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A. Amoah et al.
In addition, mice show an increase in HSC frequency with age, while the rhesus monkey, shows a decrease with age.12 Moreover, even among distinct murine inbred strains, HSC number and function is distinct upon aging, like C57BL/6 mice present with an elevated number of HSC upon aging, but not so in DBA/2 animals.13,14 For these reasons, novel studies into understanding mecha- nisms of aging of human HSC are warranted and are a pre- requisite to bolster the transition of this knowledge into the clinic.
Age-related changes in the frequency and function of HSPC have been in part previously described by a small number of groups. One study for example reported no changes in the re-population potential of aged HSC and a decreased propensity for myeloid differentiation while another recorded a decline in the reconstitution capacity of aged HSC with an increased myeloid differentiation potential.15,16 Both groups used NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) animals as recipients for in vivo xeno- transplantation to study the function of human HSC. This model requires irradiation of the recipient animal for the successful establishment of xenotransplants17 which might contribute to variable secondary effects not linked to the transplanted HSC. New and improved mouse models have been created that do not require pre-condi- tioning of the recipients for achieving human xeno- chimerism.18,19 NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) animals bear in addition to the NSG genotype a mutation in the Kit gene.19 The Kit muta- tion enables donor cells to efficiently engraft without irra- diation.20 We characterize here aged human HSC with a special focus on likely shared hallmarks of age-related changes among human and murine HSC and describe a novel approach to attenuate aging of human HSC. Our data support the possibility of rejuvenating the function of aged human HSC due to similarities between aging of murine and human HSC.
Methods
Primary cells
Bone marrow cells were isolated from young (range, 23-39 years; median age 27 years) donors acquired from Cincinnati Children’s Hospital Medical Center and aged (range, 58-82 years; median age 66 years) individuals undergoing heart sur- gery at the Ulm University Clinic, Department of Heart, Thoracic and Vascular Surgery (additional details of age strata are provided in the Online Supplementary Methods). All donors were hematologically healthy. Sample collection and investiga- tion was approved by the Internal Review Board (Ethikkomission) of Ulm University (392/16).
Flow cytometric analysis and cell sorting
Mononuclear cells (MNC) were thawed and stained in phos- phate-buffered saline (PBS) supplemented with 3% fetal bovine serum (FBS) with human specific antibodies (see the Online Supplementary Methods for details). Different cell populations were identified and sorted on a BD FACS ARIA II 4L SORP (BD Biosciences) according to the markers used by van Galen et al.21
Single cell division assay
Single HSC were sorted into Terasaki plates and checked every 12 hours under a light microscope (further details are pro- vided in the Online Supplementary Methods).
Colony forming unit assay
In order to assess the myeloid and erythroid generative poten- tial of samples, 200 HSC were seeded in methylcellulose medi- um (further details are provided in the Online Supplementary Methods).
Xenotransplantation
All animal experiments were carried out in accordance to institutional guidelines and approved by the Regierungspräsidium Tübingen (TVA 1412). Five hundred HSC were injected via the tail vein into non-conditioned or low dose irradiated (1.6 Gy) NBSGW mice. At 8 and 12 weeks, aspirates were drawn from the bone marrow (BM) of mice after adminis- tering anesthesia. Human cells were identified using human-spe- cific antibodies (see the Online Supplementary Methods for further details) and analyzed on LSR Fortessa SORP flow cytometer (BD Biosciences). Human chimerism was determined as a percentage of total CD45+ cells and mature cells, as a percentage of human CD45+ cells.
CASIN treatment
HSC were collected in serum-free expansion media and incu- bated at 37°C, 3% oxygen for 1 hour. Cells were then trans- ferred into media ± CASIN, incubated for 4 hours and washed. Cells were then used in subsequent experiments.
Immunofluorescent staining
Cells were seeded in serum-free expansion media, fixed and polarity for Cdc42 or tubulin assessed as previously described by Florian et al.9 Cdc42-GTP in HSC was determined using the anti- body described by Althoff et al.22 (see the Online Supplementary Methods for further details).
Western blot
A Rac/Cdc42 assay reagent (# 14-325, Millipore) was used in pull down assays according to the manufacturer’s protocol (see the Online Supplementary Methods for further details).
Statistical analysis
Statistical analyses were performed with GraphPad Prism 8 (version 8.1.2) and are presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) and box plots as minimum and maximum points. Kendall’s correlation analy- sis was performed with R version 4.0.3, RStudio Team (2020) version 1.3.1093. *P<0.05, **P<0.005, ***P<0.0005.
Results
Changes in the immunophenotypic frequencies of hematopoietic populations occur with age
Data on whether there are changes in the frequency of HSC in the BM of humans upon aging remains controver- sial. This might be, at least in part, due to different gating strategies employed to identify human HSC.15,16 Using a more recently established and improved marker profile for the identification of human HSC21 (Online Supplementary Figure S1A), we first determined the fre- quency of HSC (Lin-CD34+SSc low CD38-CD90+CD45ra-), HSPC (Lin-CD34+SSc low CD38-) and Lin-CD34+SSc low cells in BM cells from the sternum of the elderly. While the fre- quency of the Lin-CD34+SSc low population did not change with age within our cohort, the HSPC frequency within Lin-CD34+SSc low population and HSC frequency within HSPC population increased with age (Figure 1A to C).
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haematologica | 2022; 107(2)