Small Number Of Hematopoietic Stem Cells Research Articles

3133 - Characterizing the Developmental Origins of the Adult Blood System

Blood production in the adult mouse is sustained by long-lived progenitors thought to arise from hematopoietic stem cells (HSCs) some time during development. HSCs are themselves born in the large arteries of the mouse embryo, after which they mature and expand in other developmental niches. These large arteries (aorta, umbilical, and vitelline) are characterized by cell clusters that contain a small number of HSCs that go on to establish the entire adult blood system. Very little is known about the composition of these clusters–the number of HSCs they harbor, their clonal contribution to adult blood, and the identity and fate of the other cluster cells. Here, we utilize a combination of transposon barcoding and Flt3-CreER lineage tracing to characterize the contribution of embryonic progenitors to adult blood. In contrast to the notion that all cells are derived from HSCs, we identify a population of long-lived progenitors that emerge within hematopoietic clusters and that significantly contribute to adult blood. Finally, we provide estimates of the number of embryonic progenitors throughout development that ultimately comprise the adult blood system.

A metabolic map of hematopoietic stem cells

Background A general problem in biology is whether different types of cells in the same tissue are metabolically different from each other, and whether such differences are important for cellular function. Experiments that can comprehensively measure the cellular metabolome typically require millions of cultured cells and cannot be used with small numbers of rare cells freshly isolated from tissues. In particular, hematopoietic stem cells (HSCs), the blood forming stem cells of the bone marrow, have been intensively studied for decades but their metabolic composition is largely unknown. Results We have developed a method to measure metabolites in small numbers of HSCs. The HSC isolation and mass spectrometry methods have been optimized for maintenance of the metabolome during purification, sensitivity and robustness. About 50 metabolites can be quantified from 10,000 HSCs, covering a wide spectrum of the cellular metabolome. Several metabolic differences exist between HSCs and other bone marrow cells, including restricted progenitors. I am investigating the role of these metabolic adaptations in HSCs. Conclusion

Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment

The vast majority of hematopoietic stem cells (HSCs) reside in specialized niches within the bone marrow during steady state, maintaining lifelong blood cell production. A small number of HSCs normally traffic throughout the body; however, exogenous stimuli can enhance their release from the niche and entry into the peripheral circulation. This process, termed mobilization, has become the primary means to acquire a stem cell graft for hematopoietic transplant at most transplant centers. Currently, the preferred method of HSC mobilization for subsequent transplantation is treatment of the donor with granulocyte colony-stimulating factor. The mobilizing effect of granulocyte colony-stimulating factor is not completely understood, but recent studies suggest that its capacity to mobilize HSCs, at least in part, is a consequence of alterations to the hematopoietic niche. The present article reviews some of the key mechanisms mediating HSC mobilization, highlighting recent advances and controversies in the field.

Long-Term, Cytokine-Free Ex Vivo Expansion of Human Cord Blood Mononuclear Cells Using a Novel Closed-Loop 3D Dual Hollow Fibre Perfused Bioreactor

AbstractAbstract 828Human umbilical cord blood (CB) is a good source of hematopoietic stem cells (HSCs), but its use is limited by the low volume of the product and the relatively small number of HSCs. Conventional attempts at ex vivo expansion of HSCs and other hematopoietic cells have used two-dimensional (2D) tissue culture flasks or well-plates, which require feeder cells and/or various combinations of cytokines for long-term hematopoiesis. These conditions differ drastically from that provided by the natural bone marrow (BM) microenvironment wherein a complex network within the stem cell niche modulates self-renewal and pluripotency. Moreover 2D cultures have other limitations including lack of mixing, critical concentration gradients for pH, dissolved oxygen, cytokines, nutrients and metabolites, and the low number of cells that can be supported by a given surface area. Previously, we have shown that CB mononuclear cells (MNCs) can be expanded in a three dimensional (3D) static culture in cytokine-free medium for 4 weeks with a total expansion of 53-fold using polyurethane (PU) scaffolds coated with collagen type I. We hypothesized that combining these scaffolds with a perfused dual hollow fibre bioreactor (DHFB) would form a functional automatable BM biomimicry that could sustain long-term hematopoiesis and enhance the expansion of CBMNCs in a cytokine-free environment. The bioreactor includes a) a selective ceramic hollow fibre membrane system, which enables fast-flow perfusion with fresh medium for the supply of nutrients and removal of metabolites, b) a slow-flow, selectively-permeable membrane system for the supply of cytokines and the continuous harvest of mature red blood cells (RBCs) and, c) a PU scaffold rendered bioactive by collagen coating which provides a 3D in vitro culture system mimicking the natural BM microenvironment. CBMNCs were isolated using Ficoll-Paque density centrifiugation and seeded in the bioreactor at a density of 1.6×107cells/cm3. The DHFB was perfused at a flow rate of 160 ml/day in recirculation mode in order to allow for the accumulation of products. Nutrients and metabolites were monitored in the cell culture including glucose, glutamine, glutamate, lactate and ammonia and, the data used to perfuse the DHFB at optimal conditions. The hollow-fibre membranes allowed the selective mass transport of nutrients (glucose), metabolites (lactate), proteins (albumin) as well as RBCs. CBMNCs were cultured without the addition of exogenous cytokines for 3 weeks inside the DHFB, within which time cells were expanded by 300-fold, compared with 53-fold in 3D static culture correlates. Immunophenotypic analysis of extracted cells after 3 weeks showed an increase of CD71+CD45- erythroid progenitors as well as a maintained CD34+ hematopoietic progenitor cell population. Within the perfused DHFB, cells migrated into the pores of the scaffold establishing “niche-like” areas of growth identified by scanning electron microscopy, similar to those observed in static 3D culture conditions. After the 3D DHFB cultures had established, we “flexed” the system with the addition of 100ng/ml stem cell factor (SCF) and 2U/ml erythropoietin (EPO) in order to expand and mature erythrocytes and attempt selective harvesting via the closed-loop automated DHFB system. With this cocktail, mature enucleated RBCs were observed exiting the scaffold through the selectively permeable membrane, whilst other cell types and progenitors were maintained within the scaffold. This observation was confirmed by surface immunophenotype of the cells collected in the harvesting system. In conclusion, we have shown that the 3D DHFB system allowed for higher cytokine-free expansion of CBMNCs than that observed in the static 3D correlate, with evidence of directed differentiation of HSCs into erythroid progenitors in the absence of exogenous cytokines. This expansion and selective harvesting of RBCs was accomplished in a closed-loop system with monitoring and control optimisation of nutrient-waste exchange. The erythroid maturation and selective harvesting of RBCs was enhanced by the addition of SCF and EPO to the culture system. This technology enables a functional BM biomimicry and could be used as a tool to study the hematopoietic microenvironment in 3D ex vivo and for the eventual expansion of hematopoietic cell components for therapeutic use. Disclosures:No relevant conflicts of interest to declare.

BMP4: its role in development of the hematopoietic system and potential as a hematopoietic growth factor.

Blood formation occurs throughout the life of an individual in a process driven by hematopoietic stem cells (HSCs). The ability of bone marrow (BM) and cord blood (CB) HSC to undergo self-renewal and develop into multiple blood lineages has made these cells an important clinical resource. Transplantation with BM- and CB-derived HSCs is now used extensively for treatment of hematological disorders, malignancies, and immunodeficiencies. An understanding of the embryonic origin of HSC and the factors regulating their generation and expansion in vivo will provide important information for the manipulation of these cells ex vivo. This is critical for the further development of CB transplantation, the potential of which is limited by small numbers of HSC in the donor population. Although the origins of HSCs have become clearer and progress has been made in identifying genes that are critical for the formation and maintenance of HSCs, less is known about the signals that commit specific populations of mesodermal precursors to hematopoietic cell fate. Critical signals acting on these precursor cells are likely to be derived from visceral endoderm in yolk sac and from underlying stroma in the aorta-gonad-mesonephros region. Here we summarize briefly the origin of yolk sac and embryonic HSCs before detailing evidence that bone morphogenic protein-4 (BMP4) has a crucial role in Xenopus and mammalian HSC development. We discuss evidence that BMP4 acts as a hematopoietic growth factor and review its potential to modulate HSC in ex vivo expansion cultures from cord blood.