We investigate the mechanisms that direct ‘cell fate’ in human embryos and stem cells. This means studying the different factors that tell embryonic cells which type of cell to become.
After a human egg is fertilised, the cells multiply as the embryo grows. After five days there are around 100 cells, under 10 of which are embryonic epiblast cells that go on to form the foetus – these are pluripotent cells, as they are capable of becoming any type of cell in the body. The remaining 90 or so cells will go on to form either the placenta or the yolk sac.
We’re trying to understand how these early human pluripotent embryonic cells are established, how they remain pluripotent and how this process if turned off when the cells specialise. We’re trying to map out the complex hierarchy of different genes that control cell activity in early development, determine the influence of factors outside of the cells and understand the similarities and differences between human and mouse development.
The processes that underpin early development and stem cell pluripotency are fundamental to human biology. If we knew how these processes worked, this knowledge could inform the understanding and treatment of infertility and developmental disorders. We could also use this knowledge to improve our use of stem cells in both science and medicine.
The allocation of cells to a specific lineage is regulated by the activities of key signalling pathways and developmentally regulated transcription factors. The focus of our research is to understand the influence of signalling and transcription factors on differentiation during early human development. During preimplantation development, totipotent human zygotes undergo subsequent rounds of mitotic cell divisions leading to the divergence of pluripotent embryonic cells, which form the foetus, and extra-embryonic cells, which contribute to the placenta and yolk sac.
The central question we are addressing is what are the molecular mechanisms that regulate embryonic pluripotency and how is it disengaged during cellular differentiation? We seek to define the genetic hierarchy acting during differentiation, the influence of extracellular signalling and the extent to which these mechanisms are conserved between humans and mice.
The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here we describe GIANI (General Image Analysis of Nuclei-based Images; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.
Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions.
The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
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