Learning from early waves of hematopoietic precursors

Science Spotlight

Learning from early waves of hematopoietic precursors

from the Bernstein lab, Clinical Research Division

July 17, 2017
The figure represent the model proposed by the authors that Hematopoiesis from hematopoietic stem cells isolated from embryo between days 9 and 11.5 could happen in multiple waves contributing to B-1a B Cells. Endothelial cells from the embryonic vessels (hemogenic endothelial cells) give rise to early multilineage progenitors producing erythroid, myeloid, and innate-immune lymphoid. Next, the early pre-HSCs produce more developmentally restricted HSCs with innate-immune biased lymphoid potential. Finally,  late pre-HSCs lead to the apparition of the fetal HSCs with a limited B-1a potential eventually lost in the adult bone marrow.

Hematopoiesis from hematopoietic stem cells isolated from embryo between days 9 and 11.5 could happen in multiple waves contributing to B-1a B Cells. Endothelial cells from the embryonic vessels (hemogenic endothelial cells) give rise to early multilineage progenitors producing erythroid, myeloid, and innate-immune lymphoid. Next, the early pre-HSCs produce more developmentally restricted HSCs with innate-immune biased lymphoid potential. Finally, late pre-HSCs lead to the apparition of the fetal HSCs with a limited B-1a potential eventually lost in the adult bone marrow.

Figure provided by Dr. Brandon Hadland.

Hematopoietic stem cells (HSCs) are undifferentiated cells whose progeny reconstitute blood cells lineages such as monocytes/macrophages or T and B lymphocytes through a process called hematopoiesis. HSCs possess an indefinite self-renewal potential explaining the interest in these cells for transplantation for the sustained reconstitution of blood cells. B cells are a type of lymphocytes responsible for the humoral immunity (immunity mediated by antibodies). Several subtypes of B cells have been identified such B1 or B2 cells. B1 cells generally produce low-affinity antibodies with broad reactivity against their antigens, while B2 cells produce higher affinity antibodies with more restricted reactivity.

Several sources of hematopoietic stem cells are available for transplantation such as adult bone marrow, umbilical cord blood, or fetal liver cells. However, adult cells do not contribute significantly to the B1 cell lineage involved in innate immune response, accentuating the need to identify and better characterize other sources of HSCs. A recent study published in Stem Cell Reports from Drs. Brandon Hadland and Irwin Bernstein (Clinical Research Division) investigated the potential of hematopoietic stem cells from early embryonic stages to reconstitute the B cell repertoire. Previous studies from the group identified a niche of endothelial cells (EC) derived from the mouse embryo aorta-gonad-mesophrenos region as capable of supporting the ex vivo development of HSCs with long-term and multi-lineage engraftment potential. The authors developed a transgenic mouse model expressing the Zs-Green fluorochrome inserted in the Fgd5 locus, to facilitate the isolation of both endothelial and hematopoietic stem cells from this niche. Cells of interest were then isolated by flow cytometry and grew in culture for 5 days after which, one half was used for phenotypic characterization by flow cytometry while the other half was transplanted for in vivo engraftment assessment in lethally irradiated mice.

Twelve to sixteen weeks post-transplant, the analysis of engrafted cells in the peripheral blood and bone marrow indicated that hematopoietic cells with a VE-cadherin- Gr1-F4/80-Sca1+EPCR+Fgd5-ZsGr+ phenotype provided long-term engraftment as well as robust growth ex vivo, while other cells did not engraft and failed to form colonies in ex vivo culture. This was confirmed by secondary transplants, a procedure in which bone marrow HSCs were harvested from the primary transplanted mice between 12 and 16 weeks after transplant and transplanted into another mouse for further engraftment assessment. Serial transplantation and hematopoietic reconstitution is the gold standard for HSC function.

HSCs isolated from embryos at day 11.5 (E11.5) gave rise to a complete B cell repertoire constituted of B1a, B1b and B2 cells in the peritoneum and in the spleen, two anatomic locations containing immune cell populations crucial for immune response. As a comparison, the same experiment performed with purified HSCs from adult bone marrow did not reconstitute such a complete B cells lineage as B1a cells were poorly detected in the peritoneum. HSCs isolated from E9.5 stage also gave rise to hematopoietic cells providing long-term and multilineage engraftment but were less homogenous and with a lower self-renewal potential compared with the E11.5 stage. These cells also reconstituted a complete B cell lineage similar to the E11.5 cells but with a higher tendency to develop B1a cells while more B2 cells arose from E11.5 stages cells. These data support a model in which HSCs potential evolves in waves as cells collected at different stages of the embryonic development present variations in their potential for hematopoietic differentiation. However, as the cells mature they lose the ability to reconstitute these lineages.

Interestingly, isolated pre-HSCs presented some heterogeneity in their phenotype, ability to engraft in vivo and self-renewal potential leading to variability in engraftment efficiencies.  This suggests that not all cells possess the potential to engraft and differentiate. To allow identification of these cells the authors characterized two cell surface markers: EPCR, a marker previously identified for the characterization of 30-50% of fetal and adult HSCs, and the Notch ligand D114 (Delta-like-4). Whereas precursors with HSC potential expressed both Dll4 and high levels of EPCR, those expressing high levels of EPCR but low level of D114 lack the HSC potential. This was confirmed in vivo by an increased engraftment at 16 weeks post-transplant when using D114+ cells relative to D114- hematopoietic precursors.

“Generating true long-term repopulating HSC from human pluripotent stem cells has been a central but elusive challenge in the field.  To solve this, we are focused on identifying the physiologic signals required for embryonic HSC development, which can in turn be applied to generate HSC from human PSC-derived precursors for therapeutic applications.  In this study, we utilized a novel approach to interrogate, at the single cell level, the unique phenotypic and functional properties of embryonic precursors to HSC, providing new insights into HSC development.  We are currently extending this approach to also study the transcriptional profiles of individual HSC precursors by single cell RNA-sequencing, in order to identify signaling pathways and gene regulatory networks that drive HSC development. Moving forward, we expect that these studies will inform methods to generate HSC de novo from patient-specific pluripotent stem cells, which can be used for a variety of clinical applications including disease modeling, drug discovery, and gene and cell therapies for benign and malignant hematologic disorders.”, concluded Drs. Hadland and Bernstein.

 

Funding for this study was provided by the National Institute of Allergy and Infectious Diseases (NIAID-NIH), National Heart, Lung, and Blood Institute (NHLBI-NIH) and by the Alex’s Lemonade Stand Foundation and Hyundai Hope on Wheels Foundation.

 

Hadland BK, Varnum-Finney B, Mandal PK, Rossi DJ, Poulos MG, Butler JM, Rafii S, Yoder MC, Yoshimoto M, Bernstein ID. 2017. A common origin for B-1a and B-2 lymphocytes in clonal pre-hematopoietic stem cells. Stem cell Reports. 6;8(6):1563-1572.