Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

Abstract

Summary Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation.

Origin and segregation of the human germline

“…Specification of PGCLCs in vitro occurs within a 3D aggregate that consists of a hitherto poorly characterized fraction of somatic components. Currently, PGCLCs can be induced in various 2D aggregates but more efficiently in 3D embryoids, highlighting the importance of the structure, cell-cell interactions or signalling from adjacent tissues (Minn et al, 2020(Minn et al, , 2021). Here we have shown that these somatic cells collectively represent those in the posterior region of the embryo during gastrulation.…”

New developments in the biology of fibroblast growth factors

“…At the early time points of gastruloid development (0–12 h), FGF2, FGFR1, and FGFR signaling antagonists, SPRY1, DUSP6, and CBL were expressed at high levels. During differentiation (44 h) these FGF signaling components decreased, and FGF17 increased in mesoderm and endoderm along with expression of BMP, WNT, NODAL, and HIPPO signaling components (Minn et al, 2021). Comparison of mouse gastrulation and human gastruloids showed that mouse expresses high levels of Fgf8 , which is required for cell migration, while human gastruloid mesenchyme expressed high FGF17 and very low FGF8 (Minn et al, 2020).…”

Cardiac Organoids and Gastruloids to Study Physio-Pathological Heart Development

“…A human gastruloid protocol was then improved by Minn et al [14] using hES cells cultured on micropatterned surfaces of 500 µm diameter. When challenged by BMP4, cells differentiated into the three germ layers, as well as extraembryonic-like and primordial germ cells.…”

“…They reported that these gastruloids follow the developmental kinetic of in vivo gastrulation. Epiblast and ectoderm precursors emerged after 12 h, and mesendoderm progenitors were detected by 24 h. These cells give rise to the mesoderm and endoderm by 44 h, The CS7 (Carnegie Stage 7) human gastrula [55] (featuring the same kinetic of gene expression as their in vitro counterparts [14]).…”

“…A few trajectory inference analyses could be applied to the human single cell datasets available so far, as only one study reported such an analysis [14]; more data are thus required. Such further analyses should be very informative, specifically as to the possibility of pushing forward gastruloids in more advanced stages of heart formation.…”