Figure 2

Characterization of adult peripheral nerve-derived human SCs and fibroblasts. (a) Discrimination of SCs and fibroblasts via double immunolabeling with S100β and fibronectin antibodies showing strong cytoplasmic expression of S100β in the SCs and extracellular location of fibronectin in the fibroblasts. Images taken at low (upper panels) and high magnification (lower panels) are provided to illustrate the impact of fibroblast overgrowth in a typical human SC culture (passage-2, 18 year old, male) deprived of mitogenic factors for 10 days. SCs in confluent cultures have a tendency to display an elongated, spindle-shaped phenotype. Although morphological differences can aid in SC versus fibroblast identification, immunostaining is needed for clear discrimination, especially in those areas where the SCs intermingle with the fibroblasts (a, lower right panel). (b) Fibroblast proliferation in human SC cultures. SC cultures were maintained in SC growth medium (standard conditions) and allowed to incorporate EdU for 3 days before fixation and detection of EdU in the nucleus (red) of SCs (red arrowheads) and fibroblasts (white arrowheads), as determined by p75^NGFR immunostaining (green). Quantitative data from 3 independent early passage SC cultures is shown to illustrate the variability in the extent of fibroblast proliferation under optimal conditions for SC growth. Donors were identified as D1 (18 year old, male), D2 (10 year old, female) and D3 (51 year old, male). (c) Mitogenic effect of purified growth factors and serum in human fibroblasts obtained by differential adhesion to plastic. Fibroblasts were plated in multiwell dishes to assess the incorporation of [³H]-thymidine in the absence (control) and presence of the indicated mitogenic factors (see Methods). Statistical significance was obtained from a One-Way ANOVA. Experimental treatments showing non-significant differences (ns) with respect to the control (no growth factors added, left bars) are indicated. (d) Lack of senescence in contaminating human fibroblasts. Late passage cultures (passage-4) enriched in senescent human SCs were used to determine SA-βGAL activity (shown in red as artificial color). Two areas (insets) were selected to depict the distribution of SA-βGAL activity (red) in p75^NGFR positive cells (green). Quantitative data and level of significance (T-test) from 2 independent cultures (D1, 18 year old, male, and D2, 51 year old, male) are shown on the right panel. DAPI or Hoechst (blue) was used to label cell nuclei in these and all subsequent fluorescence microscopy images.