title: Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential.
abstract: The derivation of osteogenic cells from human embryonic stem cells (hESC) has been hampered by the absence of easy and reproducible protocols. hESC grown in feeder-free conditions, often show a sub population of fibroblast-like, stromal cells growing between the colonies. Thus, we examined the possibility that these cells represent a population of stromal (mesenchymal) stem cells (hESC-stromal). Two in house derived hES cell lines (Odense3 and KMEB3) as well as an externally derived cell line (Hues8) were transitioned to feeder-free conditions. A sub population of fibroblast-like cells established between the hESC colonies were isolated by selective adherence to hyaluronic acid-coated plates (100 μg/ml) and were characterized using a combination of FACS analysis and staining. The cells were CD44(+), CD29(+), CD73(+), CD166(+), CD146(+), and CD105(+); and, Oct4⁻, CD34⁻, CD45⁻ and CXCR4⁻. When cultured in osteogenic differentiation media, up regulation of osteoblastic lineage markers (DLX5, MSX2, RUNX2, SPARC, ALP, COL1a1, BGLAP, IBSP, DCN, LOX-L4) and production of in vitro mineralized matrix was detected. hESC-stromal cells loaded on a carrier and implanted either subcutaneously or in a critical size calvarial defect in immune deficient mice for 10 weeks, resulted in new bone formation and partial repair of the calvarial defect. In conclusion, hESC-stromal can be isolated from hESC cultures and represent a good source for obtaining cells with osteogenic differentiation potential suitable for regenerative medicine protocols.
authors.affiliation: Department of Endocrinology & Metabolism, Laboratory for Molecular Endocrinology (KMEB), Medical Biotechnology Centre (MBC), Winsløwparken 25, University of Southern Denmark, 5000 Odense C, Denmark. lharkness@health.sdu.dk
authors.forename: Linda
authors.initials: L
authors.lastname: Harkness
authors.forename: Amer
authors.initials: A
authors.lastname: Mahmood
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ditzel
authors.forename: Basem M
authors.initials: BM
authors.lastname: Abdallah
authors.forename: Jens V
authors.initials: JV
authors.lastname: Nygaard
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
mesh_terms: D017667:Adipocytes / Q000166:cytology; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D010006:Osteoblasts / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology*