Correction: Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells

<p>Correction: Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells</p

Chemokine Ligand 5 (CCL5) Derived from Endothelial Colony-Forming Cells (ECFCs) Mediates Recruitment of Smooth Muscle Progenitor Cells (SPCs) toward Critical Vascular Locations in Moyamoya Disease

<div><p>The etiology and pathogenesis of moyamoya disease (MMD) are still obscure. Previous studies indicated that angiogenic chemokines may play an important role in the pathogenesis of the disease. Recently, it was discovered that peripheral blood-derived endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SPCs) have defective functions in MMD patients. Therefore, the interaction of ECFCs and SPCs, the precursors of two crucial cellular components of vascular walls, with some paracrine molecules is an intriguing subject. In this study, co-culture of ECFCs and SPCs from MMD patients and healthy normal subjects revealed that MMD ECFCs, not SPCs, are responsible for the defective functions of both ECFCs and SPCs. Enhanced migration of SPCs toward MMD ECFCs supported the role for some chemokines secreted by MMD ECFCs. Expression arrays of MMD and normal ECFCs suggested that several candidate cytokines differentially produced by MMD ECFCs. We selected chemokine (C-X-C motif) ligand 6 (CXCR6), interleukin-8 (IL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 for study, based on the relatively higher expression of these ligands in MMD ECFCs and their cognate receptors in MMD SPCs. Migration assays showed that only CCL5 significantly augmented the migration activities of SPCs toward ECFCs. Treatment with siRNA for the CCL5 receptor (CCR5) abrogated the effect, confirming that CCL5 is responsible for the interaction of MMD ECFCs and SPCs. These data indicate that ECFCs, not SPCs, are the major players in MMD pathogenesis and that the chemokine CCL5 mediates the interactions. It can be hypothesized that in MMD patients, defective ECFCs direct aberrant SPC recruitment to critical vascular locations through the action of CCL5.</p></div

Trans-well migration assays (original magnification ×200).

<p>Migration of SPCs is enhanced when MMD ECFCs are placed in the bottom well (n = 4 for each group). The cell intensity value for combination of normal SPCs and MMD ECFCs is 166 ± 58.9% of control (combination of normal SPCs and normal ECFCs) (p = 0.029). The cell intensity for combination of MMD SPCs and MMD ECFCs are 539 ± 161.8% of control (p<0.001).</p

Differentially expressed chemokines in MMD ECFCs.

<p>(A) Expression of various chemokines are compared between normal ECFCs (n = 4) and MMD ECFCs (n = 7). (B) KEGG pathway enrichment analysis; upregulated genes are involved in chemokine signaling pathway. (C) Four most-up-regulated chemokines in MMD ECFCs are selected for further analysis. (D) Confirmation of mRNA expression of select gene in ECFCs by RTq-PCR (n = 4 for each group). Significantly higher levels of CLCL6, IL8, and CCL5 mRNA are expressed in MMD-ECFCs (CXCL6, p = 0.038; IL8, p = 0.021; CCL2 p = 0.239; CCL5, p = 0.008).</p

mRNA expression of cyto-chemokine receptors in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) after co-culture with brain tumor-initiating cells (BTICs).

<p>Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of cyto-chemokine receptors after co-culture of (A) medulloblastoma-BTICs, (B) atypical teratoid/rhabdoid tumors (AT/RT)-BTICs and (C) glioblastoma-BTICs. The mRNA level of each cytokine receptor was normalized to the level of GAPDH. All data are representative of three independent experiments, and each value represents the mean ± SD. *Significant difference from control (P < 0.05).</p

In vitro tube formation assays (original magnification ×100).

<p>(A and B) MMD ECFCs (green colored) make fewer tubes per unit area than normal ECFCs (n = 6, 11.9 ± 4.0% of control; p<0.001). MMD SPCs (red colored) also make less number of tubes (n = 6, 51.8 ± 21.5% of control; p<0.001). (C) The tube walls made by MMD ECFCs are thinner (n = 5, 14.4% of control; p< 0.001), but the tube walls composed of MMD SPCs are thicker than those of controls (n = 5, 763.4% of control; p<0.001) (*p<0.05, **p<0.01, ***p<0.001).</p

Induced cytokine levels in the BTICs after co-cultured with HFF1 or hAT-MSCs (pg/ml)^*.

<p>BTICs: brain tumor initiating cells, HFF1: human foreskin fibroblast, hAT-MSCs: human adipose-derived mesenchymal stem cells, AT/RT: atypical teratoid rhabdoid tumor, MCP-1: monocyte chemoattractant protein 1, SDF-1: stromal cell-derived factor 1, RANTES: regulated on activation, normal T cell expressed and secreted, IL-6: interleukin-6 ligand, IL-8: interleukin-8, IGF-1: insulin-like growth factor 1ligand, PDGF-bb: platelet-derived growth factor, VEGF: vascular endothelial growth factor, Ang-1: angiopoietin1</p><p>*[induced cytokine level in the BTICs with HFF1] = [total cytokine level in the BTICs with HFF1]-[cytokine level in the HFF1 only]</p><p>[induced cytokine level in the BTICs with hAT-MSCs] = [total cytokine level in the BTICs with hAT-MSCs]-[cytokine level in the hAT-MSCs only]</p><p>Induced cytokine levels in the BTICs after co-cultured with HFF1 or hAT-MSCs (pg/ml)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129292#t001fn002" target="_blank">*</a>}.</p

Migratory ability of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) toward brain tumor-initiating cells (BTICs) <i>in vivo</i>.

<p>Fluorescence images of the brains were taken at the indicated times. (A) In the absence of tumor, the fluorescent signals of hAT-MSCs (blue) are weakened at 2 weeks and are not detectable at 3 weeks. (B) <i>In vivo</i> sequential tracking was performed by injecting both NEO-LIVE 675-labeled BTICs (red) and NEO-LIVE 797-labeled hAT-MSCs (blue). hAT-MSCs gradually migrate toward the tumor site, and strong fluorescent signals are observed at 4 weeks post-injection at the tumor site. (C) Mice were injected the NEO-LIVE 797-labeled hAT-MSCs or HFF1. Representative <i>in vivo</i> fluorescence images show that the HFF1 signals are decreased or fade out. (D) On the contrary, the hAT-MSC signals are widened at all BTIC-derived mouse brain tumor sites at 3 weeks post-injection when compared with HFF1 cells. The color bar indicates radiant efficiency.</p

Co-culture experiments of ECFCs (green colored) and SPCs (red colored) (original magnification ×100).

<p>(A) In co-culture system (n = 7 for each group), both ECFCs and SPCs contribute to the tube formation. (B and C) MMD ECFCs when co-cultured with either normal SPCs or MMD SPCs make significantly less number of tubes than normal ECFCs (p = 0.168 for [normal ECFCs + normal SPCs] vs. [normal ECFCs + MMD SPCs]; p<0.001 for [normal ECFC+ normal SPC] vs. [MMD ECFC+ normal SPC]; p<0.001 for [normal ECFC+ normal SPC] vs. [MMD ECFC+ MMD SPC]).</p

Knock down of cytokine receptors on hAT-MSCs.

<p>After siRNAs treatment to each cytokine receptors on hAT-MSCs, (A) the protein expressions were confirmed in hAT-MSCs by western blot and (B) the migratory ability toward BTICs is assessed using trans-well assay. (C) The quantified results show inhibition of migration by selective knock down of cytokine receptors. ×50 magnification. All data are representative of three independent experiments, and each value represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.</p