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, 21 (3-4), 767-81

Effects of a Ceramic Biomaterial on Immune Modulatory Properties and Differentiation Potential of Human Mesenchymal Stromal Cells of Different Origin

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Effects of a Ceramic Biomaterial on Immune Modulatory Properties and Differentiation Potential of Human Mesenchymal Stromal Cells of Different Origin

Giulio Bassi et al. Tissue Eng Part A.

Abstract

The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs), and cord blood (CB-MSCs) in the presence of a hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as a scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types toward T, B, and NK cells; in addition, when primed with inflammatory cytokines, MSCs similarly increased their suppressive capacities in the presence or absence of HA/TCP. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with upregulation of OSTERIX and OSTEOCALCIN, similar to what was obtained with dexamethasone and, to a higher extent, with bone morphogenetic protein 4 (BMP-4) treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of indoleamine-2,3 dioxygenase (IDO), which plays a central role in T-cell inhibition, but also cyclooxygenase-2 (COX-2) that was not significantly involved in the immune modulatory effect of human undifferentiated MSCs. Since COX-2 is significantly involved in bone healing, its induction by HA/TCP could also contribute to the therapeutic activity of MSCs for bone tissue engineering.

Figures

<b>FIG. 1.</b>
FIG. 1.
Effect of hydroxyapatite (HA)/tricalcium-phosphate (TCP) discs on survival and proliferation of T, B, and NK cells. (A) Relative percentage of live (CD45+/active-caspase 3) and dead cells (CD45+/active-caspase 3+) after culture in absence or presence of HA/TCP discs (n=8); (B) Percentage of live CFSE-labeled dividing cells after 4–6 days of culture in absence or presence of HA/TCP (n=8); (C) mesenchymal stromal cells (MSCs) were collected after 5 days of culture in absence or presence of HA/TCP and stained with appropriate antibodies (empty histogram) or isotype-matched controls (gray histogram). One bone marrow (BM)-MSC sample, representative of 14 MSC batches (5 BM-MSCs, 5 ASCs, and 4 cord blood [CB]-MSCs), is shown for MSC-related markers and, in (D), for immunological markers. Wilcoxon paired test was used to compare different groups, ns p>0.05.
<b>FIG. 2.</b>
FIG. 2.
Immune modulatory features of BM-MSCs, ASCs, and CB-MSCs toward different immune effector cells in presence of HA/TCP scaffold. (A) Immune effector cells (IEC) were co-cultured in absence or presence of BM-MSCs (n=5), ASCs (n=5), and CB-MSCs (n=4), and seeded in standard culture setting or in presence of HA/TCP. After 4–6 days of co-culture, T, B, and NK cells were collected and stained with anti-CD45-APC and active-caspase-3 antibody-PE. Percentages of live (clear histograms) and dead (gray histograms) cells were calculated on total CD45+ events. (B) CFSE-labeled T, B, and NK cells were stimulated and co-cultured with different MSC types (BM, ASC, CB, and interferon-γ (IFN-γ)+tumor necrosis factor-α (TNF-α) treated MSC: pBM, pASC, and pCB) in a standard culture setting and in combination with HA/TCP discs. After 4–6 days of co-culture, cells were collected and stained with CD45-PerCP and TO-PRO-3. Percentage of divided cells in absence of MSCs was set as 100% (dotted lines), and each condition was normalized on this value. Relative proliferation of immune effector cells in co-culture with MSCs of different origins in a standard culture setting (white box) was analyzed in parallel with the relative proliferation in co-culture with different MSC types in association with HA/TCP discs (gray box). (C) CFSE-labeled T cells were stimulated and co-cultured with BM-MSCs (n=5) in presence of L-1MT, NS-398, and anti-IFN-γ blocking antibody in standard culture condition and in presence of HA/TCP scaffold. After 6 days of co-culture, cells were collected and stained with CD45-PerCP and TO-PRO-3. The percentage of live T cells that underwent at least one cell division was represented in each condition. Dotted line represented the mean of divided T cells in absence of MSCs. (D) MSCs (n=9) were stimulated or not by IFN-γ +TNF-α for 48 h in absence or presence of HA/TCP discs. Culture supernatants were collected for indoleamine-2,3 dioxygenase (IDO) activity quantification by HPLC. One-way ANOVA test was used to compare different groups. ns p>0.05; *p<0.05; **p<0.01; and ***p<0.001.
<b>FIG. 3.</b>
FIG. 3.
Differentiation potential of BM-MSCs, ASCs, and CB-MSCs in presence of dexamethasone (DXM) or bone morphogenetic protein 4 (BMP-4) in standard culture conditions. After a 21 day culture in osteogenic medium, cells were fixed and stained for determination of alkaline phosphatase activity (upper), mineral deposition through Von Kossa (VK) staining (middle; representative image of lipid droplet detection in ASCs as indicated by the arrow), and calcium nodule formation by means of Alizarin Red (AR) staining (lower). Images were acquired by Axiovert Z1 microscope (Zeiss) at 10× magnification. The representative images are derived from five different donors for BM-MSCs, five different donors for ASCs, and four different donors for CB-MSCs. Color images available online at www.liebertpub.com/tea
<b>FIG. 4.</b>
FIG. 4.
Osteoblastic differentiation of BM-MSCs, ASCs, and CB-MSCs with DXM or BMP-4-inducing media in a standard culture setting and in presence of HA/TCP scaffold. (A) BM-MSCs, ASCs, and CB-MSCs were cultured for 21 days in presence of osteogenic medium supplemented with dexamethasone or BMP-4. Relative gene expression was represented as fold change (Y axis) as compared with control condition without inducers. (B) Synergistic effect of HA/TCP discs on osteocalcin (OSC) expression in BM-MSCs. Y axis represents fold change of OSC gene expression in BM-MSCs+BMP-4 in association with HA/TCP as compared with BM-MSCs cultured in presence of BMP-4 in a standard culture setting. (C) Osterix and osteocalcin relative expression in BM-MSCs, ASCs, and CB-MSCs cultured in presence of HA/TCP, as compared with cells cultured in standard culture conditions, in absence of dexamethasone and BMP-4 addition. (D) Western blotting analysis of runt-related transcription factor 2 (RUNX2) expression of MSCs cultured in expansion medium, according to each cell factory protocol (upper panel). Western blotting analysis of RUNX2 protein expression after 21 days of culture in differentiation medium containing DXM or BMP-4 in BM-MSCs, ASCs, and CB-MSCs. β-actin was used as loading control. (E) Quantification of RUNX2 expression in expanded MSCs (BM-MSC, n=3; ASC n=3; CB-MSC n=3) and in differentiated MSCs (BM-MSC, n=3; ASC n=3; CB-MSC n=3). For quantification of RUNX2 expression, relative protein level was normalized to β-actin. Nonparametric paired Wilcoxon test was used to compare different groups. *p<0.05 and **p<0.01.
<b>FIG. 5.</b>
FIG. 5.
Immune modulatory features of predifferentiated BM-MSCs, ASCs, and CB-MSCs toward different immune effector cells in presence of HA/TCP scaffold. (A) After differentiation process of MSCs, purified T, B, and NK cells were thawed and seeded in 24-well plates in presence of undifferentiated or BMP-4-differentiated MSCs. After 4–6 days of co-culture T, B, and NK cells were collected and stained with anti-CD45-APC and active-caspase-3 antibody-PE. Percentage of live (clear histograms) and dead (gray histograms) cells was calculated in total CD45+ events. (B) CFSE-labeled T, B, and NK cells were stimulated and co-cultured with undifferentiated and predifferentiated BM-MSCs, ASCs, and CB-MSCs. After 4–6 days of co-culture, cells were collected and stained with CD45-PerCP and TO-PRO-3. Percentage of dividing cells in absence of MSCs was set as 100% (dotted lines), and each condition was normalized on this value. Relative proliferation of immune effector cells in co-culture with undifferentiated MSCs (white box) was analyzed in parallel with the relative proliferation in co-culture with BMP-4-differentiated MSCs (gray box). The data obtained are represented as relative proliferation of stimulated-T, -B, or -NK cells in comparison with the control condition (immune effector cells without MSCs). (C) CFSE-labeled T cells were stimulated and co-cultured with undifferentiated and predifferentiated BM-MSCs in presence of L-1MT and NS-398. After 6 days of co-culture, cells were collected and analyzed by means of flow cytometry. Percentage of dividing cells in absence of MSCs was set as 100% (dotted lines), and each condition was normalized to this value. The percentage of proliferation of T cells co-cultured with MSCs in absence of inhibitor was considered as zero. Relative rescue of proliferation was calculated by comparing the percentage of divided cells with the control. (D) Culture supernatants of differentiated and/or primed BM-MSCs (n=5) were collected for IDO activity quantification by HPLC and for prostaglandin E2 (PGE2) quantification by ELISA assay. One-way ANOVA test was used to compare different groups; *p<0.05; **p<0.01; and ***p<0.001.
<b>FIG. 6.</b>
FIG. 6.
IDO1 and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in primed BM-MSCs in control medium or in presence of BMP-4. (A) BM-MSCs were cultured for 21 days in presence of either osteogenic medium alone or supplemented with BMP-4. After differentiation process, cells were cultured for 48 h with inflammatory cytokines before RNA extraction. Relative gene expression is represented (Y axis) using GADPH as a reference gene. (B) Effect of inflammatory priming on RUNX2 expression in BM-MSCs cultured for 21 days in presence of BMP-4. After differentiation process, cells were exposed to inflammatory cytokines for 48 h before RNA extraction. Relative gene expression is represented as fold change (Y axis) as compared with GADPH expression. (C) Western blotting analysis of RUNX2, NF-κB, phospho-NF-κB, and phospho-Smad-1, -5, and -8 expression in BM-MSCs. After 21 days of induction with either control medium, DXM-based medium or BMP-4-based medium, cells were lysed and proteins were collected and detected through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). β-Actin was used as loading control. One representative experiment was shown for BM-MSCs. pC=48 h IFN-γ and TNF-α +21 days control medium; C=21 days control medium; Cp=21 days control medium+48 h IFN-γ and TNF-α; pD=48 h IFN-γ and TNF-α +21 days DXM-based medium; D=21 days DXM-based medium; Dp=21 days DXM-based medium+48 h IFN-γ and TNF-α; pB=48 h IFN-γ and TNF-α +21 days BMP-4-based medium; B=21 days BMP-4-based medium; and Bp=21 days BMP-4-based medium+48 h IFN-γ and TNF-α. Nonparametric paired Wilcoxon test was used to compare different groups. *p<0.05 and **p<0.01.

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