Activation of Wnt Pathway Suppresses Growth of MUG-Chor1 Chordoma Cell Line.

Chordoma as a malignant bone tumor, occurs along the axial skeleton and does not have an effective therapy. Brachyury, which is a crucial player for the formation of early embryonic notochord, is abundantly found in both sporadic and familial chordoma. During embryonic development, Brachyury expression was reported to be regulated by the Wnt pathway. The objective of the study is to investigate the role of Wnt signaling in a human chordoma cell line in terms of proliferation, survival, and invasiveness. We tried to elucidate the signaling events that regulate Chordoma cancer. In this regard, Wnt pathway was activated or inhibited using various strategies including small molecules, siRNA-based knockdown and overexpression applications. The results indicated the negative regulatory effect of Wnt signaling activity on proliferation and migration capacity of the chordoma cells. It was revealed that when GSK3ß was inhibited, the Wnt pathway was activated and negatively regulated T/Bra expression. Activity of the Wnt pathway caused cell cycle arrest, reduced migration potential of the cells, and led to cell death. Therefore, the present study suggests that the Wnt pathway plays a key role in suppressing the proliferation and invasive characteristics of human chordoma cells and has a great potential as a therapeutic target in further clinical studies.

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells.

BACKGROUND: The heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/ß-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature. METHODS: Toxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds. RESULTS: Obtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels. CONCLUSION: This study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.

Regulatory role of apelin receptor signaling in migration and differentiation of mouse embryonic stem cell-derived mesoderm cells and mesenchymal stem/stromal cells.

Mesoderm-derived cells, including bone, muscle, and mesenchymal stem/stromal cells (MSCs), constitute various parts of vertebrate body. Cell therapy with mesoderm specification in vitro may be a promising treatment for diseases affecting organs of mesodermal origin. Repair and regeneration of damaged organs with in vitro generation of mesoderm-derived tissues and MSCs hold a great potential for regenerative therapy. Therefore, understanding the signaling pathways involving mesoderm and mesoderm-derived cellular differentiation is important. Previous findings indicated the importance of Apelin receptor (Aplnr) signaling, during embryonic development, in gastrulation, cell migration, and differentiation. Nevertheless, regulatory role of Aplnr pathway in differentiation of mesoderm and mesoderm-derived MSCs remains unclear. In the current study, we tried to elucidate the role of Aplnr signaling during mesoderm cell migration and differentiation from mouse embryonic stem cells (mESCs). By activating and suppressing Aplnr signaling pathway via peptide, small molecule, and genetic modifications including siRNA- and shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout (KO), we revealed that Aplnr signaling not only induces migration of cells during germ layer formation but also enhances mesoderm differentiation through FGF/MAPK pathway. Antibody array and LC/MS protein profiling data demonstrated that Apelin-13 treatment enhanced cell cycle, EGFR, FGF, Wnt, and Integrin signaling pathway proteins. Furthermore, Aplelin-13 treatment improved MSC characteristics, with mesenchymal phenotype and high expression of MSC markers, and silencing Aplnr signaling components resulted in significantly reduced expression of MSC markers. Also, Aplnr signaling activity enhanced proliferation and survival of the cells during MSC derivation from mesoderm.

Surface coating materials regulates the attachment and differentiation of mouse embryonic stem cell derived embryoid bodies into mesoderm at culture conditions.

Abstract: Pluripotent stem cells as a promising cell source with unlimited proliferation and differentiation capacity hold great promise for cell-based therapies in regenerative medicine. Establishment of appropriate culture conditions might enable the control of cellular fate decision in cell culture. Transfer of three-dimensional (3D) embryoid bodies to two-dimensional (2D) monolayer culture systems for initiation of cell differentiation and specialization requires an adaptation of cells which can be managed by extracellular matrix (ECM) materials. Here we compare the characteristics of four different cell culture coating materials and their effect on attachment and differentiation of cells spreading from mouse embryonic stem cell (mESC) derived embryoid bodies (EBs) in mesoderm inducing culture conditions. Atomic force microscope (AFM) and scanning electron microscope (SEM) analysis along with Water Contact Angle technique were used to analyze physical properties of ECM materials and to evaluate cellular behavior on surfaces. Cell migration and differentiation were performed initially by using mesoderm inducing culture conditions and then three germ layer specification conditions. We investigated properties of coating materials such as roughness and wettability control cell attachment, migration and differentiation of mESCs. Matrigel-Gelatin combination is suitable for cell attachment and migration of cells spreading from 3D EBs followed by transfer onto coated surfaces. Matrigel-Gelatin coating enhanced differentiation of cells into mesoderm like cells via EMT process. Our data demonstrated that the Matrigel-Gelatin combination as a cell culture coating matrix might serve as a suitable platform to transfer EBs for differentiation and might influence pluripotent stem cell fate decision into mesoderm and further mesoderm derivative cell populations. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00529-z.

Protective role of Cytoglobin and Neuroglobin against the Lipopolysaccharide (LPS)-induced inflammation in Leydig cells ex vivo.

Leydig cells are responsible for testosterone production in male testis upon stimulation by luteinizing hormone. Inflammation and oxidative stress related Leydig cell dysfunction is one of the major causes of male infertility. Cytoglobin (CYGB) and Neuroglobin (NGB) are two globin family member proteins which protect cells against oxidative stress. In the current study, we established a Lipopolysaccharide (LPS)-induced inflammation model in TM3 Leydig cell culture to study the function of CYGB and NGB proteins under inflammatory conditions. CYGB and NGB were downregulated using siRNA and shRNA based experimental strategies. Overexpression was conducted using lentiviral pLenti-III-CYGB-2A-GFP, and pLenti-III-NGB-2A-GFP vector systems. As testicular macrophages regulate immune function upon inflammation and steroidogenesis of Leydig cells, we generated direct/indirect co-culture systems of TM3 and mouse macrophage (RAW264.7) cells ex vivo. Downregulation of CYGB and NGB induced nitride oxide (NO) release, blocked cell cycle progression, reduced testosterone production and increased inflammatory and apoptotic pathway gene expression in the presence and absence of LPS. On the other hand, CYGB and NGB overexpression reduced TNFα and COX-2 protein expressions and increased the expression of testosterone biogenesis pathway genes upon LPS stimulation. In addition, CYGB and NGB overexpression upregulated testosterone production. The present study successfully established an inflammatory interaction model of TM3 and RAW264.7 cells. Suppression of CYGB and NGB in TM3 cells changed macrophage morphology, enhanced macrophage cell number and NO release in co-culture experiments upon LPS exposure. In summary, these results demonstrate that globin family members might control LPS induced inflammation by regulating apoptotic mechanisms and macrophage response.

Parathyroid Cell Differentiation from Progenitor Cells and Stem Cells: Development, Molecular Mechanism, Function, and Tissue Engineering.

Parathyroid glands are endocrine organs which are located posterior to thyroid glands and control secretion of parathyroid hormone (PTH) in order to regulate blood calcium level. PTH maintains calcium homeostasis by acting on the bone, kidney, and small intestine. PTH deficiency leads to chronic hypocalcemia, organ calcinosis, kidney and heart failure, painful muscle spasms, neuromuscular problems, and memory problems. Since parathyroid cells have inadequate proliferation potential in culture conditions, their utilization as a cellular therapy option is very limited. Although studies conducted so far include parathyroid cell differentiation from various cell types, problems related to successful cellular differentiation and transplantation still remain. Recently, parathyroid tissue engineering has attracted attention as a potential treatment for the parathyroid-related diseases caused by hypoparathyroidism. Although major progression is made in the construction of tissue engineering protocols using parathyroid cells and biomaterials, PTH secretion to mimic its spontaneous harmony in the body is a challenge. This chapter comprehensively defines the derivation of parathyroid cells from various cell sources including pluripotent stem cells, molecular mechanisms, and tissue engineering applications.

Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro.

Hypothalamic-pituitary-adrenal (HPA) axis regulates stress response in the body and abnormal increase in oxidative stress contributes to the various disease pathogenesis. Although hypothalamic distribution of Apelin receptor (APLNR) has been studied, the potential regulatory role in hormone releasing function of hypothalamus in response to stress is not well elucidated yet. To determine whether APLNR is involved in the protection of the hypothalamus against oxidative stress, gonadotropin-releasing hormone (GnRH) cells were used as an in vitro model system. GT1-7 mouse hypothalamic neuronal cell line was subjected to H2O2 and hypoxia induced oxidative stress under various circumstances including APLNR overexpression, knockdown and knockout. Overexpression and activation of APLNR in GnRH producing neurons caused an increase in cell proliferation under oxidative stress. In addition, blockage of APLNR function by siRNA reduced GnRH release. Activation of APLNR initiated AKT kinase pathway as a proliferative response against hypoxic culture conditions and blocked apoptosis. Although expression and activation of APLNR have not been related to GnRH neuron differentiation during development, positive contribution of activated APLNR signaling to GnRH release in mouse embryonic stem cell derived GnRH neurons was observed in the present study. Sustained overexpression and complete deletion of APLNR in mouse embryonic stem cell derived GnRH neurons reduced GnRH release in vitro. The present findings suggest that expression and activation of APLNR in GnRH releasing GT1-7 neurons might induce a protective mechanism against oxidative stress induced cell death and APLNR signaling may play a role in GnRH neurons.

Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol.

Mouse embryonic stem cells (mESCs) were first derived and cultured nearly 30 years ago and have been beneficial tools to create transgenic mice and to study early mammalian development so far. Fibroblast feeder cell layers are often used at some stage in the culture protocol of mESCs. The feeder layer-often mouse embryonic fibroblasts (MEFs)-contribute to the mESC culture as a substrate to increase culture efficiency, maintain pluripotency, and facilitate survival and growth of the stem cells. Various feeder-dependent and feeder-independent culture and differentiation protocols have been established for mESCs. Here we describe the isolation, culture, and preparation feeder cell layers and establishment of feeder-dependent/independent protocol for mESC culture. In addition, basic mESC protocols for culture, storage, and differentiation were described.

Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs).

Mesenchymal Stem Cells (MSCs), as an adult stem cell type, are used to treat various disorders in clinics. However, derivation of homogenous and adequate amount of MSCs limits the regenerative treatment potential. Although mesoderm is the main source of mesenchymal progenitors during embryonic development, neuromesodermal progenitors (NMPs), reside in the primitive streak during development, is known to differentiate into paraxial mesoderm. In the current study, we generated NMPs from human embryonic stem cells (hESC), subsequently derived MSCs and characterized this cell population in vitro and in vivo. Using a bFGF and CHIR induced NMP formation protocol followed by serum containing culture conditions; here we show that MSCs can be generated from NMPs identified by not only the expression of T/Bra and Sox 2 but also FLK-1/PDGFRα in our study. NMP-derived MSCs were plastic adherent fibroblast like cells with colony forming capacity and trilineage (osteo-, chondro- and adipo-genic) differentiation potential. In the present study, we demonstrate that NMP-derived MSCs have an endothelial tendency which might be related to their FLK-1+/PDGFRα + NMP origin. NMP-derived MSCs displayed a protein expression profile of characterized MSCs. Growth factor and angiogenesis related pathway proteins were similarly expressed in NMP-derived MSCs and characterized MSCs. NMP-derived MSCs keep characteristics after short-term and long-term freeze-thaw cycles and localized into bone marrow followed by tail vein injection into NOD/SCID mice. Together, these data showed that hESC-derived NMPs might be used as a precursor cell population for MSC derivation and could be used for in vitro and in vivo research.

Organoids in Tissue Transplantation.

Improvements in stem cell-based research and genetic modification tools enable stem cell-based tissue regeneration applications in clinical therapies. Although inadequate cell numbers in culture, invasive isolation procedures, and poor survival rates after transplantation remain as major challenges, cell-based therapies are useful tools for tissue regeneration.Organoids hold a great promise for tissue regeneration, organ and disease modeling, drug testing, development, and genetic profiling studies. Establishment of 3D cell culture systems eliminates the disadvantages of 2D models in terms of cell adaptation and tissue structure and function. Organoids possess the capacity to mimic the specific features of tissue architecture, cell-type composition, and the functionality of real organs while preserving the advantages of simplified and easily accessible cell culture models. Thus, organoid technology might emerge as an alternative to cell and tissue transplantation. Although transplantation of various organoids in animal models has been demonstrated, liöitations related to vascularized structure formation, cell viability and functionality remain as obstacles in organoid-based transplantation therapies. Clinical applications of organoid-based transplantations might be possible in the near future, when limitations related to cell viability and tissue integration are solved. In this review, the literature was analyzed and discussed to explore the current status of organoid-based transplantation studies.

Gene Editing in Human Pluripotent Stem Cells: Recent Advances for Clinical Therapies.

The identification of human embryonic stem cells and reprogramming technology to obtain induced pluripotent stem cells from adult somatic cells have provided unique opportunity to create human disease models, gene editing strategies and cell therapy options.Development of pluripotent stem cells from somatic cells and genomic manipulation tools enabled to use site specific nucleases in the cell therapy research. Identification of efficient gene manipulation, safe differentiation and use will provide a novel strategy to treat many diseases in the near future. Current available registered clinical trials clearly indicate the need for pluripotent stem cell and gene therapy treatment options. Although gene editing based pluripotent stem cell research is a popular field for research worldwide, improvement of clinical approaches for treatment still remains to be investigated. In this review, we summarized the current situation of gene editing based pluripotent cell therapy developments and applications in clinics.