Low-affinity Nerve Growth Factor Receptor (CD271) Heterogeneous Expression in Adult and Fetal Mesenchymal Stromal Cells

Abstract

Human multipotent mesenchymal stromal cells (MSC) are isolated from a plethora of tissue sources for cell therapy purposes. In 2006, the International Society for Cellular Therapy (ISCT) published minimal guidelines to define MSC identity. Nevertheless, many independent studies demonstrated that cells meeting the ISCT criteria possessed heterogeneous phenotypes and functionalities, heavily influenced by culture conditions. In this study, human MSC derived from many adult (bone marrow and adipose tissue) or fetal (cord blood, Wharton's jelly, umbilical cord perivascular compartment and amniotic fluid) tissues were investigated. Their immunophenotype was analyzed to define consistent source-specific markers by extensive flow cytometry analysis and real-time qRT-PCR. CD271⁺ subpopulations were detected in adult MSC, whereas NG2 was significantly more expressed in fetal MSC but failed validation on independent samples coming from an external laboratory. The highest number of CD271⁺ adult MSC were detected soon after isolation in serum-based culture conditions. Furthermore, heterogeneous percentages of CD271 expression were found in platelet lysate-based or serum-free culture conditions. Finally, CD271⁺ adult MSC showed high clonogenic and osteogenic properties as compared to CD271^- cells. To conclude, in this phenotype-function correlation study CD271⁺ subpopulation confers heterogeneity on adult MSC, confirming the need of more specific markers to address MSC properties.

Figure 1

Fetal and adult multipotent mesenchymal stromal cell (MSC) morphology. (a) Representative bright-field microscopy images of cultured MSC isolated from fetal and adult tissue sources, and of human skin fibroblasts (HSF). Fetal and adult MSC clonogenic potential. (b) The histogram shows the percentage of cells with clonogenic capability under low-density seeding conditions for fetal and adult MSC (n = 3 for each cell population). Statistical analysis was by non-parametric two-tailed Mann-Whitney-Wilcoxon test (P = 0.5333); ns, statistically not significant difference. ADMSC, adipose tissue MSC; BMMSC, bone marrow MSC; CBMSC, cord blood MSC; WJMSC, Wharton’s jelly MSC; PVC, perivascular cell; AFC, amniotic fluid cell.

Figure 2

Adult multipotent mesenchymal stromal cells (MSC) exhibit CD271-positive subpopulation. (a) Representative dot plots of CD271 expression by adult and fetal MSC and of human skin fibroblast (HSF). (b) CD271 surface marker expression and (c) gene expression, both normalized to stromal non-stem control (HSF). Mean and standard deviation for single MSC types (n = 3) or for adult (n = 6) vs. fetal (n = 12) MSC groups (separated by dotted line). Statistical analysis was by non-parametric two-tailed Mann-Whitney-Wilcoxon test (P = 0.0004) or by Kolmogorov-Smirnov normality test (α = 0.05), followed by unpaired two-tailed t-test (P = 0.012); **P < 0.01, ***P < 0.001. ADMSC, adipose tissue MSC; BMMSC, bone marrow MSC; CBMSC, cord blood MSC; WJMSC, Wharton’s jelly MSC; PVC, perivascular cell; AFC, amniotic fluid cell; adult, MSC from adult sources (black bar); fetal, MSC from fetal sources (white bar).

Figure 3

Fetal multipotent mesenchymal stromal cells (MSC) show high positivity for NG2. (a) Representative histograms of NG2 expression by adult and fetal MSC and of human skin fibroblast (HSF). (b) NG2 surface marker expression and (c) gene expression, both normalized to stromal non-stem control (HSF). Mean and standard deviation for single MSC types (n = 3) or for adult (n = 6) vs. fetal (n = 12) MSC groups (separated by dotted line). Statistical analyses were by non-parametric two-tailed Mann-Whitney-Wilcoxon test (b, P = 0.001), or by Kolmogorov-Smirnov normality test (α = 0.05) followed by unpaired two-tailed t-test (c, P = 0.0001); ***P < 0.001. ADMSC, adipose tissue MSC; BMMSC, bone marrow MSC; CBMSC, cord blood MSC; WJMSC, Wharton’s jelly MSC; PVC, perivascular cell; AFC, amniotic fluid cell; adult, MSC from adult sources (black bar); fetal, MSC from fetal sources (white bar).

Figure 4

Fetal and adult multipotent mesenchymal stromal cells (MSC) expression of progenitor- and stemness-associated surface markers. (a) Representative histograms of surface marker expression (CD56, PDGFRβ, CD146 and SSEA4) and (b) gene expression (CD56, PDGFRβ, CD146; not applicable for SSEA4), both normalized to stromal non-stem control (human skin fibroblasts, HSF). Mean and standard deviation for single MSC types (n = 3) or for adult (n = 6) vs. fetal (n = 12) MSC groups (separated by dotted line). Statistical analysis was by Kolmogorov-Smirnov normality test (α = 0.05), followed by unpaired two-tailed t-test for CD56 (a, P = 0.021; b, P = 0.0397), CD146 (b, P = 0.598) and SSEA4 (a, P = 0.0775), by non-parametric two-tailed Mann-Whitney-Wilcoxon test for CD146 (a, P = 0.0976), PDGFRβ (a, P = 0.0007; b, P = 0.0032); *P < 0.05, **P < 0.01, ***P < 0.001; ns, statistically not significant difference. ADMSC, adipose tissue MSC; BMMSC, bone marrow MSC; CBMSC, cord blood MSC; WJMSC, Wharton’s jelly MSC; PVC, perivascular cell; AFC, amniotic fluid cell; adult, MSC from adult sources (black bar); fetal, MSC from fetal sources (white bar).

Figure 5

Hierarchical clustering of multipotent mesenchymal stromal cell (MSC) immunophenotypes separates fetal and adult harvest. (a) Dendrogram generated by single linkage clustering. Height represents relative distance between clusters. (b) Principal component (PC) analysis showing grouping of MSC by source, centered on human skin fibroblasts (HSF) as controls, relative to PC1 and PC2 variables. ADMSC, adipose tissue MSC; BMMSC, bone marrow MSC; CBMSC, cord blood MSC; WJMSC, Wharton’s jelly MSC; PVC, perivascular cell; AFC, amniotic fluid cell.

Figure 6

Adult MSC harbor a CD271⁺ subpopulation. (a) Representative dot plots showing CD271 expression in additional MSC populations isolated by an independent laboratory. (b) Representative histograms showing NG2 expression in additional MSC populations isolated by an independent laboratory. (c) Validation of CD271 expression in n = 8 samples for each MSC source (adult MSC, n = 16; fetal MSC, n = 32). (d) Percentage of colony forming unit-fibroblasts (CFU-Fs) in sorted CD271⁺ (adult CD271⁺) and CD271⁻ (adult CD271⁻) adult MSC. (e) Representative cultures of sorted CD271⁺ (adult CD271⁺) and CD271⁻ (adult CD271⁻) adult MSC, which received osteogenic stimuli. (f) CD271 expression in adult and fetal MSC under different culture conditions. (g) CD271 expression in adult and fetal MSC at early and late passages. Statistical analysis was by unpaired two-sided Mann-Whitney-Wilcoxon test for c (P = 0.0001) and g (Adult P1 vs P5, P = 0.0286; fetal P1 vs P5, P = 0.5143), by unpaired one-sided Mann-Whitney-Wilcoxon test for d (P = 0.0429), by two-way ANOVA (α = 0.05) followed by Tukey’s multiple comparisons test for f (FBS vs PL, P < 0.0001; FBS vs defined, P < 0.0001; PL vs defined, P > 0.05); *P < 0.05, ****P < 0.0001; ns, statistically not significant difference. ADMSC, adipose tissue MSC; BMMSC, bone marrow MSC; CBMSC, cord blood MSC; WJMSC, Wharton’s jelly MSC; adult, adult MSC; fetal, fetal MSC.