Mean and CV values of log2 FI for each of 246 investigated markers. (DOCX 22 kb

Eliana Amati (3690244)

Francesco Rodeghiero (3690250)

Gianluca Rotta (4773177)

Giuseppe Astori (38488)

Katia Chieregato (3690253)

Marco Ruggeri (3690226)

Martina Bernardi (747956)

Omar Perbellini (3690223)

Sabrina Sella (3690241)

Stem Cell Research & Therapy

Neural marker classification according to unsupervised HCL. Heat-map expression of the markers identified by Okolicsanyi [27]: CD24, CD200, CD271, CD56, CD146, and CD73. Antigen expression is color coded from white (no expression) to red (strong expression). Data are presented as log2 FI (median fluorescence intensity on the isotype control). (TIFF 1568 kb

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Additional file 5: Figure S3. of 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

Eliana Amati

Omar Perbellini

Gianluca Rotta

Martina Bernardi

Katia Chieregato

Sabrina Sella

Francesco Rodeghiero

Marco Ruggeri

Giuseppe Astori

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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

List of the lowest variable markers (n = 59, CV cut-off of log2 FI ≤ 0.5) sorted by function and cluster expression. Markers with no expression were assigned to cluster 1, low-expressed markers to cluster 2, and intermediate markers to cluster 3, while a total of 18 markers clustering as very strong were assigned to cluster 4. (PPTX 50 kb

Additional file 3: Table S2. of 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

Additional file 2: Table S1. of 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

Differentially expressed markers between bulk cultures and single derived clones. The expression of each marker (n = 246) was compared by paired t test (n = 3; p < 0.01). (TIFF 1629 kb

Additional file 4: Figure S2. of 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

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