Human germline in vitro models for development and the epigenetic program

Germ cells, the precursors of sperm and egg, are unique and considered to be immortal in the sense that they generate a new organism, and theoretically at least, give rise to endless generations. By contrast, somatic (body) cells perish with each generation. Germ cells transmit genetic instructions that are passed on from one generation to the next. The germline also transmits non-genetic (epigenetic) information, which can regulate gene expression in developing embryos and adults, but without alterations to the genetic information. Epigenetic modifications can be induced and erased but some can persist intergenerationally, and potentially, transgenerationally. The parent-of-origin specific genomic imprints that have an essential role in human development persist intergenerationally. Faulty imprints result in a number of diseases that affect behavior, growth and obesity.
Environmental factors, including diet can also apparently induce epigenetic modifications in germ cells according to some studies, which could be transmitted transgenerationally with potential consequences for human health. To gain insight into mechanisms of induction and transmission of epigenetic information, it is essential to understand the underlying mechanisms of how the information might accrue, and evade the robust germline reprogramming to be transmitted through the germline. Studies on human germ cells are challenging. Since many dynamic events for germ cell development occur in early developmental stages in the uterus, where human germ cells are not readily accessible or testable experimentally, except for the rare germ cells that can be obtained from aborted fetuses. Moreover, the extensively used mouse model for germ cell development does not accurately reflect mechanisms of human germline development, since critical differences have become evident recently. For this reason, it is essential to develop tractable experimental models in culture that can be manipulated and tested in culture.
Aberrant germ cells and mutations can also be the cause of infertility and germ cell tumors. Purification of mitochondria also occurs in early germ cells before their transmission through eggs. An increase in the proportion of defective mitochondria causes many human diseases. Research on germ cells is required to understand the underlying mechanisms, for the development of diagnostic methods, and potential treatment of germline associated diseases.
A robust and quantitative culture model for human germline development using induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), including some derived from affected patients would represent a major advance. Patient derived iPSCs provide a powerful tool if they carry mutations affecting genomic and epigenomic information that might be the cause of specific germline disorders. Recently, we established a robust culture system for early PGCs differentiation using human iPSCs and ESCs (Irie et al., 2015); this provided information on the key regulators of the human germline for the first time. This also showed that extrapolation from studies in mice to humans may not provide valid information for the latter. However, under the current culture system, PGCs develop up to the early stages of initiation of the epigenetic program but not its completion. With the additional information we now have on later stages of germ cells isolated from aborted fetuses, we can exploit this knowledge to develop efficient culture model for more advanced PGC development.
The latest efficient genome editing (CRISPR/Cas9) technique combined with the patient specific iPSCs will allow us to confirm the precise genomic evidence for the diseases. These studies may also be informative for the causes of germ cell tumors such as seminomas, embryonal carcinomas, and the pediatric brain tumors called germinomas that originate from early germ cells.

We have carried out detailed computational analysis of the transcriptome and epigenome of in vitro (~Wk2.5), and in vivo Wk5. 5-Wk9 PGCs. This has provided an initial experimental framework, and testable predictions for extrinsic and intrinsic factors needed for further development of PGCs. PGC specific reporter cell lines will provide development-monitoring assays for advanced PGCs by high-throughput live cell imaging. Long-term culture with hourly quantitative information of the fluorescence activation allows for simultaneous testing of 576 conditions.
We will screen for cytokines and small compounds to stimulate development of PGCs, using the candidates identified by bioinformatic modeling, complemented by unbiased screening with commercial compound libraries, including small molecules to lower the epigenetic barriers for development. For intrinsic factors, we will focus on transcriptional factors that are specifically activated sequentially in vivo PGCs. We will conduct co-culture of nascent PGCs with human/porcine gonadal or Sertoli cells to provide a favorable niche, combined with extrinsic and/or intrinsic approaches to increase the probability of finding the best conditions for PGC development.
Our immediate aim is combine all the information to generate equivalent of ~Wk10 PGCs in culture, which covers a period of significant epigenetic program leading to comprehensive erasure of DNA methylation. These PGCs will be compared bioinformatically with in vivo gonadal counterparts, gametes and seminoma. We aim to understand the overall mechanisms that regulate PGC development, together with the mechanisms of germ cell programming, as well as causes of disorders, by using patient specific iPSCs and gene modifications. We will also perform global gene inhibition and activation screening with CRISPR/Cas9. When appropriate, we will collaborate on some aspects, for example on the mitochondrial bottleneck and purification in early PGCs.

Advances in germ cells, development and stem cell biology will benefit the areas of health science, regenerative medicine, assisted reproduction, pharmaceutical industry and biotechnology. These areas are within the strategic priorities of the MRC in improving health. Research on germ cells and underlying mechanisms, including epigenetic programming, could provide knowledge for advances in human health.

The in vitro production of germ cells could provide assays for pharmaceutical companies, for drug screening and toxicological studies as follows: 1- screening for the effects of environmental pollutants, 2- screening for compounds that can modulate germ cell development and for the treatment of infertility, 3- as a potential source of synthetic oocytes with the capacity to reprogram somatic cells to pluripotency, with applications in regenerative medicine.

Second, our studies could lead to advances in the field of epigenetic mechanisms that can induce, propagate or erase these modifications. They have a role in cell fate decisions and in human diseases. Epigenetic modifiers have emerged as significant targets for therapeutic agents for treatment of human diseases, including cancers. Pharmaceutical companies have established research in this field, but they seek collaborations with academics for cell-based assays, for screening chemical libraries. Our study has the potential to generate a wealth of information on epigenetic modifiers that could be exploited for drug discoveries. This includes treatment of cancers of germ cell origin that could contribute to advancing the overall understanding of tumourigenesis, leading to better diagnosis and treatment.

Advances in in vitro derived germ cells has attracted considerable public attention, as this can potentially lead to development of gametes from somatic cells via iPSCs, for the treatment of germ cell diseases and infertility. However, this area of research raises many ethical issues. Engagement with the public is therefore vital and in which we participate actively. Studies on germ cells have also come to the fore in the context of advances in gene editing tools, particularly CRISPR/Cas9. Potential applications can include correction of mutations in spermatogonial stem cells and the generation of viable and functional sperm thereafter. While these applications are not imminent and would require legislation, there is public awareness of these possibilities. We consider it important to provide information on the cutting edge science to the general public.

Recent reports have also suggested that environmental factors have the potential to induce heritable epigenetic modifications in germ cells, with consequences for human health that could impact transgenerationally. This area has also attracted considerable public interest, sometimes through false or exaggerates media reports while the underlying mechanisms remain unclear. It is important to convey accurate information on the current state of knowledge to the general public.

For the applications of our research, we have many contacts with the biotechnology and pharmaceutical sectors, particularly in the Cambridge area. We are, for example, examining the role of i-BET that regulates expression of BRD4, a reader of epigenetic modifications. This compound was discovered by GSK that is in clinical trial for the treatment of blood cancers; we are currently examining its role in the context of mouse and human PGC specification, and seminomas, and have made some interesting preliminary observations. At the same time, we publish all our findings in peer reviewed open access journals that can be widely accessed by academics and commercial sectors.

Since our studies on 'synthetic' gametes and gene editing techniques are of great public interest, and to the regulators and government agencies for the use of human embryos and gametes, the PI participates actively in public debates.