Studying human germline development presents many challenges. In particular, primordial germ cell (PGC) specification and PGC migration occur shortly after implantation when the developing embryo is largely inaccessible. Profiling of later stage gonadal PGCs has allowed comparison with mouse and other species, revealing not only many similarities but also notable differences. Recently, in vitro systems to generate functional PGCs from pluripotent stem cells have been established in mouse. These protocols are easily adapted to human, suggesting remarkable conservation in PGC specification mechanisms. Such in vitro systems may now provide an important tool in which to study early human germline development and disease. Here, we review the recent advances both in vivo and in vitro, and how these may influence the study of the human germline in the years to come.
Single-cell sequencing is a powerful methodology for revealing traits of individual cells in heterogeneous populations such as embryonic stem cell cultures or whole tissues. As cellular behaviors typically depend on coordinated expression of many genes and translated proteins, unbiased sequencing methods that can extract genome-wide profiles from single cells without prior knowledge about the initial cellular sample are needed. In recent years, single-cell RNA sequencing was used to identify complex cell populations, reconstruct developmental trajectories, and model transcriptional dynamics. As the DNA representation in a cell is much lower than that of RNA, single-cell DNA-based methods such as genomic sequencing, ChIP-seq, ATAC-seq, and methods for studying the 3-D architecture of genomes are limited by the low number of molecules that can be detected making them less sensitive and more challenging for computational analysis. In this chapter, we review current single-cell sequencing techniques, discuss the power and limitations of these methods, and discuss their implementation in the context of pluripotency and differentiation studies.
Here, we discuss how epigenetic processes, including posttranslational modifications of histones and posttranscriptional modifications of RNA, differ in pluripotent embryonic stem cells (ESCs) in comparison with their differentiated counterparts. Additionally, we discuss the specificity of DNA repair epigenetics in ESCs, in which the OCT4 protein play an important role, not only in pluripotency maintenance but also in the DNA damage response. Additionally, in ESC biology, the N6-methyladenosine (m6A) modification of RNA was found to be functionally essential. It is known that m6A RNAs regulate the balance between the self-renewal capability and differentiation properties of ESCs. This posttranscriptional modification, regulating gene expression and the DNA damage response, appears to also be crucial, especially during embryonic development.
Chromosomes must be tightly folded within cell nuclei but in a manner ensuring accessibility to the machinery regulating transcription, replication, and repair. Recent studies have identified the hierarchical organization principles of chromosome folding, which impact on transcriptional regulation of gene programs. Differentiation of pluripotent stem cells is characterized by a transition from generally open and transcriptionally permissive chromatin to a more polarized state, whereby lineage-specific genes are maintained in an "open" configuration and genes in other lineages are shut down within repressive, more compacted chromatin. Despite such an apparent difference between pluripotent and differentiated chromatin states, the underlying architectural organization principles are very similar. Here, we provide an overview on our understanding of how chromosome folding is linked to cell fate control via transcriptional regulation, focusing on the similarities and differences between pluripotent and somatic cells and special cases (e.g., mitosis) where chromatin folding appears very different.
During mammalian development the pluripotency appears in cells within the inner cell mass (ICM) of the blastocyst and disappears during gastrulation, corresponding to a 4- to 5-day period in mice and approximately 2 weeks in humans. During this period the pluripotent tissue, the epiblast, expands from about 10 homogeneous cells to several hundred. This rapid increase in cell number correlates with the transcriptional and epigenetic rewiring of pluripotent escapees that differentiate during gastrulation. The differentiation processes have long been attributed mainly as a coordinated response to extrinsic signaling cues. However, in the last years, the cell cycle has emerged as an intrinsic determinant that contributes to cell fate decision-making. In this review, we discuss the recent discoveries in this area and, in particular, how the cell cycle is regulated and impacts in the biology of the pluripotent cells.
