Repli-seq Sample Preparation using Cell Sorting with Cell-Permeant Dyes.

Replication timing is significantly correlated with gene expression and chromatin organization, changes dynamically during cell differentiation, and is altered in diseased states. Genome-wide analysis of replication timing is performed in actively replicating cells by Repli-seq. Current methods for Repli-seq require cells to be fixed in large numbers. This is a barrier for sample types that are sensitive to fixation or are in very limited numbers. In this article, we outline optimized methods to process live cells and intact nuclei for Repli-seq. Our protocol enables the processing of a smaller number of cells per sample and reduces processing time and sample loss while obtaining high-quality data. Further, for samples that tend to form clumps and are difficult to dissociate into a single-cell suspension, we also outline methods for isolation, staining, and processing of nuclei for Repli-seq. The Repli-seq data obtained from live cells and intact nuclei are comparable to those obtained from the standard protocols. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Live cell isolation and staining Alternate Protocol: Nuclei isolation and staining.

In Silico Stage-Matching of Human, Marmoset, Mouse, and Pig Embryos to Enhance Organ Development Through Interspecies Chimerism.

Currently, there is a significant shortage of transplantable organs for patients in need. Interspecies chimerism and blastocyst complementation are alternatives for generating transplantable human organs in host animals such as pigs to meet this shortage. While successful interspecies chimerism and organ generation have been observed between evolutionarily close species such as rat and mouse, barriers still exist for more distant species pairs such as human-mouse, marmoset-mouse, human-pig, and others. One of the proposed barriers to chimerism is the difference in developmental stages between the donor cells and the host embryo at the time the cells are introduced into the host embryo. Hence, there is a logical effort to stage-match the donor cells with the host embryos for enhancing interspecies chimerism. In this study, we used an in silico approach to simultaneously stage-match the early developing embryos of four species, including human, marmoset, mouse, and pig based on transcriptome similarities. We used an unsupervised clustering algorithm to simultaneously stage-match all four species as well as Spearman's correlation analyses to stage-match pairs of donor-host species. From our stage-matching analyses, we found that the four stages that best matched with each other are the human blastocyst (E6/E7), the gastrulating mouse embryo (E6-E6.75), the marmoset late inner cell mass, and the pig late blastocyst. We further demonstrated that human pluripotent stem cells best matched with the mouse post-implantation stages. We also performed ontology analysis of the genes upregulated and commonly expressed between donor-host species pairs at their best matched stages. The stage-matching results predicted by this study will inform in vivo and in vitro interspecies chimerism and blastocyst complementation studies and can be used to match donor cells with host embryos between multiple species pairs to enhance chimerism for organogenesis.

Interspecies Chimeric Barriers for Generating Exogenic Organs and Cells for Transplantation.

A growing need for organs and novel cell-based therapies has provided a niche for approaches like interspecies chimeras. To generate organs from one donor species in another host species requires techniques such as blastocyst complementation and gene editing to successfully create an embryo that has cells from both the donor and the host. However, the task of developing highly efficacious and competent interspecies chimeras is met by many challenges. These interspecies chimeric barriers impede the formation of chimeras, often leading to lower levels of chimeric competency. The barriers that need to be addressed include the evolutionary distance between species, stage-matching, temporal and spatial synchronization of developmental timing, interspecies cell competition and the survival of pluripotent stem cells and embryos, compatibility of ligand-receptor signaling between species, and the ethical concerns of forming such models. By overcoming the interspecies chimera barriers and creating highly competent chimeras, the technology of organ and cellular generation can be honed and refined to develop fully functioning exogenic organs, tissues, and cells for transplantation.

Cell Reprogramming for Regeneration and Repair of the Nervous System.

A persistent barrier to the cure and treatment of neurological diseases is the limited ability of the central and peripheral nervous systems to undergo neuroregeneration and repair. Recent efforts have turned to regeneration of various cell types through cellular reprogramming of native cells as a promising therapy to replenish lost or diminished cell populations in various neurological diseases. This review provides an in-depth analysis of the current viral vectors, genes of interest, and target cellular populations that have been studied, as well as the challenges and future directions of these novel therapies. Furthermore, the mechanisms by which cellular reprogramming could be optimized as treatment in neurological diseases and a review of the most recent cellular reprogramming in vitro and in vivo studies will also be discussed.

Microglia and Macrophages in Neuroprotection, Neurogenesis, and Emerging Therapies for Stroke.

Stroke remains the number one cause of morbidity in the United States. Within weeks to months after an ischemic event, there is a resolution of inflammation and evidence of neurogenesis; however, years following a stroke, there is evidence of chronic inflammation in the central nervous system, possibly by the persistence of an autoimmune response to brain antigens as a result of ischemia. The mechanisms underlying the involvement of macrophage and microglial activation after stroke are widely acknowledged as having a role in ischemic stroke pathology; thus, modulating inflammation and neurological recovery is a hopeful strategy for treating the long-term outcomes after ischemic injury. Current treatments fail to provide neuroprotective or neurorestorative benefits after stroke; therefore, to ameliorate brain injury-induced deficits, therapies must alter both the initial response to injury and the subsequent inflammatory process. This review will address differences in macrophage and microglia nomenclature and summarize recent work in elucidating the mechanisms of macrophage and microglial participation in antigen presentation, neuroprotection, angiogenesis, neurogenesis, synaptic remodeling, and immune modulating strategies for treating the long-term outcomes after ischemic injury.