A Senescent Subset in Human Hematopoietic Stem Cell (HSC) Compartment As Target for Senotherapy

Background: In mouse model for aging studies, clearance of senescent cells in the hematopoietic stem cell (HSC) compartment has been shown to rejuvenate the hematopoietic system. To identify the contribution of senescent cells to aging of human hematopoiesis, we have performed a direct comparison of aged and young human hematopoietic stem and progenitor cells (HSPCs). In a subsequent step, we compared the alterations in transcriptome profiles upon aging between humans and mice to determine the mechanistic differences in the aging process between the two species.

Methods:Humanspecimens: HSPCs (CD34+) were isolated from the bone marrow of healthy human subjects (old = 59 to 74 years, n=7; young = 21 to 31 years, n=5). Mousespecimens: Bone marrow cells were harvested from 10 young (6 to 8 weeks old) and 10 aged (100 weeks old) mice. Murine hematopoietic stem cells (HSCs) were isolated via flow cytometry sorting (characterized as Lin- Kit+ Sca1+ CD150+ cells). Single cell transcriptome studies of these specimens were performed, followed by functional clustering and developmental trajectory analyses.

Results: We have identified two novel subsets of HSPCs in aged human subjects. A cluster, designated as multipotent progenitor 2A (MPP2A), is found in the early phase of HSC aging in old subjects, and another cluster “PreB_X” downstream in the lymphoid trajectory. The MPP2A cluster is highly enriched in senescence signature, as demonstrated by their transcriptome profile showing increased telomere attrition, DNA damage, activation of P53 pathway and cell cycle arrest, with significant elevations of the following genes: ATF3, CDKN1A, CLU, FOSB, ID1, JUN, MAFF, PLK3. In mouse model, the accumulation of an analogous subset with senescence signature (MPP2A analogs) was confirmed in the aged LT-HSC population, with significantly increased expressions of Atf3, Clu, Fosb, Id1, Jun, Plk3. Elimination of this subset has been shown to rejuvenate hematopoiesis in the same animal model. Remarkable is a consistent and significant upregulation of P53 pathway, formation of senescence-associated heterochromatin foci (SAHF), and increased expression of CDKN1A in aged human HSC compartment, and specifically in the MPP2A cluster. All these results indicate an activation of the P53-P21^WAF1/CIP1 pathway as a main mechanism for cell cycle arrest in human senescent HSCs. This is in contrast to the findings in mouse model, where increased levels of Cdkn2a, Cdkn2b, and activation of the p16^INK4a pathway represent the hallmarks of senescence. Applying our analysis algorithm and parameters, we were able to validate an accumulation of MPP2A analogous subset upon aging in other human HSC datasets published in the literature.

Conclusion: Aging in human HSC compartment is mainly caused by the clonal evolution and accumulation of a senescent cell cluster MPP2A. The latter is characterized by increased telomere attrition, cell cycle arrest, and a remarkable up-regulation of CDKN1A. The identification of a senescent subset in human HSC compartment, in analogy to a similar population in LT-HSC in mice indicate that clearance of this senescent population with senotherapy is feasible and potentially beneficial. For humans, however, the P53-P21^CIP1 positive cells, and not the p16INK4a positive cells as in mice, represent the main target.

Zhou:Dialectic Therapeutics: Other: Co-founder and shareholder of Dialectic Therapeutics, a company that is developing Bcl-xL PROTACs to treat cancers; Co-inventor of the Bcl-xL PROTACs disclosed in this study.