Amit Grover

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding—a common and life-threatening side effect of many cancer therapies—and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin−Sca-1+c-Kit+CD34+Flt3hi) and common myeloid progenitors (CMPs) (Lin−Sca-1+c-Kit+CD34+CD41hi) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre–granulocyte-macrophage progenitor (pre-GM) (Lin−Sca-1−c-Kit+CD41−FcγRII/III−CD150−CD105−). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA‐1 and FOG‐1 transcription factors to identify FOG‐1 as required for the formation of all committed Mk‐ and E‐lineage progenitors, whereas GATA‐1 was observed to be specifically required for E‐lineage commitment. FOG‐1‐deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG‐1 and GATA‐1 as required for formation of bipotent Mk/E progenitors and their E‐lineage commitment, respectively, and show that FOG‐1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E‐lineage commitment. Finally, C/EBPs and FOG‐1 exhibited transcriptional cross‐regulation in early myelo‐erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.

Erythropoietin guides multipotent hematopoietic progenitor cells toward an erythroid fate.

Erythropoietin suppresses non-erythroid cell fate options to induce an erythroid lineage bias at all lineage bifurcations between HSCs and committed erythroid progenitors.

Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells

Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis.

Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.

A transit amplifying population underpins the efficient regenerative capacity of the testis

The spermatogonial stem cell (SSC) that supports spermatogenesis throughout adult life resides within the GFR&agr;1-expressing A type undifferentiated spermatogonia. The decision to commit to spermatogenic differentiation coincides with the loss of GFR&agr;1 and reciprocal gain of Ngn3 (Neurog3) expression. Through the analysis of the piRNA factor Miwi2 (Piwil4), we identify a novel population of Ngn3-expressing spermatogonia that are essential for efficient testicular regeneration after injury. Depletion of Miwi2-expressing cells results in a transient impact on testicular homeostasis, with this population behaving strictly as transit amplifying cells under homeostatic conditions. However, upon injury, Miwi2-expressing cells are essential for the efficient regenerative capacity of the testis, and also display facultative stem activity in transplantation assays. In summary, the mouse testis has adopted a regenerative strategy to expand stem cell activity by incorporating a transit-amplifying population to the effective stem cell pool, thus ensuring rapid and efficient tissue repair.

Human adult HSCs can be discriminated from lineage-committed HPCs by the expression of endomucin

Key Points EMCN is a novel marker of human HSCs. EMCN is a more specific marker of HSCs than CD34 as it can discriminate HSCs from lineage-committed HPCs.

Studying BDNF/TrkB Signaling: Transcriptome Analysis from a Limited Number of Purified Adult or Aged Murine Brain Neurons

It is recognized by now that the basal ganglia contain some of the circuits most vulnerable to age-related effects. However, it is still unknown how these changes are regulated during aging. We have recently shown that loss of TrkB signaling in striatopallidal enkephalinergic (ENK) neurons lead to age-dependent spontaneous hyperlocomotion, associated with reduced striatopallidal activation, demonstrating that BDNF-TrkB signaling in striatal ENK neurons contributes to the inhibitory control of locomotor behavior exerted by the indirect pathway. Hence, we have established a unique mouse model that provides a rare example of an age-dependent locomotor defect. Identification of the genes and associated molecular pathways relevant to the maintenance of locomotor control requires systematic, unbiased gene expression profiling of the aging striatal circuit from young adult and aged mouse brain, both in normal and TrkBdeficient conditions. For this purpose, we have chosen whole transcriptome analysis by RNA sequencing (RNA-Seq) that offers higher resolution than other methods. To achieve this we have established a protocol that allows for the isolation of fluorescently labeled neurons from adult (3 months) or aged (8 months) mouse brain for whole transcriptome analysis by RNA-Seq using a limited number (<200) of neurons. Neuronal subsets were genetically labeled in vivo with a fluorescent marker and isolated using a sucrose artificial cerebrospinal fluid (aCSF) solution and differential centrifugation before fluorescent activated cell sorting (FACS)-based purification. This was followed by direct cDNA synthesis using an optimized SmartSeq method, resulting in the generation of robust libraries for Illumina sequencing. In contrast to previous methods used for neuronal gene profiling, this protocol can be used for high-throughput gene expression profiling from limited numbers of adult or aged brain neurons at moderate costs. The whole protocol described here takes 3–4 days from neuronal purification to preparation of cDNA libraries ready for Illumina sequencing.