High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells reveals common and differentially expressed markers: identification of angiotensin-converting enzyme (CD143) as a marker differentially expressed between adult and perinatal tissue sources

Abstract

Background: Mesenchymal stromal cells (MSC) are a heterogeneous population of multipotent progenitors used in the clinic because of their immunomodulatory properties and their ability to differentiate into multiple mesodermal lineages. Although bone marrow (BM) remains the most common MSC source, cord blood (CB) can be collected noninvasively and without major ethical concerns. Comparative studies comprehensively characterizing the MSC phenotype across several tissue sources are still lacking. This study provides a 246-antigen immunophenotypic analysis of BM- and CB-derived MSC aimed at identifying common and strongly expressed MSC markers as well as the existence of discriminating markers between the two sources.

Methods: BM-MSC (n = 4) were expanded and analyzed as bulk (n = 6) or single clones isolated from the bulk culture (n = 3). CB-MSC (n = 6) were isolated and expanded as single clones in 5/6 samples. The BM-MSC and CB-MSC phenotype was investigated by flow cytometry using a panel of 246 monoclonal antibodies. To define the markers common to both sources, those showing the smallest variation between samples (coefficient of variation of log₂ fold increase ≤ 0.5, n = 59) were selected for unsupervised hierarchical cluster analysis (HCL). Differentially expressed markers were identified by directly comparing the expression of all 246 antigens between BM-MSC and CB-MSC.

Results: Based on HCL, 18 markers clustered as strongly expressed in BM-MSC and CB-MSC, including alpha-smooth muscle antigen (SMA), beta-2-microglobulin, CD105, CD13, CD140b, CD147, CD151, CD276, CD29, CD44, CD47, CD59, CD73, CD81, CD90, CD98, HLA-ABC, and vimentin. All except CD140b and alpha-SMA were suitable for the specific identification of ex-vivo expanded MSC. Notably, only angiotensin-converting enzyme (CD143) was exclusively expressed on BM-MSC. CD143 expression was tested on 10 additional BM-MSC and CB-MSC and on 10 umbilical cord- and adipose tissue-derived MSC samples, confirming that its expression is restricted to adult sources.

Conclusions: This is the first study that has comprehensively compared the phenotype of BM-MSC and CB-MSC. We have identified markers that could complement the minimal panel proposed for the in-vitro MSC definition, being shared and strongly expressed by BM- and CB-derived MSC. We have also identified CD143 as a marker exclusively expressed on MSC derived from adult tissue sources. Further studies will elucidate the biological role of CD143 and its potential association with tissue-specific MSC features.

Keywords: Angiotensin-converting enzyme; Bone marrow; CD143; Cord blood; Flow cytometry; Hematopoietic progenitor cell marker; High-throughput screening; Immunophenotype; Lyoplate; Mesenchymal stromal cells.

Fig. 1

Common MSC marker identification. a Marker classification according to unsupervised HCL. Heat-map expression of the 59 markers selected by HCL on both bone marrow (BM)- and cord blood (CB)-MSC samples. Antigen expression level is color coded from white (no expression) to red (strong expression). Data are presented as log₂ FI (median fluorescence intensity on the isotype control). Markers are shown according to four clusters of expression (no expression: cluster 1; low expression: cluster 2; intermediate expression: cluster 3; strong expression: cluster 4), with clusters identified with different shades of gray; CB-MSC samples are shown in blue; BM-MSC samples in green; bulk samples in red; single clones in yellow. b Common MSC marker identification. Boxes extend from 25th percentile to the 75th percentile, the line in the middle represents the median value and the whiskers extend from minimum to maximum values. ISCT-positive and negative markers are shown in grey. Abbreviations: HCL, hierarchical clustering; FI, fold increase

Fig. 2

Marker refinement according to % of positive cells and robust coefficient of variation (rCV). The common and highly expressed markers were further selected according to a % of positivity higher than 80% and a rCV < 4% for each sample

Fig. 3

Differentially expressed markers between bone marrow mesenchymal stromal cells (BM-MSC) and cord blood mesenchymal stromal cells (CB-MSC). a Heat-map expression of the three differentially expressed markers: CD130, CD141, and hematopoietic progenitor cell marker (CD143/ACE). Antigen expression level is color coded from white (no expression) to red (very strong expression). b Boxes of expression extend from 25th percentile to the 75th percentile, the line in the middle represents the median value and the whiskers extend from minimum to maximum values. The boxes referred to the log₂ fold increase (Log₂FI) and % of positive (% pos) cells. The differences were computed by Mann-Whitney U test, p < 0.01. Mean fluorescence intensity histograms of each marker are reported on the right. White histograms: BM-MSC; grey histograms: CB-MSC

Fig. 4

CD143 verification as a discriminating marker between adult and perinatal MSC sources. Dot plots of CD143 expression as FI in all the investigated MSC sources (adipose tissue (AT), umbilical cord (UC), bone marrow (BM), and cord blood (CB)). The differences were computed by Mann-Whitney U test, ***p < 0.0001. Black circles: adult MSC sources; white circles: perinatal MSC. ns not significant