Characterization of Senescence of Culture-expanded Human Adipose-derived Mesenchymal Stem Cells

Abstract

Background and objectives: Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates in regenerative medicine. The need for in vitro propagation to obtain therapeutic quantities of the cells imposes a risk of impaired functionality due to cellular senescence. The aim of the study was to analyze in vitro senescence of previously cryopreserved human ADSCs subjected to serial passages in cell culture.

Methods and results: ADSC cultures from 8 donors were cultivated until proliferation arrest was reached. A gradual decline of ADSC fitness was observed by altered cell morphology, loss of proliferative, clonogenic and differentiation abilities and increased β-galactosidase expression all of which occurred in a donor-specific manner. Relative telomere length (RTL) analysis revealed that only three tested cultures encountered replicative senescence. The presence of two ADSC subsets with significantly different RTL and cell size was discovered. The heterogeneity of ADSC cultures was supported by the intermittent nature of aging seen in tested samples.

Conclusions: We conclude that the onset of in vitro senescence of ADSCs is a strongly donor-specific process which is complicated by the intricate dynamics of cell subsets present in ADSC population. This complexity needs to be carefully considered when elaborating protocols for personalized cellular therapy.

Keywords: Cell aging; Heterogeneity; Human adipose-derived mesenchymal stem cells; Relative telomere length; Serial passage; Subpopulations.

Fig. 1

Proliferation capacity and morphology of ADSCs during in vitro long-term cultivation. (A) Cumulative population doublings (PD). (B) ADSC proliferation curves showing population doubling time (PDT) at each passage. (C) ADSC morphology during long-term cultivation. Monolayer cells from early (upper-left) and late (upper-right) passage, and suspended cells from early (lower-left) and late (lower-right) passage are shown. (D) Clonogenicity potential of ADSCs. Upper panel –relative CFU test values; lower panel – an image of CFU test wells reflecting donor variance. OD: optical density.

Fig. 2

Bilineage potential of ADSCs. Shown is the comparison of early (P3) and late (P9) passage of two representative samples (CS-3 and CS-8) reflecting donor-specific differences. (A) Adipogenesis detected by Oil Red O staining of lipid inclusions. (B, C) Osteogenesis detected by (B) Alizarin Red S staining of extracellular calcium, and (C) alkaline phosphatase activity.

Fig. 3

Senescence-related changes of ADSC phenotype. (A) SA-β-gal expression was tested histochemically. Images were subjected to visual evaluation by two observers with regard to color intensity and percentage of stained cells per field of view. A typical view of increased SA-β-gal expression in ADSCs during long-term culture is shown (lower panel) with the respective visual evaluation data (upper panel) (B) ADSC metabolic activity determined by MTT assay. Optical density (OD) readings are expressed as mean of triplicate measurements. (C) Quantity of hydrogen peroxide in ADSCs. H₂O₂ standard curve is shown (left panel). H₂O₂ measurements of samples are expressed as mean of two replicates (right panel). AU: arbitrary units; P: passage. Error bars indicate standard deviation.

Fig. 4

Relative telomere length (RTL) of culture-expanded ADSCs and identification of cell subpopulations. (A) Flow FISH analysis of RTL dynamics during long-term culture. Each data point is expressed as mean±standard deviation of duplicate measurements. Continuous line represents mean RTL of five (P3–9), three (P11), and two (P13) samples. (B) The presence of two ADSC subpopulations (S and L) during RTL measurement was detected (upper panel). In contrast, only a single population of control Jurkat cells existed (lower panel, J). The S and L populations differed with respect to their RTL (C) and cell size (D) (data expressed as mean of all analyzed passages of five samples CS-1,2,3,6,8±standard deviation; *p<0.05, **p=0.001). AU: arbitrary units; P: passage.