Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors.

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

Direct role of FLT3 in regulation of early lymphoid progenitors.

Given that FLT3 expression is highly restricted on lymphoid progenitors, it is possible that the established role of FLT3 in the regulation of B and T lymphopoiesis reflects its high expression and role in regulation of lymphoid-primed multipotent progenitors (LMPPs) or common lymphoid progenitors (CLPs). We generated a Flt3 conditional knock-out (Flt3fl/fl) mouse model to address the direct role of FLT3 in regulation of lymphoid-restricted progenitors, subsequent to turning on Rag1 expression, as well as potentially ontogeny-specific roles in B and T lymphopoiesis. Our studies establish a prominent and direct role of FLT3, independently of the established role of FLT3 in regulation of LMPPs and CLPs, in regulation of fetal as well as adult early B cell progenitors, and the early thymic progenitors (ETPs) in adult mice but not in the fetus. Our findings highlight the potential benefit of targeting poor prognosis acute B-cell progenitor leukaemia and ETP leukaemia with recurrent FLT3 mutations using clinical FLT3 inhibitors.

Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.

Human monocyte subtype expression of neuroinflammation and regeneration-related genes is linked to age and sex.

Stroke is a leading cause of disability and the third cause of death. The immune system plays an essential role in post-stroke recovery. After an ischemic stroke, monocytes infiltrate the injured brain tissue and can exacerbate or mitigate the damage. Ischemic stroke is more prevalent in the aged population, and the aging brain exhibits an altered immune response. There are also sex disparities in ischemic stroke incidence, outcomes, and recovery, and these differences may be hormone-driven and determined by genetic and epigenetic factors. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of neuronal inflammation- and regeneration-related genes depends on age and sex. A FACS analysis of blood samples from 44 volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of natural killer cells increased in females. The proportion of B-cells decreased in both sexes with age, and subtypes of monocytes were not linked to age or sex. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly, ANXA1 and CD36 showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1ß, S100A8, TNFα, CD64, CD33, TGFß1, TLR8, CD91) were differentially changed in monocyte subtypes with increased aging. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes.

Human monocyte subtype expression of neuroinflammation- and regeneration-related genes is linked to age and sex.

Aging profoundly affects the immune system leading to an increased propensity for inflammation. Age-related dysregulation of immune cells is implicated in the development and progression of numerous age-related diseases such as: cardiovascular diseases, neurodegenerative disorders, and metabolic syndromes. Monocytes and monocyte-derived macrophages, being important players in the inflammatory response, significantly influence the aging process and the associated increase in inflammatory disease risk. Ischemic stroke is among age-related diseases where inflammation, particularly monocyte-derived macrophages, plays an important deteriorating role but could also strongly promote post-stroke recovery. Also, biological sex influences the incidence, presentation, and outcomes of ischemic stroke, reflecting both biological differences between men and women. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of genes implicated in stroke-related inflammation and post-stroke tissue regeneration depends on age and sex. A flow cytometry analysis of blood samples from 44 healthy volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of NK-cells increased in females. The proportion of B-cells decreased in both sexes with age. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly, ANXA1 and CD36 showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1ß, S100A8, TNFα, CD64, CD33, TGFß1, TLR8, CD91) were differentially changed in monocyte subtypes with increasing age. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes.

Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation.

Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent.

In vitro clonal multilineage differentiation of distinct murine hematopoietic progenitor populations.

Here we describe an in vitro co-culture system that can differentiate hematopoietic progenitor populations to all major hematopoietic lineages at clonal level. We present both a sensitive single-cell switch-culture system as well as a less laborious alternative barcoding protocol more convenient for larger cell numbers. Importantly, generation of all lineages from single long-term hematopoietic stem cells are described, following 21 days of culture. This protocol represents an efficient tool for validation experiments for single-cell genomics data. For complete details on the use and execution of this protocol, please refer to Safi et al. (2022).1.

Single-cell multiomics of human fetal hematopoiesis define a developmental-specific population and a fetal signature.

Knowledge of human fetal blood development and how it differs from adult blood is highly relevant to our understanding of congenital blood and immune disorders and childhood leukemia, of which the latter can originate in utero. Blood formation occurs in waves that overlap in time and space, adding to heterogeneity, which necessitates single-cell approaches. Here, a combined single-cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), the molecular profile of established immunophenotype-gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs), such as CD90 and CD49F, were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow data set revealed that the HSC state was less frequent in FL, whereas cells with a lymphomyeloid signature were more abundant. An erythromyeloid-primed multipotent progenitor cluster was identified, potentially representing a transient, fetal-specific population. Furthermore, differentially expressed genes between fetal and adult counterparts were specifically analyzed, and a fetal core signature was identified. The core gene set could separate subgroups of acute lymphoblastic leukemia by age, suggesting that a fetal program may be partially retained in specific subgroups of pediatric leukemia. Our detailed single-cell map presented herein emphasizes molecular and immunophenotypic differences between fetal and adult blood cells, which are of significance for future studies of pediatric leukemia and blood development in general.

Expression and role of FLT3 in regulation of the earliest stage of normal granulocyte-monocyte progenitor development.

Mice deficient in c-fms-like tyrosine kinase 3 (FLT3) signaling have reductions in early multipotent and lymphoid progenitors, whereas no evident myeloid phenotype has been reported. However, activating mutations of Flt3 are among the most common genetic events in acute myeloid leukemia and mice harboring internal tandem duplications within Flt3 (Flt3-ITD) develop myeloproliferative disease, with characteristic expansion of granulocyte-monocyte (GM) progenitors (GMP), possibly compatible with FLT3-ITD promoting a myeloid fate of multipotent progenitors. Alternatively, FLT3 might be expressed at the earliest stages of GM development. Herein, we investigated the expression, function, and role of FLT3 in recently identified early GMPs. Flt3-cre fate-mapping established that most progenitors and mature progeny of the GM lineage are derived from Flt3-expressing progenitors. A higher expression of FLT3 was found in preGMP compared with GMP, and preGMPs were more responsive to stimulation with FLT3 ligand (FL). Whereas preGMPs and GMPs were reduced in Fl(-/-) mice, megakaryocyte-erythroid progenitors were unaffected and lacked FLT3 expression. Notably, mice deficient in both thrombopoietin (THPO) and FL had a more pronounced GMP phenotype than Thpo(-/-) mice, establishing a role of FL in THPO-dependent and -independent regulation of GMPs, of likely significance for myeloid malignancies with Flt3-ITD mutations.

Regulome analysis in B-acute lymphoblastic leukemia exposes Core Binding Factor addiction as a therapeutic vulnerability.

The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFß results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFß-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

Concurrent stem- and lineage-affiliated chromatin programs precede hematopoietic lineage restriction.

The emerging notion of hematopoietic stem and progenitor cells (HSPCs) as a low-primed cloud without sharply demarcated gene expression programs raises the question on how cellular-fate options emerge and at which stem-like stage lineage priming is initiated. Here, we investigate single-cell chromatin accessibility of Lineage-, cKit+, and Sca1+ (LSK) HSPCs spanning the early differentiation landscape. Application of a signal-processing algorithm to detect transition points corresponding to massive alterations in accessibility of 571 transcription factor motifs reveals a population of LSK FMS-like tyrosine kinase 3 (Flt3)intCD9high cells that concurrently display stem-like and lineage-affiliated chromatin signatures, pointing to a simultaneous gain of both lympho-myeloid and megakaryocyte-erythroid programs. Molecularly and functionally, these cells position between stem cells and committed progenitors and display multi-lineage capacity in vitro and in vivo but lack self-renewal activity. This integrative molecular analysis resolves the heterogeneity of cells along hematopoietic differentiation and permits investigation of chromatin-mediated transition between multipotency and lineage restriction.

Defining the Emerging Blood System During Development at Single-Cell Resolution.

Developmental hematopoiesis differs from adult and is far less described. In the developing embryo, waves of lineage-restricted blood precede the ultimate emergence of definitive hematopoietic stem cells (dHSCs) capable of maintaining hematopoiesis throughout life. During the last two decades, the advent of single-cell genomics has provided tools to circumvent previously impeding characteristics of embryonic hematopoiesis, such as cell heterogeneity and rare cell states, allowing for definition of lineage trajectories, cellular hierarchies, and cell-type specification. The field has rapidly advanced from microfluidic platforms and targeted gene expression analysis, to high throughput unbiased single-cell transcriptomic profiling, single-cell chromatin analysis, and cell tracing-offering a plethora of tools to resolve important questions within hematopoietic development. Here, we describe how these technologies have been implemented to address a wide range of aspects of embryonic hematopoiesis ranging from the gene regulatory network of dHSC formation via endothelial to hematopoietic transition (EHT) and how EHT can be recapitulated in vitro, to hematopoietic trajectories and cell fate decisions. Together, these studies have important relevance for regenerative medicine and for our understanding of genetic blood disorders and childhood leukemias.

In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signaling.

Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia.

FLT3 ligand and not TSLP is the key regulator of IL-7-independent B-1 and B-2 B lymphopoiesis.

Phenotypically and functionally distinct progenitors and developmental pathways have been proposed to exist for fetally derived B-1 and conventional B-2 cells. Although IL-7 appears to be the primary cytokine regulator of fetal and adult B lymphopoiesis in mice, considerable fetal B lymphopoiesis and postnatal B cells are sustained in the absence of IL-7; in humans, B-cell generation is suggested to be largely IL-7-independent, as severe combined immune-deficient patients with IL-7 deficiency appear to have normal B-cell numbers. However, the role of other cytokines in IL-7-independent B lymphopoiesis remains to be established. Although thymic stromal lymphopoietin (TSLP) has been proposed to be the main factor driving IL-7-independent B lymphopoiesis and to distinguish fetal from adult B-cell progenitor development in mice, recent studies failed to support a primary role of TSLP in IL-7-independent fetal B-cell development. However, the role of TSLP in IL-7-independent adult B lymphopoiesis and in particular in regulation of B-1 cells remains to be established. Here we demonstrate that, rather than TSLP, IL-7 and FLT3 ligand are combined responsible for all B-cell generation in mice, including recently identified B-1-specified cell progenitors. Thus, the same IL-7- and FLT3 ligand-mediated signal-ing regulates alternative pathways of fetal and adult B-1 and B-2 lymphopoiesis.

Generation and trapping of a mesoderm biased state of human pluripotency.

We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explore mesoderm lineage-bias within the human pluripotent stem cell compartment. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes. Functionally these cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we 'trap' and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of 'cross-antagonistic' signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, biomanufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.