LumenFormationIs anIntrinsicPropertyofIsolatedHumanPluripotentStem Cells

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cystswithasharedapicaldomainwithin20 hrof plating;thesecystscollapseto formmonolayers after5days.Expressionof pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCz, is surrounded by apicallytargetedorganelles(earlyendosomesandGolgi).Molecularly,actinpolymerization,regulatedbyARP2/3andmammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.