Functional sensory circuits built from neurons of two species.

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.

Metformin decelerates aging clock in male monkeys.

In a rigorous 40-month study, we evaluated the geroprotective effects of metformin on adult male cynomolgus monkeys, addressing a gap in primate aging research. The study encompassed a comprehensive suite of physiological, imaging, histological, and molecular evaluations, substantiating metformin's influence on delaying age-related phenotypes at the organismal level. Specifically, we leveraged pan-tissue transcriptomics, DNA methylomics, plasma proteomics, and metabolomics to develop innovative monkey aging clocks and applied these to gauge metformin's effects on aging. The results highlighted a significant slowing of aging indicators, notably a roughly 6-year regression in brain aging. Metformin exerts a substantial neuroprotective effect, preserving brain structure and enhancing cognitive ability. The geroprotective effects on primate neurons were partially mediated by the activation of Nrf2, a transcription factor with anti-oxidative capabilities. Our research pioneers the systemic reduction of multi-dimensional biological age in primates through metformin, paving the way for advancing pharmaceutical strategies against human aging.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Time matters: Human blastoids resemble the sequence of blastocyst development.

In a recent issue of Nature, Kagawa et al. reported a highly efficient and robust protocol for generating human blastoids from naive human pluripotent stem cells. The blastoids resemble human blastocysts, follow the sequential lineage specification of blastocyst development, and can attach to endometrial cells with the polar trophectoderm to model implantation.

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Single-Cell Transcriptomic Atlas of Primate Ovarian Aging.

Molecular mechanisms of ovarian aging and female age-related fertility decline remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from young and aged non-human primates (NHPs) and identified seven ovarian cell types with distinct gene-expression signatures, including oocyte and six types of ovarian somatic cells. In-depth dissection of gene-expression dynamics of oocytes revealed four subtypes at sequential and stepwise developmental stages. Further analysis of cell-type-specific aging-associated transcriptional changes uncovered the disturbance of antioxidant signaling specific to early-stage oocytes and granulosa cells, indicative of oxidative damage as a crucial factor in ovarian functional decline with age. Additionally, inactivated antioxidative pathways, increased reactive oxygen species, and apoptosis were observed in granulosa cells from aged women. This study provides a comprehensive understanding of the cell-type-specific mechanisms underlying primate ovarian aging at single-cell resolution, revealing new diagnostic biomarkers and potential therapeutic targets for age-related human ovarian disorders.

Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures.

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.

Keeping the Rhythm while Changing the Lyrics: Circadian Biology in Aging.

Aging and circadian rhythms have been linked for decades, but their molecular interplay has remained obscure. Sato et al. and Solanas et al. now reveal that, while core clock components remain nearly unaltered, aging is associated with tissue-specific rewiring, which can be prevented by calorie restriction.

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Stem Cells: A Renaissance in Human Biology Research.

The understanding of human biology and how it relates to that of other species represents an ancient quest. Limited access to human material, particularly during early development, has restricted researchers to only scratching the surface of this inherently challenging subject. Recent technological innovations, such as single cell "omics" and human stem cell derivation, have now greatly accelerated our ability to gain insights into uniquely human biology. The opportunities afforded to delve molecularly into scarce material and to model human embryogenesis and pathophysiological processes are leading to new insights of human development and are changing our understanding of disease and choice of therapy options.

In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming.

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells.

Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.

Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing.

The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies.

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

Navigating the epigenetic landscape of pluripotent stem cells.

Pluripotent stem cells, which include embryonic stem cells and induced pluripotent stem cells, use a complex network of genetic and epigenetic pathways to maintain a delicate balance between self-renewal and multilineage differentiation. Recently developed high-throughput genomic tools greatly facilitate the study of epigenetic regulation in pluripotent stem cells. Increasing evidence suggests the existence of extensive crosstalk among epigenetic pathways that modify DNA, histones and nucleosomes. Novel methods of mapping higher-order chromatin structure and chromatin-nuclear matrix interactions also provide the first insight into the three-dimensional organization of the genome and a framework in which existing genomic data of epigenetic regulation can be integrated to discover new rules of gene regulation.