Age-related yield of adipose-derived stem cells bearing the low-affinity nerve growth factor receptor

Abstract

Adipose-derived stem cells (ADSCs) are a heterogeneous cell population that may be enriched by positive selection with antibodies against the low-affinity nerve growth factor receptor (LNGFR or CD271), yielding a selective cell universe with higher proliferation and differentiation potential. This paper addresses the need for determining the quantity of ADSCs positive for the CD271 receptor and its correlation with donor's age. Mononuclear cells were harvested from the lower backs of 35 female donors and purified using magnetic beads. Multipotency capacity was tested by the expression of stemness genes and through differentiation into preosteoblasts and adipocytes. A significant statistical difference was found in CD271(+) concentrations between defined age intervals. The highest yield was found within women on the 30-40-year-old age range. CD271(+) ADSCs from all age groups showed differentiation capabilities as well as expression of typical multipotent stem cell genes. Our data suggest that the amount of CD271(+) cells correlates inversely with age. However, the ability to obtain these cells was maintained through all age ranges with a yield higher than what has been reported from bone marrow. Our findings propose CD271(+) ADSCs as the primary choice for tissue regeneration and autologous stem cell therapies in older subjects.

Figure 1

Frequency of CD271⁺ ADSCs per mL of initial adipose tissue taken from the lower backs of 30 healthy female donors with similar body mass index. (a) Distribution of CD271⁺ stem cell/mL of initial adipose tissue and donor's age. Data appear normal and a straight line is adjusted with a goodness of fit of R ² = 0.71. The fitted regression equation and its P value are shown. (b) Box and whisker plots representing the median, lower, and upper quartiles of CD271⁺ ADSC yield/mL of processed adipose tissue for each age range.

Figure 2

Representative results of flow cytometry analysis are shown for samples of 3 × 10⁵ CD271⁺ ADSCs passage 1 from a 42-year-old donor. (a) The CD271⁺ ADSC population indicated with gage P1 comprises 75.4% of the 10,000 events analyzed. (b) Histogram showing that 73% of cells from gage P1 yield positive for the CD271-APC receptor. (c) Histogram depicting that the CD271⁺ ADSC population is negative for CD45-FITC receptor. (d) Histogram demonstrating that the CD271⁺ ADSC population is positive for the CD105-PE receptor. (e) The histogram shows that 87% of the CD271⁺ ADSC population is positive for the CD34-PE. (f) Histogram demonstrating that 97% of the CD271⁺ ADSC population yields positive for the CD90-FITC receptor.

Figure 3

Morphological changes through different induction media. (a) CD271⁺ ADSCs passage 1 after 21 days in culture without induction (20x). (b) Morphological changes visible at day 21 of adipogenic differentiation (20x). (c) Osteogenic differentiation observed at induction day 21 (20x). (d) Microphotograph showing extracellular mineralization unveiled through Alizarin Red staining in CD271⁺ ADSCs after 21 days of osteogenic induction (20x).

Figure 4

Gene expression analysis. The upper image shows a representative agarose gel electrophoresis showing the expression of selected stem cell genes from the following donor age groups: (a) 30–40 years old, (b) 41–50 years old, and (c) 51–65 years old; (d) shows the average of the GADPH relative gene expression and the error bars indicate the standard deviation in samples from the same age group.