Letters to the Editor

Comparison of trophic factors secreted from human adipose-derived stromal vascular fraction with those from adipose-derived stromal/stem cells in the same individuals

Cell therapy for several different diseases has been experimentally investigated for regeneration of damaged tissues. Adipose tissue in the human body is abundant, and can be easily and safely harvested in large quantities, thus it has attracted attention as a source for cell therapy. Stromal vascular fraction (SVF) is currently a major source for cell therapy after isolation from adipose tissues without the need for culture using a Celution System (Cytori Therapeutics) [1], [2]. In our previous studies, we found that periurethral injection of autologous SVFs may represent a safe and feasible treatment modality for male stress urinary incontinence [3], [4]. However, characterization of the SVFs used in our experiments has yet to be reported.

Mesenchymal stromal cells (MSCs) are well known as a potential source for cell therapy and their therapeutic effect reportedly occurs through secretion of trophic factors [5], [6]. SVFs are composed of heterogeneous cell populations, including adipose-derived stromal/stem cells (ASCs), granulocytes, monocytes, lymphocytes, endothelial cells and pericytes [7]. Unfortunately, there is scant information available from comprehensive cytokine or chemokine quantitative analyses of conditioned medium samples isolated from SVFs. In this letter, we show trophic factors released by SVFs and compared them with those released by ASCs in the same individuals.

SVFs were previously isolated from tissues obtained from three male subjects (77, 69 and 75 years old) with a Celution System and used in our clinical research [3]. For the present study, SVFs were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; AusGenex) for isolation of ASCs and SVFs were also cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% FBS as culture of SVFs in 24-well plates. ASCs were detached after reaching 60–70% confluence by incubation with 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA; Life Technologies) and subcultured at a 1:4 dilution under the same conditions. Furthermore, after reaching 60–70% confluence following the second passage (Figure 1A), the culture medium was switched to DMEM without serum and medium samples were collected 24 h later. For SVFs, on day 2 of the primary culture, floating cells and culture media were collected and centrifuged, then the cell pellets were suspended in RPMI 1640 without serum and re-plated in 24-well plates containing adherent cells (Figure 1B), then medium samples were collected after 24 h. Collected medium samples from both ASCs and SVFs were concentrated approximately 20-fold by use of an Amicon Ultra-15 centrifugal filter unit with an Ultracel-3 membrane (Millipore). The concentrated samples were placed in 96-well plates for analyses of cytokine and chemokine expressions (Milliplex MAP; Human Cytokine/Chemokines Panel-Premixed 41 Plex; Millipore), in accordance with the manufacturer's guidelines. Secreted factors were quantified using a Bio-Plex 200 reader and Bio-Plex Pro wash station (Bio-Rad). Findings regarding the secreted factors were analyzed using Bio-Plex Manager Version 3.0 software. All measurements were normalized to pg/mL/10⁶. The present study was approved by the Ethics Committee of the Nagoya University Graduate School of Medicine, and by the committee of the Japanese Ministry of Health, Labor and Welfare according to the Guidelines on Clinical Research using Human Stem Cells. Written informed consent was obtained from the patients.

As compared with ASCs, SVFs released a greater variety of cytokines (Figure 1C, 1D and 1E), including macrophage inflammatory protein 1 alpha (MIP-1α), granulocyte colony-stimulating factor (G-CSF), macrophage-derived chemokine (MDC), interleukin (IL)-6, IL-8, growth-related oncogene (GRO), monocyte chemoattractant protein-1 (MCP-1), IL-1 receptor antagonist (IL-1ra), macrophage inflammatory protein 1 beta (MIP-1β), interferon-inducible protein (IP-10), platelet-derived growth factor BB (PDGF-BB), IL-12p40, granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-α). The distribution of secreted factors from ASCs was similar to that noted from dental pulp stem cells, as detailed in our previous study [8].

Fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), G-CSF and GM-CSF have been clinically used for wound healing [9]. FGF and VEGF, secreted from both ASCs and SVFs, are well-known trophic factors that have angiogenesis and anti-apoptosis effects [5], [6]. On the other hand, G-CSF and GM-CSF are multifunctional cytokines [9] secreted by SVFs alone. We also found that SVFs secreted many cytokines or soluble protein at significantly higher amounts as compared with ASCs, indicating that SVFs are a more multifunctional source for cell therapy. In other words, factors secreted from SVFs may harbor various paracrine effects, such as immunomodulation, anti-apoptosis, angiogenesis, support of growth and differentiation of local stem cells and progenitor cells, anti-scarring and chemoattraction.