Page 58 - 2022_02-Haematologica-web

A. Amoah et al.
Aged HSC treated with CASIN at 5 mM or 10 mM were more similar in their contribution to chimerism to young HSC at 8 weeks post transplantation. Aged HSC treated with the higher concentration of CASIN, 10 mM, were able to sustain a significantly lower, young-like contribu- tion to chimerism at 12 weeks post transplantation. This mirrors our finding that aged HSC, treated with 10 mM of CASIN, showed the highest increase in the repolarization of aged HSC (Figure 4F). Assessment of the frequency of human myeloid (CD33+) and B cells (CD19+) in the BM of recipient mice revealed no significant differences among any of the experimental groups (Figure 5C and D) implying that aged human HSC, at least in NBSGW ani- mals, show no differentiation skewing as reported for aged murine HSC.
Colony forming unit assays also revealed the myelo- erythroid potential of aged and CASIN-treated aged HSC were preserved (Online Supplementary Figure S6F to H) which is in line with the report by Pang et al.16
In summary, our xenotransplantation experiments demonstrate that, to our surprise, aged human HSC are able to confer an elevated level of chimerism upon trans- plantation into NBSGW animals, which is likely a result of the elevated activity of Cdc42 in aged HSC, as aged HSC treated with 10 mM of CASIN are more similar to young HSC than to aged HSC.
Discussion
In this study, we characterized aging-related changes in human HSC and tested an intervention to target these aging-related changes. Using a well-established marker panel for the identification of primitive hematopoietic cells in BM, we show that there is an increase in the HSC fre- quency and a decrease in lymphoid progenitors in BM of the sternum of the elderly. We specifically selected the low side scatter CD34+ cells as our reference population in accordance to the guidelines of the International Society of Hematotherapy and Graft Engineering (ISHAGE).35 Additionally, the gating of low side scatter CD34+ cells minimizes variation across labs thereby increasing repro- ducibility and reliability.35 It is a possibility that the dispar- ities in previous reports on changes in the frequency of human HSC15,16 may result from the use of different refer- ence populations. Our data also demonstrates a delayed entry of aged HSC into division, an observation similarly made by Flach but not us for aged murine HSC,8,36 but no overall delay in division kinetics once the cells start to divide. This finding is in contrast to observations by Pang et al.16 which concluded that aged HSC are more prone to divide. Here, Pang determined the proportion of G0 and non-G0 cells using Hoechst 33342 and Pyronin Y while we used Hoechst 33342 and Ki-67. While Pyronin Y correlates with RNA content, Ki-67 is seen as a marker for prolifera- tion in itself. It is thus a possibility that upon aging the cor- relation between elevated RNA content and proliferation might be somewhat diminished. The molecular mecha- nisms of the delayed entry of aged human HSC into cell division upon stimulation might be linked to elevated Cdc42 activity as cells treated with a pharmacological inhibitor of Cdc42 activity (CASIN), proceeded into divi- sion ahead of their untreated counterparts. In mutant yeast, hyperactive levels of Cdc42 result in the formation of multiple budding sites, however in wild- type cells, foci
competition is quickly resolved, with only one axis matur- ing and recruiting other proteins such as Bem1, to facilitate division.37 It is therefore a possibility that also in aged human HSC, the correct allocation of proteins to distinct positions within cells that are critical for proper initiation of division, might be altered upon elevated activity of Cdc42, resulting in a delay.
Knowledge on the molecular mechanisms underlying aging of HSC have supported the development of thera- peutic approaches to mitigate aging of HSC. For example, CASIN shows great promise in rejuvenating aged HSC9 and also in reverting aging-associated immune remodel- ing (AAIR) in murine models.32 A multivariate analysis model of gene expression profiles and biological age iden- tified Cdc42 as a strong predictor of survival and that a higher Cdc42 level is associated with higher mortality.38 We showed previously that the level of Cdc42 activity in human blood cells correlates positively with age39 and in this study, we identified a similar association in LDBM cells. Aged human HSC also show elevated activity of Cdc42 and a low frequency of HSC polar for polarity pro- teins. We also report a negative correlation between the activity of Cdc42 in individual donors and the frequency of HSC polar for polarity proteins like Cdc42 in that donor. We could demonstrate that the inhibition of the activity of Cdc42 is sufficient to increase the frequency of chronologically aged HSC polar for Cdc42 but not for tubulin which suggests in human cells, Cdc42 and tubulin axes may not be closely linked as shown in murine cells.9 Furthermore, the number of HSC in a given donor and the activity of Cdc42 in hematopoietic cells of that donor are positively correlated, which suggests that Cdc42 activity may affect the frequency of human HSC upon aging and could therefore, directly or indirectly, con- tribute to the increased HSC frequency observed. Given that in mice, apolar distribution of Cdc42 (a consequence of elevated activity of Cdc42) drives HSC to divide sym- metrically,8 and that elevated activity of Cdc42 in human leukemia stem cells is linked to more symmetric divi- sions,40 we postulate here an increased frequency of sym- metric divisions also for HSC of the elderly, which though will require further investigations.
In order to assess changes in function upon aging, HSC were transplanted into NBSGW recipients. By employing this model, secondary elements that may be introduced due to irradiation were avoided.41 We noted higher chimerism levels in non-conditioned recipients of aged HSC whereas conditioned recipients showed no increase. These observations also corroborate findings that irradia- tion, even at low doses, induces functional changes in mes- enchymal stromal cells which influences their hematopoi- etic-supporting ability.42 Our data suggests that the elevat- ed Cdc42 activity in aged HSC drives the age-related high level of chimerism observed in non-irradiated NBSGW recipients since recipients of young and 10 mM CASIN- treated aged HSC exhibited close similarities in the level of human chimerism. The higher level of chimerism driven by aged human HSC further supports a model in which human aged HSC might predominantly undergo symmet- rical divisions. Another possibility remains that an elevated repopulation potential of aged human HSC could be driven by the presence of individual clones43 linked to aging-relat- ed clonal hematopoiesis reported for a certain percentage of the elderly.44 Most human studies though demonstrated clonal hematopoiesis only in peripheral blood and not
400
haematologica | 2022; 107(2)