Human adipose-derived stromal vascular fraction: characterization, safety and therapeutic potential in an experimental mouse model of articular injury

Abstract

Due to their capacity to self-renew, proliferate and generate multi-lineage cells, adult-derived stem cells offer great potential in regenerative therapies to treat maladies such as diabetes, cardiac disease, neurological disorders and orthopedic injuries. Commonly derived from adipose tissue, the stromal vascular fraction (SVF), a heterogeneous cell population enriched with mesenchymal stem cells (MSCs), has garnered interest as a cellular therapy due to ease of accessibility as an autologous, point-of-care application. However, the heterogeneous cell population within SVF is not historically taken into consideration when injecting into patients. Here, we characterized SVF, determined its safety and verify its therapeutic effects in a NOD/scid mouse model of articular injury. SVF were isolated from lipoaspirates utilizing a commercially available system (InGeneron Inc.), while MSCs were isolated from SVF via cell culture. Flow cytometry showed that neither age nor BMI affects the frequency of progenitor cells-like (CD31+CD34+), immune cells-like (CD4+) T cells, (CD14+) monocytes and total number of cells obtained. However, there was a negative correlation between donor BMI and MSC frequency within the SVF. ELISAs showed that following LPS activation in SVF, there were low levels of TNF-α and high levels of IL-10 secreted. However, T cell activation with anti-CD3 or anti-CD3+ anti-CD28, while leading to expected high levels of IFN-γ, did not lead to significant levels of TGF-β. PCR analysis showed no significant numbers of cells outside the joint 1-hour post injection, moreover, no engraftment or abnormal growth in other organs 60-days post injection. Finally, both cell populations were able to ameliorate disease progression, as confirmed by the increase in movement of treated groups compared to injured groups. Noteworthy, the histological analysis indicated that there was no cartilage growth, suggesting an alternative therapeutic mechanism to cartilage regeneration.

Keywords: Adipose; Autologous stem cells; Cartilage; Mesenchymal stem cell.

Figure 1.. Number and frequency of different cell populations within the SVF. Nucleated SVF cells and SVF subpopulations (CD4+ T cells, CD14+ monocytes, CD31+ CD34+ epithelial progenitors, CD31- CD45- CD34+ MSCs) were plotted against donor age (Top) and BMI (Bottom) (n=30, squared Pearson's correlation = R²).

Figure 2.. SVF activation. (a) SVF isolated from human adipose were activated in 96-well plates (5x10⁴ cells/well) with LPS (100 ng/ml) or with anti-CD3/anti-CD28 coated beads, and the supernatant was collected 18 hours and 7 days post activation, respectively. Levels of both pro-(TNF-α and IFN-γ) and anti-(IL-10 and TGF-β) inflammatory cytokines were measured using ELISAs. (b) Following LPS, there were low levels of TNF-α and high levels of IL-10 being secreted. (c) T cell activation with beads coated in either anti-CD3 or anti-CD3 + anti-CD28, while leading to expected high levels of IFN-γ, did not lead to significant levels of TGF-β. (Mean ± 3 SD, one-Way ANOVA, ** p≤0.01 compared to Control).

Figure 3.. Biodistribution of SVF and MSC. (a) PCR of human β-globin versus mouse GAPDH-specific primers revealing Primer 1 -KM (arrow) as human-specific (b) PCR results of human and mouse DNA quantification using the human-specific primer KM and non-specific primer GAPDH. (c) Flow cytometry profile of SVF population compared to isolated MSCs. (d) MSC detection in the lung using RNAScope. (e) Representative PCR for detection of intravenously injected MSC and SVF (5x10⁵ cells). (f) Quantification of lung engraftment (arrow) by densitometry relative to GAPDH signals (N=2).

Figure 4:. SVF biodistribution and therapeutic outcome following articular injury. (a) Articular injury study scheme. (b) Representative pictures of injured knees. (c) No organ engraftment following intra-articular injury. (d) Distance travelled measured with force plate actimeter for sham, articular injury mice that received PBS, articular injury mice treated with SVF/MSCs. (e) Representative histological images of knee joints. (f) 60-days post-injection analysis of engraftment in various mouse organs (Mean ± SD, Friedman Test, * p≤0.05 compared to Control).