Infrapatellar Fat Pad Stem Cells Responsiveness to Microenvironment in Osteoarthritis: From Morphology to Function

Abstract

Recently, infrapatellar fat pad (IFP) has been considered as a source of stem cells for cartilage regeneration in osteoarthritis (OA) due to their ability for differentiation into chondrocytes. However, stressful conditions, like that related to OA, may induce a pathogenic reprograming. The aim of this study was to characterize the structural and functional properties of a new population of stem cells isolated from osteoarthritic infrapatellar fat pad (OA-IFP). Nine OA patients undergoing total knee arthroplasty (TKA) were enrolled in this study [median age = 74 years, interquartile range (IQR) = 78.25-67.7; median body mass index = 29.4 Kg/m², IQR = 31.7-27.4]. OA-IFP stem cells were isolated and characterized for morphology, stemness, metabolic profile and multi-differentiative potential by transmission electron microscopy, flow cytometric analysis, gene expression study and cytochemistry. OA-IFP stem cells displayed a spindle-like morphology, self-renewal potential and responsiveness (CD44, CD105, VEGFR2, FGFR2, IL1R, and IL6R) to microenvironmental stimuli. Characterized by high grade of stemness (STAT3, NOTCH1, c-Myc, OCT-4, KLF4, and NANOG), the cells showed peculiar immunophenotypic properties (CD73⁺/CD39⁺/CD90⁺/CD105⁺/CD44^-/+/CD45^-). The expression of HLA-DR, CD34, Fas and FasL was indicative of a possible phenotypic reprograming induced by inflammation. Moreover, the response to mechanical stimuli together with high expression level of COL1A1 gene, suggested their possible protective response against in vivo mechanical overloading. Conversely, the low expression of CD38/NADase was indicative of their inability to counteract NAD⁺-mediated OA inflammation. Based on the ultrastructural, immunophenotypic and functional characterization, OA-IFP stem cells were hypothesized to be primed by the pathological environment and to exert incomplete protective activity from OA inflammation.

Keywords: inflammation; infrapatellar fat pad; osteoarthritis; reprograming; stem cells.

FIGURE 1

Osteoarthritic infrapatellar fat pad (OA-IFP) and derived cells. (A) Magnetic resonance imaging of the knee in sagittal section, showing the infrapatellar fat pad (IFP – white arrow) before total knee arthroplasty(TKA). (B) Gross appearance of the IFP after surgical excision and before tissue enzymatic digestion for cells isolation. (C) Representative optical microscope image of the IFP stem cells at P0 in culture. P, passage. Scale bar, 100 μm.

FIGURE 2

Proliferative potential and metabolic activity. (A) Optical microscope images of OA-IFP cells in proliferative medium at P8 and P20. P: passage. Scale bars, 100 μm. (B) Population Doubling Level (PDL) calculated throughout 12 generations; cells performed 11.9 ± 8.3 population doublings. (C) Vitality/necrosis/apoptosis test after treatment with fluorescent dyes. Blue dye stained viable cells while red elements corresponded to necrotic cells (scale bar, 100 μm); apoptotic cells were colored in green (scale bar, 50 μm). The images are representative of cells at 8th, 14th, and 20th generations. (D) Metabolic activity (MTT assay) of OA-IFP cells from 24 h to 14 days in culture. For the panels (B,D), results are the average of 3 technical replicates each referring to 9 patients.

FIGURE 3

Ultrastructural characterization of OA-IFP stem cells. Toluidine Blue staining images (A) and TEM micrographs (B–E) of OA-IFP cells at P8 in culture. Black arrows in (C) and (D) show electron dense lysosomes and empty vesicles. Black asterisk in (E) shows secretion material resembling collagen fibrils outside the cytoplasm. M, mitochondria; N, nucleus; G, golgi apparatus; RER, rough endoplasmic reticulum. Scale bars, (A) 20 μm; (B) and (C) 2 μm; (D) and (E) 1 μm. (F) mRNA expression levels of CD38 and CALR in OA-IFP stem cells compared to human leukocytes (^∗P < 0.5; ^∗∗P < 0.01). For panel (F), results are the average of 3 technical replicates referring to 9 patients.

FIGURE 4

Analysis of OA-IFP by qPCR, FCM and immunohistochemistry. OA-IFP subcultures from 8th generation and under proliferative conditions were analyzed at a sub-confluence state for stemness (A,B) or cellular responsivity markers (C,D) by qPCR (A) and FCM [FacsDiva and FlowJo software (B,D)] (B–D) where the histograms show the mean percentage of positive cells; samples treated with only secondary antibodies or isotype control antibodies were used as references. Three technical replicates for each sample were analyzed. (E) Double immunohistochemical analysis of OA-IFP tissue samples showing cellular elements positive to HLA-DR (red) and negative to CD45 (brown). Lymphocytic elements, positive to only CD45 (brown), were considered as an internal control of the method (black arrow, E,a) (Scale bar: 25 μm). FCM, flow cytometry analysis.

FIGURE 5

OA-IFP stem cells responsiveness to environmental stimuli. (A) qPCR analysis of mRNA expression levels of COL1A1, SPARC, GLUT1, and CTTN gene on OA-IFP stem cells at a sub-confluence state; null-expression of GLUT4, CTTN, COL2A1, COL9A3, COL10A1 mRNAs was detected. (B) Response of OA-IFP stem cells to adipogenic stimuli. After 7 and 14 days from stimulation, the cells showed cytoplasmic red-stained lipid droplets; scale bars, 100 μm. (C) Response of OA-IFP stem cells to soluble (endothelial medium enriched of angiogenic factors) and mechanical stimuli (stiff and soft support) at 3 and 7 days from stimulation. OA-IFP stem cells seeded on plastic surface and cultured in proliferation medium represented the control-group; scale bars, 100 μm. (D) Image processing steps of images referring to OA-IFP stem cells cultured on stiff support, at 3 and 7 days from stimulation. After edge detection and threshold binary tools (a,e) the skeletons were calculated (b,f), before the analysis of branches (i.e., number and length) (c,g) and branching points (d,h). The processed images highlight the formation of the cord-like structures. For panel (A), results are the average of 3 technical replicates for each sample referring to 9 patients.