Angiogenic and anti-inflammatory properties of micro-fragmented fat tissue and its derived mesenchymal stromal cells

Abstract

Background: Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population.

Methods: Human LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1.

Results: Our results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1.

Conclusions: Our data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.

Figures

Fig. 1

Histological and immunohistochemical analysis of LG and LP. H&E staining shows higher content of microvessels in LG (a) compared to LP (b). The immunohistochemistry shows an intense staining (black arrows) for the endothelial marker CD31 (c and d) and mural cell marker SMA (e and f). Staining with NG2 (g and h) was associated to microvessels and showed the presence of pericytes, that were more abundant in LG compared to LP (black arrows). Staining for CD146 (i and j), a marker present on both pericytes and endothelial cells, was more diffuse and intense in LP, while in LG was more associated to microvessels (black arrows). VEGF staining was present in the connective of both LG (k) and LP (l); LG showed less connective matrix than LP (Magnification 20x)

Fig. 2

Cultures of LG-MSCs upon collagenase dissociation. Representative of five different Lipogems®-derived MSCs (LG-MSC) cultured from 1 h (a) to 21 days (f). Upon enzymatic digestion of human LG specimens, the obtained clusters were seeded in IMDM + 5 % FBS (a) in order to allow the adhesion and the proliferation of the released cells. After 72 h (b) all cultures showed the capacity to adhere on a plastic substrate and the typical stromal morphology. Starting from 5 days of culture (c) clusters of endothelial cells (black arrow) were highly distinguishable among the stromal cells and after 10 days (d) were removed from the culture by using CD31+ magnetic beads. Stromal and endothelial cell population was cultured separately and at 21 days reached complete confluence (e) and (f) respectively. (Magnification 10x)

Fig. 3

Immunostaining of early culture of cells obtained after collagenase digestion of LG. Endothelial, pericyte and mesenchymal markers were used to characterize the cells extracted from LG. Cells analyzed upon 48-72 h of culture, showed the presence of a significant number of cell colonies positive for CD31, CD34 endothelial markers, surrounded by fibroblastic negative cells (black arrows). Conversely, several fibroblastic-like cells were positive for the pericyte marker NG2, and the mesenchymal markers CD105, CD44 and SMA. Interestingly, CD146 stained very intensely both colonies and fibroblastic cells; very few cells were negative. Fibroblastic-like cells were also positive for TGFβ1 and VEGF, growth factors that are usually secreted by MSCs. (Magnification 20x)

Fig. 4

Immunocytochemistry of secondary culture of LG-derived MSCs. Immunocytochemistry was performed on LG-MSCs after 2–3 in vitro passages. Under these experimental conditions, LG-MSCs lost the expression of endothelial markers CD31, CD34 and KDR (a receptor for VEGF), while retaining pericyte and mesenchymal markers NG2, CD105 and SMA. Cells were still positive for TGFβ1 and VEGF. To note, CD146 positive cells (black arrow) were very few, suggesting that this marker in LG was probably mostly expressed by the cells of endothelial origin (Magnification 20x)

Fig. 5

Characterization of Lipogems®-derived MSCs. The levels of CD44-PE, CD73-PE, CD90-PE, CD105-PE, CD31-PE markers in LG-MSCs were analyzed by flow cytometry. The percentage indicate the positivity of the cells for their relative surface markers, while the gate indicate the isotypic control (Magnification 10x)

Fig. 6

Angiogenic properties of LG and LG-MSCs. The angiogenic properties of LG and LG-MSCs were investigated by testing their CM. In (a) the effect of LG-CM and LG-MSCs-CM on HUVECs proliferation. EGM medium was used as positive control for proliferation, EGM medium without growth factors the negative control (CTRL) (* p < 0.05; ** p < 0.01 versus CTRL). In (b) the quantification of cord formations on MTG by HUVECs under the influence of LG-CM and LG-MSCs-CM at 12 h and at 24 h in (c) (* p < 0.05; ** p < 0.01 versus CTRL). In (d) the analysis of angiogenic molecules present in the secretome of LG-CM in comparison to LG-MSCs-CM. MMP2 was significantly reduced in LG-CM, while ANG-1 and ANG-2 were increased (* p < 0.05; ** p < 0.01 versus LG-MSCs-CM). In (e) and in (f) the effect of LG-CM and LG-MSCs-CM on the expression of HUVEC ICAM-1 and VCAM-1 respectively (* p < 0.05; ** p < 0.01 versus CTRL; °p < 0.05, °° p < 0.01 versus TNFα treatment)

Fig. 7

LG-CM and LG-MSCs possess anti-inflammatory properties. The capacity of LG-CM and LG-MSCs to affect inflammatory cell functions was tested on the U937 monocyte cell line. In (a) the effect of LG-CM and LG-MSCs-CM on U937 migration. Different dilutions of LG-CM and LG-MSCs-CM were placed in the lower compartment of a transwell insert (5um pore size). The chemokine MCP-1 at (10 ng/ml) was used as a positive stimuli for U937 migration. RPMI+ 0.2 % BSA was the control medium (CTRL) (* p < 0.05; ** p < 0.01 versus CTRL; °p < 0.05, versus MCP-1 treatment). In (b) adhesion of U937 to HUVECs monolayer in the presence of LG-CM and LG-MSCs-CM. TNFα at 20 ng/ml was used to activate HUVECs (* p < 0.05; ** p < 0.01 versus CTRL; °p < 0.05, °° p < 0.01 versus TNFα treatment). The photos below the figure show the morphological appearance of U937 attached to HUVECs primed with TNFα, LG-CM and LG-MSCs-CM after 30’ incubation (5x magnifications). In (c) and in (d) the release of RANTES and MCP-1 by U937 under the influence of LG-CM and LG-MSCs-CM stimulated or not with LPS (1ug/ml) respectively (*p < 0.05; **p < 0.01 versus CTRL; °p < 0.05, °° p < 0.01 versus LPS treatment)