title: Derivation of stromal (skeletal and mesenchymal) stem-like cells from human embryonic stem cells.
abstract: Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for functional osteogenic cells.
authors.affiliation: Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University and King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia. ammahmood@ksu.edu.sa
authors.forename: Amer
authors.initials: A
authors.lastname: Mahmood
authors.forename: Linda
authors.initials: L
authors.lastname: Harkness
authors.forename: Basem M
authors.initials: BM
authors.lastname: Abdallah
authors.forename: Mona
authors.initials: M
authors.lastname: Elsafadi
authors.forename: May S
authors.initials: MS
authors.lastname: Al-Nbaheen
authors.forename: Abdullah
authors.initials: A
authors.lastname: Aldahmash
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis; D013724:Teratoma / Q000378:metabolism / Q000473:pathology; D014746:Vimentin / Q000378:metabolism
chemical_list: C525718:BMP2 protein, human; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D014746:Vimentin