Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells

Abstract

Background: Mesenchymal stem cells (MSC) are pluripotent cells, present in the bone marrow and other tissues that can differentiate into cells of all germ layers and may be involved in tissue maintenance and repair in adult organisms. Because of their plasticity and accessibility these cells are also prime candidates for regenerative medicine. The contribution of stem cell aging to organismal aging is under debate and one theory is that reparative processes deteriorate as a consequence of stem cell aging and/or decrease in number. Age has been linked with changes in osteogenic and adipogenic potential of MSCs.

Results: Here we report on changes in global gene expression of cultured MSCs isolated from the bone marrow of mice at ages 2, 8, and 26-months. Microarray analyses revealed significant changes in the expression of more than 8000 genes with stage-specific changes of multiple differentiation, cell cycle and growth factor genes. Key markers of adipogenesis including lipoprotein lipase, FABP4, and Itm2a displayed age-dependent declines. Expression of the master cell cycle regulators p53 and p21 and growth factors HGF and VEGF also declined significantly at 26 months. These changes were evident despite multiple cell divisions in vitro after bone marrow isolation.

Conclusions: The results suggest that MSCs are subject to molecular genetic changes during aging that are conserved during passage in culture. These changes may affect the physiological functions and the potential of autologous MSCs for stem cell therapy.

Figure 1

Isolation of Bone Marrow Stem Cells. Bone marrow was aspirated from tibia and fibula of mice (4 per group) at age 2m, 8m, and 26m. 1(A-C) shows cells at the first passage from 2m, 8m, and 26m ages respectively. Morphologies of the cells was heterogenous at this stage. 1(D-F) shows cells after 10 passages again a 2, 8 and 26m respectively, cells at this passage were homogeneous with a flattened morphology.

Figure 2

FACS analyses indicate similar cell surface antigen profiles of MSC from different age groups. Cells were labeled with fluorescent antibodies for Sca-1, CD44, CD11b, and CD45 and analyzed by FACS. Consistent with a mesenchymal phenotype, all cells were positive for the markers Sca-1 and CD44 (>99% and 98% respectively). Cells were negative for the monocyte marker CD11b and the hematopoietic lineage marker CD45 (<2%).

Figure 3

Expression of stem cell markers. Immunofluoresence of isolated bone marrow stem cells. Isolated MSC passage 11 were fixed and stained with anti-Sca-1 and CD44 antibodies. All cells were positive for these stem cell markers.

Figure 4

Osteogenic Differentiation. MSC, passage 11 from each age were grown to confluence and exposed to osteogenic induction medium for 14 days and stained with Alizarin Red. Cells from each age group were positive after 14 days (A-C; 2mo, 8mo, 26mo) whereas no staining was seen in cells treated with control medium (D,E, F; 2mo, 8mo, 26mo).

Figure 5

Age-related Decline of Alkaline phosphatase (AP) activity. Osteogenic differentiation involves increased secretion of AP. Alkaline phosphataes was measured in the media of differentiating cell at day 10 as described in Methods. Significantly less AP was secreted from cells taken from mice at age 26 mo (n=4; p<0.01).

Figure 6

Adipogenic differentiation. MSCs from 2, 8, or 26-month old mice were grown to confluence and exposed to adipogenic induction medium (D) or culture medium (UD) as described in Methods. Cell from each age group were Oli-Red positive and displayed intracellular fat droplettes.

Figure 7

Heirarchical Clustering of MSC mRNA. Clustering was implemented as described in Methods. Pearson correlation coefficients taken from plotting signal intensity values of duplicate chips across all genes validated that triplicate experiments were similar (typical correlation coefficients for previous double amplification experiments have been r2 ~ .988).

Figure 8

Comparisons of down and up-regulated transcripts. Venn Diagram illustrating fold gene expression differences between 2m, 8m, and 26m (t-test <2-fold, p<0.01) of 40,359 transcripts. Expression levels of 2111 transcripts decreased from 2m to 8m and 2547 transcripts decreased from 8m to 26m. Only 86 transcripts commonly decreased over both age groups. Analysis of up-regulated genes in the same manner revealed 1487 transcripts that were increased from 2m to 8m and 2402 increased from 8m to 26m. There was no overlap.

Figure 9

Scatter Plot Analysis Indicating up and down regulated genes from microarray. This figure indicates the global changes in gene expression during two phases of aging and growth. While the two figures look similar, the identification of the genes reveals two different gene sets. Some of the genes that undergo some of the largest changes are indicated.

Figure 10

RT-PCR confirmation of age-related changes of cell cycle and apoptosis genes. A RT-PCR (cancer) SuperArray of 84 cell cycle and apoptosis genes was used to compare mRNA from 2m and 26m mouse MSCs. The analysis confirms 2-fold and 50-fold decrease of p53 and p21 respectively and significant decrease of apoptosis transcripts with age.

Figure 11

Western blot of p53 expression. (a) Cell lysates from each age group were analyzed by western blot as described in Methods. Consistent with microarray and RT-PCR analysis, MSCs derived from 26-month mice did not express the p53 protein. (B) Spleen lysates from progressively aged mice showed no change of p53 protein.

Figure 12

Microarray profiles of pro-angiogenic cytokines and growth factor genes. Expression levels of IGF-1, VEGF-A, VEGF-B, VEGF-C and HGF were from microarray.

Figure 13

RT-PCR measurement of Growth factor transcripts. RT-PCR was performed as described in Methods. These analysis confirmed marked down regulation of the growth factor cytokine genes with age. The largest changes were the HGF gene (>272-fold) and IGF-1 gene transcripts.

Figure 14

ELISA measurement of VEGF secretion. ELISA was performed as described in Methods. MSC from each age were grown to confluence in parallel and VEGF measured in the medium 24h after replacement. Significantly less VEGF was secreted from cells taken from mice at age 26 mo (n=4; p<0.02).