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Abstract Objectives The endogenous repairing based on the activation of neural stem cells (NSCs) is impaired by neurodegenerative diseases. The present study aims to characterize human stem cells from the apical papilla (hSCAPs) with features of mesenchymal stem cells (MSCs) and to demonstrate the neuronal differentiation of hSCAPs into NSCs through the formation of three-dimensional (3D) neurospheres, verifying the structural, immunophenotyping, self-renewal, gene expression and neuronal activities of these cells to help further improve NSCs transplantation. Methodology The hSCAPs were isolated from healthy impacted human third molar teeth and characterized as MSCs. They were then induced into 3D-neurospheres using a specific neural induction medium. Subsequently, the intra-neurospheral cells were confirmed to be NSCs by the identification of Nissl substance and the analysis of immunofluorescence staining, self-renewal ability, and gene expression of the cells. Moreover, the neuronal activity was investigated using intracellular calcium oscillation. Results The isolated cells from the human apical papilla expressed many markers of MSCs, such as self-renewal ability and multilineage differentiation. These cells were thus characterized as MSCs, specifically as hSCAPs. The neurospheres induced from hSCAPs exhibited a 3D-floating spheroidal shape and larger neurospheres, and consisted of a heterogeneous population of intra-neurospheral cells. Further investigation showed that these intra-neurospheral cells had Nissl body staining and also expressed both Nestin and SOX2. They presented a self-renewal ability as well, which was observed after their disaggregation. Their gene expression profiling also exhibited a significant amount of NSC markers (NES, SOX1, and PAX6). Lastly, a large and dynamic change of the fluorescent signal that indicated calcium ions (Ca2+) was detected in the intracellular calcium oscillation, which indicated the neuronal activity of NSCs-derived hSCAPs. Conclusions The hSCAPs exhibited properties of MSCs and could differentiate into NSCs under 3D-neurosphere generation. The present findings suggest that NSCs-derived hSCAPs may be used as an alternative candidates for cell-based therapy, which uses stem cell transplantation to further treat neurodegenerative diseases.
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The non-classical MHC class I (MHC-Ib) proteins are important modulators of immune system during pregnancy favoring survival of the fetus. Contrary to ubiquitously expressed classical MHC-I proteins, MHC-Ib proteins are oligomorphic, and expressed in specific cell/tissue types thus minimizing maternal immune-mediated rejection of fetal-allograft and a successful pregnancy. A unique characteristic of MHC-Ib glycoproteins is expression of surface and soluble isoforms due to alternative splicing phenomenon. Bovine fetal trophoblast cells, during the third trimester of pregnancy, express non-classical bovine leukocyte antigen class Ib (BoLA-Ib) antigens. BoLA-NC3*00101, is a non-classical class I allele from cattle AH11 haplotype and is expressed as cell surface isoform. However, lack of monoclonal antibody (mAb) hinders the development of specific assay to detect the soluble/secreted BoLA-I antigens released by fetal trophoblast cells. The objective of this study is to synthesize mAb specific to NC3*00101 molecule to develop an isotype-matched ELISA to assess BoLA-I protein(s). We demonstrated that majority of NC3*00101 hybridoma-supernatants were low-titer and showed inappreciable reactivity in ELISA and flow cytometry assays. We identified a clone of hybridoma (NC3-3*2) that secreted mAb specific to BoLA-NC3*00101 as demonstrated with flow cytometry. NC3-3*2 clone secreted supernatant that captured BoLA-NC3*00101 protein in ELISA. Unfortunately, despite several efforts we could not recover the cryopreserved hybridoma. Non-recoverability and instability, thus, limited production of NC3*00101-mAbs. This study provides the evidence that mice immunized with bovine class Ib proteins elicit a specific immunogenic response and warrants future studies into generation of stable hybridoma secreting antibodies against BoLA-Ib proteins using robust methods of fusion and cryopreservation of hybridomas.