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AimsInfantile haemangioma is a tumour of the microvasculature characterised by aggressive angiogenesis during infancy and spontaneously gradual involution, often leaving a fibro-fatty residuum. The segmental distribution of a subgroup of infantile haemangioma, especially those associated with midline structural anomalies that constitute posterior fossa malformations–hemangiomas–arterial anomalies–cardiac defects–eye abnormalities–sternal cleft and supraumbilical raphe syndrome (PHACES), led us to investigate whether neural crest cells might be involved in the aetiology of this tumour.MethodsImmunohistochemical staining on paraffin embedded infantile haemangioma sections and immunocytochemical staining on cells derived from proliferating haemangioma cultures were performed.ResultsThe endothelium of proliferating infantile haemangioma contains abundant cells that express the neurotrophin receptor (p75), a cell surface marker that identifies neural crest cells, and also for brachyury, a transcription factor expressed in cells of the primitive mesoderm. The endothelium is also immunoreactive for the haematopoietic stem cell marker, CD133; the endothelial-haematopoietic stem/progenitor marker, CD34; the endothelial cell markers, CD31 and VEGFR-2; and the mesenchymal stem cell markers, CD29 and vimentin. Additionally, immunoreactivity for the transcription factors, Sox 9 and Sox 10, that are expressed by prospective neural crest cells was also observed. Cells from microvessel-like structures were isolated from in vitro cultured haemangioma tissue explants embedded in a fibrin matrix. Immunostaining of these cells showed that they retained expression of the same lineage-specific markers that are detected on the paraffin embedded tissue sections.ConclusionsThese data infer that infantile haemangioma is derived from primitive mesoderm and that the cells within the lesion have a neural crest stem cell phenotype, and they express proteins associated with haematopoietic, endothelial, neural crest and mesenchymal lineages. The authors propose a model to account for the natural progression of infantile haemangioma based upon the multipotent expression profile of the primitive mesoderm and their neural crest stem cell phenotype to form all the cell lineages detected during infantile haemangioma proliferation and involution.

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AimTo identify and characterize cancer stem cells (CSC) in glioblastoma multiforme (GBM).MethodsFour-micrometer thick formalin-fixed paraffin-embedded GBM samples from six patients underwent 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers NANOG, OCT4, SALL4, SOX2, and pSTAT3. IF IHC staining was performed to demonstrate co-expression of these markers with GFAP. The protein expression and the transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, and STAT3 were investigated using Western blotting (WB) and NanoString gene expression analysis, respectively.ResultsDAB and IF IHC staining demonstrated the presence of a CSC population expressing NANOG, OCT4, SOX2, SALL4, and pSTAT3 with the almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4, within GBM. The expression of NANOG, SOX2 and, pSTAT3 but, not OCT and SALL4, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of NANOG, OCT4, SALL4, STAT3, and SOX2 in GBM.ConclusionThis study demonstrated a population of CSCs within GBM characterized by the expression of the CSC markers NANOG, SALL4, SOX2, pSTAT3 and OCT4 at the protein and mRNA levels. The almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4 would support the putative existence of a stem cell hierarchy within GBM.

AimTo identify and characterize cancer stem cells (CSC) in moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC).MethodsFour micrometer-thick, formalin-fixed, paraffin-embedded MDBMSCC samples from six patients underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers, NANOG, OCT4, SALL4, SOX2, and pSTAT3; cancer stem cell marker, CD44; squamous cell carcinoma (SCC) marker, EMA; and endothelial marker, CD34. The transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, STAT3, and CD44 were studied using NanoString gene expression analysis and colorimetric in situ hybridization (CISH) for NANOG, OCT4, SOX2, SALL4, and STAT3.ResultsDiaminobenzidine and immunofluorescent (IF) IHC staining demonstrated the presence of (1) an EMA+/CD44+/SOX2+/SALL4+/OCT4+/pSTAT3+/NANOG+ CSC subpopulation within the tumor nests; (2) an EMA−/CD44−/CD34−/SOX2+/OCT4+/pSTAT3+/NANOG+ subpopulation within the stroma between the tumor nests; and (3) an EMA−/CD44−/CD34+/SOX2+/SALL4+/OCT4+/pSTAT3+/NANOG+ subpopulation on the endothelium of the microvessels within the stroma. The expression of CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG was confirmed by the presence of mRNA transcripts, using NanoString analysis and NANOG, OCT4, SOX2, SALL4, and STAT3 by CISH staining.ConclusionThis study demonstrated a novel finding of three separate CSC subpopulations within MDBMSCC: (1) within the tumor nests expressing EMA, CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG; (2) within the stroma expressing SOX2, SALL4, OCT4, pSTAT3, and NANOG; and (3) on the endothelium of the microvessels within the stroma expressing CD34, SOX2, SALL4, OCT4, pSTAT3, and NANOG.