The multiple roles of Thy-1 in cell differentiation and regeneration.

Thy-1 is a 25-37 kDa glycophosphatidylinositol (GPI)-anchored cell surface protein that was discovered more than 50 years ago. Recent findings have suggested that Thy-1 is expressed on thymocytes, mesenchymal stem cells (MSCs), cancer stem cells, hematopoietic stem cells, fibroblasts, myofibroblasts, endothelial cells, neuronal smooth muscle cells, and pan T cells. Thy-1 plays vital roles in cell migration, adhesion, differentiation, transdifferentiation, apoptosis, mechanotransduction, and cell division, which in turn are involved in tumor development, pulmonary fibrosis, neurite outgrowth, and T cell activation. Studies have increasingly indicated a significant role of Thy-1 in cell differentiation and regeneration. However, despite recent research, many questions remain regarding the roles of Thy-1 in cell differentiation and regeneration. This review aimed to summarize the roles of Thy-1 in cell differentiation and regeneration. Furthermore, since Thy-1 is an outer leaflet membrane protein anchored by GPI, we attempted to address how Thy-1 regulates intracellular pathways through cis and trans signals. Due to the complexity and mystery surrounding the issue, we also summarized the Thy-1-related pathways in different biological processes, and this might provide novel insights in the field of cell differentiation and regeneration.

CD90 promotes cell migration, viability and sphere­forming ability of hepatocellular carcinoma cells.

Cluster of differentiation (CD)90 (Thy­1) was proposed as a marker for the liver cancer stem cells that are responsible for tumorigenic activity, however its involvement in the progression of hepatocellular carcinoma (HCC) remains unknown. The aim of the present study was to determine the effect of CD90 on the biological functions of HCC and to investigate the associated circular RNA (circRNA) involved in this process. The analysis of the in vitro data demonstrated that CD90+ cells isolated from SK­Hep­1 cells exhibited increased viability, migration and invasive abilities compared with CD90­ cells. In addition, circRNA expression profiles in CD90+ and CD90­ cells were screened using a microarray assay and hsa_circ_0067531 and hsa_circ_0057096 were identified to be expressed at significantly different levels. It was additionally demonstrated that the expression of hsa_circ_0067531 in HCC tissues was significantly decreased compared with normal adjacent tissues. Overall, the results of the present study suggested that CD90 may be used as a potential biomarker for HCC. Furthermore, it was demonstrated that hsa_circ_0067531 may affect the biological functions of CD90+ HCC cells and may be a promising candidate to aid in the diagnosis and therapy of HCC.

Multiple roles of CD90 in cancer.

THY1 (CD90) is a 25-37-kDa heavily N-glycosylated, glycophosphatidylinositol (GPI) anchored cell surface protein. It is usually expressed on thymocytes, mesenchymal stem cells, hematopoietic stem cells, natural killer cells, neurons, endothelial cells, renal glomerular mesangial cells, follicular dendritic cells, fibroblasts, and myofibroblasts. It has been found to regulate cell adhesion, migration, apoptosis, axon growth, cell-cell and cell-matrix interactions, T-cell activation, and fibrosis. Several reports have shown that CD90 has an important role in cancer in regulating cancer cell proliferation, metastasis, and angiogenesis. There are also evidences that CD90 is an important prognostic marker in many cancers. Consequently, therapies that target CD90 have great promise in treating many cancers. However, several studies also indicate a contradictory role for CD90, where it acts as a tumor suppressor. In this review, we summarize the expression, function of CD90 in different cancers and its possible use as a biomarker or a therapeutic target in cancer. The challenges and future prospects for the use of CD90 for clinical applications are also discussed in this review.

Rabbit olfactory stem cells. Isolation protocol and characterization.

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Mechanism of mast cell adhesion to human tenocytes in vitro.

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5ß1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5ß1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5ß1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin.

Thy-1 modulates neurological cell-cell and cell-matrix interactions through multiple molecular interactions.

Thy-1, or CD90, is a glycosylphosphatidylinositol-linked cell surface glycoprotein expressed on multiple cell types, including neurons, thymocytes, fibroblasts, endothelial cells, mesangial cells, and some hematopoietic and stromal stem cells. Thy-1 is developmentally regulated and evolutionarily conserved. Its cellular effects vary between and in some cases within cell types, tissues, and species, indicating that its biological role is context dependent. However, it most often seems to affect cell-cell or cell-matrix interactions and cellular adhesion and migration. In the nervous system, Thy-1 mediates bidirectional cell-cell communication, which modulates cell-matrix adhesion. Neurons express high levels of Thy-1, which interacts with alpha(v)beta3 integrin present in astrocytes and stimulates increased astrocyte adhesion to the underlying surface (trans signaling) and in neurites, the same ligand-receptor association triggers neurite retraction and inhibition of axonal growth (cis signaling). Although Thy-1 lacks a cytoplasmic domain, it affects multiple intracellular signaling cascades through interaction with a number of molecules within lipid raft microdomains. Improved understanding of how this enigmatic adhesion molecule modulates signaling and cell phenotype may yield novel insights into neurodevelopment and nerve recovery after injury.

Canine intra-articular multipotent stromal cells (MSC) from adipose tissue have the highest in vitro expansion rates, multipotentiality, and MSC immunophenotypes.

OBJECTIVE: To identify the optimum intra-articular multipotent stromal cell (MSC) tissue source in the canine stifle. STUDY DESIGN: Experimental. SAMPLE POPULATION: Infrapatellar adipose tissue, synovium lining the joint capsule, and synovium surrounding the cranial cruciate ligament (CrCL) from normal stifles of 6 dogs. METHODS: Nucleated cell density for each tissue was determined, and cell doublings (CD) and doubling times (DT) were quantified for expansion rates. Adipogenic, osteogenic, and chondrogenic differentiation was confirmed with light microscopy. Fibroblastic, adipogenic, and osteogenic colony forming unit frequencies were determined for multipotentiality. Tissue-specific target gene expression was assessed, and percentages of CD29(+) , CD34(+) , CD44(+) , CD45(+) , and CD90(+) cells quantified. RESULTS: Adipose tissue had the highest MSC density (ASC). The CD decreased with increasing passages for all cell types, and ASC values tended to be higher. Multipotentiality decreased with passage, but remained highest in ASC. Tissue-specific target gene expression was higher in induced versus noninduced cells, and ASCs had the highest upregulation across passages. Most cells were CD29(+) , CD44(+) , CD90(+) , and percentages decreased with passage. Within cell types, there were more CD29(+) ASC in early passages and more CD44(+) and CD90(+) ASC in later passages. CONCLUSIONS: ASC had the highest in vitro expansion rates, CFU frequencies, tissue-specific target gene expression, and percentages of MSC immunophenotypes.

Melanoma cells use Thy-1 (CD90) on endothelial cells for metastasis formation.

The cell adhesion molecule Thy-1 (CD90) mediates the adhesion of melanoma cells to activated human endothelial cells (EC) via the interaction with the αvß3-integrin on the tumor cells in vitro. Here, we report a strong expression of Thy-1 on both blood vessel and lymphatic EC in melanoma and melanoma metastases. Vascular endothelial growth factor and tumor necrosis factor-α were identified as inducers of Thy-1 expression on EC in vitro. The physiological role of Thy-1 for lymphogenic and hematogenic metastasis of melanoma cells was substantiated in an experimental metastasis model using B16/F10 melanoma cells. Mice lacking Thy-1 showed markedly diminished experimental lung metastasis after injection of B16/F10 melanoma cells compared to wild-type littermate controls. In addition, on generation of a primary subcutaneous tumor, metastasis to regional lymph nodes was clearly reduced in Thy-1(-/-) mice. However, Thy-1 deletion did not affect subcutaneous primary tumor growth, tumor-induced recruitment of inflammatory cells or T cells, angiogenesis, or T-cell activation. In conclusion, Thy-1 contributes to metastasis of melanoma cells by mechanisms likely involving a Thy-1-mediated adhesion of melanoma cells to EC.

Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1-) subsets of mouse embryonic fibroblasts (MEF). TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK) only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma.

THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)/G(1) phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC.

Roles and regulation of Thy-1, a context-dependent modulator of cell phenotype.

Thy-1 or CD90 is a glycophosphatidylinositol-linked glycoprotein expressed on the surface of neurons, thymocytes, subsets of fibroblasts, endothelial cells, mesangial cells and some hematopoietic cells. Thy-1 is evolutionarily conserved, developmentally regulated, and often has dramatic effects on cell phenotype; however, the effects vary between and in some cases within cell types and tissues, and between similar tissues in different species, indicating that the biological role of Thy-1 is context-dependent. Thy-1 exists in soluble form in some body fluids; however, the mechanisms of its shedding are unknown. In addition, Thy-1 expression can be regulated by epigenetic silencing. Because Thy-1 modulates many basic cellular processes and is involved in the pathogenesis of a number of diseases, it is important to better understand its regulation.

Dermal fibroblasts induce maturation of dendritic cells.

To trigger an effective T cell-mediated immune response in the skin, cutaneous dendritic cells (DC) migrate into locally draining lymph nodes, where they present Ag to naive T cells. Little is known about the interaction of DC with the various cellular microenvironments they encounter during their migration from the skin to lymphoid tissues. In this study, we show that human DC generated from peripheral blood monocytes specifically interact with human dermal fibroblasts via the interaction of beta(2) integrins on DC with Thy-1 (CD90) and ICAM-1 on fibroblasts. This induced the phenotypic maturation of DC reflected by expression of CD83, CD86, CD80, and HLA-DR in a TNF-alpha- and ICAM-1-dependent manner. Moreover, fibroblast-matured DC potently induced T cell activation reflected by CD25 expression and enhanced T cell proliferation. Together these data demonstrate that dermal fibroblasts that DC can encounter during their trafficking from skin to lymph node can act as potent regulators of DC differentiation and function, and thus may actively participate in the regulation and outcome of DC-driven cutaneous immune responses.

[Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells].

OBJECTIVE: To construct THY1 eukaryotic expression plasmid and study its effects on the growth of epithelial ovarian cancer cell line SKOV3. METHODS: THY1 gene fragment was obtained from normal human ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1(+)-THY1, which was transformed into E. coli JM109 followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. SKOV3 cells divided into SKOV3-THY1, SKOV-3-Null and SKOV3 groups were transfected via liposome with the recombinant plasmid pcDNA3.1(+)-THY1, empty plasmid, or not transfected, respectively. The expression of THY1 mRNA and its protein were examined by RT-PCR and Western blot methods. The cell growth and apoptosis were evaluated by MTT assay and flow cytometry. RESULTS: The gene fragment of exogenous THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1(+) as verified by PCR, restriction endonucleases digestion and DNA sequencing, and recombinant expression plasmid pcDNA3.1(+)-THY1 transfection resulted in stable expression in SKOV3 cells as shown by RT-PCR and Western blotting. The cell growth inhibition rate of SKOV3-THY1 group (56.6% at the fifth day) was significantly higher than that of the SKOV3-Null group (12.5%, P<0.05), and the cell apoptosis rate in SKOV3-THY1 group (31.8%) was significantly higher than those in SKOV3-Null group (10.5%) and SKOV3 group (9.8%, P<0.05), but the apoptosis rate between the latter two groups was similar (P>0.05). CONCLUSIONS: The recombinant plasmid pcDNA3.1(+)-THY1 can be expressed stably in human ovarian cancer cell line SKOV3. THY1 transfection can inhibit the growth of SKOV3 cells in vitro, suggesting the important role of THY1 gene in pathogenesis and development of ovarian cancer.

Sca-1 and Thy-1 accelerate neuron-like differentiation in bone marrow stromal cells.

Bone marrow stromal cells taken from EGFP transgenic mice were sorted by magnetic beads with surface markers for Sca-1 and Thy-1. The cells were then co-cultured on organotypic hippocampal slice or with neuronal cell feeder in dish. On hippocampus, both Sca-1 and Thy-1 positive cells showed 4- 8 folds higher potential to show neuron-like morphology than negative cells. In dish, negative cells fewly survived but each positive cells survived and showed neuron-like differentiation. In both culture condition, retinoic acid supplement accelerate differentiation. Differentiated Sca-1 and Thy-1 positive cells were immunohistochemically GFAP- and NeuN-negative but nestin-, neurofilament- and NSE-positive. Neuron-like differentiation of bone marrow cells can be enhanced by selection using cell surface proteins.

Thy-1, a versatile modulator of signaling affecting cellular adhesion, proliferation, survival, and cytokine/growth factor responses.

Thy-1 is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored protein involved in T cell activation, neurite outgrowth, apoptosis, tumor suppression, wound healing, and fibrosis. To mediate these diverse effects, Thy-1 participates in multiple signaling cascades. In this review, we discuss Thy-1 signaling primarily in non-immunologic cell types, including neurons, mesangial cells, ovarian cancer cells, nasopharyngeal carcinoma cells, endothelial cells, and fibroblasts. We review the current literature regarding Thy-1 signaling via integrins, protein tyrosine kinases, and cytokines and growth factors; and the roles of these signaling pathways in cellular adhesion, apoptosis, cell proliferation, and cell adhesion and migration. We also discuss the role of Thy-1 localization to lipid rafts, and of the GPI anchor in Thy-1 signaling. Ongoing research on the mechanisms of Thy-1 signaling will add to our understanding of the diverse physiologic and pathologic processes in which Thy-1 plays a role.

Thy-1 as a regulator of cell-cell and cell-matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis.

Thy-1 (CD90) is a 25-37 kDa glycosylphosphatidylinositol (GPI) -anchored glycoprotein expressed on many cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts. Activation of Thy-1 can promote T cell activation, and this role of Thy-1 is reviewed elsewhere. Thy-1 also affects numerous nonimmunologic biological processes, including cellular adhesion, neurite outgrowth, tumor growth, migration, and cell death. In reviewing the nonimmunologic functions of Thy-1, we discuss the phenotype of the Thy-1 null mouse, signaling pathways modulated by Thy-1, the role of the GPI anchor in Thy-1 localization to lipid rafts and signaling, and regulation of Thy-1 expression. Thy-1 is an important regulator of cell-cell and cell-matrix interactions, with important roles in nerve regeneration, metastasis, inflammation, and fibrosis.

Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines.

The fate of tumor-specific CD4(+) T cells is central to the outcome of the host immune response to cancer. We show that tumor antigen recognition by a subset of CD4(+) T cells led to their differentiation into cells capable of suppressing naive and Th1 effector cells. Such tumor-induced regulatory T cells (TMTregs) arose both from precommitted "natural" regulatory T cells and CD4(+)CD25(-)GITR(low) precursors. Once induced, TMTregs were capable of maintaining suppressor activity long after transfer into antigen-free recipients. Suppression was mediated by GITR(high) cells residing within both CD25(+) and CD25(-) subsets. Vaccination of the tumor-bearing host concomitantly expanded TMTregs and effector cells, but suppression was dominant, blunting the expansion of naive tumor-specific T cells and blocking the execution of effector function in vitro and in vivo. These studies illustrate the possibility that therapeutic vaccination could actually worsen host tolerance to tumor antigens and support treatment paradigms that seek to not only increase the frequency of tumor-specific T cells, but to do so in conjunction with strategies that inactivate or remove regulatory T-cell populations.

Hematopoietic stem cells do not engraft with absolute efficiencies.

Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here, we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition, SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore, through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether, our results indicate that even an extremely homogeneous HSC population, based on phenotype and dye efflux, cannot reconstitute mice at absolute efficiencies.