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350. Identification and Characterization of Chondrogenic Progenitor Cells in Adult Skeletal Muscle
STEM CELL THERAPIES I 350. Identification and Characterization of Chondrogenic Progenitor Cells in Adult Skeletal Muscle Guangheng Li, Bo Zheng, Laura B. Meszaros, Joseph B. Vella, Karin A. Corsi, Arvydas Usas, Tomoyuki Matsumoto, Johnny Huard. Stem Cell Research Center, Children’s Hospital of Pittsburgh, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh.
Introduction: When ectotopic bone formation is induced in skeletal muscle by the addition of bone morphogenic protein (BMP), a chondrogenic phase is typically observed. This suggests that there exists chondrogenic cells in skeletal muscle. Previously, multipotent cells have been isolated from skeletal muscle vasculature (e.g. pericytes, myoendothelial cells). In the present study, we sought to identify these chondrogenic cells and to determine if endothelial cells participate in chondrogenesis. METHODS: Transgenic mice with LacZ expressing endothelial cells (FVB/N-Tg(TIE2-lacZ)182Sato/J) were induced to generate ectopic bone in skeletal muscle by injection of AAV-BMP4. Muscle derived cells (MDCs) were then isolated from Fischer rats and sorted by flow cytometry (FACS) and evaluated for their chondrogenic and myogenic potential using immunostaining (desmin, vimentin and MyoD). RESULTS: 1. Endothelial cells (blue) in skeletal muscle vasculature were not observed to participate in the chondrogenic phase.
Legend: (a) Surface marker profile of freshly isolated MDCs. (b) Phenotypic characterization of MDCs. (c) Chondrogenic assay demonstrates positive chondrogenic differentiation of MDCs. (d, e) Typical chondrocytes which occupy lacunae can be observed in the MDCs pellet. DISCUSSION: Despite the isolation of multipotent cells from muscle vasculature our results suggest that endothelial cells do not participate in the chondrogenic phase of ectopic bone formation. However, chondrogenic cells have previously been isolated from rat MDCs, particularly the fibrogenic cells residing in the muscle fascia (FDCs) (ORS 2007 abstract). In the present study, FACS sorting and subsequent characterization of MDCs demonstrated that MDCs contain at least two populations of cells, one that is chondrogenic and one that is myogenic. These chondrogenic cells are the likely candidate that participates in the chondrogenic phase of ectopic bone formation.
351. In Vitro Induction of the Hematopoietic Progenitor/Stem Cells from Human ES Cells Legend: a. AAV-BMP4 virus was injected into the gluteofemoral muscle pocket of a FVB/N-Tg (TIE2-lacZ)182Sato/J mouse. b. X-gal staining showed lacZ positive endothelial cells stained in blue. c. lacZ positive cells were not observed in the newly formed cartilage tissue at 17 days following injection of AAV-BMP4 virus. d. No lacZ positive cells were observed with higher magnification . 2. MDCs contain myogenic cells as well as chondrogenic cells that may arise from peri- and endomysium.
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Ryo Kurita, Rui Kageyama, Yoshie Miura, Takafumi Hiramoto, Tomoko Yokoo, Michiyo Okada, Atsushi Takahashi, Hiroyuki Inoue, Kenzaburo Tani. Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. The human embryonic stem (ES) cells are considered to differentiate to three lineage cells in vitro and in vivo as mouse ES cells. However, the best condition to induce these cells into the target cells/tissues and the safety of these differentiated cells/tissues remains unknown. We have recently reported that the lentivirally transduced ES cells of common marmoset, small nonhuman primate, differentiated efficiently into hematopoietic progenitor cells in vitro without the requirement of stromal cells. In this study, we examined whether the lentivirally transduction of tal1/scl induced human ES cells into hematopoietic progenitor cells in vitro, too. First of all, we determined the optimal culture condition to induce multilineage hematopoietic progenitor cells from human ES cells of hES-1 cells based on the expressions of Brachyury, Flk1 and CD34. Then we established four human ES cell lines lentivirally expressing tal1/ scl gene. The expression of hematopoietic cell markers including CD34, CD235a and CD133 was significantly increased in the embryoid bodies derived from tal1/scl-expressing ES cells cultured in the optimal growth factor condition. The number of hematopoietic Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Introduction: When ectotopic bone formation is induced in skeletal muscle by the addition of bone morphogenic protein (BMP), a chondrogenic phase is typically observed. This suggests that there exists chondrogenic cells in skeletal muscle. Previously, multipotent cells have been isolated from skeletal muscle vasculature (e.g. pericytes, myoendothelial cells). In the present study, we sought to identify these chondrogenic cells and to determine if endothelial cells participate in chondrogenesis. METHODS: Transgenic mice with LacZ expressing endothelial cells (FVB/N-Tg(TIE2-lacZ)182Sato/J) were induced to generate ectopic bone in skeletal muscle by injection of AAV-BMP4. Muscle derived cells (MDCs) were then isolated from Fischer rats and sorted by flow cytometry (FACS) and evaluated for their chondrogenic and myogenic potential using immunostaining (desmin, vimentin and MyoD). RESULTS: 1. Endothelial cells (blue) in skeletal muscle vasculature were not observed to participate in the chondrogenic phase.
Legend: (a) Surface marker profile of freshly isolated MDCs. (b) Phenotypic characterization of MDCs. (c) Chondrogenic assay demonstrates positive chondrogenic differentiation of MDCs. (d, e) Typical chondrocytes which occupy lacunae can be observed in the MDCs pellet. DISCUSSION: Despite the isolation of multipotent cells from muscle vasculature our results suggest that endothelial cells do not participate in the chondrogenic phase of ectopic bone formation. However, chondrogenic cells have previously been isolated from rat MDCs, particularly the fibrogenic cells residing in the muscle fascia (FDCs) (ORS 2007 abstract). In the present study, FACS sorting and subsequent characterization of MDCs demonstrated that MDCs contain at least two populations of cells, one that is chondrogenic and one that is myogenic. These chondrogenic cells are the likely candidate that participates in the chondrogenic phase of ectopic bone formation.
351. In Vitro Induction of the Hematopoietic Progenitor/Stem Cells from Human ES Cells Legend: a. AAV-BMP4 virus was injected into the gluteofemoral muscle pocket of a FVB/N-Tg (TIE2-lacZ)182Sato/J mouse. b. X-gal staining showed lacZ positive endothelial cells stained in blue. c. lacZ positive cells were not observed in the newly formed cartilage tissue at 17 days following injection of AAV-BMP4 virus. d. No lacZ positive cells were observed with higher magnification . 2. MDCs contain myogenic cells as well as chondrogenic cells that may arise from peri- and endomysium.
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Ryo Kurita, Rui Kageyama, Yoshie Miura, Takafumi Hiramoto, Tomoko Yokoo, Michiyo Okada, Atsushi Takahashi, Hiroyuki Inoue, Kenzaburo Tani. Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. The human embryonic stem (ES) cells are considered to differentiate to three lineage cells in vitro and in vivo as mouse ES cells. However, the best condition to induce these cells into the target cells/tissues and the safety of these differentiated cells/tissues remains unknown. We have recently reported that the lentivirally transduced ES cells of common marmoset, small nonhuman primate, differentiated efficiently into hematopoietic progenitor cells in vitro without the requirement of stromal cells. In this study, we examined whether the lentivirally transduction of tal1/scl induced human ES cells into hematopoietic progenitor cells in vitro, too. First of all, we determined the optimal culture condition to induce multilineage hematopoietic progenitor cells from human ES cells of hES-1 cells based on the expressions of Brachyury, Flk1 and CD34. Then we established four human ES cell lines lentivirally expressing tal1/ scl gene. The expression of hematopoietic cell markers including CD34, CD235a and CD133 was significantly increased in the embryoid bodies derived from tal1/scl-expressing ES cells cultured in the optimal growth factor condition. The number of hematopoietic Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy