David Ryan

Establishment of mouse expanded potential stem cells

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

Targeted gene correction of FKRP by CRISPR/Cas9 restores functional glycosylation of α-dystroglycan in cortical neurons derived from human induced pluripotent stem cells

Mutations in genes required for functional glycosylation of α-dystroglycan cause a group of congenital muscular dystrophies associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic, physiology-relevant human cellular models has limited our understanding of the cortical abnormalities in dystroglycanopathies. Here we generate induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous mutations in the ribitol-5-phosphate transferase gene, FKRP. We carry out targeted gene correction in FKRP-iPSCs using CRISPR/Cas9-mediated genome editing. We characterise the directed differentiation of FKRP- and corrected-iPSCs to neural stem cells, cortical progenitors and cortical neurons. Importantly, we show that targeted gene correction of FKRP restores functional glycosylation of α-dystroglycan in iPSC-derived cortical neurons. We independently validate this result by showing targeted gene mutation of FKRP disrupts functional glycosylation of α-dystroglycan. This work demonstrates the feasibility of using CRISPR/Cas9-engineered human iPSCs for modelling dystroglycanopathies and provides a foundation for therapeutic development. Highlights Generation of FKRP-iPSCs for modelling cortical abnormalities in dystroglycanopathies Precise gene correction by CRISPR/Cas9-mediated genome editing Directed differentiation of isogenic control and FKRP-iPSC to cortical neurons Functional glycosylation of α-dystroglycan is restored in cortical neurons derived from CRISPR/Cas9-corrected iPSCs Targeted gene mutation of FKRP disrupts functional glycosylation of α-dystroglycan in cortical neurons

Establishment In Culture Of Expanded Potential Stem Cells

Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts1,2 and contribute to both the somatic lineage and germline when returned to the blastocyst3 but are normally excluded from the trophoblast lineage and primitive endoderm4–6. Here, we report that cultures of expanded potential stem cells (EPSCs) can be established from individual blastomeres, by direct conversion of mouse embryonic stem cells (ESCs) and by genetically reprogramming somatic cells. Remarkably, a single EPSC contributes to the embryo proper and placenta trophoblasts in chimeras. Critically, culturing EPSCs in a trophoblast stem cell (TSC) culture condition permits direct establishment of TSC lines without genetic modification. Molecular analyses including single cell RNA-seq reveal that EPSCs share cardinal pluripotency features with ESCs but have an enriched blastomere transcriptomic signature and a dynamic DNA methylome. These proof-of-concept results open up the possibility of establishing cultures of similar stem cells in other mammalian species.

In Vitro Models of Brain Cancer

Glioblastoma multiforme (GBM) is the most common primary brain malignancy in adults. Despite continuing advances in surgical treatment and combined chemoradiotherapy, little improvement in overall median survival has been seen. Therapeutic advances in neuro-oncology are likely to arise through the systematic dissection of the fascinating tumor biology that exists in GBM. If we are to tackle questions such as “What is the cell of origin in brain cancer?” and “How do these cells evade standard treatment methods and ultimately identify the Achilles’ heel of this aggressive disease?” a scientific prerequisite is the availability of a robust and reliable in vitro model of glioma. What follows in this chapter is a discussion of the current state of knowledge in the generation of in vitro models of glioblastoma. Past and current models will be considered with their advantages and shortcomings highlighted. We will discuss the principles of in vitro cytotoxic assays and how translatable therapies emerge from this approach. We discuss the cell of mutation and cell of origin in GBM and how modeling oncogenic transformation can shed new light on this controversial topic. This chapter, we hope, will function as a timeline in the evolution of in vitro models of brain cancer. It illustrates how far we have come in our understanding of brain cancer but additionally highlights the barriers we face and must overcome to ensure that a cure remains within sight.

Laboratory Investigations in Support of Carbon Dioxide-in-Water Emulsions Stabilized by Fine Particles for Ocean and Geologic Sequestration of Carbon Dioxide

This semi-annual progress report includes our latest research on deep ocean sequestration of CO{sub 2}-in-Water (C/W) emulsions stabilized by pulverized limestone (CaCO{sub 3}). We describe a practical system that could be employed for the release of a dense C/W emulsion. The heart of the system is a Kenics-type static mixer. The testing and evaluation of a static mixer in the NETL High-Pressure Water Tunnel Facility was described in the previous semi-annual report. The release system could be deployed from a floating platform over the open ocean, or at the end of an off-shore pipe laying on the continental slope. Because the emulsion is much denser than ambient seawater, modeling shows that upon release the plume will sink much deeper from the injection point, increasing the sequestration time for CO{sub 2}. When released in the open ocean, a plume containing the output of a 500 MW{sub el} coal-fired power plant will typically sink hundreds of meters below the injection point. When released from a pipe on the continental shelf, the plume will sink about twice as much because of the limited entrainment of ambient seawater when the plume flows along the sloping seabed. Furthermore, the plume is slightly alkaline, not acidic. The disadvantage is that the creation of the emulsion requires significant amounts of pulverized limestone, on the order of 0.5-0.75 weight ratio of limestone to CO{sub 2}. While pulverized limestone with particle size appropriate for creating C/W emulsions can be purchased for

Derivation and Maintenance of Mouse Expanded Potential Stem Cells

38 per ton, it is shown in this report that it may be more economic to purchase raw limestone from quarries and pulverize it in situ using grinding mills. In this case the major cost elements are the capital and operating costs of the grinding mills, resulting in a total cost of about

Conserved transcriptional programmes regulating blood cell development

11 per ton of pulverized limestone, including the cost of raw material and shipping. Because we need approximately 0.75 ton of pulverized limestone per ton of liquid CO2 to create a stable C/W emulsion, the total cost of preparing the emulsion on site is about

LABORATORY INVESTIGATIONS IN SUPPORT OF CARBON DIOXIDE-LIMESTONE SEQUESTRATION IN THE OCEAN

8.5 per ton of liquid CO{sub 2}, not including the cost of the emulsion mixer. Currently, the cost estimates of capturing and liquefying CO{sub 2} at a coal-fired power plant range from