Probes for NANOG

Immunohistochemical staining demonstrated cytoplasmic and nuclear expression of OCT4 on the endothelium and the media of the lesional vessels with nuclear staining of the cells within the stroma (Figure 3A). Cytoplasmic and nuclear expression of SOX2 was present on the endothelium of the lesional vessels and the cells within the stroma with strong expression in the media of the lesional vessels (Figure 3B). Weak cytoplasmic expression of KLF4 was observed on the endothelium of the lesional vessels (Figure 3C). Nuclear expression of c-MYC was demonstrated on the endothelium and the media of the lesional vessels and within the cells in the stroma (Figure 3D). NANOG was not expressed in any of the 15 samples (Figure 3E). [Figure 3.]Figure 3.: Representative immunohistochemical-stained images of hypertrophic port-wine stain tissue samples, demonstrating the expression of OCT4 (A, brown), SOX2 (B, brown), KLF4 (C, brown), and c-MYC (D, brown). Cytoplasmic staining of the endothelium was demonstrated for SOX2, OCT4, and KLF4. Nuclear staining of SOX2, OCT4, and c-MYC was demonstrated on the endothelium and the cells within the stroma. E, NANOG was not expressed in any of the 15 samples. Nuclei were counterstained with hematoxylin (A–E, blue). Original magnification: 200×. Positive staining was demonstrated on human control tissues: seminoma for OCT4 (Supplemental Digital Content Figure 1A, http://links.lww.com/JV9/A2), skin epidermis for SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2), breast carcinoma for KLF4 (Supplemental Digital Content Figure 1C, http://links.lww.com/JV9/A2), normal colon mucosa for c-MYC (Supplemental Digital Content Figure 1D, http://links.lww.com/JV9/A2), and seminoma for NANOG (Supplemental Digital Content Figure 1E, http://links.lww.com/JV9/A2). Immunohistochemical staining of normal skin showed no expression of OCT4 (Supplemental Digital Content Figure 1F, http://links.lww.com/JV9/A2) and NANOG (Supplemental Digital Content Figure 1G, http://links.lww.com/JV9/A2). SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2), KLF4 (Supplemental Digital Content Figure 1H, http://links.lww.com/JV9/A2), and c-MYC (Supplemental Digital Content Figure 1I, http://links.lww.com/JV9/A2) were present in the epidermis of the normal skin. In addition, SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2) and KLF4 (Supplemental Digital Content Figure 1H, http://links.lww.com/JV9/A2) were expressed by some cells but not blood vessels within the stroma. A negative stain using combined Flex Negative Control Mouse and Flex Negative Control Rabbit on a section of HPWS (Supplemental Digital Content Figure 1J, http://links.lww.com/JV9/A2) showed no staining, confirming the specificity of the primary antibodies.

AIM: To investigate the expression of embryonic stem cell (ESC) markers in microcystic lymphatic malformation (mLM). METHODS AND RESULTS: Cervicofacial mLM tissue samples from nine patients underwent 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining for ESC markers octamer-binding protein 4 (OCT4), homeobox protein NANOG, sex determining region Y-box 2 (SOX2), Krupple-like factor (KLF4), and proto-oncogene c-MYC. Transcriptional activation of these ESC markers was investigated using real-time polymerase chain reaction (RT-qPCR) and colorimetric in situ hybridization (CISH) on four and five of these mLM tissue samples, respectively. Immunofluorescence (IF) IHC staining was performed on three of these mLM tissue samples to investigate localization of these ESC markers. DAB and IF IHC staining demonstrated the expression of OCT4, SOX2, NANOG, KLF4, and c-MYC on the endothelium of lesional vessels with abundant expression of c-MYC and SOX2, which was also present on the cells within the stroma, in all nine mLM tissue samples. RT-qPCR and CISH confirmed transcriptional activation of all these ESC markers investigated. CONCLUSIONS: These findings suggest the presence of a primitive population on the endothelium of lesional vessels and the surrounding stroma in mLM. The abundant expression of the progenitor-associated markers SOX2 and c-MYC suggests that the majority are of progenitor phenotype with a small number of ESC-like cells.

The sequence of pathological events in feline hypertrophic cardiomyopathy (fHCM) is still largely unknown, although we know that fHCM is characterized by interstitial remodeling in a macrophage-driven pro-inflammatory environment and that myocardial ischemia might contribute to its progression. This study aimed to gain further insights into the structural changes associated with interstitial remodeling in fHCM with special focus on the myocardial microvasculature and the phenotype of the interstitial cells. Twenty-eight hearts (16 hearts with fHCM and 12 without cardiac disease) were evaluated in the current study, with immunohistochemistry, RNA-in situ hybridization, and transmission electron microscopy. Morphometrical evaluations revealed a statistically significant lower microvascular density in fHCM. This was associated with structural alterations in capillaries that go along with a widening of the interstitium due to the accumulation of edema fluid, collagen fibers, and mononuclear cells that also proliferated locally. The interstitial cells were mainly of fibroblastic or vascular phenotype, with a substantial contribution of predominantly resident macrophages. A large proportion expressed CD34 mRNA, which suggests a progenitor cell potential. Our results indicate that microvascular alterations are key events in the pathogenesis of fHCM and that myocardial interstitial cell populations with CD34+ phenotype play a role in the pathogenesis of the disease.

The stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC are expressed in numerous cancer types suggesting the presence of cancer stem cells (CSCs). Immunohistochemical (IHC) staining performed on 12 lung adenocarcinoma (LA) tissue samples showed protein expression of OCT4, NANOG, SOX2, KLF4 and c-MYC, and the CSC marker CD44. In situ hybridization (ISH) performed on six of the LA tissue samples showed mRNA expression of OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence staining performed on three of the tissue samples showed co-expression of OCT4 and c-MYC with NANOG, SOX2 and KLF4 by tumor gland cells, and expression of OCT4 and c-MYC exclusively by cells within the stroma. RT-qPCR performed on five LA-derived primary cell lines showed mRNA expression of all the markers except SOX2. Western blotting performed on four LA-derived primary cell lines demonstrated protein expression of all the markers except SOX2 and NANOG. Initial tumorsphere assays performed on four LA-derived primary cell lines demonstrated 0-80% of tumorspheres surpassing the 50 µm threshold. The expression of the stemness-associated markers OCT4, SOX2, NANOG, KFL4 and c-MYC by LA at the mRNA and protein level, and the unique expression patterns suggest a putative presence of CSC subpopulations within LA, which may be a novel therapeutic target for this cancer. Further functional studies are required to investigate the possession of stemness traits.