Age-Related Changes in Bone-Marrow Mesenchymal Stem Cells

Abstract

The use of stem cells is part of a strategy for the treatment of a large number of diseases. However, the source of the original stem cells for use is extremely important and determines their therapeutic potential. Mesenchymal stromal cells (MSC) have proven their therapeutic effectiveness when used in a number of pathological models. However, it remains an open question whether the chronological age of the donor organism affects the effectiveness of the use of MSC. The asymmetric division of stem cells, the result of which is some residential stem cells acquiring a non-senile phenotype, means that stem cells possess an intrinsic ability to preserve juvenile characteristics, implying an absence or at least remarkable retardation of senescence in stem cells. To test whether residential MSC senesce, we evaluated the physiological changes in the MSC from old rats, with a further comparison of the neuroprotective properties of MSC from young and old animals in a model of traumatic brain injury. We found that, while the effect of administration of MSC on lesion volume was minimal, functional recovery was remarkable, with the highest effect assigned to fetal cells; the lowest effect was recorded for cells isolated from adult rats and postnatal cells, having intermediate potency. MSC from the young rats were characterized by a faster growth than adult MSC, correlating with levels of proliferating cell nuclear antigen (PCNA). However, there were no differences in respiratory activity of MSC from young and old rats, but young cells showed much higher glucose utilization than old ones. Autophagy flux was almost the same in both types of cells, but there were remarkable ultrastructural differences in old and young cells.

Keywords: aging; glucose utilization; glycolysis; mesenchymal stromal cells; mitochondria; oxidative phosphorylation; proliferation; senescence; stem; therapy; traumatic brain injury.

Figure 1

Effect of MSC transplantation on the volume of lesion in a rat traumatic brain: (A) Representative T2-weighted MR-images from coronal brain sections (0.8 mm thick, from rostral (top) towards caudal (bottom)), obtained on the 14th day after TBI. Light regions refer to ischemic areas after TBI. (B) Damage volume evaluated by MRI with analysis of T2-weighted images. * Denotes significant difference from the TBI + saline (p < 0.05) (One-way ANOVA, followed by Tukey’s post hoc analysis).

Figure 2

Effect of MSC on the neurological deficit of animals: (A) Neurological status scores estimated in the limb-placing test 1 day before and days 1, 2, 4, 7 and 14 after TBI. * p < 0.05 denotes significant difference from TBI + saline group; # p < 0.05 denotes significant difference from TBI + aMSC group (two-way ANOVA). (B) TBI induced limb asymmetry, measured by the cylinder test 14 days after the surgery. * p < 0.05 denotes a significant difference from TBI + saline group (Kruskal–Wallis test with the Mann–Whitney). Data are expressed as mean ± SEM.

Figure 3

Estimation of proliferative potential and senescence of mesenchymal stromal cells, obtained from perinatal and old rats’ bone marrow. Representative images of SA-β-gal activity staining in pMSC (A) and aMSC (B). Senescent cells are stained blue. (C) Evaluation of β-galactosidase activity (SABG) normalized on cell area in pMSC and aMSC. (D) Mean cell area from donors of different ages. (E) Real-time curve of cell index and (F) the cell growth rate measured with the RTCA iCELLigence over 160 h. (G) Immunoblots for proliferating cell nuclear antigen (PCNA) in MSC and densitometry analysis of protein bands (diagram), respectively. P, perinatal, a, adult MSC. Actin staining was used as a loading control. Number of individual cultures for Western blot was 3. *: p < 0.05, (aMSC vs. pMSC).

Figure 4

Glucose utilization and mitochondrial respiratory function in perinatal and adult MSC. Real-time proliferation and growth rate of MSC obtained from neonatal and adult rats under conditions of glycolysis inhibition with 20 mM 2-Deoxy-D-glucose (2-DG) (A) and during hypoxia (C). Cell growth rate (slope of cell index) measured after 2-DG treatment (B) and in normoxia/hypoxia conditions (D). (E) Representative oxygen consumption traces and (F) basal and maximal oxygen consumption rate (OCR) analysis in MSC in response to oligomycin (4.5 µM), CCCP (10 µM) and rotenone (2.5 µM). (G) Time course of extracellular acidification rate (ECAR), at least partially reflecting glycolytic function in MSC. Kinetics of extracellular acidification rate (ECAR) in pMSC and aMSC cultures in response to glucose (10 mM) and 2-DG (50 mM) treatment. (H) Calculated non-glycolytic acidification and glycolytic capacity in MSC. * p < 0.05 denotes a significant difference between the “pMSC” and “aMSC”; # p < 0.05 denotes a significant difference between the normoxia and hypoxia conditions for pMSC.

Figure 5

Evaluation of the mitochondrial potential, the autophagic activity and the levels of ROS in the perinatal and adult MSC obtained from the femoral bones. Flow cytometry of primary cultures of MSC loaded with MitoTracker Red (A) and Green (B) for the estimation of mitochondrial transmembrane potential and mitochondria content, correspondently. (C) ROS production as measured by DCF fluorescence in pMSC and aMSC. (D) Cyto-ID assay and (E) LysoTracker were used to evaluate the activity of autophagy and lysosomes in MSC using flow cytometry. (F) The autophagy markers levels of Beclin-1, LAMP-1 and LC-3-I/II, in pMSC and aMSC. A total of 50,000 cells and 3 samples were used for flow cytometry and Western blot analysis correspondingly.

Figure 6

Electron microscopic images of the perinatal (A–D) and adult (E–H) MSC from rat bone marrow. Vesicular structures on the cell border are visible in (A), with a high zoom of the same cell in (B). pMSC contain mitochondria as dark extended profiles existing in the orthodox configuration, while mitochondria in cells from adult MSC preferentially had round profiles, demonstrating partial fragmentation of mitochondrial reticulum. Additionally, slight local (shown by an arrow in (G)) and general (shown by arrowhead in (G)) swelling of intracristae space and matrix in these mitochondria are obvious.