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

Abstract

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.

Figure 1.

The apicosome is a unique cellular structure. (A–D) Fluorescent confocal images of single H9 cells stained with indicated markers. (E) Fluorescent confocal images of single H9 cells stained for phalloidin (green, F-ACTIN). Image in E′ is the optical y-z section (indicated by white lines) of the 3D rendered image of the cell in E. (F and G) Singly isolated H9 cells are stained for membrane (green, WGA) as well as antibodies specific to distinct organelles (red) as indicated. Bar, 10 µm. For all images, blue indicates DNA staining (HOECHST).

Figure 2.

The apicosome has features of the cellular exterior. (A and B) TEM images of single H9 cells grown under feeder-free conditions on a Geltrex-coated Thermanox substrate (A) or on a tissue culture plate (B) for 20 h. (B-i) Magnified image of the boxed region in B, showing a primary cilium within the apicosome. (C) Fluorescent confocal image of a single H9 cell stained with ciliary (ARL13B) and centrosomal (γ-TUBULIN) markers as well as HOECHST (blue). (D) H9 cell expressing EZRIN–EGFP (top), stained with ER-Tracker (red); isolated H9 cell stained with Fluo-3-AM (bottom, green) and ER-Tracker (red). (E) H9 cells expressing m-TdTomato (top) or Lifeact–mCherry (bottom), stained with Fluo-3-AM. (F) Schematic of an isolated hPSC with an internal apicosome, containing microvilli (green protrusions), a primary cilium (red), and accumulated Ca²⁺. Bars: (A and B) 5 µm; (B-i) 500 nm; (C and E) 10 µm.

Figure 3.

Formation of the apicosome in singly isolated cells. (A–C) Live imaging of H9 cells expressing EZRIN–GFP (A) or PODXL–GFP (B and C) during apicosome formation (see the detailed legend of Video 4 for C). (D) Quantitation of the total volume of the central perinuclear complex in C during imaging, revealing increase in volume over time (a representative of six independent live-imaged samples). Values were obtained from the high-threshold VR image (C, bottom left) acquired every 15 s. (E) H9 cells dissociated from monolayers incubated for 30 min with mCLING–ATTO647N to label apical membrane. Singly plated cells were examined at several time points (30 min to 20 h after plating), after immunostaining for PODXL and p-ERM. (F) Schematic of an isolated hPSC undergoing apicosome formation. Blue indicates DNA staining (HOECHST). Bars: (A and B) 25 µm; (C and E) 10 µm; (E-i) 3 µm. Time stamp: (A and B) h:min; (C) min:s.

Figure 4.

Dynamics of the apicosome in mitotic and postmitotic isolated cells. (A and B) Live imaging of mitotic and postmitotic H9 cells expressing EZRIN–GFP (A) or PODXL–GFP (B). Time stamp, h:min. Although most PODXL–GFP is segregated to one daughter cell (top), a fraction of the PODXL–GFP material is occasionally seen in the other daughter cell (bottom), revealing an unequal segregation of PODXL. (C–G) Confocal images of mitotic H9 cells (C and D) and two-cell H9 clones (E–G) stained with indicated markers. (G-i) Magnified image of the boxed region in G, revealing the presence of two primary cilia within a mature lumen shared by two cells. Bars: (A and B) 25 µm; (C–G) 10 µm; (G-i) 3 µm. For all images, blue indicates DNA staining (HOECHST).

Figure 5.

Dynamics of the apicosome in cell aggregates. (A) Schematic of the hPSC-aggregate lumen formation assay (see the legend to Fig. S3 K for a detailed description). (B–E) hESC aggregates at 0 h, stained with apical markers (PODXL [B], aPKCζ [B], p-ERM [C and D], and EZRIN [E]) in addition to markers of adherence junction (E-CADHERIN [C]), recycling endosome (RAB11 [D]), and primary cilia (ARL13B [E]). Dotted circles in green channel outline radially patterned cells. In E, the boxed regions indicate clustered EZRIN⁺ apicosomes in the center of radially patterned cells (i) and an EZRIN⁺ lumen studded with two primary cilia (ii). (F) Time-lapse imaging of H9 PODXL–GFP aggregates. Imaging began immediately after ROCK-i withdrawal (0 h), and the first image is time stamped (00:00). Time stamp, h:min. Bars: (B–F) 50 µm; (E-i) 5 µm; (E-ii) 2 µm. For all images, blue indicates DNA staining (HOECHST).