Comparative Evaluation of Human Mesenchymal Stem Cells of Fetal (Wharton's Jelly) and Adult (Adipose Tissue) Origin during Prolonged In Vitro Expansion: Considerations for Cytotherapy

Abstract

Mesenchymal stem cells (MSCs) are somatic cells with a dual capacity for self-renewal and differentiation, and diverse therapeutic applicability, both experimentally and in the clinic. These cells can be isolated from various human tissues that may differ anatomically or developmentally with relative ease. Heterogeneity due to biological origin or in vitro manipulation is, nevertheless, considerable and may equate to differences in qualitative and quantitative characteristics which can prove crucial for successful therapeutic use. With this in mind, in the present study we have evaluated the proliferation kinetics and phenotypic characteristics of MSCs derived from two abundant sources, that is, fetal umbilical cord matrix (Wharton's jelly) and adult adipose tissue (termed WJSC and ADSC, resp.) during prolonged in vitro expansion, a process necessary for obtaining cell numbers sufficient for clinical application. Our results show that WJSC are derived with relatively high efficiency and bear a substantially increased proliferation capacity whilst largely sustaining the expression of typical immunophenotypic markers, whereas ADSC exhibit a reduced proliferation potential showing typical signs of senescence at an early stage. By combining kinetic with phenotypic data we identify culture thresholds up to which both cell types maintain their stem properties, and we discuss the practical implications of their differences.

Figure 1

Proliferation kinetics of WJSC and ADSC over extended in vitro propagation. (a) Number of cumulative population doublings (PD) as a function of time in culture; (b) cell population doubling times—PDT—(hrs) at respective passages; (c) proliferation index (PI; ratio of PD over respective PDT); (d) growth curve of WJSC and ADSC within a single passage (3 to 4). The ∗ and # symbols highlight the points when confluence was reached for WJSC and ADSC, respectively. Dashed and long dashed horizontal lines denote mean values for ADSC and WJSC, respectively.

Figure 2

Variation in cell size (surface area in μm²) over time in culture and its association with proliferation kinetics. (a) Cell size (mean area ± SEM) as a phenotypic property of WJSC and ADSC during serial propagation. Dashed and long dashed horizontal lines denote mean cell size for ADSC (3876 μm²) and WJSC (2926 μm²), respectively; (b) regression analysis of ADSC size versus PDT at respective passages (P < 0.001, Spearman r correlation coefficient = 0.915). Trendline equation and goodness of fit (R ²) are shown. Boxed data points correspond to ADSC with sizes over 3,880 μm² (top 25% of cells); (c) regression analysis of WJSC size versus PDT at respective passages (P < 0.01, r = 0.6). Open triangles, p.1–10; solid triangles, p.11–20. Trendline equation and goodness of fit (R ²) are shown. Boxed data points correspond to WJSC with sizes over 4,600 μm² (top 25%).

Figure 3

Phenotypic properties of WJSC and ADSC at early and late passages. (a) Light microscopy photos illustrating cell morphology; arrows show cells with type III morphology. Top row, phase contrast. Mag. = 25x. (b) Senescence, as depicted by β-gal staining. Mag. = 25x. Subpanel shows a giant cell (>650 μm in length) with typical senescent phenotype. (c) Crystal violet staining of CFU-F (self-renewal capacity). (d) Formation of CFU-ALP (osteoprogenitor colonies). (e) Calcified bone nodule formation in response to osteogenic stimulation. Subpanel illustrates a typical alizarin red-stained bone nodule at high magnification (25x). Scale bars: (a) and (b) 100 μm, (c) and (d) 10 mm, and (e) 4 mm.

Figure 4

Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surface markers (first five and last three markers, resp.) during subculture. The vertical axes of the histograms depict event counts, and the horizontal axes represent fluorescence intensities of surface-bound conjugates, that is, marker expression levels. Staining of WJSC and ADSC with specific monoclonal antibody conjugates is illustrated by solid dark histograms (MSC markers: CD-29 and -44 FITC; CD-73, -90, and -105 PE) and by open histograms (HSC markers: CD-14 and -34 ECD; CD-45 PC5). Stripped histograms correspond to isotype-matched (negative) controls.