Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

Abstract

We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases.

Carrageenan enhances chondrogenesis and osteogenesis in human bone marrow stem cell culture

“…Further, by day 14, the cells adopted a rounded morphology with a ring around the nucleus, which is a sign of chondrogenesis (Moghadam et al, 2014). www.ecmjournal.org Also, in the present study, the longest time point was day 14, whilst previous studies have shown formation of cartilage-like tissue from BMSCs (with or without a scaffold) after 4 weeks in culture (Meinel et al, 2004;Xue et al, 2012). Sox-9 expression is also of particular importance, as it has been shown to inhibit bone morphogenetic protein 2 (BMP-2)-induced expression of osteopontin, to enhance collagen type II expression in mesenchymal stem cells (Zhao et al, 2017) and chondrocytes (Bell et al, 1997;Zhao et al, 1997) and to play a pivotal role in chondrogenesis and chondrocyte differentiation (Akiyama et al, 2002;Bi et al, 1999;Ikegami et al, 2011).…”

“…In accordance with previous studies (Benny et al, 2015;Chen et al, 2009;Cigognini et al, 2016;Kumar et al, 2015a;Kumar et al, 2015b;Lareu et al, 2007a;Lareu et al, 2007b;Magno et al, 2017;Peng et al, 2012;Prewitz et al, 2015;Rashid et al, 2014;Satyam et al, 2014;Zeiger et al, 2012), MMC prior to differentiation resulted in a significant increase in ECM deposition, without affecting cell metabolic activity (between 70 % and 80 % at days 4 and 7) and morphology. TGF-β1 and VEGF are secreted by BMSCs (Burlacu et al, 2013;Kwon et al, 2014;Xie et al, 2017) and are involved in chondrogenic and osteogenic differentiation, respectively. Although ECM binds and presents growth factors in a non-canonical way (Droguett et al, 2006;van der Kraan et al, 2002;Wilgus, 2012) and ECM sheets produced under MMC, compared to standard culture conditions, retain larger amount of VEGF, fibroblast growth factor 2 (FGF-2) and hepatocyte growth factor (HGF) (Prewitz et al, 2015), a significant increase in growth factor retention was not observed, possibly attributed to the short time point (2 d).…”

Aggregation of Human Mesenchymal Stromal Cells Eliminates Their Ability to Suppress Human T Cells

“…MSC immunomodulatory phenotype has been studied for years using 2D-adherent culture on tissue culture plastic; however, MSCs administered in vivo experience a very different environment. Specifically, there is evidence that in particular local delivery contexts, MSCs aggregate in vivo (14,17), which alters the repertoire of trophic factors the cells produce (18,19,40). This raises the question: Are spheroid MSCs more immunomodulatory potent than their adherent counterparts, or do they have a different immunomodulatory profile?…”

“…However, the size of the spheroids studied are critically different. While we used 20,000 MSCs in all of our spheroids, Zimmermann et al used spheroids containing only 500 cells, and aggregate size has been shown to substantially affect spheroid secretome (19,42). While such small sizes are controllable in the lab, we chose larger aggregates to more closely approximate spontaneous aggregation that would occur upon local injection of a concentrated cell suspension in vivo (14).…”

“…Alterations in secretome change MSCs interactions with immune cells. Studies to date have shown aggregation into spheroids causes MSCs to upregulate PGE2, TSG-6, IL-1α/β, and STC1, as well as several matrix factors (14,18,19). In trans-well experiments with immortalized mouse macrophages, human spheroid MSCs reduced macrophage production of TNF-α and increased IL-10 more than adherent MSCs (14,18,20).…”