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Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties

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Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties

Anastazja Andrzejewska et al. Front Immunol.

Abstract

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

Keywords: bone marrow stromal cell; cellular therapy; comorbidity; in vitro potency assay; in vivo and in vitro aging; mesenchymal stromal cell.

Figures

Graphical Abstract
Graphical Abstract
Overview on the molecular and functional assays used for the characterization of biobanked bone marrow stromal cells (BMSC) with respect to in vivo and in vitro aging, with primary assessment of starting material composition, cell morphology, immunophenotype, gene expression profile, multilineage differentiation capacity, immunomodulation, endothelial tube formation and inflammatory response.
Figure 1
Figure 1
Primary isolation and growth kinetics of BMSCs. (A–C) Quality control of BM samples: (A) bone marrow sample weight (in grams) and (B) number of BM-MNCs per sample (cells/cm3), (C) number of BMSCs per sample (cells/cm3), and (D) growth kinetics of BMSCs, with population doublings determined at different passages (P3-6), were quantified for BMSC preparations from adult vs. elderly (n = 9 vs. 12) and for non-diabetic vs. predominantly non-insulin-dependent early-stage diabetic donors (n = 14 vs. n = 7). Data are shown as mean ± SD, and the statistics were evaluated with a Student's t-test.
Figure 2
Figure 2
Morphology and immunophenotype of BMSCs. (A) Representative bright-field microscopy images of BMSC cultures at passage 3 and 6, comparing adult vs. elderly and non-diabetic vs. diabetic donors, showing typical fibroblast-like morphology with a trend toward more irregular morphology in diabetic donors; (B) analysis of cell size and cell volume of trypsin-detached BMSCs from adult vs. elderly (n = 9 vs. 12) and non-diabetic vs. diabetic donors (n = 14 vs. 7). Adult and elderly have similar cell size and volume at passage 3, and the cell size of adult donor-derived BMSCs does not increase with passages, while elderly donor-derived BMSCs display cell enlargement with increasing passage; and (C) flow cytometry analysis of BMSCs (% positive cells; n = 6 random adult or elderly donors at early passage 1–3) with representative histograms shown to the right (unlabeled controls are shown in solid gray). The cells highly express typical BMSC-associated markers CD44, CD73, CD90, and CD105 while exhibiting no/weak expression of non-MSC-associated markers CD14, CD31, CD34, and CD45. Data are shown as mean ± SD, and statistical evaluation was performed by Student's t-test (*p < 0.05 and ***p < 0.001).
Figure 3
Figure 3
Gene-expression-profiling of BMSCs with RNA-sequencing. (A) Principle component analysis (PCA) was employed for the visualization of group separation. Groups were stratified either according to donor age (left panel; n = 9 adult vs. n = 12 elderly donors), health status (central panel: n = 14 non-diabetic vs. n = 7 diabetic donors), and passage number (right panel; n = 6 random donors at passage 3 vs. 6). The PCA showed clear separation when comparing passage 3 vs. 6 (right panel), but no separation for the comparison of adult vs. elderly or diabetic vs. non-diabetic donors, with a random dot distribution throughout the graph. (B–D) passage 3 vs. 6 comparisons: (B) hierarchical clustering heat map with expression values sorted according to donor (rows) and gene (columns), where low expression is denoted by blue and high expression by red; (C) gene ontology (GO) enrichment analysis, with the “Top 10 Results” for changes in biological process (e.g., cell cycle, nuclear and cell division, and chromosome segregation) shown on the left y-axis and the size of the dots representing the counts of genes involved, while the color of the circle (scaled blue/lowest to red/highest) indicates significance expressed as adjusted p-value. (D) Combined David and GO “Top 8 Results” database analysis for changes in biological process (left panel; e.g., mitotic cell cycle and nuclear division) and molecular function (right panel; e.g., cytoskeletal protein binding and microtubule binding), with the z-score indicating the overall decrease or increase in expression for certain GO terms and the log-FC analysis indicating the actual number of down- or up-regulated genes within a certain GO term.
Figure 4
Figure 4
Multilineage differentiation potential of BMSCs. The differentiation capacity of MSCs from adult and elderly (n = 9 vs. 12) and non-diabetic and predominantly non-insulin-dependent early-stage diabetic donors (n = 14 vs. 7) was assessed with different in vitro assays. (A) Osteogenic differentiation was assessed by quantification of BMSC matrix mineralization upon osteogenic induction for 14, 18, and 22 days, with representative images for Alizarin red staining shown at the top. Quantification revealed a time-dependent increase in mineralization for all groups (day 14 vs. day 22) both at passages 3 and 6, while there was no difference between adult vs. elderly or non-diabetic vs. diabetic donors at either time point or passage but a strong reduction in mineralization for higher vs. lower passage cells (P6 vs. P3). (B) Adipogenic differentiation of BMSCs upon in vitro adipogenic induction for 10 and 14 days was quantified by staining of lipid-rich vacuoles with Nile Red, with representative images for vacuole formation shown at the top. Quantification revealed similar vacuole formation for both time points (day 10 vs. day 14), with a trend of modest reduction when comparing higher vs. lower passage cells (P6 vs. P3). There was again no difference between adult vs. elderly or non-diabetic vs. diabetic donors at either time point but a stronger passage-dependent reduction for cells from elderly or diabetic donor donors (P3 vs. P6), though this was less notable for cells from adult or non-diabetic donors. (C) Chondrogenic differentiation of BMSCs upon induction with TGF-beta was quantified as the ratio of proteoglycan synthesis relative to protein content, with representative histology images for alcian blue proteoglycan staining of pellet sections shown to the right. There was no difference between BMSCs from adult vs. elderly or non-diabetic vs. diabetic donors. Data are shown as mean ± SD, and statistical evaluation was performed by using a Student's t-test or ANOVA followed by post-tests (*p < 0.05, **p < 0.01, and ***p < 0.001).
Figure 5
Figure 5
Cytokine challenge reveals altered gene regulation in BMSCs from elderly donors with multiple comorbidities. (A) Comparison of unstimulated vs. cytokine-stimulated BMSCs (n = 7 donors each): hierarchical clustering heat map with expression values sorted according to donors (rows) and genes (columns), where low expression is denoted by blue and high expression by red, and corresponding gene ontology (GO) enrichment analysis with the “Top 10 Results” for changes in biological process (e.g., immune system process and immune and defense response) shown on the left y-axis, and the size of the dots representing the counts of genes involved, while the color of the circle (scaled blue/lowest to red/highest) indicating significance expressed as adjusted p-value. (B) Substratified gene expression analysis focusing only on stimulated samples (n = 7 donors): BMSCs from elderly donors with multiple comorbidities (P777 and P821, with n = 4 and n = 6 comorbidities, respectively) cluster separately from both elderly and adult donors with few comorbidities (P265 and P374; and P264, P784, and P819, respectively, n = 0–1 comorbidities), indicating that the accumulation of multiple comorbidities during advanced age results in a detectable in vivo imprint in the transcriptome of BMSCs. This was accompanied by a decline in cell proliferation and a progression from regular to irregular morphology in culture (representative images at the bottom), resulting in a progressive loss of contact inhibition and cell aggregation. Analysis of the biological processes, showing that P777 and P821 differ under stimulating conditions from the other donors, identified significant downregulation of processes associated with cell proliferation (e.g., mitotic cell cycle, cell division, and chromosome organization), while upregulated processes entailed categories associated with cell differentiation (e.g., multicellular organism process, anatomic structure development, morphogenesis, and ECM organization).
Figure 6
Figure 6
Immunomodulatory and paracrine activity of BMSCs. (A) Immunomodulatory activity of BMSCs (n = 9 adult vs. n = 12 elderly and n = 14 non-diabetic vs. n = 7 diabetic donors, passage 3) to suppress anti-CD3/CD28- or phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cell (PBMC)-proliferation. The PBMCs were labeled with the cell proliferation-tracker dye CFSE, activated with either of the two different stimuli, and cocultured for 5 days with BMSCs from adult or elderly donors, in order to assess their capacity to inhibit the proliferation of CD4+ and CD8+ T-cells with flow cytometry, with representative histograms shown to the right. The quantitative assessment of T-cell proliferation is expressed as the percentage proliferation of CD4+ and CD8+ T cells relative to the positive control without BMSCs. Both adult vs. elderly and non-diabetic vs. diabetic donor-derived MSCs are equally potent in inhibiting CD4 and CD8 T-cell proliferation. (B,C) Paracrine and proangiogenic activity of BMSCs (n = 9 adult vs. n = 12 elderly and n = 14 non-diabetic vs. n = 7 diabetic donors) with and without cytokine stimulation (10 ng/mL of TNF-alpha and IFN-gamma for 24 h): (B) secretion of IL-6 and VEGF (pg/mL) in BMSC-conditioned culture media was assessed with ELISA, detecting elevated levels of IL-6 secretion by BMSCs obtained from elderly or diabetic donors, and (C) proangiogenic activity of BMSC-conditioned media compared to blank group (medium only) in an endothelial tube formation assay, with a representative image for the quantification of endothelial network formation by quantification of the total master segment length (TMSL/field, with 3–5 images assessed per test condition); TMSL/field was slightly increased for adult and non-diabetic donors. Results are given as box plot ± min-max whiskers. Statistical analysis was performed using either a Student's t-test or ANOVA followed by post-tests (*p < 0.05, **p < 0.01, and ***p < 0.001).

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