Individual Progenitor Research Articles

Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease

The contribution of progenitor subtypes to generating the billions of neurons produced during human cortical neurogenesis is not well understood. We developed the cortical organoid lineage-tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all excitatory neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic CTNNB1 variant demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play roles in generating the diverse neurons of the human cerebral cortex.

A cell fate decision map reveals abundant direct neurogenesis bypassing intermediate progenitors in the human developing neocortex

The human neocortex has undergone strong evolutionary expansion, largely due to an increased progenitor population, the basal radial glial cells. These cells are responsible for the production of a diversity of cell types, but the successive cell fate decisions taken by individual progenitors remain unknown. Here we developed a semi-automated live/fixed correlative imaging method to map basal radial glial cell division modes in early fetal tissue and cerebral organoids. Through the live analysis of hundreds of dividing progenitors, we show that basal radial glial cells undergo abundant symmetric amplifying divisions, and frequent self-consuming direct neurogenic divisions, bypassing intermediate progenitors. These direct neurogenic divisions are more abundant in the upper part of the subventricular zone. We furthermore demonstrate asymmetric Notch activation in the self-renewing daughter cells, independently of basal fibre inheritance. Our results reveal a remarkable conservation of fate decisions in cerebral organoids, supporting their value as models of early human neurogenesis.

Supermultipotency and unpredictability in the developing superior colliculus

A recent study by Cheung, Pauler, Koppensteiner et al. combining lineage tracing with single-cell RNA sequencing (scRNA-seq) has revealed unexpected features of the developing superior colliculus (SC). Extremely multipotent individual progenitors generate all types of SC neurons and glial cells that were found to localize in a non-predetermined pattern, demonstrating a remarkable degree of unpredictability in SC development.

Abstract TP42: Single Stem Cell Modification With Biomaterials and Oligopeptide for Stroke Targeting and Recovery

Stem cell transplantation enhances stroke recovery. A primary mechanism is the release of trophic factors, such as VEGF, BDNF, and CNTF, which are thought to stimulate brain plasticity and recovery mechanisms. While promising, the treatment efficacy of stem cell transplantation is restricted by inadequate stem cell survival and insufficient trophic factor release at the stroke site. To address this, we have developed a single stem cell encapsulation method (SingleStem) using the naturally occurring glycolysis process and click chemistry strategy to modify individual neural progenitor cells (NPCs) with polymeric materials and the stroke targeting oligopeptide (E-selectin binding oligopeptide (Esbp)). Our data demonstrate that the polymeric biomaterial coating provided uniform cell protection and control of individual NPC, which increased transplanted NPC survival and enabled trophic factor release modulation (such as VEGFA and VEGFB). Additionally, the stroke targeting oligopeptide Esbp is able to bind to components on activated endothelial cells to facilitated NPC homing to the stroke region and improve stroke recovery efficacy. These advantages enable SingleStem to be an effective stroke recovery tool to improve stem cell efficacy and understand stroke recovery mechanisms.

Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics.

To meet the physiological demands of the body, organs need to establish a functional tissue architecture and adequate size as the embryo develops to adulthood. In the liver, uni- and bipotent progenitor differentiation into hepatocytes and biliary epithelial cells (BECs), and their relative proportions, comprise the functional architecture. Yet, the contribution of individual liver progenitors at the organ level to both fates, and their specific proportion, is unresolved. Combining mathematical modelling with organ-wide, multispectral FRaeppli-NLS lineage tracing in zebrafish, we demonstrate that a precise BEC-to-hepatocyte ratio is established (i) fast, (ii) solely by heterogeneous lineage decisions from uni- and bipotent progenitors, and (iii) independent of subsequent cell type-specific proliferation. Extending lineage tracing to adulthood determined that embryonic cells undergo spatially heterogeneous three-dimensional growth associated with distinct environments. Strikingly, giant clusters comprising almost half a ventral lobe suggest lobe-specific dominant-like growth behaviours. We show substantial hepatocyte polyploidy in juveniles representing another hallmark of postembryonic liver growth. Our findings uncover heterogeneous progenitor contributions to tissue architecture-defining cell type proportions and postembryonic organ growth as key mechanisms forming the adult liver.

Comprehensive spatiotemporal mapping of single-cell lineages in developing mouse brain by CRISPR-based barcoding.

A fundamental interest in developmental neuroscience lies in the ability to map the complete single-cell lineages within the brain. To this end, we developed a CRISPR editing-based lineage-specific tracing (CREST) method for clonal tracing in Cre mice. We then used two complementary strategies based on CREST to map single-cell lineages in developing mouse ventral midbrain (vMB). By applying snapshotting CREST (snapCREST), we constructed a spatiotemporal lineage landscape of developing vMB and identified six progenitor archetypes that could represent the principal clonal fates of individual vMB progenitors and three distinct clonal lineages in the floor plate that specified glutamatergic, dopaminergic or both neurons. We further created pandaCREST (progenitor and derivative associating CREST) to associate the transcriptomes of progenitor cells in vivo with their differentiation potentials. We identified multiple origins of dopaminergic neurons and demonstrated that a transcriptome-defined progenitor type comprises heterogeneous progenitors, each with distinct clonal fates and molecular signatures. Therefore, the CREST method and strategies allow comprehensive single-cell lineage analysis that could offer new insights into the molecular programs underlying neural specification.

Purification and functional characterization of novel human skeletal stem cell lineages.

Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.

Assay optimization for the objective quantification of human multilineage colony-forming units

Colony-forming unit (CFU) assays are a powerful tool in hematopoietic research because they allow researchers to functionally test the lineage potential of individual stem and progenitor cells. Assaying for lineage potential is important for determining and validating the identity of progenitor populations isolated by methods such as fluorescence-activated cell sorting (FACS). However, current methods for CFU assays are limited in their ability to robustly assay multipotent progenitors with the ability to differentiate down the myeloid, erythroid, and megakaryocytic lineages because of the lack of specific growth factors necessary for certain lineage outputs. In addition, manual counting of colony types is subjective resulting in user to user variability in assessments of cell types based on colony and cell morphologies. We demonstrate that the addition of granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF into a collagen-based MegaCult medium containing IL-3, IL-6, SCF, EPO, and TPO allows for the differentiation of common myeloid progenitors into expected proportions of colonies containing granulocytic (G), monocytic (M), erythroid (E), and megakaryocytic (Mk) cells. Additionally, we demonstrate an objective method using in situ immunofluorescence (IF) with anti-CD66b, anti-CD14, anti-CD235a, and anti-CD41 to detect G, M, E, and Mk cells, respectively. IF stained colonies can be analyzed individually at a microscope or using high-throughput microscopy. Thus, our improvements to the culture conditions and method for assay readout increase the accuracy, reproducibility, and throughput of the myeloid CFU assay.

Homophilic interaction of cell adhesion molecule 3 coordinates retina neuroepithelial cell proliferation.

Correct cell number generation is central to tissue development. However, in vivo roles of coordinated proliferation of individual neural progenitors in regulating cell numbers of developing neural tissues and the underlying molecular mechanism remain mostly elusive. Here, we showed that wild-type (WT) donor retinal progenitor cells (RPCs) generated significantly expanded clones in host retinae with G1-lengthening by p15 (cdkn2a/b) overexpression (p15+) in zebrafish. Further analysis showed that cell adhesion molecule 3 (cadm3) was reduced in p15+ host retinae, and overexpression of either full-length or ectodomains of Cadm3 in p15+ host retinae markedly suppressed the clonal expansion of WT donor RPCs. Notably, WT donor RPCs in retinae with cadm3 disruption recapitulated expanded clones that were found in p15+ retinae. More strikingly, overexpression of Cadm3 without extracellular ig1 domain in RPCs resulted in expanded clones and increased retinal total cell number. Thus, homophilic interaction of Cadm3 provides an intercellular mechanism underlying coordinated cell proliferation to ensure cell number homeostasis of the developing neuroepithelia.

Mapping of clonal lineages across developmental stages in human neural differentiation.

The cell lineages across developmental stages remain to be elucidated. Here, we developed single-cell split barcoding (SISBAR) that allows clonal tracking of single-cell transcriptomes across stages in an invitro model of human ventral midbrain-hindbrain differentiation. We developed "potential-spective" and "origin-spective" analyses to investigate the cross-stage lineage relationships and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. We uncovered many previously uncharacterized converging and diverging trajectories. Furthermore, we demonstrate that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multilineage fates of a progenitor cell-type represent the collective results of distinct rather than similar clonal fates of individual progenitors, each with distinct molecular signatures. Specifically, we uncovered a ventral midbrain progenitor cluster as the common clonal origin of midbrain dopaminergic (mDA) neurons, midbrain glutamatergic neurons, and vascular and leptomeningeal cells and identified a surface marker that can improve graft outcomes.

Unravelling the mass spectrum of destroyed dwarf galaxies with the metallicity distribution function

ABSTRACTAccreted stellar populations are comprised of the remnants of destroyed galaxies, and often dominate the ‘stellar haloes’ of galaxies such as the Milky Way (MW). This ensemble of external contributors is a key indicator of the past assembly history of a galaxy. We introduce a novel statistical method that uses the unbinned metallicity distribution function (MDF) of a stellar population to estimate the mass spectrum of its progenitors. Our model makes use of the well-known mass–metallicity relation of galaxies and assumes Gaussian MDF distributions for individual progenitors: the overall MDF is thus a mixture of MDFs from smaller galaxies. We apply the method to the stellar halo of the MW, as well as the classical MW satellite galaxies. The stellar components of the satellite galaxies have relatively small sample sizes, but we do not find any evidence for accreted populations with L > Lhost/100. We find that the MW stellar halo has N ∼ 1−3 massive progenitors (L ≳ 108L⊙) within 10 kpc, and likely several hundred progenitors in total. We also test our method on simulations of MW-mass haloes, and find that our method is able to recover the true accreted population within a factor of 2. Future data sets will provide MDFs with orders of magnitude more stars, and this method could be a powerful technique to quantify the accreted populations down to the ultra-faint dwarf mass scale for both the MW and its satellites.

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis.

Clonal cell analysis outlines the ontogenic potential of single progenitor cells, allowing the elucidation of the neural heterogeneity among different cell types and their lineages. In this work, we analyze the potency of retinal stem/progenitor cells through development using the chick embryo as a model. We implemented in ovo the clonal genetic tracing strategy UbC-StarTrack for tracking retinal cell lineages derived from individual progenitors of the ciliary margin at E3.5 (HH21-22). The clonal assignment of the derived-cell progeny was performed in the neural retina at E11.5-12 (HH38) through the identification of sibling cells as cells expressing the same combination of fluorophores. Moreover, cell types were assessed based on their cellular morphology and laminar location. Ciliary margin derived-cell progenies are organized in columnar associations distributed along the peripheral retina with a limited tangential dispersion. The analysis revealed that, at the early stages of development, this region harbors multipotent and committed progenitor cells.

3073 – HUMAN HAEMATOPOIETIC STEM AND PROGENITOR CELLS FORM KINSHIPS THAT ARE SIMILAR IN FATE AND SYNCHRONOUS IN DIVISION

While the heterogeneity of human haematopoietic stem and progenitor cells has become apparent, linking this heterogeneity to functional properties at the single-cell level remains a challenge. Here, we have developed a combination of flow cytometry, live-cell imaging, and mathematical modelling to track the cell division properties and cell commitment in human haematopoietic stem and progenitor cells. We identified two key properties: synchronicity in division and similarity in fate among progenies of individual stem and progenitor cells. Each human stem and progenitor cell give rise to a cellular kinship that share a strong resemblance in fate and divide synchronously, suggesting an intrinsic cellular memory transmitted from mother to daughter cells. Interestingly, we observe that the time to divide shared by two sibling cells is not correlated with the division time of its mother cell. These behaviours are observed and shared across the entire lifespan, from cord blood to aged bone marrow cells. Moreover, we collected evidence that these behaviours are altered in haematological malignancies such as acute myeloid leukemia. Using a multi-agent mathematical model, we were able to recapitulate the cellular early commitment, where two sister cells descending from the same ancestor share a strong correlation in fate. In conclusion, we propose a model that describe the human haematopoiesis as a heterogeneous system formed by homogeneous cell families. While the heterogeneity of human haematopoietic stem and progenitor cells has become apparent, linking this heterogeneity to functional properties at the single-cell level remains a challenge. Here, we have developed a combination of flow cytometry, live-cell imaging, and mathematical modelling to track the cell division properties and cell commitment in human haematopoietic stem and progenitor cells. We identified two key properties: synchronicity in division and similarity in fate among progenies of individual stem and progenitor cells. Each human stem and progenitor cell give rise to a cellular kinship that share a strong resemblance in fate and divide synchronously, suggesting an intrinsic cellular memory transmitted from mother to daughter cells. Interestingly, we observe that the time to divide shared by two sibling cells is not correlated with the division time of its mother cell. These behaviours are observed and shared across the entire lifespan, from cord blood to aged bone marrow cells. Moreover, we collected evidence that these behaviours are altered in haematological malignancies such as acute myeloid leukemia. Using a multi-agent mathematical model, we were able to recapitulate the cellular early commitment, where two sister cells descending from the same ancestor share a strong correlation in fate. In conclusion, we propose a model that describe the human haematopoiesis as a heterogeneous system formed by homogeneous cell families.

Individual human cortical progenitors can produce excitatory and inhibitory neurons.

The cerebral cortex is a cellularly complex structure comprising a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes-excitatory neurons that use the neurotransmitter glutamate, and inhibitory interneurons that use γ-aminobutyric acid (GABA). Previous developmental studies in rodents have led to a prevailing model in which excitatory neurons are born from progenitors located in the cortex, whereascortical interneurons are born from a separate population of progenitors located outside the developing cortex in the ganglionic eminences1-5. However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that, in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called 'single-cell-RNA-sequencing-compatible tracer for identifying clonal relationships' (STICR), we were able to carry out clonal lineage tracing of 1,912 primary human cortical progenitors from six specimens, and to capture both the transcriptional identities and the clonal relationships of their progeny. A subpopulation of corticallyborn GABAergic neurons was transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence, and these cells were frequently related to excitatory neurons and glia. Our results show that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex.

Endoderm development requires centrioles to restrain p53-mediated apoptosis in the absence of ERK activity.

Centrioles comprise the heart of centrosomes, microtubule-organizing centers. To study the function of centrioles in lung and gut development, we genetically disrupted centrioles throughout the mouse endoderm. Surprisingly, removing centrioles from the endoderm did not disrupt intestinal growth or development but blocked lung branching. In the lung, acentriolar SOX2-expressing airway epithelial cells apoptosed. Loss of centrioles activated p53, and removing p53 restored survival of SOX2-expressing cells, lung branching, and mouse viability. To investigate how endodermal p53 activation specifically killed acentriolar SOX2-expressing cells, we assessed ERK, a prosurvival cue. ERK was active throughout the intestine and in the distal lung buds, correlating with tolerance to centriole loss. Pharmacologically inhibiting ERK activated apoptosis in acentriolar cells, revealing that ERK activity protects acentriolar cells from apoptosis. Therefore, centrioles are largely dispensable for endodermal growth and the spatial distribution of ERK activity in the endoderm shapes the developmental consequences of centriolar defects and p53 activation.

Gliogenic Potential of Single Pallial Radial Glial Cells in Lower Cortical Layers.

During embryonic development, progenitor cells are progressively restricted in their potential to generate different neural cells. A specific progenitor cell type, the radial glial cells, divides symmetrically and then asymmetrically to produce neurons, astrocytes, oligodendrocytes, and NG2-glia in the cerebral cortex. However, the potential of individual progenitors to form glial lineages remains poorly understood. To further investigate the cell progeny of single pallial GFAP-expressing progenitors, we used the in vivo genetic lineage-tracing method, the UbC-(GFAP-PB)-StarTrack. After targeting those progenitors in embryonic mice brains, we tracked their adult glial progeny in lower cortical layers. Clonal analyses revealed the presence of clones containing sibling cells of either a glial cell type (uniform clones) or two different glial cell types (mixed clones). Further, the clonal size and rostro-caudal cell dispersion of sibling cells differed depending on the cell type. We concluded that pallial E14 neural progenitors are a heterogeneous cell population with respect to which glial cell type they produce, as well as the clonal size of their cell progeny.

Abstract 3051: On the origin of pediatric leukemia: studying somatic mutations in human blood

Abstract The acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis and aging is the biggest risk factor for cancer. However, children can also develop cancers even though their cells are less damaged by age. Here, we studied the cellular origin and time of onset during development of pediatric acute myeloid leukemia (pAML) to increase our understanding of why children can get cancer. We compared by whole genome sequencing individual hematopoietic stem and progenitor cells (HSPCs) and pAML blasts of the same leukemic bone marrow as well as nonmatching healthy HSPCs. We found that the number of clonal mutations in the majority of pAML cases was increased and mutational process contribution altered as compared to normal HSPCs, showing activity of specific processes that are either unique to fetal development or normally not observed in healthy HSPCs. These observations could be explained by altered selection dynamics early in life. Surprisingly, pAML cases with an overall mutation burden comparable to age matched healthy HSPCs showed worse overall and event-free survival. Identifying shared somatic mutations between individual HSPC clones and pAML using single-cell genotyping allowed us to pinpoint the developmental lineage that gave rise to the pAML. Our study provides new insights into the etiology of pAML and underscores the clinical potential of whole genome sequencing in pediatric leukemia. Citation Format: Arianne Brandsma. On the origin of pediatric leukemia: studying somatic mutations in human blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3051.

Commitment to dendritic cells and monocytes.

Dendritic cells (DCs) and monocytes are widely conserved immune cells in vertebrates that arise from hematopoietic stem cells via intermediate progenitors. The progenitors that strictly give rise to DCs or monocytes have been recently identified both in humans and in mice, thereby revealing their differentiation pathways. Advances in analysis technologies have further deepened our understanding of the development of DCs and monocytes from progenitor population-based to individual progenitor cell-based commitment. Since DC-committed progenitors, common DC progenitors (CDPs) and precursor conventional DCs (pre-cDCs) do not differentiate into monocytes, DCs are a distinct lineage from monocytes, although monocytes can acquire DC functions upon activation at tissues where they arrive.

Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum.

Brain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.

Lineage tracing of human development through somatic mutations.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.