Date of Award
Master of Science (MS)
Gastrulation is an early morphogenetic process that is conserved across most metazoans and lays out the future body plan through the formation and shaping of the three germ layers: endoderm, mesoderm, and ectoderm. Despite its importance, not much is known about the events surrounding human gastrulation that occurs in utero due to ethical and technical limitations on studying human embryos. Therefore, many researchers have devised protocols for creating in vitro models of gastrulation using embryonic stem cells. Initially starting with mouse embryonic stem cells, the field of in vitro embryo models has advanced rapidly, with protocols using human embryonic stem cells being only recently published in 2020. However, validation of these models through comparison with other in vitro models or with animal gastrulae has only started. As such, this study investigates one of the first 3D gastruloid protocol using human embryonic stem cells to create an optimized protocol using the H1 embryonic stem cell line, which is then validated with immunofluorescent staining for germ layer markers. And lastly, cells from 3D gastruloids generated from H1 and RUES2-GRL cell lineswere tested for the ability to self-aggregate through a dissociation and reseeding experiment onto 2D micropatterns. Whereas cells from dissociated RUES2-GRL 3D gastruloids showed clear evidence of self-aggregation comparable to what was reported in a similar experiment with 2D gastruloids, cells from H1 3D gastruloids did not. However, further optimization of the protocol is needed. Overall, the results reported in this thesis have identified conditions to generate in vitro H1 3D elongating gastruloids with three germ layers, as well as showing the evolutionarily conserved sorting ability of 3D RUES2-GRL gastruloid cells.