Cells of the adult human heart.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

A reference profile-free deconvolution method to infer cancer cell-intrinsic subtypes and tumor-type-specific stromal profiles.

BACKGROUND: Patient stratification based on molecular subtypes is an important strategy for cancer precision medicine. Deriving clinically informative cancer molecular subtypes from transcriptomic data generated on whole tumor tissue samples is a non-trivial task, especially given the various non-cancer cellular elements intertwined with cancer cells in the tumor microenvironment. METHODS: We developed a computational deconvolution method, DeClust, that stratifies patients into subtypes based on cancer cell-intrinsic signals identified by distinguishing cancer-type-specific signals from non-cancer signals in bulk tumor transcriptomic data. DeClust differs from most existing methods by directly incorporating molecular subtyping of solid tumors into the deconvolution process and outputting molecular subtype-specific tumor reference profiles for the cohort rather than individual tumor profiles. In addition, DeClust does not require reference expression profiles or signature matrices as inputs and estimates cancer-type-specific microenvironment signals from bulk tumor transcriptomic data. RESULTS: DeClust was evaluated on both simulated data and 13 solid tumor datasets from The Cancer Genome Atlas (TCGA). DeClust performed among the best, relative to existing methods, for estimation of cellular composition. Compared to molecular subtypes reported by TCGA or other similar approaches, the subtypes generated by DeClust had higher correlations with cancer-intrinsic genomic alterations (e.g., somatic mutations and copy number variations) and lower correlations with tumor purity. While DeClust-identified subtypes were not more significantly associated with survival in general, DeClust identified a poor prognosis subtype of clear cell renal cancer, papillary renal cancer, and lung adenocarcinoma, all of which were characterized by CDKN2A deletions. As a reference profile-free deconvolution method, the tumor-type-specific stromal profiles and cancer cell-intrinsic subtypes generated by DeClust were supported by single-cell RNA sequencing data. CONCLUSIONS: DeClust is a useful tool for cancer cell-intrinsic molecular subtyping of solid tumors. DeClust subtypes, together with the tumor-type-specific stromal profiles generated by this pan-cancer study, may lead to mechanistic and clinical insights across multiple tumor types.

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype.

The Crandell-Rees Feline Kidney Cell (CRFK) is an immortalised cell line derived from the feline kidney that is utilised for the growth of certain vaccinal viruses. Confusion exists as to whether CRFK are epithelial or mesenchymal in phenotype. The aim of this study was to characterise CRFK cells via immunofluorescence, enzyme cytochemistry, western blotting, RT-qPCR for S100A4 and comparison to primary feline proximal tubular epithelial cells (FPTEC) and feline cortical fibroblasts (FCF). CRFK cells were of fusiform morphology and appeared similar to FCF. CRFK expressed the mesenchymal intermediate filament (IF) protein vimentin together with two cell adhesion molecules associated with feline fibroblasts (CD29 and CD44), and lacked expression of the epithelial IF cytokeratin, myogenic IF desmin and endothelial marker von Willebrand factor (vWF). In addition, CRFK did not demonstrate brush border enzyme activity typical of FPTEC. S100A4 gene expression, implicated in both neoplastic transformation and epithelial to mesenchymal transition, was highly upregulated in CRFK in comparison to the primary feline renal cells. CRFK appear phenotypically similar to fibroblasts, rather than tubular epithelial cells, and may have undergone neoplastic transformation or epithelial-to-mesenchymal transition after extensive passaging. This finding may have potential implications for future research utilising this cell line.

Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature.

The International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell (ISCT MSC) committee offers a position statement to clarify the nomenclature of mesenchymal stromal cells (MSCs). The ISCT MSC committee continues to support the use of the acronym "MSCs" but recommends this be (i) supplemented by tissue-source origin of the cells, which would highlight tissue-specific properties; (ii) intended as MSCs unless rigorous evidence for stemness exists that can be supported by both in vitro and in vivo data; and (iii) associated with robust matrix of functional assays to demonstrate MSC properties, which are not generically defined but informed by the intended therapeutic mode of actions.

Integrated tumor stromal features of hepatocellular carcinoma reveals two distinct subtypes with prognostic/predictive significance.

Current clinical classification of hepatocellular carcinoma (HCC) is unable to predict prognosis efficiently. Our aim is to classify HCC into clinically/biologically relevant subtypes according to stromal factors. We detected seven types of stromal features in tumors from 161 HCC patients by immunohistochemical staining and Hematoxylin-eosin staining. Five stromal features were selected out of seven types of stromal features to construct stromal type based on LASSO COX regression model. Then, integrating multiple clinicopathologic characteristics and stromal type, we built two nomograms for overall survival (OS) and disease-free survival (DFS). Further validation of the stromal type and nomograms were performed in the testing cohort (n = 160) and validation cohort (n = 120). Using the LASSO model, we classified HCC patients into stromal type A subgroup (CD34lowTIL-stromal-ratiohighStromal-tumor-ratiolowα-SMAweakStromamature) and stromal type B subgroup (CD34highTIL-stromal-ratiolowStromal-tumor-ratiohighα-SMAstrongStromaimmature). The stromal type was an independent prognostic factor for OS and DFS in the training, testing and validation cohorts. Two nomograms (for OS and DFS) that integrated the stromal type and clinicopathologic risk factors also showed good predictive accuracy and discriminatory power. In addition, immune cell recruitment in the tumor microenvironment (TME) was conditioned by the tumor stromal type. In conclusion, the newly developed tumor stromal type was an effective predictor of OS and DFS. Furthermore, the stromal type is associated with the immune phenotype in the TME.

Nintedanib treatment delays prostate dorsolateral lobe cancer progression in the TRAMP model: contribution to the epithelial-stromal interaction balance.

Prostate cancer (PCa) progression mechanism has been linked to epithelial proliferation, tumor invasion ability, and growth factors. Nintedanib (BIBF 1120) has been reported as being FGF and VEGF pathway inhibitors, exhibiting antitumor activity. Thus, the objective herein was to characterize the early Nintedanib treatment effects on the structure and molecules involved in the basal membrane, the extracellular matrix (ECM) maintenance, in addition to the angiogenesis and mitogenic processes at different grades of prostatic tumor development in TRAMP mice. Therefore, 45 male TRAMP mice were divided into control groups: 8-week-old mice (TC8), 12-week-old mice (TC12), and 16-week-old mice (TC16); and treated groups with 10 mg/kg/day Nintedanib dose for 4 weeks. The treated groups were euthanized at 12 (TN12) and 16 (TN16) weeks of age. Samples from the dorsolateral lobe were collected and processed for light microscopy, immunohistochemistry, Western blotting, and microvessel density analysis. The results showed that early Nintedanib treatment led to an increase of healthy epithelium frequency and a reduction of LGPIN and a maximum vascularization density in the TN12 group. Also, treatment led to a well-differentiated adenocarcinoma decrease and an α and ß dystroglycan and also laminin 1 increase in the TN16 group. IGFR1 decreased in the TN16 group. To conclude, early Nintedanib treatment led to a reduction in cancer severity, interfering in both ECM compounds and angiogenesis process to then contribute to a balance, not only in the prostatic epithelium and stroma, but also in the epithelial-stromal interaction during PCa progression.

Therapeutically targeting SELF-reinforcing leukemic niches in acute myeloid leukemia: A worthy endeavor?

A tight relationship between the acute myeloid leukemia (AML) population and the bone marrow (BM) microenvironment has been convincingly established. The AML clone contains leukemic stem cells (LSCs) that compete with normal hematopoietic stem cells (HSCs) for niche occupancy and remodel the niche; whereas, the BM microenvironment might promote AML development and progression not only through hypoxia and homing/adhesion molecules, but also through genetic defects. Although it is still unknown whether the niche influences treatment results or contains any potential target for treatment, this dynamic AML-niche interaction might be a promising therapeutic objective to significantly improve the AML cure rate.

Perspectives on signet ring stromal cell tumor and related signet ring cell lesions of the gonads.

In this article, we discuss advances in our knowledge of the pathology of signet ring stromal cell tumor and related signet ring cell lesions of the ovary and a single case of signet ring stromal cell tumor of the testis. We divide ovarian signet ring cell lesions into 3 categories that reflect differences in their pathogenesis and histologic appearance. With 1 exception, all authentic cases of signet ring stromal cell tumor have been unilateral. Cases of ovarian signet ring stromal cell tumor from the literature can arise in 2 ways. The majority of cases arise multifocally from fibroma, whereas the remainder likely arise directly from the ovarian stroma. In difficult cases, immunocytochemistry provides improved diagnostic accuracy in distinguishing signet ring stromal cell tumor and its mimics from Krukenberg tumor. The most useful antibodies in this regard are epithelial membrane antigen and vimentin.

"Mesenchymal" stem cells.

Two opposing descriptions of so-called mesenchymal stem cells (MSCs) exist at this time. One sees MSCs as the postnatal, self-renewing, and multipotent stem cells for the skeleton. This cell coincides with a specific type of bone marrow perivascular cell. In skeletal physiology, this skeletal stem cell is pivotal to the growth and lifelong turnover of bone and to its native regeneration capacity. In hematopoietic physiology, its role as a key player in maintaining hematopoietic stem cells in their niche and in regulating the hematopoietic microenvironment is emerging. In the alternative description, MSCs are ubiquitous in connective tissues and are defined by in vitro characteristics and by their use in therapy, which rests on their ability to modulate the function of host tissues rather than on stem cell properties. Here, I discuss how the two views developed, conceptually and experimentally, and attempt to clarify the confusion arising from their collision.

Histologic categorization of fibrotic cancer stroma in the primary tumor is an independent prognostic index in resectable colorectal liver metastasis.

Although the molecular mechanism of desmoplastic reaction (DR) for providing aggressive tumor characteristics is increasingly recognized, the prognostic role of DR has not been investigated in colorectal liver metastasis (CRLM). A pathologic review of 412 patients who underwent hepatectomy for CRLM at 2 independent institutions was conducted. DR in primary tumors was classified as mature, intermediate, or immature on the basis of the existence of keloid-like collagen and myxoid stroma-distinctive histologic products of extracellular matrix remodeling. With respect to DR, 137, 122, and 153 patients were classified as mature, intermediate, and immature, respectively. Immature DRs were associated with higher T and N stages, higher primary tumor grade, synchronous and larger size of liver metastasis, and extrahepatic disease (P≤0.0001 to 0.002). DR significantly influenced the rate of recurrence in extrahepatic sites, including the lung, peritoneum, and local region in the primary tumor (P≤0.0001 to 0.03), rather than the remnant liver. Five-year overall survival rates after hepatectomy were the highest in the mature group (58.9%), followed by intermediate (42.1%) and immature (26.7%) groups. A significant prognostic impact of DR was observed in subset analyses for institutions, primary tumor location, and timing and number of liver metastases. Multivariate analysis revealed that DR was an independent prognostic factor along with T stage of the primary tumor, size of liver metastasis, and extrahepatic disease. Characterizing DR in the primary tumor on the basis of histologic products of cancer-associated fibroblasts is valuable in evaluating prognostic outcome after hepatectomy in CRLM patients.

Analysis of APC types involved in CD4 tolerance and regulatory T cell generation using reaggregated thymic organ cultures.

Tolerance to self-Ags is generated in the thymus. Both epithelial and hematopoietic thymic stromal cells play an active and essential role in this process. However, the role of each of the various stromal cell types remains unresolved. To our knowledge, we describe the first comparative analysis of several types of thymic hematopoietic stromal cells (THSCs) for their ability to induce CD4 tolerance to self, in parallel with the thymic epithelium. The THSCs--two types of conventional dendritic cells (cDCs), plasmacytoid dendritic cells, macrophages (MΦs), B lymphocytes, and eosinophils--were first characterized and quantified in adult mouse thymus. They were then examined in reaggregated thymic organ cultures containing mixtures of monoclonal and polyclonal thymocytes. This thymocyte mixture allows for the analysis of Ag-specific events while avoiding the extreme skewing frequently seen in purely monoclonal systems. Our data indicate that thymic epithelium alone is capable of promoting self-tolerance by eliminating autoreactive CD4 single-positive thymocytes and by supporting regulatory T cell (Treg) development. We also show that both non-Treg CD4 single-positive thymocytes and Tregs are efficiently deleted by the two populations of cDCs present in the thymus, as well as to a lesser extent by MΦs. Plasmacytoid dendritic cells, B lymphocytes, and eosinophils were not able to do so. Finally, cDCs were also the most efficient THSCs at supporting Treg development in the thymus, suggesting that although they may share some characteristics required for negative selection with MΦs, they do not share those required for the support of Treg development, making cDCs a unique cell subset in the thymus.

Prospective identification and isolation of murine bone marrow derived multipotent mesenchymal progenitor cells.

Enormous confusion still exists in the scientific community regarding the in vivo identity of putative bone marrow (BM) multipotent mesenchymal progenitor cells (MPCs). There is still lack of consensus between laboratories on this issue but recent advancements in this field have shed light on the identity of these cells in humans. However, in mice there are limited and reproducible data available that convincingly define prospectively these cells in vivo. In this review we will critically address: 1) important considerations on how to interpret MPC nomenclature, heterogeneity and differentiation abilities; 2) potential surface antigens that could aid in the isolation of MPC from mouse BM; 3) and their topography and prospective cellular relationship with pericytes, adventitial reticulocytes (ARCs) and vascular smooth muscle cells (VSMCs).

Expressão de MMP-2 e MMP-9 no endométrio de éguas saudáveis e portadoras de endometrite crônica / Expression of MMP-2 and MMP-9 in helth endometrium and in chronic endometritis of mares

Avaliaram-se, por método imunoistoquímico, a expressão e distribuição das metaloproteinases (MMP) 2 e 9 em amostras de endométrio hígido e de éguas portadoras de endometrite crônica. Foram utilizadas 60 biópsias endometriais. A MMP-2 foi observada na parede vascular, nas células estromais e no epitélio glandular, e a imunorreatividade mais intensa foi obtida nas células do epitélio glandular nas endometrites da categoria III e na parede vascular nos endométrios da categoria I. A marcação imunoistoquímica para MMP-9 mostrou-se difusa pelo endométrio e foi observada no epitélio luminal e glandular, na região da parede vascular, nas células estromais, endoteliais e do infiltrado inflamatório. Houve diminuição da marcação imunoistoquímica na região da parede vascular conforme aumentou o grau das lesões endometriais concomitante à diminuição da intensidade da reação. Não houve relação na expressão imunoistoquímica das metaloproteinases estudadas com o tipo de endometrite.

The expression and distribution of matrix metalloproteinases (MMP) 2 and 9 were evaluated in helth endometrium and in chronic endometrites of mares by means of immunohistochemistry method. Sixty endometrial biopsies were utilized. Expression of MMP-2 was observed in vascular wall, stromal cells, and glandular epithelium. More intense immune reaction was seen in glandular epithelial cells in category III endometritis and in vascular wall in category I endometrium. MMP-9 immune reaction was diffuse and was seen in luminal and glandular epithelium; vascular wall region; and stromal, endotelial, and inflammatory cells. There was a decreased of the immunohistochemical marking in vascular wall region as increased the degree of endometrial injury, as well as reducing the intensity of reaction in this compartment. There was no relation in immunohistochemistry expression of metalloproteinases with the type of endometritis.

Isolation, characterization and differentiation potential of rat bone marrow stromal cells.

BACKGROUND: Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with varying degrees of plasticity in humans. However, there are a few reports on rat-derived cells, which could be good models for the research purpose. We describe here a simple method of establishing the rat bone marrow stromal cells by the principle of adhesion and document their phenotype along with their differentiation potential to other lineages. MATERIALS AND METHODS: Rat bone marrow stromal cells were isolated by three methods: direct plastic adherence, ficoll hypaque separation and a combination of both. The stromal cells obtained by these methods were characterized by fluorescent activating cell sorting (FACS) for established hematopoietic and non-hematopoietic markers. The cells obtained by combination method (combination of ficoll density gradient centrifugation and plastic adherence) were cultured and serially passaged. Transcriptional confirmation was done by reverse transcription polymerase chain reaction (RT-PCR) for vimentin and collagen type 1 alpha 1. Attempts were made to differentiate the marrow stromal cells into adipocytes, osteocytes and neuronal like cells. RESULTS: Bone marrow samples from 10 rats yielded 4-5 million bone marrow mononuclear cells /ml per femur. Of the three methods tested, a combination method yielded good growth of spindle cells. The cells obtained by combined method showed high percentage of positivity for vimentin, fibronectin and CD90 and negative for hematopoietic markers. Further, RT-PCR confirmed vimentin and collagen type - 1 alpha 1 expression. Oil red O staining and Alizarin red staining confirmed adipocytic and osteogenic differentiation. On immunocytochemical analysis, the cells expressed nestin, beta-tubulin III, neurofilament and synaptophysin. CONCLUSION: Adequate quantities of rat marrow stromal cell cultures can be established by a simple method based on adhesion properties. Their phenotypic characteristics and plasticity support the evidence that they are mesenchymal stem cells with a distinct tendency for neural lineage.

Adipose tissue: a new source for cardiovascular repair.

Along with angiogenesis and gene therapy, stem cell transplantation is one of the newest treatment modalities proposed to improve the outcome of patients with heart failure or infarction. In this context, much interest has stemmed from experimental studies showing that cardiac transfer of unfractionated or partially purified bone marrow cells, or stem cells and progenitor cells derived from the bone marrow or peripheral blood, can enhance functional recovery after an acute myocardial infarction. On the basis of these data, stem cells and progenitor cells derived from the bone marrow have been proposed for use in the repair of cardiac tissue after acute myocardial infarction in patients. However, the relatively low abundance, small tissue volume, difficult accessibility and disease-related malfunction of bone marrow-derived stem cells make their clinical usefulness difficult in some situations. Recently it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including endothelial cells, smooth muscle cells and cardiomyocytes. The similarities between stem cells extracted from the bone marrow and the adipose tissue suggest the potential for the adipose tissue to act as an alternative, and perhaps preferable, cell source for repairing damaged tissues, such as the ischemic or infarcted heart. In this review we discuss molecular and functional characterization, preclinical results and currently ongoing clinical trials using adipose-derived stem cells in cardiovascular repair.

Stroma classification for neuroblastoma on graphics processors.

Neuroblastoma is one of the most common childhood cancers. We are developing an image analysis system to assist pathologists in their prognosis. Since this system operates on relatively large-scale images and requires sophisticated algorithms, computerised analysis takes a long time to execute. In this paper, we propose a novel approach to benefit from high memory bandwidth and strong floating-point capabilities of graphics processing units. The proposed approach achieves a promising classification accuracy of 99.4% and an execution performance with a gain factor up to 45 times compared to hand-optimised C++ code running on the CPU.

Cellular diversity of human placental stem villi: an ultrastructural and immunohistochemical study.

The aim of the study was to investigate the distribution and differentiation of cell types in the stroma of human placental stem villi (SV). A total of 14 human term placental tissues were studied. Double immunolabeling was performed for desmin-vimentin, desmin-alpha-smooth actin and vimentin-alpha-smooth actin. Cytokeratin 7, proliferating cell nuclear antigen immunolabeling was also performed. Parallel tissue samples were examined by transmission electron microscopy. HSCORE was performed for the semi-quantitative analysis of distribution of cells in the stroma of SV. Vimentin-labeled cells were mostly distributed in the subtrophoblastic area. Desmin-vimentin double immunolabeling was mainly localized in the triangular area and to a lesser degree in the perivascular area and vessel walls (p=or<0.001). However, desmin-alpha smooth actin labeling was observed predominantly in the vessel wall and perivascular area. Vimentin-alpha smooth actin immunoreactivity was significantly stronger in the triangular and perivascular areas compared to the vessel walls (p=0.003). Ultrastructurally, cells in the stroma of SV were mesenchyme cells, reticulum cells, fibroblasts, myofibroblasts, smooth muscle cells, and Hofbauer cells, filamented and vacuolated cells. The differentiation of myofibroblasts in the triangular and perivascular areas may play a role in maturation of SV and villous contractility, modulation of the intervillous space and this may have effects on maternofetal placental circulation.

Fas antigen (CD95) mediates cell survival signals to regulate functional cellular subpopulations in normal human endometrial stromal cells.

Fas antigen (CD95) is expressed on almost all types of human cells and is believed to mediate receptor-specific apoptotic signals. In human endometrial tissues, high Fas and Fas ligand expressions and Fas-mediated apoptosis in endometrial epithelial cells have been discussed in many reports but no study has examined Fas-mediated signals in endometrial stromal cells. In this study we investigated Fas expression and Fas-mediated signals of normal human endometrial stromal cells. Flow cytometric analysis revealed Fas antigen expression on the stromal cells and their Fas expression was enhanced by 8-Br-cAMP, a strong inducer of decidualization. Neither short-term nor long-term cultures with anti-Fas IgM affected proliferation or viability of the stromal cells. Anti-Fas IgM alone affected neither viable cell numbers nor PRL release of unstimulated stromal cells. However, anti-Fas IgM dose-dependently stimulated viable cell numbers of stromal cells co-stimulated with 8-Br-cAMP and anti-Fas IgM, whereas PRL secretion of the co-stimulated cells was not affected. Anti-Fas IgM dose-dependently stimulated viable cell numbers of 8-Br-cAMP-stimulated cells but did not affect PRL secretion of 8-Br-cAMP-induced decidualized cells. These results indicate that Fas antigen on human endometrial stromal cells cannot mediate receptor-specific apoptotic signals, and that Fas-mediated signals stimulate survival of 8-Br-cAMP-stimulated non-decidualized stromal cells. Thus, stimulation of Fas-antigen on the endometrial stromal cells enhances anti-apoptotic/survival signals in certain stromal cells, autoregulating functional cellular subpopulations of human endometrial stromal cells in a paracrine manner.

Revision of the 1992-edition of the WHO histological typing of odontogenic tumours. A suggestion.

Classification of odontogenic tumours is an academic exercise that has developed over the last 150 years. It was not until 1971 when a 5-year collaborated effort, organized by the World Health Organization (WHO), resulted in the first consensus on taxonomy of odontogenic tumours. The appearance of this first authoritative guide to the classification of odontogenic tumours marked the start of an era of quite intensive interest for studying this particular field of oral pathology. An updated 2nd edition of the WHO classification was published in 1992.