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Stem Cells International
Volume 2016, Article ID 1319578, 13 pages
Research Article

Expression of CD24 in Human Bone Marrow-Derived Mesenchymal Stromal Cells Is Regulated by TGFβ3 and Induces a Myofibroblast-Like Genotype

1Trauma Department, Hannover Medical School, 30625 Hannover, Germany
2Institute of Laboratory Animal Science, Hannover Medical School, 30625 Hannover, Germany
3Cellular Neurophysiology, Hannover Medical School, 30625 Hannover, Germany

Received 15 June 2015; Revised 11 August 2015; Accepted 12 August 2015

Academic Editor: Franca Fagioli

Copyright © 2016 Luisa Marilena Schäck et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplemental Table 1. Gender and age of the donors whose hBMSCs were used for microarray analysis.

Supplemental Table 2. Number of genes regulated by group stimulus in hBMSCs relative to respective control hBMSCs.

Supplemental Table 3. List of top 10 up- and downregulated genes from CD24 Up Group, CD24 Down Group, and TGFβ3 Group

Supplemental Table 4. Genes with opposed expression in the CD24 Down sample and in the CD24 Up sample relative to the respective controls (fold induction ≥ 1.5). CD24 is not found in this list because the sequence of CD24 detected by the microarray probe lies in the non-coding region of CD24. CD24 was cloned from the CD24 cDNA, therefore the non-coding region was not cloned. The up- and downregulation of CD24 was instead verified by qRT-PCR using a probe with a target sequence in the coding region of CD24 (see Supplemental Figure 3).

Supplemental Table 5. Complete –log(p-value) values of the top 5 canonical pathways of CD24 Down, CD24 Up and TGFβ3 for all three groups as determined by Ingenuity Pathway Analysis.

Supplemental Table 6. Microarray data of myofibroblast-marker genes significantly regulated in all three microarray groups (CD24 Up, CD24 Down, and TGFβ3).

Supplemental Figure 1. Immunocytochemical staining of CD24 with two different antibodies. Staining of hBMSCs with either a monoclonal anti-CD24 antibody (clone ML-5) or a polyclonal anti-CD24 antibody (both 2 µg/ml) after fixation and permeabilization of hBMSCs led to similar staining patterns with a diffuse cytosolic and a strong nuclear reactivity for CD24 (red). Nuclear staining with DAPI is shown in blue.

Supplemental Figure 2. Comparison of CD24 expression between hBMSCs cultivated with hBMSC (FBS and FGF2) and hBMSC-AB (AB-Serum) medium. A. Intracellular immunocytochemical analysis of CD24 expression revealed a similar staining pattern for CD24 in hBMSCs after culture in hBMSC or hBMSC-AB medium. Monoclonal anti-CD24 antibody is shown in green, and DAPI staining is shown in blue. Scale: 20 µm. B. Flow cytometric analysis of intracellular CD24 expression revealed that CD24 expression is not caused by either FBS or FGF2.

Supplemental Figure 3. qRT-PCR analysis of CD24 mRNA expression after knockdown of CD24, after overexpression of CD24,) or after stimulation with 10 ng/ml TGFβ3. mRNA expression changes relative to the respective controls were as follows: CD24 knockdown led to a 0.30 fold induction (± 0.06 , n = 2 biological replicates, measured with 2 technical replicates each), CD24 overexpression led to a 31065.11 fold induction (n = 1 biological replicate, measured with 2 technical replicates), and TGFb3 led to a 16.29 fold induction (± 4.01 , n = 2 biological replicates, measured with 2 technical replicates each).

Supplemental Figure 4. qRT-PCR validation of microarray expression data of CD24 Up and CD24 Down Group relative to respective controls.

Supplemental Figure 5. This is the high resolution image of Figure 2D.

  1. Supplementary Material