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First published online 18 March 2003
doi: 10.1242/jcs.00369

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Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow

Stan Gronthos1,*, Andrew C. W. Zannettino2, Shelley J. Hay3, Songtao Shi4, Stephen E. Graves5, Angela Kortesidis1 and Paul J. Simmons6

1 Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia
2 Myeloma and Mesenchymal Research Group, Matthew Robert's Foundation laboratory, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia
3 Department of Orthopaedics and Trauma, Adelaide University, Adelaide, South Australia, Australia
4 Craniofacial and Skeletal Diseases Branch, National Institute of Dental & Craniofacial Research, National Institutes of Health, Maryland, USA
5 Department of Orthopaedics, Royal Melbourne Hospital, Melbourne, Victoria, Australia
6 Stem Cell Laboratory, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia



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  		Fig. 1. Isolation and purification of BMSSCs. (A) Flow cytometric analysis of MACS-sorted STRO-1+ marrow cells demonstrated varying levels of STRO-1 expression. There were approximately three distinct populations: dull (DULL), intermediate (INT) and bright (BRT). (B) Dual-color flow cytometric analysis of VCAM-1 (PE) expression by STRO-1+ (FITC) marrow cells isolated by MACS depicting a discrete population of STRO-1BRIGHT/VCAM-1+ cells (region: R1), approximately 0.02% of the total BMMNC population. (C) The incidence of clonogenic cell colonies (>50 cells) + clusters (>10<50 cells) based on STRO-1BRIGHT/VCAM-1+ expression was determined by limiting dilution analysis of 24 replicates per cell concentration using Poisson distribution analysis (data derived from six independent experiments).

 


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  		Fig. 2. Characterization of BMSSCs in vivo. (A) Light microscopic examination of cytospins representing freshly sorted STRO-1BRIGHT/VCAM-1+ marrow cells (1000x) counterstained with heamatoxylin. (B) Transmission electron micrograph depicting the ultrastructure of freshly sorted STRO-1BRIGHT/VCAM-1+ cells (15000x). Immunohistochemical staining of cytospin preparations of the sorted STRO-1BRIGHT/VCAM-1+ cells (1000x) with collagen type I (C) and {alpha}-SMA (D). (E) Dual-color flow cytometric analysis of Ki67 (FITC) expression by STRO-1+ (PE) marrow cells isolated by MACS. The majority of the STRO-1BRIGHT marrow cells lack detectable binding of Ki67 relative to that of the isotype-matched control antibody, indicated by the vertical quad-stat marker. (F) Telomerase activity in different sorted cell populations was examined using a modified TRAP assay. TRAP products derived from CHAPS extracts of non-denatured (-) and denatured (+) total BM (lanes 1), STRO-1BRIGHT/VCAM-1+ cells sorted fraction (lanes 2) and CD34+-sorted BM cells (lanes 3). TRAP products were resolved on a 12% polyacrylamide gel, stained with SYBR green fluorescent dye, and visualised using a fluorescence scanning system. (G) A typical light microscopic view of a single purified STRO-1BRIGHT/VCAM-1+ cell, allowed to adhere to fibronectin-coated culture plates (400x). (H) A representative example of a day 14 CFU-F colony stained with toluidine blue is shown (200x). (I) Immunohistochemical staining showing all the cells that comprise the CFU-F colony express collagen type I (200x).

 


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  		Fig. 3. Proliferation potential of BMSSC clones. A total of 35 CFU-F colonies derived from STRO-1BRIGHT/VCAM-1+ single sorted cells from two BM samples were analysed for their cumulative production of cells. A marked variation in proliferative capacity between individual BMSSCs is evident. The majority of clones (29/35, 83%) exhibited only moderate growth potential, which did not persist beyond 20 population doublings. 6/35 clones (17%) demonstrated continued growth extending beyond 20 population doublings.

 


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  		Fig. 4. Gene expression profile of BMSSCs in vivo and following differentiation in vitro. RT-PCR analysis of gene expression in STRO-1BRIGHT/VCAM-1+-purified BMSSCS isolated directly from marrow aspirates (1), and when cultured in regular growth medium (2) or medium inductive for either for bone, fat or cartilage development (3) as described in Materials and Methods. The expression of a range of markers characteristic of each tissue is shown. Bone: transcription factors CBFA1 and osterix (OSX), collagen type I (COL-1), osteopontin (OPN), osteocalcin (OCN) and parathyroid hormone receptor (PTH-R). Fat: lipoprotein lipase (LPL), transcription factor PPAR{gamma}2 and leptin. Cartilage: collagen type II (COL-II), collagen type X (COL-X) and aggrecan (AGGN). Reaction mixes were subjected to electrophoresis on a 1.5% agarose gel and visualised by ethidium bromide staining. RNA integrity was assessed by GAPDH expression.

 


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  		Fig. 5. Developmental potential of BMSSCs in vitro. Primary cultures of cells derived from STRO-1BRIGHT/VCAM-1+ marrow cells were induced under osteogenic, adipocytic or chondrocytic conditions. (A) Mineralized deposits stained positively with the von Kossa reagent (arrow) formed within 4 weeks of culture under osteoinductive conditions (200x). (B) The presence of clusters of lipid containing adipocytes were also detected by Oil red-O staining (arrow) within 2 weeks of adipogenic induction (200x). (C) In aggregate cultures, collagen type II was present throughout the cellular mass following 3 weeks of chondrogenic induction (arrow) (200x).

 


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  		Fig. 6. Developmental potential of BMSSCs clones in vivo. Immunoselected STRO-1BRIGHT/VCAM-1+ marrow-derived clones were expanded in vitro, then implanted subcutaneously into SCID mice using HA/TCP carrier. Implants were harvested 8 weeks after the transplant. (A) New bone formation containing osteocytes (arrow) was observed for a proportion of clones developing at the HA/TCP particle surfaces together with surrounding fibrous and hematopoietic tissue (BM). The sections were counter stained with haematoxylin and eosin (200x). (B) The osteocytes and bone lining cells (arrow) were found to be of human origin, as demonstrated by in situ hybridization using a DNA probe specific to the human alu repeat sequence (200x).

 

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© The Company of Biologists Ltd 2003