Beyond "to divide or not to divide": Kinetics matters in hematopoietic stem cells.

Lifelong blood production is ensured by a population of rare and largely quiescent, long-lived hematopoietic stem cells (HSCs). The advent of single-cell technologies has recently highlighted underlying molecular and functional heterogeneity within the HSC pool. Despite heterogenous HSC behaviors, quiescence remains as the most uncontroversial and unifying property of HSCs. Nonetheless, a multifaceted and complex continuum of states has recently been identified within what was previously described as just "quiescent." Here we review such evidence and discuss how it challenges preconceived ideas on the contribution of cell cycle kinetics to HSC function. Specifically, we detail how both the frequency and kinetics of HSC division, largely determined by a network of molecular regulators linked to early G1, influence long-term HSC functionin vivo. In addition, we present data that indicate lengthening the duration of G1 by inhibiting CDK6 decreases lymphoid differentiation of a subset of lymphoid-primed human HSCs, thus linking cell cycle kinetics to cell fate decisions in HSCs. Finally, we reflect on how these new insights can be helpful to fully harness HSC potential in clinical applications that require ex vivo culture.We would like to thank the Cambridge Blood and Stem Cell Biobank, specifically Joanna Baxter and the team of nurses consenting and collecting cord bloos samples; the Cambridge NIHR BRC Cell Phenotyping Hub for their flow cytometry services. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA, BIRAX, Royal Society and by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute (203151/Z/16/Z). C.J. is supported by an MRC iCASE PhD studentship and S.B. by a CRUK Cambridge Cancer Centre PhD fellowship

INFLUÊNCIA DO pH NA LIBERAÇÃO DE CÉLULAS PROBIÓTICAS ENCAPSULADAS EM ÁGAR-ÁGAR

O objetivo deste estudo foi analisar a resistência do probiótico livre- Saccharomyces cerevisiae - e sua liberação sob diferentes valoresde pH (4,5, 6,0 e 7,5) e tempos (5, 30 e 50 min) quando encapsuladoem ágar-ágar, além de avaliar a micro e a macro estrutura dasesferas. O probiótico livre mostrou-se resistente em todo o períodode avaliação, apresentando média de 93,6 % de viabilidade. Alevedura encapsulada em ágar-ágar foi gradualmente liberadadas esferas, com média de 86,6 % de células difundidas para omeio externo nas diferentes soluções tampão. As inúmeras fi ssurase poros na matriz da esfera, observadas pelas microimagens, ointumescimento das cápsulas em meio aquoso e a concentraçãointracapsular empregada podem ter comprometido o totalaprisionamento das células no interior da cápsula. As esferas foramfi sicamente resistentes, pois não se solubilizaram e se mantiveramintactas durante o procedimento. Conclui-se que o ágar-ágar nãoconstitui material encapsulante adequado para conferir liberaçãocontrolada do agente probiótico nas condições experimentaispropostas neste estudo

Molecular landscapes of human hematopoietic stem cells in health and leukemia.

Blood cells are organized as a hierarchy with hematopoietic stem cells (HSCs) at the root. The advent of genomic technologies has opened the way for global characterization of the molecular landscape of HSCs and their progeny, both in mouse and human models, at the genetic, transcriptomic, epigenetic, and proteomics levels. Here, we outline our current understanding of the molecular programs that govern human HSCs and how dynamic changes occurring during HSC differentiation are necessary for well-regulated blood formation under homeostasis and upon injury. A large body of evidence is accumulating on how the programs of normal hematopoiesis are modified in acute myeloid leukemia, an aggressive adult malignancy driven by leukemic stem cells. We summarize these findings and their clinical implications.The authors would like to thank Emily Calderbank for critical review of the manuscript. Research in EL laboratory is supported by a Wellcome Trust Sir Henry Dale Fellowship and core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1111/nyas.1298

Estimating dormant and active hematopoietic stem cell kinetics through extensive modeling of bromodeoxyuridine label-retaining cell dynamics.

Bone marrow hematopoietic stem cells (HSCs) are responsible for both lifelong daily maintenance of all blood cells and for repair after cell loss. Until recently the cellular mechanisms by which HSCs accomplish these two very different tasks remained an open question. Biological evidence has now been found for the existence of two related mouse HSC populations. First, a dormant HSC (d-HSC) population which harbors the highest self-renewal potential of all blood cells but is only induced into active self-renewal in response to hematopoietic stress. And second, an active HSC (a-HSC) subset that by and large produces the progenitors and mature cells required for maintenance of day-to-day hematopoiesis. Here we present computational analyses further supporting the d-HSC concept through extensive modeling of experimental DNA label-retaining cell (LRC) data. Our conclusion that the presence of a slowly dividing subpopulation of HSCs is the most likely explanation (amongst the various possible causes including stochastic cellular variation) of the observed long term Bromodeoxyuridine (BrdU) retention, is confirmed by the deterministic and stochastic models presented here. Moreover, modeling both HSC BrdU uptake and dilution in three stages and careful treatment of the BrdU detection sensitivity permitted improved estimates of HSC turnover rates. This analysis predicts that d-HSCs cycle about once every 149-193 days and a-HSCs about once every 28-36 days. We further predict that, using LRC assays, a 75%-92.5% purification of d-HSCs can be achieved after 59-130 days of chase. Interestingly, the d-HSC proportion is now estimated to be around 30-45% of total HSCs - more than twice that of our previous estimate

A track of the clones: new developments in cellular barcoding.

International experts from multiple disciplines gathered at Homerton College in Cambridge, UK from September 12-14, 2018 to consider recent advances and emerging opportunities in the clonal tracking of hematopoiesis in one of a series of StemCellMathLab workshops. The group included 35 participants with experience in the fields of theoretical and experimental aspects of clonal tracking, and ranged from doctoral students to senior professors. Data from a variety of model systems and from clinical gene therapy trials were discussed, along with strategies for data analysis and sharing and challenges arising due to underlying assumptions in data interpretation and communication. Recognizing the power of this technology underpinned a group consensus of a need for improved mechanisms for sharing data and analytical protocols to maintain reproducibility and rigor in its application to complex tissues.We thank the BBSRC (BB/R021465/1), the German Stem Cell Network, the Wellcome-MRC Cambridge Stem Cell Institute and the Labex CelTisPhyBio (No. ANR-10-LBX-0038) for funds to support this workshop and the conference staff at Homerton Colleg

Chronic Lymphocytic Leukemia increases the pool of peripheral blood hematopoietic stem cells and skews differentiation

Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy invariably infiltrating the bone marrow. Although treatment options for patients with advanced disease have significantly improved in the past years, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary bone marrow failure. The underlying mechanisms impairing normal hematopoiesis in patients with CLL are poorly defined.We would like to express our deepest gratitude to patients who donated blood for this research. Samples were obtained with assistance from the Cambridge Blood and Stem Cell Biobank, funded by the Cambridge Cancer Centre and Cambridge Stem Cell Institute. This work was funded by the Cancer Research UK (CRUK; C49940/A17480). I.R. is a senior CRUK fellow. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA and Royal Society. Research in I.R. and E.L. laboratories is supported by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute

Intercellular network structure and regulatory motifs in the human hematopoietic system.

The hematopoietic system is a distributed tissue that consists of functionally distinct cell types continuously produced through hematopoietic stem cell (HSC) differentiation. Combining genomic and phenotypic data with high-content experiments, we have built a directional cell-cell communication network between 12 cell types isolated from human umbilical cord blood. Network structure analysis revealed that ligand production is cell type dependent, whereas ligand binding is promiscuous. Consequently, additional control strategies such as cell frequency modulation and compartmentalization were needed to achieve specificity in HSC fate regulation. Incorporating the in vitro effects (quiescence, self-renewal, proliferation, or differentiation) of 27 HSC binding ligands into the topology of the cell-cell communication network allowed coding of cell type-dependent feedback regulation of HSC fate. Pathway enrichment analysis identified intracellular regulatory motifs enriched in these cell type- and ligand-coupled responses. This study uncovers cellular mechanisms of hematopoietic cell feedback in HSC fate regulation, provides insight into the design principles of the human hematopoietic system, and serves as a foundation for the analysis of intercellular regulation in multicellular systems

Immature acute leukaemias: lessons from the haematopoietic roadmap

Funder: European Hematology Association; Id: http://dx.doi.org/10.13039/100008594Funder: La Ligue contre le cancer; Id: http://dx.doi.org/10.13039/501100004099Funder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100004440Funder: Association Laurette Fugain; Id: http://dx.doi.org/10.13039/100007394Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268Funder: Assistance Publique ‐ Hôpitaux de Paris; Id: http://dx.doi.org/10.13039/501100002738Funder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100000265Funder: Institut National Du Cancer; Id: http://dx.doi.org/10.13039/501100006364It is essential to relate the biology of acute leukaemia to normal blood cell development. In this review, we discuss how modern models of haematopoiesis might inform approaches to diagnosis and management of immature leukaemias, with a specific focus on T‐lymphoid and myeloid cases. In particular, we consider whether next‐generation analytical tools could provide new perspectives that could improve our understanding of immature blood cancer biology

Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B.

The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress

Distinct routes of lineage development reshape the human blood hierarchy across ontogeny

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult ‘two-tier’ hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.This work was supported by Postdoctoral Fellowship Awards from Canadian Institute of Health Research (CIHR) to FN and SZ. SZ is supported by (Aplastic Anemia). FN is a recipient of a scholar’s research award from the Ontario Institute of Cancer Research (OICR), through generous support from the Ontario Ministry of Research and Innovation. Research in EL laboratory is supported by a Wellcome Trust Sir Henry Dale Fellowship and core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. Work in the Dick laboratory is supported by grants from the CIHR, Canadian Cancer Society, Terry Fox Foundation, Genome Canada through the Ontario Genomics Institute, OICR with funds from the province of Ontario, a Canada Research Chair and the Ontario Ministry of Health and Long Term Care (OMOHLTC).This is the author accepted manuscript. The final version is available from AAAS via http://dx.doi.org/10.1126/science.aab211