ABSTRACT
Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.
Subject(s)
Blastocyst/metabolism , Embryonic Development/genetics , Gene Expression , Histones/genetics , Oogenesis/genetics , Animals , Female , Histones/metabolism , Immunohistochemistry , Meiosis , Mice , Pregnancy , Protein TransportABSTRACT
Development and selection of an ideal scaffold is of importance for tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible bioresorbable copolymer that belongs to the polyhydroxyalkanoate family. Because of its good biocompatibility, PHBHHx has been widely used as a cell scaffold for tissue engineering. This review focuses on the utilization of PHBHHx-based scaffolds in tissue engineering. Advances in the preparation, modification, and application of PHBHHx scaffolds are discussed.
Subject(s)
3-Hydroxybutyric Acid/chemistry , Biocompatible Materials/chemistry , Caproates/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , 3-Hydroxybutyric Acid/therapeutic use , Biocompatible Materials/therapeutic use , Bone and Bones/physiology , Caproates/therapeutic use , Cartilage/physiology , Freeze Drying , Humans , Muscle, Smooth/physiology , Regeneration , Surface PropertiesABSTRACT
Development and selection of an ideal scaffold is of importance for tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible bioresorbable copolymer that belongs to the polyhydroxyalkanoate family. Because of its good biocompatibility, PHBHHx has been widely used as a cell scaffold for tissue engineering. This review focuses on the utilization of PHBHHx-based scaffolds in tissue engineering. Advances in the preparation, modification, and application of PHBHHx scaffolds are discussed.
Subject(s)
Humans , /chemistry , Biocompatible Materials/chemistry , Caproates/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , /therapeutic use , Biocompatible Materials/therapeutic use , Bone and Bones/physiology , Caproates/therapeutic use , Cartilage/physiology , Freeze Drying , Muscle, Smooth/physiology , Regeneration , Surface PropertiesABSTRACT
SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.
Subject(s)
Humans , Cell Differentiation/genetics , Chondrogenesis/genetics , Fetal Blood/cytology , Mesenchymal Stem Cells/cytology , SOX9 Transcription Factor/genetics , Aggrecans/biosynthesis , Blotting, Western , Cartilage/metabolism , Cell Proliferation/genetics , Chondrocytes/metabolism , Collagen Type II/biosynthesis , Flow Cytometry , Green Fluorescent Proteins , Gene Expression Regulation/physiology , Human Umbilical Vein Endothelial Cells/cytology , Immunohistochemistry , Immunophenotyping , Primary Cell Culture , Reverse Transcriptase Polymerase Chain Reaction , Tissue Engineering , TransfectionABSTRACT
SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.
Subject(s)
Cell Differentiation/genetics , Chondrogenesis/genetics , Fetal Blood/cytology , Mesenchymal Stem Cells/cytology , SOX9 Transcription Factor/genetics , Aggrecans/biosynthesis , Blotting, Western , Cartilage/metabolism , Cell Proliferation/genetics , Chondrocytes/metabolism , Collagen Type II/biosynthesis , Flow Cytometry , Gene Expression Regulation/physiology , Green Fluorescent Proteins , Human Umbilical Vein Endothelial Cells/cytology , Humans , Immunohistochemistry , Immunophenotyping , Primary Cell Culture , Reverse Transcriptase Polymerase Chain Reaction , Tissue Engineering , TransfectionABSTRACT
Typically, production of induced pluripotent stem cells requires direct contact with feeder cells. However, once the stem cells have reached the appropriate maturation point, it is difficult to separate them from feeder cells, which must be irradiated with γ-rays or treated with the antibiotic mitomycin-C. We used a microporous poly-membrane-based indirect contact co-culture system with mouse embryonic fibroblasts to induce mouse pluripotent stem cells without radiation or antibiotics. We found that induced pluripotent stem cells induced by this co-culture method had a reprogramming efficiency and time similar to those induced using traditional methods. Furthermore, strongly expressed pluripotent markers showed a normal karyotype and formation and contained all three germ layers in a teratoma.