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A single small molecule-based human embryo model reveals V-ATPase requirement in mammalian blastocyst cavitation

Samhan Alsolami^1,† , Arun Pandian Chandrasekaran^1,†,* , Yiqing Jin^1,† , Yibo Wang^2,3,† , Ling Zhang^4,5 , Ismail M. Shakir^1,6 , Yingzi Zhang^1,6 , Aisha Siddique^1,6 , Gerardo Ramos-Mandujano¹ , Baolei Yuan^1,7 , Maya Ayach⁸ , Alfonso Saera-Vila⁹ , Zejun Fan¹⁰ , Siyi Fu¹ , Huoming Zhang⁸ , Saige Xin¹ , Kholoud Khalid AlDakhil¹¹ , Juan Carlos Izpisua Belmonte^1,7 , Jin Zhang^5,12,13 , Yang Yu^2,3,* , Mo Li^1,6,10,*

¹Bioscience Program, Biomedical Sciences Division (BioMed), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
²The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China
³Beijing Key Laboratory of Collaborative Innovation in Frontier Technologies for Population Quality, State Key Laboratory of Female Fertility Promotion, Beijing Advanced Center of Cellular Homeostasis and Aging-Related Diseases, Institute of Advanced Clinical Medicine, Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China
⁴Center for Reproductive Medicine, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
⁵Liangzhu Laboratory, Zhejiang University, Hangzhou, Zhejiang, China
⁶KAUST Center of Excellence for Smart Health (KCSH), Thuwal, Saudi Arabia
⁷Altos Labs, San Diego, CA, USA
⁸KAUST Core Labs, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
⁹Sequentia Biotech Inc., Barcelona, Spain
¹⁰Bioengineering Program, Biomedical Sciences Division (BioMed), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
¹¹Cytogenetics and Molecular Cytogenetics Section, Pathology and Clinical Laboratory Medicine, Riyadh, Saudi Arabia
¹²Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
¹³Center of Gene and Cell Therapy and Genome Medicine of Zhejiang Province, Hangzhou, Zhejiang, China
^†These authors contributed equally: Samhan Alsolami, Arun Pandian Chandrasekaran, Yiqing Jin, Yibo Wang
* Correspondence: Arun Pandian Chandrasekaran(arun.chandrasekaran@kaust.edu.sa)Yang Yu(yuyangbysy@126.com)Mo Li(mo.li@kaust.edu.sa)

Human naïve pluripotent stem cells (nPSCs) can be induced by various combinations of signaling factors to generate blastocyst-like structures, termed blastoids. Despite rapid progress in human blastoid models, their potential to uncover fundamental mechanisms of early human development remains limited, leaving key morphogenetic processes poorly understood. Here, we describe a simple and robust system in which dimethyl sulfoxide (DMSO) alone induces blastoid formation from human nPSCs. This model recapitulates key pre- and post-implantation features and exhibits enhanced polar trophectoderm (TE) organization, more efficient attachment within an implantation-relevant window, improved epiblast lumenogenesis associated with amniotic cavity formation, and more robust, sustained expansion of embryonic lineages following attachment. Using this system, we reveal a previously unrecognized mechanism underlying TE cavitation and identify lysosome-associated genes — particularly subunits of the proton pump V-ATPase — as essential regulators of blastoid cavitation. DMSO treatment upregulates key V-ATPase subunits (ATP6V0A4 and ATP6V1B1), which are also enriched in the TE of human embryos. Genetic or pharmacological inhibition of V-ATPase activity disrupts lysosomal acidification, blocks intracellular vacuole formation, and impairs blastoid cavitation, whereas overexpression of V-ATPase subunits rescues this phenotype. Furthermore, genetic and pharmacological perturbations of V-ATPase function significantly compromise cavitation in both mouse and human blastocysts. Finally, DMSO treatment induces membrane biomechanical changes characteristic of early embryonic development, suggesting a mode of action distinct from conventional small-molecule, signaling pathway-based induction strategies. This simple DMSO-based blastoid model recapitulates key aspects of human blastocyst development and reveals a conserved requirement for V-ATPase-mediated lysosomal acidification during early mammalian embryogenesis.

https://doi.org/10.1038/s41422-026-01239-3

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