MR/V005405/1

The transplantation of adult stem cells offers the potential to regenerate tissue. Haematopoietic stem cell transplantation has been long-established as a radical therapy for the treatment of blood cancers, while for other stem cell types, such as skin, transplantation is becoming increasing trialled in the clinic. In the context of sperm production, stem cell transplantation promises a wide range of potential applications, from the restoration of fertility of cancer patients who are affected by the sterilising effects of their treatments, to the preservation of genetic diversity of farm animals and endangered species. Yet, the low efficiency with which stem cells engraft in tissue following transplantation makes it currently unviable as a practical technology. Here, by resolving the regulatory programmes that control the fate of spermatogonial stem cells, the aim of this proposal is to harness the flexible behaviour of stem cells and to identify chemical compounds that could increase regeneration efficiency, opening new horizons in the treatment of fertility and animal conservation.

Previously, through collaborative studies, we have used genetic cell labelling approaches and in vivo live-imaging to resolve the dynamics and fate behaviour of spermatogonial stem cells in mouse testes both in steady-state and during regeneration following injury. These studies have shown that stem cells are heterogeneous and dynamic both in their gene expression pattern and their renewal potential. Further, functional studies by our team have found evidence that the regulation of stable stem cell density during homeostasis involves a "quorum sensing"-like mechanism, similar to that encountered in bacterial populations or ecological settings, that allows stem cells to switch reversibly between states biased towards renewal or poised for differentiation. Yet the underlying gene regulatory programmes controlling this state choice remain in question.

To address this, we propose a multi-disciplinary strategy that will exploit advances in single-cell technology to profile the heterogeneity of individual spermatogonial stem cell populations and their early differentiating progenies, both in steady-state conditions and during regeneration. By combining these approaches with genetic in vivo cell lineage tracing in the mouse testis, we will identify the key gene regulatory networks that control the balance between stem cell renewal and differentiation. Finally, to advance our understanding towards potential clinical application in humans, we will compare the activity of the key signalling pathways between mouse and human tissues by mining human single-cell datasets. We will then validate these results by performing deep-phenotypical characterisations of human spermatogonial stem cell populations using a multiplex proteomics approach.

By resolving the mechanisms that regulate stem cell renewal and their ability to transition reversibly between primed states, our long-term goal is to identify chemical modulators that can increase transplantation efficiency, opening up the potential of this technology for practical use and clinical applications.

Spermatogonial stem cell (SSC) transplantation promises a wealth of applications, from restoration of fertility in cancer patients, to the preservation of genetic diversity of farm animals and endangered species. Yet, low transplantation/engraftment efficiencies make it currently unviable as a practical technology.

Using a combination of lineage tracing and intravital imaging, our previous studies have resolved the functional identity and fate behaviour of SSCs in mouse testes, both during homeostasis and regeneration following injury. These studies have shown that SSCs are highly heterogeneous, transitioning reversibly between states biased for renewal and poised (licenced) for differentiation. Moreover, states that have entered into a differentiation pathway can reacquire renewal potential in response to injury, greatly increasing the size of the effective stem cell pool. Yet, despite a predictive understanding of cell dynamics, the regulatory programmes remain in question. Here, by delineating the mechanisms that regulate stochastic renewal and cell state flexibility, we aim to identify compounds that can modulate cell fate choices and increase transplantation efficiency, unlocking the potential of this technology.

To develop this programme, we will exploit advances in single-cell technologies (scRNA-seq and scATAC-seq) to resolve the transcriptional heterogeneity of SSCs and their progenies during steady-state and regeneration. By combining these approaches with in vivo tracing, we will use comparative genomics to identify the pathways that control fate asymmetry. As a step towards clinical application, we will mine human data and perform deep-phenotypical characterisations of human SSCs using a multiplex proteomics approach to compare the activity of signalling pathways in mouse and human testes.
These insights will allow us to identify and test chemical modulators, with the aim of increasing SSC regeneration capability and transplantation efficiency.

The transplantation of adult stem cells offers the potential to regenerate tissue. Haematopoietic stem cell transplantation has been in use for over 50 years and provides a radical therapy for the treatment of blood cancers, while for other stem cell types, such as skin, transplantation is becoming increasing trialled in the clinic. In the context of sperm production, stem cell transplantation promises a wide range of potential applications, from the restoration of fertility of cancer patients who are affected by the sterilising effects of their treatments, to the preservation of genetic diversity of farm animals and endangered species. Yet, the low efficiency with which stem cells engraft in tissue following transplantation makes it currently unviable as a practical technology.
This study aims to provide insight into the molecular mechanisms that regulate spermatogonial stem cell fate decisions in the mammalian testis, both in steady-state conditions and during regeneration following damage or transplantation. Importantly, our results will allow us to identify and test the action of chemical activators and inhibitors that can promote stem cell proliferation and increase transplantation efficiency.

With an emphasis on single-cell transcriptional and epigenetic profiling consolidated by deep phenotypical characterisation of mouse and human tissues, this study will also provide a paradigm for how comparative single-cell multi-omics approaches can be used to dissect mechanistic insights into fate decision-making programmes. Alongside its intrinsic interest to academic researchers working in the field of germ cell biology, this study will impact the wider community of stem cell biologists and clinicians interested in the application of stem cell transplantation in regenerative medicine. Moreover, the quantitative, statistical and computational methods developed in this study will be of interest to the wider community of biologists interested in mechanisms of tissue stem and progenitor cell fate.