Branching development of early post-implantation human embryonic-like tissues in 3D stem cell culture

Abstract

Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in an efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation, pre-gastrulation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), before their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size of hESCs. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density of hESCs. Conversely, a large initial cell seeding density of hESCs promoted the development of mesodermal-like tissues. Intermediate cell seeding densities of hESCs in the 3D suspension culture promoted the development of asymmetric embryonic-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models.

Keywords: Branching tissue development; Human pluripotent stem cells; Synthetic embryonic model.

Figure 1.. Guided self-organization of human pre-gastrulation embryonic-like tissues.

a, Schematics of generating and long-time culturing three types of tissues from hESCs, including asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues. b, Representative bright-field images of cells in the pyramid well array and tissues in the 3D gel matrix at different time points as indicated. Scale bars, 100 μm. c, Number of cells inside a single pyramidal well as a function of the initial cell seeding density. d, Representative confocal images of the epiblast-like tissues at 20 h stained for DAPI, NANOG, GATA3 and T. Scale bar, 50 μm. e, Representative confocal images of the three types of embryonic-like tissues at 55 h stained for DAPI, NANOG, GATA3 and T as indicated. Scale bars, 50 μm.

Figure 2.. Properties of tissues grown from an intermediate cell seeding density (n = 1166).

a, Schematics of image-based tissue profiling, which includes segmentation and feature extraction, and unsupervised trajectory inference. b, Linear multi-class SVM classifier for automatically distinguishing columnar (col), amniotic-like (amn) and asymmetric (asy) tissues. Dashed lines represent decision boundaries. c, Percentages of three types of tissues over time. d, Tissue and lumen area over culture time. e, Total cell number and GATA3+ cell number over culture time. f, Tissue area plotted against the number of cells contained in each tissue. g, Number of GATA3+ cells plotted against tissue area. h, Tissue area plotted against tissue thickness ratio. Color-coding in f-h denotes culture time of tissues.

Figure 3.. Developmental trajectories of tissues grown from an intermediate initial cell seeding density (n = 1166).

a-c, Diffusion maps showing the trifurcating development of tissues. Color-coding denotes thickness ratio, GATA3+ cell ratio and culture time as indicated. d, Bifurcating trajectory of asymmetric embryonic-like tissues and mesodermal-like tissues. Color-coding denotes trunk and branch points. e-g, NANOG+ cell ratio, GATA3+ cell intensity and tissue thickness ratio as a function of pseudotime of tissue development. h, Bifurcating trajectory of amniotic-like tissues and mesodermal-like tissues. i-k, T+ cell intensity, GATA3+ cell ratio and tissue area as a function of pseudotime of tissue development. l, Intermediate states between asymmetric and amniotic branches. m-o, GATA3+ cell number, NANOG+ cell ratio and T+ cell intensity as a function of pseudotime of tissue development.

Figure 4.. Progression of tissue development.

a, Heterogeneous tissue morphology and cell lineage composition ordered by real time. b, Pseudotime ordering and trifurcating development. c, Representative images of tissues growing along the asymmetric branch. d, Representative images of tissues growing along the amniotic branch. e, Representative images of tissues growing along the mesodermal branch. Scale bars, 50 μm.

Figure 5.. Properties and developmental trajectories of tissues grown from a small initial cell seeding density (n = 555).

a, Schematics of bifurcating tissue development. b, Tissue area and lumen area as a function of culture time. c, Total cell number and T+ cell number as a function of culture time. d, Percentages of three types of tissues over time. e, Tissue thickness ratio plotted against tissue equivalent diameter. f, T+ cell intensity plotted against tissue thickness ratio. Color-coding in e and f denotes culture time. g, Diffusion maps of tissues. Color-coding denotes tissue thickness ratio, GATA3+ cell ratio and culture time as indicated. h, Bifurcating trajectory of amniotic-like and asymmetric tissues. i, GATA3+ cell ratio, NANOG+ cell ratio, T+ cell ratio and thickness ratio as a function of psuedotime of tissue development.

Figure 6.. Properties and developmental trajectories of tissues grown from a large initial cell seeding density (n = 965).

a, Schematics of bifurcating tissue development. b, Tissue area and lumen area over culture time. c, Total cell number and T+ cell number over culture time. d, Percentages of three types of tissues over time. e, GATA3+ cell number plotted against tissue equivalent diameter. f, NANOG+ cell ratio plotted against tissue area. Color-coding in e and f denotes culture time. g, Diffusion maps of tissues. Color-coding denotes tissue thickness ratio, area and culture time. h, Bifurcating trajectory of asymmetric and mesodermal-like tissues. i, T+ cell ratio, tissue area, and tissue thickness ratio as a function of psuedotime of tissue development.