Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles

ABSTRACT: Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. KEY MESSAGE: Type of xeno-free media influences biological properties of UC-MSCs in vitro. Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs. EVs collected from xeno-free cultures of UC-MSCs are biologically active. Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells. Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00109-016-1471-7) contains supplementary material, which is available to authorized users

Semiquantitative analysis of angiogenesis-related proteins secreted by MSCs after 10 days of endothelial culture by Western blotting.

<p><b>(A)</b> Representative nitrocellulose membranes incubated with conditioned culture medium harvested from cultures of all three experimental groups of MSC (MCPIP1-overexpressing MSCs, empty vector- treated (Puro) MSCs and untreated (Control) MSCs). Pairs of duplicate spots represent each angiogenesis- related protein. Pair of duplicate spots with upregulated expression when compared with control cells were included in brackets. <b>(B)</b> Semiquantitative assessment of selected protein concentrations based on pixel density analysis with Quantity One software. All results are presented as means ± SD. Statistically significant differences (P<0.05) are shown when compared with Puro (*) and Control (#). The analysis was conducted using a mixture of conditioned media collected under cells prepared from three independent experiments. Control—untreated MSCs; Puro—empty vector-treated MSCs; MCPIP1- MSCs overexpressing MCPIP1.</p

Proteins unique or greatly expressed in MCPIP1- expressing MSCs when compared with Puro-treated MSCs.

<p>All listed proteins were identified in 2 samples based on two or more peptides identified for every protein; classified according to the fold change in expression based on global proteomic analysis. Selected functions of all listed proteins were assigned based on UniProtKB data base. ND- fold change in protein expression was not computed since the indicated proteins were not detected in Puro-treated cells.</p><p>Proteins unique or greatly expressed in MCPIP1- expressing MSCs when compared with Puro-treated MSCs.</p

Proteins not found or expressed at lower level in MCPIP1-expressing MSCs when compared with Puro-treated MSCs.

<p>All listed proteins were identified in 2 samples based on two or more peptides identified for every protein; classified in accordance with the fold change in expression based on global proteomic analysis. Selected functions of the listed proteins were assigned based on UniProtKB data base. ND- fold change in protein expression was not computed since the indicated proteins were not detected in MCPIP1-overexpressing cells.</p><p>Proteins not found or expressed at lower level in MCPIP1-expressing MSCs when compared with Puro-treated MSCs.</p

Expression of angiogenesis- related markers in MSCs after angiogenic differentiation.

<p><b>(A)</b> Expression of mRNA for Gata-2, vWF, Tie-2 and VE-cadherin genes in MCPIP1- overexpressing MSCs after 5 and 10 days of angiogenic differentiation by real time RT-PCR. Fold change in mRNA concentration in MCPIP1- transduced MSCs was computed when compared with Puro-treated cells (calculated as 1). <b>(B)</b> Representative images of angiogenic marker expression assessed with immunocytochemistry in MCPIP1-overexpressing MSCs and Puro- treated MSCs differentiated into endothelial phenotype <i>in vitro</i>. MCPIP1-overexpressing MSCs and Puro were stained against intranuclear transcription factor Gata-2 (Alexa Fluor 488, green) and VE-cadherin (Alexa Fluor 546, red), whereas nuclei were co-stained with DAPI (blue). Cells were analyzed with Leica DM-IRE fluorescent microscope. Scale bars indicate 50μm. <b>(C)</b> Quantitative analysis of angiogenic differentiation of MCPIP1- overexpressing MSCs and Puro cells after 5 and 10d of culture. Graphs represent percentages of cells expressing the indicated angiogenic marker identified by immunohistochemisty within both MSC groups. All results are presented as means ± SD. Statistically significant differences (P<0.05) are shown when compared with Puro (*). Analysis based on three independent experiments. Puro—empty vector-treated MSCs; MCPIP1- MSCs overexpressing MCPIP1.</p

Impact of MCPIP1 expression on selected functions of MSCs at 72h post transduction.

<p><b>(A)</b> Viability and apoptosis induction by flow cytometry assays. Table represents percent content of cells undergoing necrosis, early apoptosis, late apoptosis and exhibiting caspase 3 and 7 activation among MCPIP1- overexpressing MSCs, empty vectors (Puro)- treated and untreated (Control) MSCs. <b>(B)</b> Proliferation by Countess II Automated Cell Counter (Life Technologies) (left). The graph shows the relative level of proliferation of MCPIP1-overexpressing MSCs (black bar) when compared with Puro-treated cells (hatched bar; recalculated as 1) and untreated Control MSCs (white bar). Morphology of MCPIP1- overexpressing MSCs, empty vectors (Puro)- treated and untreated (Control) MSCs (right). Scale bars: 100 μm. <b>(C)</b> Antigenic profile of MSCs by flow cytometry. Expression of CD90, CD105 and Sca-1 antigens on MCPIP1-overexpressing MSCs and control cells (Control and Puro) is shown on representative dot-plots. Analyzes were performed on CD45- subsets indicating MSCs using the LSR II flow cytometer (Becton Dickinson). Right graph shows quantitative data representing percent content of each subpopulation of antigenically-defined MSCs among three experimental groups. All results are presented as mean ± SD. Statistically significant differences (P<0.05) are shown when compared with Puro (*) and Control (#). Analysis based on three independent experiments. Control—untreated MSCs; Puro—empty vector-treated MSCs; MCPIP1- MSCs overexpressing MCPIP1.</p