ALMS1-Deficient Fibroblasts Over-Express Extra-Cellular Matrix Components, Display Cell Cycle Delay and Are Resistant to Apoptosis
Figure 1
Size, shape and ultrastructural evaluation of ALMS fibroblasts.
(a) Fibroblasts of healthy control (C3) and (b) ALMS patient (PT3) were grown on standard tissue culture (2D cultures) coverslips and stained with hematoxylin-eosin (magnification 20×). (c) Cell length of about 40 fibroblasts per subject was quantified by measuring the longitudinal cell length and was plotted as mean (horizontal line), maximal and minimal length values (vertical line). **P<0.01 ALMS fibroblast length mean versus controls length mean. (d) The area covered by fibroblast cells during exponential growth was estimated by cell counting in Bürker chambers, after 0.2% trypan blue staining. Results are reported as mean ± SEM. *P<0.05. ALMS fibroblasts were compared with controls by flow cytometric analysis: (e) the resulting forward (FSC) and (f) side light scatter (SSC) mean intensity values are shown for each subject. Controls: C1, black triangle; C2, black star; C3, black diamond; Patients: PT1, white circle; PT2, white square; PT3, white triangle; PT4, white diamond (see also Table 1). Panel g reports the SSC distribution of fibroblasts from C1 (black line) and PT1 (grey line) showing the highly significant shift (***P<0.001, as determined by the Kolmogorov-Smirnov analysis according to the Macintosh CELLQuest software user's guide, Becton Dickinson). (h) Fibroblasts of healthy control (C2) and (i) ALMS patient (PT2) were cultured in HYAFF-11™ scaffolds (3D-cultures) and stained with hematoxylin-eosin (magnification 5×; * biomaterial scaffolds; → fibroblast cells). Transmission electron microscopy was performed in healthy control (C2) (l–n–p) and ALMS patient (PT2) (m–o–q) on 3D-cultured cells. Control fibroblasts showed a normal phenotype, with typical bipolar morphology (l), cytoplasm rich in perpendicular oriented microfilaments (n, ring), the presence of normal pinocytic vesicles (l arrow and p) and probably collagen fibers in the extracellular space (p, ring). In contrast, ALMS fibroblasts appeared as elongated cells tightly adherent to each other and showed well-defined long cytoplasmic extensions (m). Microfilaments (o, ring) were arranged in a unique direction, parallel to the long axis of the cells. A large amount of exocytic vesicles and probably collagen fibrils (q, arrow and ring, respectively) suggest active secretion. Magnification: l–m = 5000×; n–o = 25000×; p = 40000×; q = 30000× (see also Figure S3 and S4).