Improved blind demixing methods for recovering dense neuronal morphology from barcode imaging data

Cellular barcoding methods offer the exciting possibility of 'infinite-pseudocolor' anatomical reconstruction --- i.e., assigning each neuron its own random unique barcoded 'pseudocolor,' and then using these pseudocolors to trace the microanatomy of each neuron. Here we use simulations, based on densely-reconstructed electron microscopy microanatomy, with signal structure matched to real barcoding data, to quantify the feasibility of this procedure. We develop a new blind demixing approach to recover the barcodes that label each neuron. We also develop a neural network which uses these barcodes to reconstruct the neuronal morphology from the observed fluorescence imaging data, 'connecting the dots' between discontiguous amplicon signals.

Multiplexed optical barcoding of cells via photochemical programming of bioorthogonal host–guest recognition

Barcoding provides abilities to learn about individual species within an apparently homogeneous population. We describe a light-mediated multiplexed cellular barcoding strategy through spatial programming of cucurbit[7]uril molecular recognition.

Cellular Barcoding of HSCs during Development Reveals Long-Term Persistence of T Cell Progenitor Clones in the Thymus

Developmental hematopoietic stem and progenitor cells (HSPCs) have been thought to be largely replaced by adult hematopoietic stem cells (HSCs) after birth. Here, we performed simultaneous genetic lineage tracing by cellular barcoding and transcriptional profiling of zebrafish hematopoiesis to investigate HSPC clonal dynamics and gain insight into the developmental origin of adult blood cells. We took advantage of a recently developed dynamic lineage tracing system, scGESTALT, a CRISPR-based approach that allows for inducible cellular barcoding at two different time points during zebrafish embryonic development. We induced the first stage of lineage recording in the early embryo via microinjection at the one-cell stage and selected 28 hours post-fertilization, just before the birth of definitive blood stem cells, for additional recording activity. In this way, we were able to barcode HSCs, which will pass their unique barcodes on to their progeny, thereby enabling lineage tracing. To study the clonal dynamics and developmental origins of adult blood cells, we dissected paired adult kidney marrow and thymi tissues from barcoded fish between 3 and 7 months of age. We then performed single-cell RNA sequencing (scRNA-seq) in combination with lineage tracing by specifically amplifying the GESTALT barcode and sequencing both transcriptional and GESTALT barcode libraries. In total, we recovered transcriptional profiles from 71,109 cells across 9 fish. By optimizing promoter choice, we improved barcode recovery by almost 3-fold (17.7% vs. 6.5%) for detection in cells from the zebrafish kidney marrow. In two of these more efficient lineage recording fish, we detected 61 and 72 unique HSPC clones that contributed to adult hematopoiesis. The majority of these HSPC clones did not exhibit significant lineage biases; however, we demonstrated that 6/61 and 4/72 clones from the two fish, respectively, were significantly enriched in the myeloid lineage and 8/61 and 8/72 clones were significantly enriched in the lymphoid lineage (p< 0.05). These findings demonstrate that myeloid and lymphoid biased clones arise during normal development. From our paired thymus and kidney marrow sample, we identified 51 unique kidney marrow clones, 10 shared, and 2 unique thymus clones. This latter finding of unique thymus clones was surprising and suggests that long-lived embryonic T cell progenitors persist and contribute to adult T cell production in the zebrafish. Taken together, we have demonstrated how scGESTALT can uncover complex lineage relationships in blood, mapping the origins and contributions of HSCs and embryonic progenitors to the adult hematopoietic system. Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder.

High complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance

The impact of functional heterogeneity in response to radiation therapy is poorly understood to the present. It remains elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. We applied high-resolution lentiviral cellular barcoding for quantitative clonal tracking and deconvolution of clonal dynamics in response to IR. Clonal tracking of over 400.000 HNSCC patient-derived tumor cells and the analyses of over 1500 million sequencing reads in clonogenic survival assays reveals that fractionated IR induced a strong selective pressure for clonal reduction. This significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96-75% and correlated to the degree of radiation sensitivity. Survival and clonogenicity is characterized by an intensive clonal distortion and dominance of individual tumor cells. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (p<0.05) higher deterministic clonal selection showing that the ratio of these parameters is amenable to radiation sensitivity which correlates to prognostic biomarkers of HNSCC. Evidence for the existence of a rare subpopulation with an intrinsically radiation resistant phenotype commonly surviving IR was found at a frequency of 0.6-3.3% (p<0.001, FDR 3%). With cellular barcoding we introduce a novel functional heterogeneity associated qualitative readout for tracking dynamics of clonogenic survival in response to radiation. This enables the quantification of intrinsically radiation resistant tumor cells from patient samples and reveals the contribution of stochastic and deterministic clonal selection processes in response to IR.

The host brain is permissive to colonization by Toxoplasma gondii

SummaryPathogenic infections and the diseases they cause are defined by invasion and colonization of distinct host cell types and tissue niches. In the case of viruses and bacteria, molecular and cellular barcoding has shaped our understanding of within-host pathogen population dynamics, and informed therapeutic intervention strategies. Host brain colonization is a clinically untreatable feature of persistent infection by the eukaryotic pathogen Toxoplasma gondii, and the process remains poorly understood. The host blood-brain barrier is expected to physically restrict parasite colonization of this tissue niche and force the infection through a selection bottleneck, however tools and technologies to test this hypothesis have not been available. Here, we have developed a simple CRISPR-based method to barcode Toxoplasma parasites, and then used complex libraries of barcoded parasites to define how the different phases of an infection shape the pathogen population structure. Unexpectedly, we have discovered that the murine host brain does not restrict parasite colonization, with the population structure predominantly shaped by a bottleneck experienced during the acute phase of infection. These data support an evolutionary strategy to maximize genetic diversity of parasite persister cells within the intermediate host brain for subsequent transmission into the definitive feline host.

The clonal and molecular aetiology of emergency dendritic cell development

SummaryExtrinsic regulation of single haematopoietic stem and progenitor cell (HSPC) fate is crucial for immune cell development. Here, we examine the aetiology of Flt3 ligand (Flt3L)-mediated emergency development of type 1 conventional dendritic cells (cDC1s), which results in enhanced immunity against infections and cancer. Using cellular barcoding, we demonstrate a predominant role of enhanced clonal expansion and moderate contribution via recruitment of additional cDC1-generating HSPCs. The selective cDC1 expansion occurs primarily via multi-/oligo-potent clones, without compromising output to other lineages. To understand the molecular hallmarks early during a Flt3L response, we develop Divi-Seq to simultaneously profile cell division history, surface phenotype and transcriptional state of single HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative ‘early progenitor’-like state, which leads to selective emergence of CD11c+cKit+ transitional precursors with high cellular output to cDC1s. These findings inform the mechanistic action of Flt3L in natural immunity and immunotherapy at a clonal level.

Erythropoietin directly affects single hematopoietic stem cell differentiation after transplantation

The cytokine erythropoietin (EPO) is a potent inducer of erythrocyte development and one of the most prescribed biopharmaceuticals. The action of EPO on erythroid progenitor cells is well established, but its action on hematopoietic stem cells (HSCs) is still debated. Here, using cellular barcoding, we traced the differentiation of hundreds of single HSCs, after in vitro EPO exposure and transplantation, in five different hematopoietic cell lineages, and observed the occurrence of high-output Myeloid-Erythroid-megaKaryocyte (MEK)-biased and Myeloid-B-cell-Dendritic cell (MBDC)-biased clones. ScRNAseq analysis of in vitro EPO-exposed HSCs revealed upregulation of erythroid associated genes in a subset of HSCs. Collectively, as well as demonstrating a direct effect of EPO on HSCs, our results suggest an EPO-mediated induction of MEK-bias in multi-outcome HSCs, and that this change in output is functionally compensated by MBDC-biased HSCs.