Heterogeneity and aging of the hematopoietic stem cells

Stem cells are necessary for generating all cells in the body and are defined by two unique properties. First, their multilineage differentiation potential and second, their self-renewal ability.

The hematopoietic stem cells (HSCs) are a rare population of cells that generate all blood cells in the human body and sit at the top of the hematopoietic hierarchy. However, the exact underlying molecular mechanisms that regulate their behavior remain partially obscure, which is a crucial part to elucidating HSC behavior. Previously, it was thought that the HSC population was homogeneous and generated all blood cells without any preference. However, this was disproved by various subsequent studies showing the presence of functionally different HSCs in the HSC pool. This included myeloid-biased (M-bi) HSCs, lymphoid-biased (L- bi) HSCs, and platelet-biased HSCs which show specific lineage differentiation preferences; as well as HSCs with a balanced output. Currently, the molecular mechanisms differentiating lineage-biased HSCs are unclear because they could not be prospectively isolated since the exact immunophenotypic definition of lineage-biased HSCs is not well characterized. As HSCs cannot be characterized based on cell surface markers, transplantation assays have to be done to confirm their long-term (LT) self-renewal and multilineage capability; highlighting the need to identify and understand the factors that discriminate them.

Study I showed that CD49b is a reliable marker to further separate the conventional LT-HSC population into subpopulations that enrich for either the CD49b- M-bi or the CD49b+ L-bi HSCs. The two CD49b HSC populations differ in their chromatin accessibility even though gene expression comparisons showed no differences. This suggested that different HSCs are already primed to a preferred blood lineage before differentiation. The enrichment of lineage-biased HSCs in both CD49b populations demonstrated the usefulness of adding cell surface markers, though both CD49b subsets remain heterogeneous.

Since residual heterogeneity in the HSC pool was observed in both CD49b subsets from study I, we attempted to further purify the functional LT-HSC population by including CD229 as a cell surface marker in study II. A multipotent progenitor (MPP) population was identified when both CD49b and CD229 were used in combination to subfractionate the LT-HSC pool. This showed that the addition of CD229 can help further purify the heterogeneous HSC population by eliminating the MPPs from the conventional LT-HSC population.

Additional challenges in HSC studies, on top of a heterogeneous HSC population, is the reduction in hematopoietic system function with age, which leads to an overall decrease in HSC fitness and a higher chance of developing hematopoietic malignancies. One of the characteristics of an aging hematopoietic system is the increase in M-bi HSCs and a decrease in L-bi HSCs, but the underlying cause for such a shift in the HSC pool remains unclear.

Therefore, to identify key regulators in directing HSC lineage bias and changes in HSCs with age, we studied the aging of CD49b HSC subpopulations in study III. From the in vivo studies, it was found that with age, both CD49b HSC subsets become more myeloid-biased. From the molecular studies, it was found that chromatin accessibility increases with age, that the additional accessibility gained in old age was de novo, and this gain of chromatin accessibility progresses through the adult age. Furthermore, it was suggested that the same transcriptional factor might be regulating both HSC aging and lineage bias.

Taken together, studies I-III have shown that by adding cell surface markers in HSC selection, the HSC pool can be further purified and HSC subsets enriching for different lineage biases can be identified. Even though the subsets cannot be distinguished on the transcriptomic level, they show chromatin accessibility differences. In addition, we have found a potential transcription factor that is responsible for both HSC aging and lineage bias. These studies, taken together, not only help the field in understanding more about HSCs, but also provide the basis for future translational studies and therapeutic research.

List of scientific papers

I. Ece Somuncular, Julia Hauenstein, Prajakta Khalkar, Anne-Sofie Johansson, Özge Dumral, Nicolai S. Frengen, Charlotte Gustafsson, Giuseppe Mocci, Tsu-Yi Su, Hugo Brouwer, Christine L. Trautmann, Michael Vanlandewijck, Stuart H. Orkin, Robert Månsson, and Sidinh Luc. CD49b identifies functionally and epigenetically distinct subsets of lineage-biased hematopoietic stem cells. Stem Cell Reports. 2022 Jul 12;17(7). https://doi.org/10.1016/j.stemcr.2022.05.014

II. Ece Somuncular*, Tsu-Yi Su*, Özge Dumral, Anne-Sofie Johansson, and Sidinh Luc. Combination of CD49b and CD229 reveals a subset of multipotent progenitors with short-term activity within the hematopoietic stem cell compartment. Stem Cells Transl Med. 2023 Nov 3;12(11). https://doi.org/10.1093/stcltm/szad057

III. Tsu-Yi Su*, Julia Hauenstein*, Ece Somuncular*, Özge Dumral, Elory Leonard, Charlotte Gustafsson, Efthymios Tzortzis, Aurora Forlani, Anne-Sofie Johansson, Hong Qian, Robert Månsson, and Sidinh Luc. Aging is associated with functional and molecular changes in distinct hematopoietic stem cell subsets. Nat Commun. 2024 Sep 11;15(1):7966. https://doi.org/10.1038/s41467-024-52318-1

*Equal contribution