A combination of hematopoietic stem cell (HSC)- and endothelial cell (EC)-lineage-tracing mouse models enables us to determine blood cell origins. We present a protocol to induce cell labeling in vivo and to trace labeled hematopoietic cells to segregate their origins. We describe the steps for harvesting various hematopoietic tissues, antibody staining, and analyzing the Tomato+ percentages within each immune cell population. We also show how to estimate HSC- and EC-derived percentages of the target cell populations. For complete details on the use and execution of this protocol, please refer to Kobayashi et al.1.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.