Capturing the first haematopoietic stem cell: the needle in the haystack

The most powerful cell in the blood differentiation hierarchy is the haematopoietic stem cell (HSC). It is the only cell capable of building an entire haematopoietic system from scratch, i.e. long-term (LT) repopulating a multilineage blood system within a lethally irradiated recipient. Though primitive blood is generated at early embryonic stages, it is only from embryonic day (E)10.5 of mouse development (4-5 weeks in human gestation) onwards that the definitive adult haematopoietic system is established with the formation of LT-HSCs. These first HSCs emerge in small ventral intra-aortic haematopoietic clusters (IAHCs) in the aorta-gonad-mesonephros (AGM) region. Like with many systems, a tight regulation of gene expression by master transcription factors (TF) is what ultimately drives cell fate change. Gata2 is one such TF known to play a crucial role in mouse definitive haematopoiesis. Through the work of de Pater et al. (2013) (Chapter 1) it became clear that Gata2 is both required for the generation of the very first HSCs and also, unlike other key TFs like Runx1, the survival of HSCs. The earliest HSCs are generated through an endothelial-to-haematopoietic transition (EHT), a process where flat arterial haemogenic endothelial cells (HECs) round up into IAHCs, containing HSCs. Solaimani Kartalaei et al. (2015) (Chapter 4) discovered that Gpr56 is one of the most highly upregulated genes during EHT (in mouse) and that it is essential for HSC generation (in zebrafish). Gpr56 is also a target of the key haematopoietic ‘heptad’ TFs in both mouse and human blood progenitors. To examine another signalling pathway required in the embryo for HSC generation, we used a BMP responsive element (BRE)-GFP transgenic mouse to show that all AGM HSCs are BMP-activated (Crisan et al., 2015) (Chapter 6). At slightly later stages, in the foetal liver and bone marrow, HSC heterogeneity starts to appear with the presence of genetically distinct BMP- and non-BMP activated HSCs. Given the importance of the ‘heptad’ TFs in establishing HSC identity, we next developed a novel Gata2-Venus (G2V) reporter mouse to isolate and examine the dynamics and function of live Gata2-expressing and non-expressing cells (Kaimakis et al., 2016) (Chapter 2). Gata2 is expressed in all HSCs, however haematopoietic progenitors can be generated either Gata2-dependently or -independently, the latter being less potent and genetically distinct from the first. With Eich et al. (2018) (Chapter 3) we went further and deciphered the role of Gata2 in haematopoietic fate establishment. By using time-lapse imaging of E10.5 live aortic sections of G2V embryos, we discovered rapid pulsatile level changes in Gata2 expression, specifically in those single cells undergoing EHT. This finding indicated the highly unstable genetic state of individual cells undergoing fate change. Furthermore, aortic cells haploinsufficient for Gata2 show many fewer pulsatile and EHT events, emphasizing the importance of Gata2 levels in this process. Having defined the specific (medium) levels of Gata2(Venus) protein in the population containing the first HSCs, we used our G2V model to isolate a pure population of HSCs as they are generated for the first time in development (Vink et al., 2020) (Chapter 5). Through iterations of index-sorting, single-cell transcriptomics and functional analyses we were able to greatly enrich for HSCs (~70x compared to Eich et al. (2018)). Based on refined CD31, cKit and CD27 expression and specific physical parameters we isolated the first cells with an HSC identity and identified their precise gene expression profile. Immunohistochemistry localized these HSCs to only the smallest IAHCs. The combined effort of my research and novel tools generated to facilitate the creation of this body of work/portfolio have now led us for the first time to study the one or two cells per embryo that harbour HSC identity at the developmental time when they are beginning to be made. Now that we have cracked the genetic code of the true HSC, the field is one step closer to translating these findings and applying them to producing human clinically relevant HSCs, which will ultimately improve transplantation therapies and benefit the fight against leukaemia

Embryonic Origins of the Hematopoietic System: Hierarchies and Heterogeneity

The hierarchical framework of the adult blood system as we know it from current medical and hematology textbooks, displays a linear branching network of dividing and differentiated cells essential for the growth and maintenance of the healthy organism. This view of the hierarchy has evolved over the last 75 years. An amazing increase in cellular complexity has been realized; however, innovative single-cell technologies continue to uncover essential cell types and functions in animal models and the human blood system. The most potent cell of the hematopoietic hierarchy is the hematopoietic stem cell. Stem cells for adult tissues are the long-lived self-renewing cellular component, which ensure that differentiated tissue-specific cells are maintained and replaced through the entire adult lifespan. Although much blood research is focused on hematopoietic tissue homeostasis, replacement and regeneration during adult life, embryological studies have widened and enriched our understanding of additional developmental hierarchies and interacting cells of this life-sustaining tissue. Here, we review the current state of knowledge of the hierarchical organization and the vast heterogeneity of the hematopoietic system from embryonic to adult stages

On The Maximum Mass of Stellar Black Holes

We present the spectrum of compact object masses: neutron stars and black holes that originate from single stars in different environments. In particular, we calculate the dependence of maximum black hole mass on metallicity and on some specific wind mass loss rates (e.g., Hurley et al. and Vink et al.). Our calculations show that the highest mass black holes observed in the Galaxy M_bh = 15 Msun in the high metallicity environment (Z=Zsun=0.02) can be explained with stellar models and the wind mass loss rates adopted here. To reach this result we had to set Luminous Blue Variable mass loss rates at the level of about 0.0001 Msun/yr and to employ metallicity dependent Wolf-Rayet winds. With such winds, calibrated on Galactic black hole mass measurements, the maximum black hole mass obtained for moderate metallicity (Z=0.3 Zsun=0.006) is M_bh,max = 30 Msun. This is a rather striking finding as the mass of the most massive known stellar black hole is M_bh = 23-34 Msun and, in fact, it is located in a small star forming galaxy with moderate metallicity. We find that in the very low (globular cluster-like) metallicity environment the maximum black hole mass can be as high as M_bh,max = 80 Msun (Z=0.01 Zsun=0.0002). It is interesting to note that X-ray luminosity from Eddington limited accretion onto an 80 Msun black hole is of the order of about 10^40 erg/s and is comparable to luminosities of some known ULXs. We emphasize that our results were obtained for single stars only and that binary interactions may alter these maximum black hole masses (e.g., accretion from a close companion). This is strictly a proof-of-principle study which demonstrates that stellar models can naturally explain even the most massive known stellar black holes.Comment: 15 pages, ApJ accepte

Development and evaluation of digital twins for district-level heating energy demand simulation.

To achieve the aim of a CO2 neutral built environment in 2050, a large part of the existing housing stock will have to be energetically retrofitted. It has been noted that a neighbourhood-oriented approach will be necessary for the feasibility, affordability and timeliness of this aim. Considering that many different stakeholders are involved in renovations at the neighbourhood level, and that multiple neighbourhoods will have to be retrofitted at the same time, efficient working methods are imperative. To facilitate the design, construction and operation of the new energy infrastructure, a prototype for a digital environment (digital twin) is developed for four Dutch pilot neighbourhoods. In this contribution, the authors will describe a procedure to convert publicly available geo-information to a CityGML model, which is used to simulate the monthly and annual space heating energy demand using SimStadt. To assess model fidelity, the simulation results are compared with publicly available aggregated energy use data. A procedure will be described to split the measured natural gas use into gas usage for space heating, domestic hot water and cooking. It is found that the simulation tends to overestimate the energy demand for space heating by 4 - 125%. This difference is largely explained by the manner in which the thermal properties of the buildings are estimated. In addition, the homogeneity of the neighbourhood in terms of the different building functions present has an impact on the accuracy of the simulation. Finally, possible invalid assumptions concerning setpoint temperatures and internal heating loads are of interest. It is concluded that more accurate simulation results will be obtained through the use of current input data. Most importantly: (i) reliable information on the buildings’ current thermal properties through e.g. energy audits, and (ii) reliable information on the buildings’ setpoint temperatures and internal heating loads through on-board monitoring systems

Subregional localization and characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head

Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Gen

Modeling Personalized Adjuvant TreaTment in EaRly stage coloN cancer (PATTERN)

Aim: To develop a decision model for the population-level evaluation of strategies to improve the selection of stage II colon cancer (CC) patients who benefit from adjuvant chemotherapy. Methods: A Markov cohort model with a one-month cycle length and a lifelong time horizon was developed. Five health states were included; diagnosis, 90-day mortality, death other causes, recurrence and CC death. Data from the Netherlands Cancer Registry were used to parameterize the model. Transition probabilities were estimated using parametric survival models including relevant clinical and pathological covariates. Subsequently, biomarker status was implemented using external data. Treatment effect was incorporated using pooled trial data. Model development, data sources used, parameter estimation, and internal and external validation are described in detail. To illustrate the use of the model, three example strategies were evaluated in which allocation of treatment was based on (A) 100% adherence to the Dutch guidelines, (B) observed adherence to guideline recommendations and (C) a biomarker-driven strategy. Results: Overall, the model showed good internal and external validity. Age, tumor growth, tumor sidedness, evaluated lymph nodes, and biomarker status were included as covariates. For the example strategies, the model predicted 83, 87 and 77 CC deaths after 5 years in a cohort of 1000 patients for strategies A, B and C, respectively. Conclusion: This model can be used to evaluate strategies for the allocation of adjuvant chemotherapy in stage II CC patients. In future studies, the model will be used to estimate population-level long-term health gain and cost-effectiveness of biomarker-based selection strategies

Catching Element Formation In The Act

Gamma-ray astronomy explores the most energetic photons in nature to address some of the most pressing puzzles in contemporary astrophysics. It encompasses a wide range of objects and phenomena: stars, supernovae, novae, neutron stars, stellar-mass black holes, nucleosynthesis, the interstellar medium, cosmic rays and relativistic-particle acceleration, and the evolution of galaxies. MeV gamma-rays provide a unique probe of nuclear processes in astronomy, directly measuring radioactive decay, nuclear de-excitation, and positron annihilation. The substantial information carried by gamma-ray photons allows us to see deeper into these objects, the bulk of the power is often emitted at gamma-ray energies, and radioactivity provides a natural physical clock that adds unique information. New science will be driven by time-domain population studies at gamma-ray energies. This science is enabled by next-generation gamma-ray instruments with one to two orders of magnitude better sensitivity, larger sky coverage, and faster cadence than all previous gamma-ray instruments. This transformative capability permits: (a) the accurate identification of the gamma-ray emitting objects and correlations with observations taken at other wavelengths and with other messengers; (b) construction of new gamma-ray maps of the Milky Way and other nearby galaxies where extended regions are distinguished from point sources; and (c) considerable serendipitous science of scarce events -- nearby neutron star mergers, for example. Advances in technology push the performance of new gamma-ray instruments to address a wide set of astrophysical questions.Comment: 14 pages including 3 figure