How a 300µM Gastruloid Can Shed Light on Human Development
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Gastrulation is an early stage in fetal development, occurring two weeks after conception. During gastrulation, the fetus (or blastula, to be exact), which until then had been a hollow single-layered sphere of cells, develops into a three-layered cell structure known as a gastrula.
Figure 1: Gastrulation. A diagram of the transformation from a fertilized egg (a zygote) to a gastrula.
These three germ layers are the endoderm, mesoderm and ectoderm, from which all body systems, tissues and organs will later develop. (Figure 2)
Since the law prohibits growing embryonic cell cultures ex-vivo, beyond two weeks from the date of fertilization, it is exceedingly difficult to investigate the gastrulation phase in humans.
In order to ameliorate the ethical, legal, and technical limitations, a team of scientists from the University of Cambridge has managed to transform embryonic stem cells (ESCs) and generate gastruloids – three-dimensional multicellular aggregates, that resemble human embryos during the premature stages of development.1
Figure 2: Mesoderm, Ectoderm and Endoderm. The three germ layers and some of the cell types and tissues they give rise to (Shutterstock)
The cells chosen for this project were several human ESC lines, that were cultured routinely in NutriStem® hPSC XF medium.
A critical part of the human gastruloid process is the starting state of the cells, which must be in optimal condition before beginning. When the adherent cultures of human ESCs were at 40%-60% confluence, they were pre-treated in NutriStem supplemented with CHIR99201*. The researchers found that when the cells were cultured in an alternative pluripotency medium, including mTeSR1 or Essential-8, they did not form elongated gastruloids.
After the pre-treatment the cells were dissociated, washed, and reaggregated in basal differentiation medium, forming compact, spherical aggregates within a few hours. The aggregates progressively broke symmetry and formed elongated structures, with maximum elongation at 72-96 hours. (Fig.3 a-b)
Figure 3: Structure and morphology of human gastruloids.
a. Schematic of the human gastruloid protocol. Chi, Chiron (CHIR99201); Nutristem, NutriStem hPSC XF medium; E6, Essential 6 medium; ROCKi, ROCK inhibitor. b. Temporal morphology. Two representative examples from the MasterShef7 (mShef7) cell line are shown (n=3 experiments). Scale bars 200 µm.1
In order to unravel the enigma of whether a human gastruloid can generate derivatives of the three germ layers, the researchers tried to identify cells expressing markers of the mesoderm, endoderm and neuroectoderm. Using time-lapse videos of the aggregation and elongation they managed to follow the process of symmetry breaking and segregation of germ layer progenitors in human gustruloids. The researchers noticed extreme differences in cellular response to different signals, suggesting that the formation of human gastruloids is highly dependent on the signaling exposure of the initial cell population. Following this observation, they examined the effect of retinoic acid on the gastruloid culture. Retinoic acid is known to disrupt axial patterning and cause congenital malformations. The treatment promoted rounded gastruloids with high expression of certain genes and a strong reduction of others. The results supported the notion that the signalling environment has a critical effect on the human gastruloid formation in terms of morphology and patterning.
To examine the transcriptional complexity of human gastruloids the researchers used Tomo-sequencing (Tomo-seq), a method to obtain genome-wide expression data with spatial resolution. Two gastruloids were embedded in cryofreezing medium and sectioned along their anteroposterior (A-P) axis. Each section was processed by RNA sequencing. (Fig. 4 a-b).
Figure 4: Transcriptomics anteroposterior organization of human gastruloids.
a. Wide field imaging of 72-h RUES-GLR human gastruloids that were used for tomo-seq. scale bar, 100 µm. b. mRNA expression of the fluorescent reporter transgenes along the anteroposterior (A-P) axis.1
1,023 genes with reproducible expressions were identified between the two replicates. These genes were organized into 22 main classes of expression pattern, with representatives of all three germ layers.
The main question raised from these global patterns of gene expression was whether this organization reflects elements of a body plan. This possibility was supported by the organization of a posterior domain of gene expression in the human gastruloids (Fig. 5a) in which the researchers observed a node-like transcriptional domain (Fig. 5b). At the anterior end, the researchers observed a polarization of genes that are associated with the development of cardiac mesoderm (Fig. 5c).
Figure 5: Comparative elements of early embryogenesis.
a. Schematic of the tailbud of the mammalian embryo. meso., mesoderm; RA, retinoic acid. Raldh2 is also known as Aldh1a2; Bra is also known as T. b. Schematic of the node region of the mammalian embryo. c. Schematic of the cardiac mesoderm region of the mammalian embryo. d. Illustration of CS8 and CS9 embryos, showing gross anatomical features, including somite boundaries.1
The human gastruloid system in this experiment represents the first step towards in-vitro modelling of the emergence of a three-dimensional human body plan. The observation of an axial somitogenesis organization (expressed by genes) suggests an approximate staging of the embryo formation. A major transition occurs between Carnegie stage** 8 (CS8) and CS9 (corresponding to embryogenesis days 17-19 and 19-21, respectively) that is associated with the onset of somitogenesis. CS9 embryos have 1-3 pairs of somites*** that are absent in CS8 embryos (Fig. 5d).
this pattern of gene expression in the human gastruloid led the researchers to suggest that a 72-h human gastruloid might serve as a model for some of the late features of CS8 or early CS9 of human development.
In conclusion, human gastruloids reveal a novel opportunity for a better understanding of the mechanisms associated with embryogenesis and early human development.
*CHIR99201 is a glycogen synthase kinase 3 (GSK3) inhibitor.
**Carnegie stages are a standardized system of 23 stages used to provide a unified developmental chronology of the vertebrate embryo.
***Somites are bilaterally paired blocks of paraxial mesoderm that form along the anterior-posterior axis of the developing embryo in segmented animals. Invertebrates, somites give rise to skeletal muscle, cartilage, tendons, endothelium, and dermis.
1. N. Moris, et al. An in vitro model of early anteroposterior organization during human development. Nature 582, 410–415 (2020).