/Calretinin or Tuj1/ChAT/Calbindin),glia(GFAP/s100b)and ganglia(EDNRB/RET/TrkC)were identified.CONCLUSION: ENC progenitors give rise to excitatory neurons,glia and ganglia within the submucosal and muscular layers of ENS-HIO-TESI.These data demonstrate establishment of an ENS in HIO-TESI supplemented with ENC progenitor cells derived solely from hPSCs.