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. 2016 Mar 22;7(12):13285-96.
doi: 10.18632/oncotarget.7690.

Increased IL-6 Secretion by Aged Human Mesenchymal Stromal Cells Disrupts Hematopoietic Stem and Progenitor Cells' Homeostasis

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Free PMC article

Increased IL-6 Secretion by Aged Human Mesenchymal Stromal Cells Disrupts Hematopoietic Stem and Progenitor Cells' Homeostasis

Kelsey O'Hagan-Wong et al. Oncotarget. .
Free PMC article

Abstract

Hematopoietic stem and progenitor cell (HSPC) homeostasis declines with age, leading to impaired hematopoiesis. Mesenchymal stromal cells (MSC) are critical components of the bone marrow niche and key regulators of the balance between HSPC proliferation and quiescence. Accrual of DNA damage, a hallmark of cellular aging, occurs in aged MSC. Whether MSC aging alters the bone marrow niche triggering HSPC dysfunction is unknown. Using a human MSC-HSPC co-culture system, we demonstrated that DNA damaged MSC have impaired capacity to maintain CD34+CD38- HSPC quiescence. Furthermore, human MSC from adult donors display some hallmarks of cellular senescence and have a decreased capacity to maintain HSPC quiescence and the most primitive CD34+CD38- subset compared to MSC from pediatric donors. IL-6 neutralization restores the MSC-HPSC crosstalk in senescent and adult MSC-HSPC co-cultures highlighting the relevance of the local microenvironment in maintaining HSPC homeostasis. These results provide new evidence implicating components of the MSC secretome in HSPC aging.

Keywords: Gerotarget; aging; hematopoietic stem and progenitor cells; mesenchymal stromal cells; senescence; senescence-associated secretory phenotype.

Conflict of interest statement

CONFLICTS OF INTEREST

There is no conflict of interest.

Figures

Figure 1
Figure 1. Irradiation induces senescence biomarkers in MSC
A. MSCs were exposed to increasing doses of radiation (0 - 50 Gy) and proliferation was assessed 8 days later using LI-COR analysis (DRAQ5 fluorescence intensity (total DNA content). B. Senescence-associated beta-galactosidase activity was assessed at baseline and 9 days following 5 Gy irradiation in fibroblasts (positive controls, HCA2-hTERTs) and MSCs. C. MSC cell size was evaluated 8 days following 5 Gy irradiation using FACS. D. Direct molecular markers of DNA double strand breaks were evaluated in control and irradiated MSC using immunofluorescence (9 days following irradiation with 5 Gy). Representative images of cells harboring small nuclear DNA damage foci constituted of both 53BP1 (red) and phospho-H2AX (green) (yellow highlight red-green colocalization, the nuclei are counterstained in blue (DAPI)). The extracted 53BP1 red channel is presented in grayscale to highlight 53BP1 DNA damage foci that appears like nuclear white dots (right panels). Selected cells are boxed in white squares labeled 1-3 for magnification in E. to highlight colocalization between 53BP1 (red) and phospho-H2AX (green). Note the yellow dots in irradiated cells (box 2 and 3). F. The molecular biomarker of senescence (PML, green) was evaluated in control and irradiated MSC using immunofluorescence (nuclei counterstained in blue (DAPI)). Notice increased total levels of PML and PML nuclear bodies in irradiated cells. The extracted PML green channel is presented in grayscale to highlight the nuclear increase in PML levels (right panels) G. Representative images of cells harboring DNA-SCARS highlighted by 53BP1 (red) and PML (green) colocalization. Nuclei are counterstained in blue (DAPI). The extracted 53BP1 red channel is presented in grayscale to highlight 53BP1 DNA damage foci that appears like nuclear white dots (right panels). Selected cells are boxed in white squares labeled 1-3 for magnification in H. to highlight colocalization (yellow) between 53BP1 (red) and PML (green). Note the yellow dots in irradiated cells (box 2 and 3). I. The quantified data for the representative images presented for DNA damage foci (53BP1-gH2AX colocalization) and DNA-SCARS (53BP1-PML colocalization) is presented as the percentage of cells with > 1 colocalized nuclear foci. Mean ±SD of 4 independent experiments are reported where (*) represents p ≤ 0.0001.
Figure 2
Figure 2. Increased secretion of senescence-associated cytokines by irradiated MSC
The production of IL-6 and IL-8 in iMSC (5Gy) and controls was evaluated by ELISA 9 days after irradiation. iMSC produced significantly higher levels of IL-6 A. and IL-8 B. compared to controls. Mean (triplicate) of 7 independent experiments are reported where (**) represents p ≤ 0.01.
Figure 3
Figure 3. Senescent MSC reduce HSPC quiescence and the frequency of primitive CD34+CD38- cells
A. CD34+ expansion, B. percentage of CD34+ in generation 0, C. frequencies of CD34+CD38 subpopulations, and D. CD34+ viability were evaluated in MSC-HSPC and iMSC-HSPC (5Gy) co-cultures (1:1 ratio) following 4 days of co-culture. Open-faced circles represent MSC from pediatric donors (< 16 years-old) and black circles represent MSC from adult donors (> 40 years-old). E. CD34+ proliferation was also evaluated following HSPC culture with conditioned medium from MSC and iMSC. Mean ±SD of 6-10 independent experiments are reported where (*) represent p ≤ 0.05.
Figure 4
Figure 4. MSC from adult donors have a senescence-like phenotype
A. MSC were isolated from adult (> 40 years) and pediatric donors (< 16 years). MSC proliferation was measured by DRAQ5 intensity day 4 post seeding in adult and pediatric samples. B. Cell size was measured by flow cytometry (Mean FSC-A) in adult and pediatric MSC samples. C. IL-6 and D. IL-8 production by adult and pediatric MSC were quantified by ELISA. Mean ±SD of 7-10 independent experiments are reported where (*) represents p ≤ 0.05.
Figure 5
Figure 5. Loss of HSPC quiescence and primitive subpopulations in adult MSC-HSPC co-cultures
A. CD34+ expansion, B. percentage of CD34+ in generation 0,C. frequency of CD34+/C38 subpopulations, and D. viability were evaluated in HSC-MSC co-cultures (1:1 ratio) at day 4. MSC used were isolated from pediatric (< 16 years) or adult (> 40 years) donors. Mean of 10 independent experiments are reported where (*) represents p ≤ 0.05.
Figure 6
Figure 6. Increased production of IL-6 by senescent and aged MSC impairs HSPC quiescence
A. CD34+ proliferation, B. percentage of CD34+ in generation 0, and C. frequency of CD34+CD38 subpopulations were determined after 4 days in co-cultures with irradiated (5 Gy) and non-irradiated MSC in the presence or absence of an IL-6 neutralizing antibody (20ng/ml). Similar experiments were performed comparing the effect of IL-6 neutralization in adult and pediatric MSC-HSPC co-cultures D.-F. Results were normalized to 0 Gy control in A. -C. and Ped MSC condition in D.-F.. Mean value ± SD of 10 independent experiments are reported where (*) indicates p ≤ 0.05; (**) indicates p ≤ 0.01; (ns) indicates p > 0.05.

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