Possibility of selection of chondrogenic progenitor cells by telomere length in FGF-2-expanded mesenchymal stromal cells

Abstract

Telomere length plays an important role in regulating the proliferative capacity of cells, and serves as a marker for cell cycle history and also for their remaining replicative potential. Mesenchymal stromal cells (MSC) are known to be a significant cell source for therapeutic intervention and tissue engineering. To investigate any possible limitations in the replicative potential and chondrogenic differentiation potential of fibroblast growth factor-2-expanded MSCs (FGF(+)MSC), these cells were differentiated at various population doublings (PDs), and telomere length and telomerase activity were measured before and after differentiation. FGF(+)MSC cultured at a relatively low density maintained proliferation capability past more than 80 PD and maintained chondrogenic differentiation potential up to at least 46 PD and long telomeres up to 105 PD, despite expressing low levels of telomerase activity. Interestingly, upon chondrogenic differentiation of these cells, telomeres showed a remarkable reduction in length. This shortening was more extensive when FGF(+)MSC of higher PD levels were differentiated. These findings suggest that telomere length may be a useful genetic marker for chondrogenic progenitor cells.

Figure 1

Cell proliferation and morphological aspects. (a) Proliferation curve of FGF(+)MSC or FGF(–)MSC from MSC₁₉ and ₄₅ (donor age, 19 and 45 years old, respectively). MSCs were isolated from bone marrow aspirates of normal adult donors. After confirming cell adhesion to the plate, MSCs were cultured with 15% FBS‐DMEM with or without FGF‐2 (final conc.; 1 ng/ml). Cumulative population doublings level (PDL) is regarded as zero for culture starting immediately after the primary culture of cells, and calculated to increase according to the equation: log₂{(the number of collected cells)/(the number of seeded cells)}. (b) Representative morphological aspects. FGF+; cells cultured with media including FGF‐2, FGF−; without FGF‐2. The scale indicates 5 µm.

Figure 2

Representative microscopic views of chondrogenesis of MSC. The pellet tissue was evaluated by toluidine blue stain (a–f) and immunohistochemical analysis (type II collagen; g–m). FGF(+) MSC‐derived pellet: 9, 12 and 46 PD: (a, g) (b, h) and (c, i), respectively. FGF(–)MSC‐derived pellet: 8 PD (e, k). D⁻: control, chondrogenic differentiation media not including TGF‐β3 or dexamethasone. FGF(+)MSC‐derived:10 PD (d, j). FGF(–)MSC‐derived: 7 PD (f, l). Negative control for immunohistochemical analysis (m). Magnification was 200‐fold. The scale indicates 50 µm.

Figure 3

Effect of FGF‐2 on telomere length of MSC. Telomere length was measured by Southern blot analysis. MSC₁₄ and ₁₉: donor age, 14 and 19 years old. h‐Chon₇₉: human chondrocytes, 79 years old. Chon. F+ or Chon. F−; chondrogenesis of FGF(+)MSC or FGF(–)MSC.