02787nas a2200577   4500000000100000000000100001008004100002260001500043653001500058653001800073653002400091653001100115653002400126653001700150653003000167653003700197653002200234653002000256653004400276653004000320653003700360653004700397653002400444100001400468700002000482700002900502700001600531700002100547700001400568700002700582700001500609700001600624700001300640700001200653700001700665700001700682700001700699700001800716700001900734700002100753700002700774700001200801700001600813700001200829700001100841245012000852300001700972490000700989520119900996022001402195       2023                            d  c2023-09-0710aBlastocyst10aCell Survival10aEmbryo Implantation10aHumans10aSignal Transduction10aTrophoblasts10aendometrial stromal cells10ahuman blastocyst-like structures10ahuman blastocysts10ahuman blastoids10ahuman integrated stem cell embryo model10ahuman peri-implantation development10anaive human embryonic stem cells10anaive human induced pluripotent stem cells10asyncytiotrophoblast1 aLeqian Yu1 aDeirdre Logsdon1 aCarlos A. Pinzon-Arteaga1 aJialei Duan1 aToshihiko Ezashi1 aYulei Wei1 aAna Elisa Ribeiro Orsi1 aSeiya Oura1 aLizhong Liu1 aLei Wang1 aKun Liu1 aXiaoyun Ding1 aLinfeng Zhan1 aJunfei Zhang1 aAsrafun Nahar1 aCaitlen Stobbe1 aMandy Katz-Jaffe1 aWilliam B. Schoolcraft1 aTao Tan1 aGary C. Hon1 aYe Yuan1 aJun Wu00aLarge-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk  a1246-1261.e90 v303 aRecent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts.  a1875-9777