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Topic: Osteoblasts and bone formation
Pihlström, Sandra(1,2)*;Määttä, Kirsi(1,2);Mäkitie , Riikka(1,2,3);Aronen, Mira(2);Mäkitie, Outi(1,2,4,5);Pekkinen, Minna(1,2,5);
(1)University of Helsinki, Faculty of Medicine, Helsinki, Finland;(2)Folkhälsan Research Center, Institute of Genetics, Helsinki, Finland;(3)Helsinki University Hospital and University of Helsinki, Department of Otorhinolaryngology- Head and Neck Surgery, Helsinki, Finland;(4)Karolinska Institutet, Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Stockholm, Sweden;(5)Helsinki University Hospital and University of Helsinki, Children's Hospital, Helsinki, Finland;
Abstract Text
Several skeletal disorders exhibit abnormal osteoblast development and function along with mineralization defects, creating a demand for an osteoblastic in vitro system. However, native osteoblasts are difficult to isolate from affected patients and problematic to expand in vitro. Similar issue concerns bone marrow mesenchymal stem cells (MSCs), the original progenitors of osteoblasts. One potential alternative source of cells that are non-immunogenic, easily expandable and readily available through a minimal invasive harvesting procedure are human dermal fibroblasts. Therefore, we developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy, approved by an ethical committee, and differentiated them into osteoblast-like cells by treating them with β-glycerophosphate, ascorbic acid and dexamethasone. The concentrations and duration of the components varied during the osteogenic treatment. Additionally, we differentiated human commercial MSCs, derived from bone marrow, into osteoblasts and used as a positive control. The osteoblastic phenotype was verified by staining alkaline phosphatase (ALP) and calcium deposits (Alizarin red and von kossa staining), and measuring mRNA and protein levels for specific osteoblastic markers using RNA sequencing and proteomic analysis. After 14 days of treatment, both fibroblasts and MSCs stained positive for ALP together with a significant increase in bone specific ALP compared to untreated cells. At a later time point, both cell types deposited minerals, as confirmed by staining, indicating mineralization. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell lines. Since osteoblasts have an essential role in osteoarthritis, by e.g. producing numerous transcription factors, growth factors, and other molecules involved in osteoarthritis pathogenesis, these data are supportive of osteogenic differentiation. Together, this data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system.
Topic: Osteoblasts and bone formation
Pihlström, Sandra(1,2)*;Määttä, Kirsi(1,2);Mäkitie , Riikka(1,2,3);Aronen, Mira(2);Mäkitie, Outi(1,2,4,5);Pekkinen, Minna(1,2,5);
(1)University of Helsinki, Faculty of Medicine, Helsinki, Finland;(2)Folkhälsan Research Center, Institute of Genetics, Helsinki, Finland;(3)Helsinki University Hospital and University of Helsinki, Department of Otorhinolaryngology- Head and Neck Surgery, Helsinki, Finland;(4)Karolinska Institutet, Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Stockholm, Sweden;(5)Helsinki University Hospital and University of Helsinki, Children's Hospital, Helsinki, Finland;
Abstract Text
Several skeletal disorders exhibit abnormal osteoblast development and function along with mineralization defects, creating a demand for an osteoblastic in vitro system. However, native osteoblasts are difficult to isolate from affected patients and problematic to expand in vitro. Similar issue concerns bone marrow mesenchymal stem cells (MSCs), the original progenitors of osteoblasts. One potential alternative source of cells that are non-immunogenic, easily expandable and readily available through a minimal invasive harvesting procedure are human dermal fibroblasts. Therefore, we developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy, approved by an ethical committee, and differentiated them into osteoblast-like cells by treating them with β-glycerophosphate, ascorbic acid and dexamethasone. The concentrations and duration of the components varied during the osteogenic treatment. Additionally, we differentiated human commercial MSCs, derived from bone marrow, into osteoblasts and used as a positive control. The osteoblastic phenotype was verified by staining alkaline phosphatase (ALP) and calcium deposits (Alizarin red and von kossa staining), and measuring mRNA and protein levels for specific osteoblastic markers using RNA sequencing and proteomic analysis. After 14 days of treatment, both fibroblasts and MSCs stained positive for ALP together with a significant increase in bone specific ALP compared to untreated cells. At a later time point, both cell types deposited minerals, as confirmed by staining, indicating mineralization. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell lines. Since osteoblasts have an essential role in osteoarthritis, by e.g. producing numerous transcription factors, growth factors, and other molecules involved in osteoarthritis pathogenesis, these data are supportive of osteogenic differentiation. Together, this data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system.
Institute of Genetics, Folkhälsan Research Center, Helsinki, Finland and CAMM, RPU, Faculty of Medicine, University of Helsinki, Finland
Topic: Osteoblasts and bone formation
Pihlström, Sandra(1,2)*;Määttä, Kirsi(1,2);Mäkitie , Riikka(1,2,3);Aronen, Mira(2);Mäkitie, Outi(1,2,4,5);Pekkinen, Minna(1,2,5);
(1)University of Helsinki, Faculty of Medicine, Helsinki, Finland;(2)Folkhälsan Research Center, Institute of Genetics, Helsinki, Finland;(3)Helsinki University Hospital and University of Helsinki, Department of Otorhinolaryngology- Head and Neck Surgery, Helsinki, Finland;(4)Karolinska Institutet, Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Stockholm, Sweden;(5)Helsinki University Hospital and University of Helsinki, Children's Hospital, Helsinki, Finland;
Abstract Text
Several skeletal disorders exhibit abnormal osteoblast development and function along with mineralization defects, creating a demand for an osteoblastic in vitro system. However, native osteoblasts are difficult to isolate from affected patients and problematic to expand in vitro. Similar issue concerns bone marrow mesenchymal stem cells (MSCs), the original progenitors of osteoblasts. One potential alternative source of cells that are non-immunogenic, easily expandable and readily available through a minimal invasive harvesting procedure are human dermal fibroblasts. Therefore, we developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy, approved by an ethical committee, and differentiated them into osteoblast-like cells by treating them with β-glycerophosphate, ascorbic acid and dexamethasone. The concentrations and duration of the components varied during the osteogenic treatment. Additionally, we differentiated human commercial MSCs, derived from bone marrow, into osteoblasts and used as a positive control. The osteoblastic phenotype was verified by staining alkaline phosphatase (ALP) and calcium deposits (Alizarin red and von kossa staining), and measuring mRNA and protein levels for specific osteoblastic markers using RNA sequencing and proteomic analysis. After 14 days of treatment, both fibroblasts and MSCs stained positive for ALP together with a significant increase in bone specific ALP compared to untreated cells. At a later time point, both cell types deposited minerals, as confirmed by staining, indicating mineralization. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell lines. Since osteoblasts have an essential role in osteoarthritis, by e.g. producing numerous transcription factors, growth factors, and other molecules involved in osteoarthritis pathogenesis, these data are supportive of osteogenic differentiation. Together, this data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system.
Topic: Osteoblasts and bone formation
Pihlström, Sandra(1,2)*;Määttä, Kirsi(1,2);Mäkitie , Riikka(1,2,3);Aronen, Mira(2);Mäkitie, Outi(1,2,4,5);Pekkinen, Minna(1,2,5);
(1)University of Helsinki, Faculty of Medicine, Helsinki, Finland;(2)Folkhälsan Research Center, Institute of Genetics, Helsinki, Finland;(3)Helsinki University Hospital and University of Helsinki, Department of Otorhinolaryngology- Head and Neck Surgery, Helsinki, Finland;(4)Karolinska Institutet, Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Stockholm, Sweden;(5)Helsinki University Hospital and University of Helsinki, Children's Hospital, Helsinki, Finland;
Abstract Text
Several skeletal disorders exhibit abnormal osteoblast development and function along with mineralization defects, creating a demand for an osteoblastic in vitro system. However, native osteoblasts are difficult to isolate from affected patients and problematic to expand in vitro. Similar issue concerns bone marrow mesenchymal stem cells (MSCs), the original progenitors of osteoblasts. One potential alternative source of cells that are non-immunogenic, easily expandable and readily available through a minimal invasive harvesting procedure are human dermal fibroblasts. Therefore, we developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy, approved by an ethical committee, and differentiated them into osteoblast-like cells by treating them with β-glycerophosphate, ascorbic acid and dexamethasone. The concentrations and duration of the components varied during the osteogenic treatment. Additionally, we differentiated human commercial MSCs, derived from bone marrow, into osteoblasts and used as a positive control. The osteoblastic phenotype was verified by staining alkaline phosphatase (ALP) and calcium deposits (Alizarin red and von kossa staining), and measuring mRNA and protein levels for specific osteoblastic markers using RNA sequencing and proteomic analysis. After 14 days of treatment, both fibroblasts and MSCs stained positive for ALP together with a significant increase in bone specific ALP compared to untreated cells. At a later time point, both cell types deposited minerals, as confirmed by staining, indicating mineralization. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell lines. Since osteoblasts have an essential role in osteoarthritis, by e.g. producing numerous transcription factors, growth factors, and other molecules involved in osteoarthritis pathogenesis, these data are supportive of osteogenic differentiation. Together, this data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system.