Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency.

ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αß and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Prolonged delays in human microbiota transmission after a controlled antibiotic perturbation.

Humans constantly encounter new microbes, but few become long-term residents of the adult gut microbiome. Classical theories predict that colonization is determined by the availability of open niches, but it remains unclear whether other ecological barriers limit commensal colonization in natural settings. To disentangle these effects, we used a controlled perturbation with the antibiotic ciprofloxacin to investigate the dynamics of gut microbiome transmission in 22 households of healthy, cohabiting adults. Colonization was rare in three-quarters of antibiotic-taking subjects, whose resident strains rapidly recovered in the week after antibiotics ended. In contrast, the remaining antibiotic-taking subjects exhibited lasting responses, with extensive species losses and transient expansions of potential opportunistic pathogens. These subjects experienced elevated rates of commensal colonization, but only after long delays: many new colonizers underwent sudden, correlated expansions months after the antibiotic perturbation. Furthermore, strains that had previously transmitted between cohabiting partners rarely recolonized after antibiotic disruptions, showing that colonization displays substantial historical contingency. This work demonstrates that there remain substantial ecological barriers to colonization even after major microbiome disruptions, suggesting that dispersal interactions and priority effects limit the pace of community change.

KRAS(G12D) drives lepidic adenocarcinoma through stem-cell reprogramming.

Many cancers originate from stem or progenitor cells hijacked by somatic mutations that drive replication, exemplified by adenomatous transformation of pulmonary alveolar epithelial type II (AT2) cells1. Here we demonstrate a different scenario: expression of KRAS(G12D) in differentiated AT1 cells reprograms them slowly and asynchronously back into AT2 stem cells that go on to generate indolent tumours. Like human lepidic adenocarcinoma, the tumour cells slowly spread along alveolar walls in a non-destructive manner and have low ERK activity. We find that AT1 and AT2 cells act as distinct cells of origin and manifest divergent responses to concomitant WNT activation and KRAS(G12D) induction, which accelerates AT2-derived but inhibits AT1-derived adenoma proliferation. Augmentation of ERK activity in KRAS(G12D)-induced AT1 cells increases transformation efficiency, proliferation and progression from lepidic to mixed tumour histology. Overall, we have identified a new cell of origin for lung adenocarcinoma, the AT1 cell, which recapitulates features of human lepidic cancer. In so doing, we also uncover a capacity for oncogenic KRAS to reprogram a differentiated and quiescent cell back into its parent stem cell en route to adenomatous transformation. Our work further reveals that irrespective of a given cancer's current molecular profile and driver oncogene, the cell of origin exerts a pervasive and perduring influence on its subsequent behaviour.

Purification and characterization of human neural stem and progenitor cells.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

Intermittent fasting induces rapid hepatocyte proliferation to restore the hepatostat in the mouse liver.

Nutrient availability fluctuates in most natural populations, forcing organisms to undergo periods of fasting and re-feeding. It is unknown how dietary changes influence liver homeostasis. Here, we show that a switch from ad libitum feeding to intermittent fasting (IF) promotes rapid hepatocyte proliferation. Mechanistically, IF-induced hepatocyte proliferation is driven by the combined action of systemic FGF15 and localized WNT signaling. Hepatocyte proliferation during periods of fasting and re-feeding re-establishes a constant liver-to-body mass ratio, thus maintaining the hepatostat. This study provides the first example of dietary influence on adult hepatocyte proliferation and challenges the widely held view that liver tissue is mostly quiescent unless chemically or mechanically injured.

Concordance of MERFISH spatial transcriptomics with bulk and single-cell RNA sequencing.

Spatial transcriptomics extends single-cell RNA sequencing (scRNA-seq) by providing spatial context for cell type identification and analysis. Imaging-based spatial technologies such as multiplexed error-robust fluorescence in situ hybridization (MERFISH) can achieve single-cell resolution, directly mapping single-cell identities to spatial positions. MERFISH produces a different data type than scRNA-seq, and a technical comparison between the two modalities is necessary to ascertain how to best integrate them. We performed MERFISH on the mouse liver and kidney and compared the resulting bulk and single-cell RNA statistics with those from the Tabula Muris Senis cell atlas and from two Visium datasets. MERFISH quantitatively reproduced the bulk RNA-seq and scRNA-seq results with improvements in overall dropout rates and sensitivity. Finally, we found that MERFISH independently resolved distinct cell types and spatial structure in both the liver and kidney. Computational integration with the Tabula Muris Senis atlas did not enhance these results. We conclude that MERFISH provides a quantitatively comparable method for single-cell gene expression and can identify cell types without the need for computational integration with scRNA-seq atlases.

Molecular hallmarks of heterochronic parabiosis at single-cell resolution.

The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

Early prediction of preeclampsia in pregnancy with cell-free RNA.

Liquid biopsies that measure circulating cell-free RNA (cfRNA) offer an opportunity to study the development of pregnancy-related complications in a non-invasive manner and to bridge gaps in clinical care1-4. Here we used 404 blood samples from 199 pregnant mothers to identify and validate cfRNA transcriptomic changes that are associated with preeclampsia, a multi-organ syndrome that is the second largest cause of maternal death globally5. We find that changes in cfRNA gene expression between normotensive and preeclamptic mothers are marked and stable early in gestation, well before the onset of symptoms. These changes are enriched for genes specific to neuromuscular, endothelial and immune cell types and tissues that reflect key aspects of preeclampsia physiology6-9, suggest new hypotheses for disease progression and correlate with maternal organ health. This enabled the identification and independent validation of a panel of 18 genes that when measured between 5 and 16 weeks of gestation can form the basis of a liquid biopsy test that would identify mothers at risk of preeclampsia long before clinical symptoms manifest themselves. Tests based on these observations could help predict and manage who is at risk for preeclampsia-an important objective for obstetric care10,11.

Establishment and characterization of stable, diverse, fecal-derived in vitro microbial communities that model the intestinal microbiota.

Efforts to probe the role of the gut microbiota in disease would benefit from a system in which patient-derived bacterial communities can be studied at scale. We addressed this by validating a strategy to propagate phylogenetically complex, diverse, stable, and highly reproducible stool-derived communities in vitro. We generated hundreds of in vitro communities cultured from diverse stool samples in various media; certain media generally preserved inoculum composition, and inocula from different subjects yielded source-specific community compositions. Upon colonization of germ-free mice, community composition was maintained, and the host proteome resembled the host from which the community was derived. Treatment with ciprofloxacin in vivo increased susceptibility to Salmonella invasion in vitro, and the in vitro response to ciprofloxacin was predictive of compositional changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. These findings demonstrate that stool-derived in vitro communities can serve as a powerful system for microbiota research.

Aged skeletal stem cells generate an inflammatory degenerative niche.

Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.

Distinct skeletal stem cell types orchestrate long bone skeletogenesis.

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

Sexual and asexual development: two distinct programs producing the same tunicate.

Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs.

During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.

Ageing hallmarks exhibit organ-specific temporal signatures.

Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.

Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs.

Antibiotics alter microbiota composition and increase infection susceptibility. However, the generalizable effects of antibiotics on and the contribution of environmental variables to gut commensals remain unclear. To address this, we tracked microbiota dynamics with high temporal and taxonomic resolution during antibiotic treatment in a controlled murine system by isolating variables such as diet, treatment history, and housing co-inhabitants. Human microbiotas were remarkably resilient and recovered during antibiotic treatment, with transient dominance of resistant Bacteroides and taxa-asymmetric diversity reduction. In certain cases, in vitro sensitivities were not predictive of in vivo responses, underscoring the significance of host and community context. A fiber-deficient diet exacerbated microbiota collapse and delayed recovery. Species replacement through cross housing after ciprofloxacin treatment established resilience to a second treatment. Single housing drastically disrupted recovery, highlighting the importance of environmental reservoirs. Our findings highlight deterministic microbiota adaptations to perturbations and the translational potential for modulating diet, sanitation, and microbiota composition during antibiotics.

FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences.

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing.

Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.

Single Cell Transcriptomes Derived from Human Cervical and Uterine Tissue during Pregnancy.

This work presents the workflow for generating single cell transcriptomes derived from primary human uterine and cervical tissue obtained during planned cesarean hysterectomies. In total, a catalogue of 310 single cell transcriptomes are obtained, cell types present in these biopsies are inferred, and specific genes defining each of the cellular types present in the tissue are identified. Further validation of the inferred cell identity is also demonstrated via meta-analysis of independent repositories in literature generated by bulk sequenced data of fluorescence-activated cell sorting sorted cells.