Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age

Abstract

In the human hematopoietic system, aging is associated with decreased bone marrow cellularity, decreased adaptive immune system function, and increased incidence of anemia and other hematological disorders and malignancies. Recent studies in mice suggest that changes within the hematopoietic stem cell (HSC) population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies. Though the mouse HSC population has been shown to change both quantitatively and functionally with age, changes in the human HSC and progenitor cell populations during aging have been incompletely characterized. To elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated immunophenotypic HSC and other hematopoietic progenitor populations from healthy, hematologically normal young and elderly human bone marrow samples. We found that aged immunophenotypic human HSC increase in frequency, are less quiescent, and exhibit myeloid-biased differentiation potential compared with young HSC. Gene expression profiling revealed that aged immunophenotypic human HSC transcriptionally up-regulate genes associated with cell cycle, myeloid lineage specification, and myeloid malignancies. These age-associated alterations in the frequency, developmental potential, and gene expression profile of human HSC are similar to those changes observed in mouse HSC, suggesting that hematopoietic aging is an evolutionarily conserved process.

Fig 1.

Increased frequency of HSC in normal elderly bone marrow compared to young. (A) Gating strategy and flow cytometric profile of HSC (Lin⁻, CD34⁺, CD38⁻, CD90⁺, CD45RA⁻) in representative hematopoietically normal young (Left) and elderly (Right) bone marrow samples. The left panels for each sample are gated on lineage negative (Lin⁻) live cells, and the right panels are gated on Lin⁻CD34⁺CD38⁻ live cells. (B) Summary of HSC as frequency of total Lin⁻CD34⁺ population from multiple young (n = 13) and elderly (n = 11) bone marrow samples. *P < 10⁻⁷. (C) Summary of quiescent G₀ (Hoechst 33342^low, Pyronin Y^low, correlating with 2N DNA and low levels of RNA) and non-G₀ (Pyronin Y^high, correlating with 2N to 4N DNA and higher levels of RNA) HSC frequency out of total HSC from multiple young (n = 13) and elderly (n = 8). # P < 0.013. Error bars represent standard deviation.

Fig 2.

Decreased frequency of CLP in normal elderly bone marrow compared to young. (A) Gating strategy and flow cytometric profile of CLP (Lin⁻, CD34⁺, CD127⁺) in representative hematopoietically normal young (Left) and elderly (Right) bone marrow samples. The left panels for each sample are gated on lineage negative (Lin⁻) live cells, and the right panels are gated on Lin⁻CD34⁺ live cells. (B) Summary of CLP as frequency of total Lin⁻CD34⁺ population from multiple normal young (n = 10) and elderly (n = 7) bone marrow samples. *P < 2.0 × 10⁻⁴.

Fig 3.

Diminished lymphoid versus myeloid differentiation capacity of HSC from normal elderly bone marrow compared to young bone marrow. (A) Gating strategy and flow cytometric profile of representative progeny derived from FACS-purifed HSC from hematologically normal young (Left) and elderly (Right) bone marrow samples cultured for 14 days on AC6.2.1 stromal cell line. The panels for each sample are gated on human CD45⁺ live cells. (B) Summary of CD13⁺ and/or CD33⁺ myeloid versus CD19⁺ B-lymphoid distribution generated from 50−200 HSC from multiple normal young (n = 8) and elderly (n = 6) bone marrow samples cocultured with AC6.2.1 stromal cell line. *P < 0.011. (C) Summary of bone marrow engraftment as measured by percent human chimerism per 500 transplanted HSC from unique young (n = 10) and elderly (n = 9) bone marrow samples. Each diamond represents an individual mouse transplanted with HSC from unique human bone marrow samples, and the bar indicates the average. On average, mice transplanted with young human HSC developed approximately twofold more human chimerism than mice transplanted with elderly human HSC, but this difference was not statistically significant due to the numbers of mice transplanted with young (n = 4) or elderly (n = 3) human HSC, which did not have any detectable chimerism. However, the approximately twofold difference in percent human chimerism between mice successfully engrafted with either young (n = 6) or elderly (n = 6) human HSC is statistically significant (P < 0.02). (D) Summary of human CD13⁺ and/or CD33⁺ myeloid versus CD19⁺ B-lymphoid distribution from bone marrow of mice successfully engrafted with young (n = 6) or elderly (n = 6) human HSC. Each diamond represents an individual mouse transplanted with HSC from a unique individual and the bar indicates the average. On average, the myeloid-to-lymphoid ratio was increased in the mice transplanted with elderly HSC by 14-fold. #P < 0.004. Error bars represent standard deviation.

Fig 4.

Gene expression profiling of HSC from normal elderly and young bone marrow reveals transcriptional differences reflecting myeloid lineage-bias of elderly HSC. (A) Validation of microarray data by quantitative RT-PCR: average fold-change in the expression of selected genes as determined by microarray analysis (14 young and 8 elderly HSC samples) and quantitative RT-PCR (4 young and 4 elderly HSC samples; independent of samples analyzed by microarrays). Error bars represent standard deviation. Heat maps reflecting expression levels of (B) myeloid-specific and (C) lymphoid-specific age-regulated genes that are significantly differentially expressed between young and elderly HSC.