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Abstract

Aging is a complex process that is associated with changes in many parts of the body over the lifespan of an individual. In this work, various aspects of aging in the human bone marrow were investigated. As the regenerative power of a tissue is linked to the potential of its stem cells to replace the accumulated damages, the aging process in somatic stem cells was studied focusing on the influence of the niche in regulating stem cell aging. The hematopoietic stem cells (HSC) together with elements that constitute the bone marrow niche were investigated as a model for somatic stem cell aging as HSCs are accessible in healthy human individuals. The subjects ranged from 20 to 60 years with a median age of 33.2 years. From each bone marrow sample, the CD34+ population as HSCs and four other cell subpopulations, lymphocytes and precursors (LYM), monocytes/macrophages precursors (MON), granulocytic (GRA), and erythroid precursors (ERP) were isolated by flow cytometry. The mesenchymal stromal cells (MSC) were isolated by in vitro culture. We found that the relative proportions, cell size as well as cell granularity of the major bone marrow constituents did not correlate with the biological age of the donors. However, further downstream analysis indicated that age-associated changes were prominent on protein level in HSCs as well as in other cell types of the niche such as MSCs. The interactions between the HSCs and the niche were studied in vitro using a coculture system of CD34+ cells and mesenchymal stromal cells (MSC). As previous studies indicated that the supportive function of MSCs as well as their differentiation potentials towards adipocytes and osteocytes change significantly with age, we have examined the supportive ability of the undifferentiated MSCs versus adipogenically differentiated MSCs (ADI-MSCs) and osteogenically differentiated MSCs (OST-MSCs) for HSCs. We showed that MSCs, ADI-MSCs and OST-MSCs were able to support the proliferation of HSCs and maintain their primitive immunephenotype. Compared to undifferentiated MSCs and OST-MSCs, the co-culture with ADI-MSCs increased the proliferation of HSCs much stronger while still maintaining the HSCs at a high expression level of CD34. As the impact of the MSCs on HSCs might be caused by epigenetic changes, the aging-associated alterations in the marrow niche were studied at the chromatin level. To this end, changes in chromatin accessibility were studied in MSCs by ATAC-seq. After establishing the protocol for performing ATAC-seq using primary MSCs, we studied the MSC samples derived from 16 healthy human subjects of different ages between 21 and 59 years. A set of 122,884 ATAC-seq peaks was identified. We have demonstrated that donor age is associated with alterations in open chromatin profiles. Moreover, at a false discovery rate of 5%, we could identify 4,579 differential chromatin accessible sites upon aging. A functional analysis of these sites showed enrichment of cell development and differentiation processes. Additionally, genes of the hippo signaling pathway, TGF-beta signaling pathway, cancer pathways and cell adhesion pathways were also found to be enriched. A motif enrichment analysis suggested that TATA box motifs and binding sites for transcription factors TFAP2C, KLF16, HIC1.p2, WT1 and MTF.p2 were enriched in promoter regions of differential chromatin accessible sites upon aging. In conclusion, this study showed that the interplay with the stem cell niche controls HSC functions. The differentiation of MSCs affects the proliferation and stemness of HSCs in vitro. Furthermore, we have demonstrated that aging is associated with chromatin accessibility alterations in MSCs, which provides a foundation for further in-depth mechanistic analyses.