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

A Lungfish survivor of the end-Devonian extinction and an Early Carboniferous dipnoan radiation.

Until recently the immediate aftermath of the Hangenberg event of the Famennian Stage (Upper Devonian) was considered to have decimated sarcopterygian groups, including lungfish, with only two taxa, Occludus romeri and Sagenodus spp., being unequivocally recorded from rocks of Tournaisian age (Mississippian, Early Carboniferous). Recent discoveries of numerous morphologically diverse lungfish tooth plates from southern Scotland and northern England indicate that at least ten dipnoan taxa existed during the earliest Carboniferous. Of these taxa, only two, Xylognathus and Ballgadus, preserve cranial and post-cranial skeletal elements and are yet to be described. Here we present a description of the skull of a new genus and species of lungfish, Limanichthys fraseri gen. et sp. nov. that hails from the very lowest Tournaisian from the Ballagan Formation of Burnmouth, southern Scotland. The new specimen represents the earliest definitive Tournaisian lungfish skull material thus providing valuable insight into the response of this group, and indeed, the Sarcopterygii as a whole, immediately following the Upper Devonian Hangenberg event. Phylogenetic analysis places Limanichthys fraseri within the Devonian ‘phaneropleurid-fleurantiid’ grade of lungfish and that the Carboniferous lungfish represent forms that have their origins deep in the Mid and Late Devonian as well as those from a unique Carboniferous radiation.This work was carried out with the aid of NERC consortium grants NE/J022713/1 (Cambridge), NE/J020729/1 (Leicester) and NE/J021091/1 (Southampton)

Sea ice extent and seasonality for the Early Pliocene northern Weddell Sea

Growth increment analysis coupled with stable isotopic data (δ18O/δ13C) from Early Pliocene (ca 4.7 Ma) Austrochlamys anderssoni from shallow marine sediments of the Cockburn Island Formation, northern Antarctic Peninsula, suggest these bivalves grew through much of the year, even during the coldest parts of winter recorded in the shells. The high frequency fluctuation in growth increment width of A. anderssoni appears to reflect periodic, but year-round, agitation of the water column enhancing benthic food supply from organic detritus. This suggests that Austrochlamys favoured waters that were largely sea ice free. Our data support interpretation of the Cockburn Island Formation as an interglacial marine deposit and the previous hypothesis that Austrochlamys retreated from the Antarctic as sea ice extent expanded, this transition occurring during climate cooling in the Late Pliocene

Suboxic DOM is bioavailable to surface prokaryotes in a simulated overturn of an oxygen minimum zone, Devil’s Hole, Bermuda

Oxygen minimum zones (OMZs) are expanding due to increased sea surface temperatures, subsequent increased oxygen demand through respiration, reduced oxygen solubility, and thermal stratification driven in part by anthropogenic climate change. Devil’s Hole, Bermuda is a model ecosystem to study OMZ microbial biogeochemistry because the formation and subsequent overturn of the suboxic zone occur annually. During thermally driven stratification, suboxic conditions develop, with organic matter and nutrients accumulating at depth. In this study, the bioavailability of the accumulated dissolved organic carbon (DOC) and the microbial community response to reoxygenation of suboxic waters was assessed using a simulated overturn experiment. The surface inoculated prokaryotic community responded to the deep (formerly suboxic) 0.2 μm filtrate with cell densities increasing 2.5-fold over 6 days while removing 5 μmol L−1 of DOC. After 12 days, the surface community began to shift, and DOC quality became less diagenetically altered along with an increase in SAR202, a Chloroflexi that can degrade recalcitrant dissolved organic matter (DOM). Labile DOC production after 12 days coincided with an increase of Nitrosopumilales, a chemoautotrophic ammonia oxidizing archaea (AOA) that converts ammonia to nitrite based on the ammonia monooxygenase (amoA) gene copy number and nutrient data. In comparison, the inoculation of the deep anaerobic prokaryotic community into surface 0.2 μm filtrate demonstrated a die-off of 25.5% of the initial inoculum community followed by a 1.5-fold increase in cell densities over 6 days. Within 2 days, the prokaryotic community shifted from a Chlorobiales dominated assemblage to a surface-like heterotrophic community devoid of Chlorobiales. The DOM quality changed to less diagenetically altered material and coincided with an increase in the ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) gene number followed by an influx of labile DOM. Upon reoxygenation, the deep DOM that accumulated under suboxic conditions is bioavailable to surface prokaryotes that utilize the accumulated DOC initially before switching to a community that can both produce labile DOM via chemoautotrophy and degrade the more recalcitrant DOM

The Impact of Clinically Relevant Culture on Human Haematopoietic Stem Cells: A Kinetic Analysis at Single Cell Resolution

Haematopoietic stem cell (HSC) ex vivo gene therapy is now successfully used to treat an in-creasing number of monogenic disorders affecting the blood system. The ultimate efficacy of this therapy depends upon successful targeting of long-term haematopoietic stem cells (LT-HSCs), which are characterised by a predominantly quiescent status and sustained self-renewal capacity. Clinical ex vivo gene therapy protocols target the heterogenous mix of stem and pro-genitor cells encompassed in the CD34+ fraction with a limited understanding of how ex vivo manipulation impacts a purified LT-HSC subset. Importantly, long-term repopulation capacity is lost over culture, although the kinetics and molecular drivers of this process remain unclear. To address these questions, I perform single cell RNA-Seq, in vitro functional assays and in vivo transplantation in a time resolved manner following cord blood (CB) culture in differentiation facilitating conditions and mobilized peripheral blood (mPB) culture during a lentiviral ex vivo gene therapy protocol. First, I characterise the molecular impact of a 62 hr lentiviral ex vivo gene therapy protocol on mPB LT-HSC, short-term HSC (ST-HSC) and CD34+ cells. I reveal that the ex vivo protocol dramatically rewires the quiescent LT-HSC transcriptome and demonstrate that the majority of transcriptional changes are attributed to the culture conditions rather than lentiviral transduction. Secondly, I refine the kinetics associated with HSC functional attrition over the first complete cell cycle ex vivo. Long-term repopulation capacity is maintained for the first 6 hr irrespective of HSC source or tested culture conditions. I identify the 6 hr time-point as encompassing an adaptation period where LT-HSCs are rapidly responding to the instructive signals of culture and is molecularly underpinned by transient upregulation of cell stress response signalling. Follow-ing the 6 hr time-point, long-term repopulation capacity drops dramatically by 24 hr in CB culture and 62 hr in mPB culture. Loss of HSC function is correlated with reduced survival, cell cycle progression (late G1-M), sharp upregulation of MYC and a reduced ability to resolve proteostatic stress. Taken together, my results demonstrate that the 6 to 24 hr transition point is instrumental for the determination of LT-HSC cell fate ex vivo. Finally, using an ex vivo culture system of reversible early G1 arrest, I formally test whether cell cycle progression drives loss of self-renewal capacity in culture. Long-term in vivo transplantation approaches conclusively establish that cell cycle progression is not responsible for the loss of long-term repopulation capacity associated with clinically relevant HSC culture

A lungfish survivor of the end-Devonian extinction and an Early Carboniferous dipnoan radiation

Until recently the immediate aftermath of the Hangenberg event of the Famennian Stage (Upper Devonian) was considered to have decimated sarcopterygian groups, including lungfish, with only two taxa, Occludus romeri and Sagenodus spp., being unequivocally recorded from rocks of Tournaisian age (Mississippian, Early Carboniferous). Recent discoveries of numerous morphologically diverse lungfish tooth plates from southern Scotland and northern England indicate that at least 10 dipnoan taxa existed during the earliest Carboniferous. Of these taxa, only two, Xylognathus and Ballgadus, preserve cranial and postcranial skeletal elements, which have yet to be described. Here we present a description of the skull of a new genus and species of lungfish, Limanichthys fraseri gen. et sp. nov., which hails from the very earliest Tournaisian in the Ballagan Formation of Burnmouth, southern Scotland. The new specimen represents the earliest definitive Tournaisian lungfish skull material, thus providing an invaluable insight into the response of this group – and, indeed, Sarcopterygii as a whole – immediately following the latest Devonian Hangenberg event. Phylogenetic analysis places Limanichthys fraseri within the Devonian ‘phaneropleurid-fleurantiid’ grade of lungfish and shows that the Carboniferous lungfish represent forms that have their origins deep in the Middle and Late Devonian as well as those from a unique Carboniferous

Hyaluronic acid-GPRC5C signalling promotes dormancy in haematopoietic stem cells

Altres ajuts: The Behrens-Weise-Foundation, the German Research Foundation (DFG) under the German Excellence Strategy (CIBSS-EXC-2189, project ID 390939984), SFB1425 (Project #422681845), SFB992 (Project #192904750; B07), SFB1479 (P05); Wellcome-MRC Cambridge Stem Cell Institute (203151/Z/16/Z)Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid-GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs

Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of cell cycle

Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols reliant on culture. However, the kinetics and mechanisms by which this occurs remain incompletely characterized. Here, through time-resolved scRNA-Seq, matched in vivo functional analysis and the use of a reversible in vitro system of early G1 arrest, we define the sequence of transcriptional and functional events occurring during the first ex vivo division of human LT-HSCs. We demonstrate that the sharpest loss of LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limiting global variability in gene expression and transiently upregulating gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programmes in culture. However, contrary to current assumptions, we demonstrate that loss of HSC function ex vivo is independent of cell cycle progression. Finally, we show that targeting LT-HSC adaptation to culture by inhibiting early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrates that controlling early LT-HSC adaptation to ex vivo culture, for example via JAK inhibition, is of critical importance to improve HSC gene therapy and expansion protocols

Life Histories, Salinity Zones, and Sublethal Contributions of Contaminants to Pelagic Fish Declines Illustrated with a Case Study of San Francisco Estuary, California, USA

Human effects on estuaries are often associated with major decreases in abundance of aquatic species. However, remediation priorities are difficult to identify when declines result from multiple stressors with interacting sublethal effects. The San Francisco Estuary offers a useful case study of the potential role of contaminants in declines of organisms because the waters of its delta chronically violate legal water quality standards; however, direct effects of contaminants on fish species are rarely observed. Lack of direct lethality in the field has prevented consensus that contaminants may be one of the major drivers of coincident but unexplained declines of fishes with differing life histories and habitats (anadromous, brackish, and freshwater). Our review of available evidence indicates that examining the effects of contaminants and other stressors on specific life stages in different seasons and salinity zones of the estuary is critical to identifying how several interacting stressors could contribute to a general syndrome of declines. Moreover, warming water temperatures of the magnitude projected by climate models increase metabolic rates of ectotherms, and can hasten elimination of some contaminants. However, for other pollutants, concurrent increases in respiratory rate or food intake result in higher doses per unit time without changes in the contaminant concentrations in the water. Food limitation and energetic costs of osmoregulating under altered salinities further limit the amount of energy available to fish; this energy must be redirected from growth and reproduction toward pollutant avoidance, enzymatic detoxification, or elimination. Because all of these processes require energy, bioenergetics methods are promising for evaluating effects of sublethal contaminants in the presence of other stressors, and for informing remediation. Predictive models that evaluate the direct and indirect effects of contaminants will be possible when data become available on energetic costs of exposure to contaminants given simultaneous exposure to non-contaminant stressors