Atlas of fetal metabolism during mid-to-late gestation and diabetic pregnancy.

Mounting evidence suggests metabolism instructs stem cell fate decisions. However, how fetal metabolism changes during development and how altered maternal metabolism shapes fetal metabolism remain unexplored. We present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and liquid chromatography-mass spectrometry (LC-MS), we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Our analysis revealed metabolic features specific to a hyperglycemic environment and signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from hyperglycemic dams. Tracing 13C-glucose revealed disparate fetal nutrient sourcing depending on maternal glycemic states. Regardless of glycemic state, histidine-derived metabolites accumulated in late-stage fetal tissues. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations due to maternal hyperglycemia.

A two-front nutrient supply environment fuels small intestinal physiology through differential regulation of nutrient absorption and host defense.

The small intestine contains a two-front nutrient supply environment created by luminal dietary and microbial metabolites (enteral side) and systemic metabolites from the host (serosal side). Yet, it is unknown how each side contributes differentially to the small intestinal physiology. Here, we generated a comprehensive, high-resolution map of the small intestinal two-front nutrient supply environment. Using in vivo tracing of macronutrients and spatial metabolomics, we visualized the spatiotemporal dynamics and cell-type tropism in nutrient absorption and the region-specific metabolic heterogeneity within the villi. Specifically, glutamine from the enteral side fuels goblet cells to support mucus production, and the serosal side loosens the epithelial barrier by calibrating fungal metabolites. Disorganized feeding patterns, akin to the human lifestyle of skipping breakfast, increase the risk of metabolic diseases by inducing epithelial memory of lipid absorption. This study improves our understanding of how the small intestine is spatiotemporally regulated by its unique nutritional environment.

The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Tracing the origin of alveolar stem cells in lung repair and regeneration.

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

Identifying specific functional roles for senescence across cell types.

Cellular senescence plays critical roles in aging, regeneration, and disease; yet, the ability to discern its contributions across various cell types to these biological processes remains limited. In this study, we generated an in vivo genetic toolbox consisting of three p16Ink4a-related intersectional genetic systems, enabling pulse-chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined p16Ink4a+ cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair. Notably, clearance of p16Ink4a+ macrophages significantly mitigates hepatocellular damage, whereas eliminating p16Ink4a+ ECs aggravates liver injury. Additionally, targeted reprogramming of p16Ink4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16Ink4a+ cells and offers insights for developing cell-type-specific senolytic therapies in the future.

An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] ∼100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50] < 50 µg/mL) and total lineage-concentrations estimates of 50-200 µg/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak.

An outbreak of over 1,000 COVID-19 cases in Provincetown, Massachusetts (MA), in July 2021-the first large outbreak mostly in vaccinated individuals in the US-prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined viral genomic and epidemiological data from 467 individuals, including 40% of outbreak-associated cases. The Delta variant accounted for 99% of cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US overall, likely due to high vaccination rates and a robust public health response.

Gut bacterial nutrient preferences quantified in vivo.

Great progress has been made in understanding gut microbiomes' products and their effects on health and disease. Less attention, however, has been given to the inputs that gut bacteria consume. Here, we quantitatively examine inputs and outputs of the mouse gut microbiome, using isotope tracing. The main input to microbial carbohydrate fermentation is dietary fiber and to branched-chain fatty acids and aromatic metabolites is dietary protein. In addition, circulating host lactate, 3-hydroxybutyrate, and urea (but not glucose or amino acids) feed the gut microbiome. To determine the nutrient preferences across bacteria, we traced into genus-specific bacterial protein sequences. We found systematic differences in nutrient use: most genera in the phylum Firmicutes prefer dietary protein, Bacteroides dietary fiber, and Akkermansia circulating host lactate. Such preferences correlate with microbiome composition changes in response to dietary modifications. Thus, diet shapes the microbiome by promoting the growth of bacteria that preferentially use the ingested nutrients.

Embryonic origins of adult pluripotent stem cells.

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.

Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution.

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.

Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.

Revisiting astrocyte to neuron conversion with lineage tracing in vivo.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.

Generation of Functional Human 3D Cortico-Motor Assembloids.

Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.

Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics.

Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.

A Common Embryonic Origin of Stem Cells Drives Developmental and Adult Neurogenesis.

New neurons arise from quiescent adult neural progenitors throughout life in specific regions of the mammalian brain. Little is known about the embryonic origin and establishment of adult neural progenitors. Here, we show that Hopx+ precursors in the mouse dentate neuroepithelium at embryonic day 11.5 give rise to proliferative Hopx+ neural progenitors in the primitive dentate region, and they, in turn, generate granule neurons, but not other neurons, throughout development and then transition into Hopx+ quiescent radial glial-like neural progenitors during an early postnatal period. RNA-seq and ATAC-seq analyses of Hopx+ embryonic, early postnatal, and adult dentate neural progenitors further reveal common molecular and epigenetic signatures and developmental dynamics. Together, our findings support a "continuous" model wherein a common neural progenitor population exclusively contributes to dentate neurogenesis throughout development and adulthood. Adult dentate neurogenesis may therefore represent a lifelong extension of development that maintains heightened plasticity in the mammalian hippocampus.

Adaptive Immune Resistance Emerges from Tumor-Initiating Stem Cells.

Our bodies are equipped with powerful immune surveillance to clear cancerous cells as they emerge. How tumor-initiating stem cells (tSCs) that form and propagate cancers equip themselves to overcome this barrier remains poorly understood. To tackle this problem, we designed a skin cancer model for squamous cell carcinoma (SCC) that can be effectively challenged by adoptive cytotoxic T cell transfer (ACT)-based immunotherapy. Using single-cell RNA sequencing (RNA-seq) and lineage tracing, we found that transforming growth factor ß (TGF-ß)-responding tSCs are superior at resisting ACT and form the root of tumor relapse. Probing mechanism, we discovered that during malignancy, tSCs selectively acquire CD80, a surface ligand previously identified on immune cells. Moreover, upon engaging cytotoxic T lymphocyte antigen-4 (CTLA4), CD80-expressing tSCs directly dampen cytotoxic T cell activity. Conversely, upon CTLA4- or TGF-ß-blocking immunotherapies or Cd80 ablation, tSCs become vulnerable, diminishing tumor relapse after ACT treatment. Our findings place tSCs at the crux of how immune checkpoint pathways are activated.