Project description:Objective: Craniofacial bone defects caused by injuries and congenital diseases are a formidable challenge to clinicians. Research has shown promise in using bone marrow mesenchymal stem cells (BM-MSCs) from limb bones for craniofacial bone regeneration; yet little is known about the potential of BM-MSCs from craniofacial bones. This study compared BM-MSCs isolated from limb and craniofacial bones in pigs, a preclinical model closely resembling humans. Design: Bone marrow was aspirated from the tibia and mandible of four-month-old pigs (n=4), followed by BM-MSC isolation, culture-expansion and confirmation by flow cytometry. Proliferation rates were compared using population doubling times. Osteogenic differentiation was evaluated by quantifying alkaline phosphatase (ALP) activity. Total mRNA was extracted from freshly isolated BM-MSCs and analyzed to compare gene expressions of tibial and mandibular BM-MSCs using an Affymetrix GeneChip porcine genome array, followed by real-time RT-PCR evaluation of two neural crest markers. Results: BM-MSCs from both locations expressed MSC markers without expression of hematopoietic markers. Mandibular BM-MSCs proliferated significantly faster than tibial BM-MSCs. Without osteogenic inducers, mandibular BM-MSC alkaline phosphatase activities were 3.3-fold greater than those of tibial origin. Microarray analysis identified 383 differentially expressed genes in mandibular and tibial BM-MSCs, including higher expression of cranial neural crest-related genes nestin and BMP-4 in mandibular BM-MSCs, a trend also confirmed by real-time RT-PCR. Among differently expressed genes, only 47 showed greater than 1.5-fold differences in expression. Conclusions: These data indicate that despite many similarities in gene expression, mandibular BM-MSCs express of number of genes differently than tibial BM-MSCs and have a phenotypic profile that may make them advantageous for craniofacial bone regeneration. Bone marrow was aspirated from the mandibular symphyseal region and the tibia of 3 pigs. Mesenchymal stem cells were isolated from the bone marrow and cultured to 80% confluence. Cells were harvested for total RNA extraction and the RNA was analyzed by Affymetrix GeneChip porcine genome array.
Project description:Objective: Craniofacial bone defects caused by injuries and congenital diseases are a formidable challenge to clinicians. Research has shown promise in using bone marrow mesenchymal stem cells (BM-MSCs) from limb bones for craniofacial bone regeneration; yet little is known about the potential of BM-MSCs from craniofacial bones. This study compared BM-MSCs isolated from limb and craniofacial bones in pigs, a preclinical model closely resembling humans. Design: Bone marrow was aspirated from the tibia and mandible of four-month-old pigs (n=4), followed by BM-MSC isolation, culture-expansion and confirmation by flow cytometry. Proliferation rates were compared using population doubling times. Osteogenic differentiation was evaluated by quantifying alkaline phosphatase (ALP) activity. Total mRNA was extracted from freshly isolated BM-MSCs and analyzed to compare gene expressions of tibial and mandibular BM-MSCs using an Affymetrix GeneChip porcine genome array, followed by real-time RT-PCR evaluation of two neural crest markers. Results: BM-MSCs from both locations expressed MSC markers without expression of hematopoietic markers. Mandibular BM-MSCs proliferated significantly faster than tibial BM-MSCs. Without osteogenic inducers, mandibular BM-MSC alkaline phosphatase activities were 3.3-fold greater than those of tibial origin. Microarray analysis identified 383 differentially expressed genes in mandibular and tibial BM-MSCs, including higher expression of cranial neural crest-related genes nestin and BMP-4 in mandibular BM-MSCs, a trend also confirmed by real-time RT-PCR. Among differently expressed genes, only 47 showed greater than 1.5-fold differences in expression. Conclusions: These data indicate that despite many similarities in gene expression, mandibular BM-MSCs express of number of genes differently than tibial BM-MSCs and have a phenotypic profile that may make them advantageous for craniofacial bone regeneration.
Project description:Integrated transcriptomic and proteomic analysis of the molecular cargo of extracellular vesicles derived from porcine adipose tissue-derived mesenchymal stromal/stem cells
Project description:Adipose-derived and bone-marrow-derived mesenchymal stem cells were collected from 3 pigs and cultivated in vitro up to 3 passages. At passage 3 cells were cultured to 80% confluence and induced to differentiate in adipose and bone. Cell were harvested at 0 day of differentiation (dd) or pre-differentiation, at 2, 7, and 21dd for RNA extraction. The RNA was used for a large microarray analysis using a specific pig oligo-array with >10,000 annotated genes. The main aim of the microarray analysis was to directly compare the two transcriptomics adaptation of the two mesenchymal stem cells during osteogenic and adipogenic differentiation The mesenchymal stem cells were harvested at 0, 2, 7, and 21 day of differentiation (dd). A dye-swap reference design (reference = mixture of RNA from several porcine tissues) was used.