RESUMEN
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.
Asunto(s)
Humanos , Diferenciación Celular/genética , Condrogénesis/genética , Sangre Fetal/citología , Células Madre Mesenquimatosas/citología , Factor de Transcripción SOX9/genética , Agrecanos/biosíntesis , Western Blotting , Cartílago/metabolismo , Proliferación Celular/genética , Condrocitos/metabolismo , Colágeno Tipo II/biosíntesis , Citometría de Flujo , Proteínas Fluorescentes Verdes , Regulación de la Expresión Génica/fisiología , Células Endoteliales de la Vena Umbilical Humana/citología , Inmunohistoquímica , Inmunofenotipificación , Cultivo Primario de Células , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ingeniería de Tejidos , TransfecciónRESUMEN
OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.
Asunto(s)
Humanos , Tejido Adiposo/citología , Cartílago Articular/citología , Diferenciación Celular/genética , Condrocitos/metabolismo , Condrogénesis/genética , Colágeno/metabolismo , Células Madre Mesenquimatosas , Adipogénesis/genética , Biomarcadores/metabolismo , Células Cultivadas , Condrocitos/citología , Colágeno/genética , Elastina/genética , Elastina/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Células Madre Mesenquimatosas , Osteogénesis/genética , ARN Mensajero/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factores de TiempoRESUMEN
Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. Cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. This study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns. These findings provide novel information concerning genes involved in the chondrogenesis of human BM-MSCs.
Asunto(s)
Humanos , Células de la Médula Ósea/citología , Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis/genética , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
beta ig-h3 is a TGF-beta-induced extracellular matrix protein which is expressed in many tissues including bones and cartilages. In previous reports, we showed that beta ig-h3 mediates cell adhesion and migration and, especially in bones, negatively regulates the mineralization in the end stage of endochondral ossification. Here, to elucidate the expression pattern and role of beta ig-h3 in chondrocyte differentiation, ATDC5 chondrocytes and embryonic and postnatal mice were used for in vitro differentiation studies and in vivo studies, respectively. beta ig-h3 was strongly induced by the treatment of TGF-beta1 and the expression level of beta ig-h3 mRNA and protein were highly expressed in the early stages of differentiation but decreased in the late stages in ATDC5. Furthermore, the patterns of TGF-beta1, -beta2, and -beta3 mRNA expression were concurrent with beta ig-h3 in ATDC5. beta ig-h3 was deeply stained in perichondrium (PC), periosteum (PO), and prehypertrophic chondrocytes (PH) through the entire period of endochondral ossification in mice. beta ig-h3 was mainly expressed in PC and PH at embryonic days and obviously in PH in postnatal days. These results suggest that beta ig-h3 may play a critical role as a regulator of chondrogenic differentiation in endochondral ossification.