Kenichiro Taniguchi

Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells

Summary We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian 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.

A pluripotent stem cell-based model for post-implantation human amniotic sac development

Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.Early in human embryonic development, it is unclear how amniotic sac formation is regulated. Here, the authors use a human pluripotent stem cell-based model, termed the post-implantation amniotic sac embryoid, to recapitulate early embryogenic events of human amniotic sac development.

An apicosome initiates self-organizing morphogenesis of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+. Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.

250 Ezrin, an Apical Surface Protein, Functions in a Distinct Type of Cell Division in the Early Intestinal Epithelium to Aid Expansion of Luminal Surface

Intestinal villi provide an enormous surface area for nutrient absorption. Significant loss of intestinal surface area can compromise intestinal function, causing intestinal failure. Though short-term treatments for this life-threatening condition are available, all patients need lifelong monitoring for growth and nutritional status, and would benefit from treatments that can directly increase intestinal surface area. In mice, a large increase in surface area occurs with villus development, which begins at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found a unique type of cell division that is triggered specifically at E14.5, which we have named an e-division (lumen extending division). We propose that in an e-division, new apical surface is deposited at the cytokinetic plane such that the two daughter cells segregate onto adjacent villi. The e-division is distinct from cell divisions occurring before E14.5, which we have named g-divisions (girth building divisions); g-divisions do not involve deposition of new apical surface between daughter cells. Our data (lineage tracing, 3D reconstruction, and SEM) suggest that in mice deficient in the apical surface protein Ezrin, e-divisions fail stochastically, resulting in fused villi. We are modeling e-divisions in vitro using MDCK and Caco2 cell lines, which form luminal surfaces during the first cell division when plated in a 3D matrix. Using RNA interference, we have found that reducing Ezrin expression compromises lumen formation in our 3D cyst assay, providing a mechanistic explanation for the presence of fused villi in vivo. Further understanding of the process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding novel therapies for those with intestinal failure.