Hematopoietic stem and progenitor cell fate decisions during fetal development

Rare multipotent hematopoietic stem cells (HSCs) are responsible for the replenishment of blood cells throughout life. In the maturation process, from primitive HSCs to peripheral blood cells, the intermediate cells pass through multiple steps of increasingly lineage-restricted progenitors and cell fate decisions are made at each step. However, fetal hematopoiesis is more complex and less studied than adult hematopoiesis. The aim of this thesis was to further characterize and compare fetal, neonatal and adult mouse hematopoiesis including lineage-biased HSCs.

Single HSC transplantation studies have previously demonstrated that adult mouse HSCs are heterogeneous in their ability to replenish different blood cell lineages upon transplantation. It remains unclear to what degree the adult patterns of lineage-bias and restriction exist during fetal development.

In Study I, we investigated the potential lineage-bias of mouse HSCs from liver and bone marrow at the time of birth. Perinatal HSCs demonstrated less platelet (P)- and platelet-erythroid-myeloid (PEM)-bias/restriction and more consistent lymphoid reconstitution than adult HSCs. However, the development of P- and PEM-bias/restriction had already begun in perinatal HSCs in a distinct HSC subset expressing von Willebrand factor (Vwf-reporter+ HSCs). We compared the transcriptional landscapes of Vwf-reporter+ and Vwf-reporter liver perinatal HSCs through single-cell RNA sequencing and identified transcriptional lineage priming corresponding to their lineage bias upon transplantation. The study provides new insights into the blood reconstitution potential of HSCs at birth.

Platelets are critical for hemostasis and play a role in both the innate and adaptive immune systems. Previous studies have implied the presence of an alternative, faster differentiation pathway for platelet production, however it has not been convincingly shown that such an alternative pathway can originate from true HSCs.

In Study II, we demonstrated a non-hierarchical relationship between two different stem cell types in adult mouse bone marrow, Vwf-reporter multi-HSCs reconstituting all blood cell lineages and Vwf-reporter+ P-HSCs primarily reconstituting platelets. Single-cell RNA sequencing confirmed that platelet- restricted progenitors originating from Vwf-reporter+ P-HSCs are molecularly different from Vwf-reporter- multi-HSCs. Vwf-reporter- multi-HSCs use a slower multipotent differentiation pathway, whereas Vwf-reporter+ P-HSCs use an alternative and faster platelet-restricted pathway. After hematopoietic challenges by chemotherapy, the faster platelet-restricted pathway was activated for platelet replenishment. These discoveries could be of importance for the development of new treatments aimed at stimulating platelet recovery in clinical settings.

B-cell development has been most extensively studied in mice, where the timing and anatomical location of the first embryonic hematopoietic cells with B- lymphocyte potential have been established. However, these cells represent multipotent stem or progenitor cells. In contrast, the timing, location, and properties of the first fetal B-cell restricted progenitors have yet to be determined.

In Study III, we demonstrated that expression of Mb1 describes an earlier fetal stage of CD19- B-cell restricted progenitors than previously reported. These progenitors emerge at embryonic day 12.5 in the fetal liver and are molecularly distinct from subsequent stages of fetal B progenitor cells. Mini-bulk RNA sequencing revealed that the previously defined fetal liver and adult bone marrow PreProB cells can be subdivided by Mb1 expression. Progenitors expressing Mb1 have activated a transcriptional program compatible with B-cell specification which is not initiated in PreProB cells lacking Mb1 expression. These findings are relevant for understanding potential cellular targets and leukemia-initiating cells in the development of infant and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

List of scientific papers

I. Platelet and myeloid lineage biases of transplanted single perinatal mouse hematopoietic stem cells. Karin Belander Strålin*, Joana Carrelha*, Axel Winroth, Christoph Ziegenhain, Michael Hagemann-Jensen, Laura M. Kettyle, Amy Hillen, Kari Högstrand, Ellen Markljung, Francesca Grasso, Masafumi Seki, Stefania Mazzi, Yiran Meng, Bishan Wu, Edwin Chari, Madeleine Lehander, Rickard Sandberg, Petter S. Woll, Sten Eirik W. Jacobsen. Cell Research 2023;33(11):883-886. https://doi.org/10.1038/s41422-023-00866-4

II. Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells. Joana Carrelha*, Stefania Mazzi*, Axel Winroth*, Michael Hagemann-Jensen, Christoph Ziegenhain, Kari Högstrand, Masafumi Seki, Margs S. Brennan, Madeleine Lehander, Bishan Wu, Yiran Meng, Ellen Markljung, Ruggiero Norfo, Hisashi Ishida, Karin Belander Strålin, Francesca Grasso, Christina Simoglou Karali, Affaf Aliouat, Amy Hillen, Edwin Chari, Kimberly Siletti, Supat Thongjuea, Adam J. Mead, Sten Linnarsson, Claus Nerlov, Rickard Sandberg, Tetsuichi Yoshizato, Petter S. Woll, Sten Eirik W. Jacobsen. Nature Immunology 2024;25(6):1007-1019. https://doi.org/10.1038/s41590-024-01845-6

III. Identification of a B-cell restricted progenitor emerging early in fetal development. Karin Belander Strålin*, Masafumi Seki*, Joanna C. A. Green, Stephen Loughran, Kari Högstrand, Ellen Markljung, Axel Winroth Amy Hillen, Edwin Chari, Charlotta Boiers, Emanuele Azzoni, Joana Carrelha, Tetsuichi Yoshizato, Petter S. Woll, Sten Eirik W. Jacobsen. [Submitted]

*Equal contribution