ATEuropean El-Sera Cellsfi et aland. Materials Vol. 21 2011 Self-assembly (pages 558-567) of human DOI: foetal 10.22203/eCM.v021a42 skeletal cells into an osteo-chondrogenic ISSN 1473-2262 shell DEVELOPMENTAL PLASTICITY OF HUMAN FOETAL FEMUR-DERIVED CELLS IN PELLET CULTURE: SELF ASSEMBLY OF AN OSTEOID SHELL AROUND A CARTILAGINOUS CORE Ahmed T. El-Serafi 1,4, David I. Wilson2,3, Helmtrud I. Roach1 and Richard O.C. Oreffo1,2,5* 1Bone & Joint Research Group, 2Centre for Human Development, Stem Cells and Regeneration, 3Division of Human Genetics, Institute of Developmental Sciences, University of Southampton School of Medicine, U.K. 4Medical Biochemistry Department, Faculty of Medicine, Suez Canal University, Egypt 5Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia Abstract Introduction This study has examined the osteogenic and chondrogenic Cultured cells derived from human foetal femurs can differentiation of human foetal femur-derived cells in differentiate into osteogenic, chondrogenic and adipogenic 3-dimensional pellet cultures. After culture for 21-28 lineages (Mirmalek-Sani et al., 2006). The femur of a days in osteogenic media, the pellets acquired a unique 10 week old foetus consists of two large cartilaginous confi guration that consisted of an outer fi brous layer, an epiphyses that contain chondrocytes and a diaphysis osteoid-like shell surrounding a cellular and cartilaginous with a hypertrophic cartilage within a thin bone collar region. This confi guration is typical to the cross section (Mirmalek-Sani et al., 2006). At 10 weeks, vascular of the foetal femurs at the same age and was not observed invasion of the diaphysis has not occurred and the in pellets derived from adult human bone marrow stromal isolated cells constitute a mixed population of epiphyseal cells. Time course study showed that after 7-14 days, the and hypertrophic chondrocytes as well as osteogenic cells of the inner cellular region were viable, proliferated cells derived from the bone collar. The earliest phase rapidly, and were immuno-positive for c-myc, as well as of long bone development in the embryo consists of a for bone sialoprotein and type I collagen. After 21-28 days, rod-shaped condensation of mesenchymal cells which the cells accumulated at the inner edge of the osteoid shell. differentiate along the chondrogenic lineage centrally, The direction of osteoid formation thus differed from that while the peripheral cells become fl attened and form the of periosteal bone formation. Following micro-dissection perichondrium of the so-called cartilage ‘anlage’ (Sandell of the human foetal femurs into epiphyses, bone cylinder and Adler, 1999; Hall and Miyake, 2000; Goldring et al., and hypertrophic cartilage, epiphyseal chondrocytes and 2006). Subsequent bone formation involves two phases. osteoblasts both gave rise to osteoid-shell forming cells. The initial phase intramembranous bone formation, These studies demonstrate the developmental plasticity occurs outside the mid-shaft region of the cartilage of human foetal skeletal and epiphyseal chondrocytes rod. Cells from the perichondrium differentiate into and suggest that the microenvironment modulates lineage osteoblasts and deposit osteoid onto the cartilage anlage commitment and matrix formation. Furthermore, this ex and chondrocytes located within the central part of the vivo model offers a new approach to delineate human bone cartilage rod differentiate to hypertrophic chondrocytes. development as well as a model with potential application The second phase, endochondral ossification, takes for evaluation of therapeutic compounds for bone formation. place with vascular invasion. Endochondral ossifi cation involves apoptosis of terminal chondrocytes, invasion Keywords: Human foetal cells, stem cells, differentiation, of vascular cells into the vacated lacunae, resorption of bone development, pellet culture, bone model, tissue transverse septa and deposition of bone onto non-resorbed engineering, self assembly, 3D model. calcifi ed longitudinal septa. In the last decade tremendous advances have been made in identifying the signals, transcriptional regulators and altered gene expression patterns that accompany the various stages of endochondral ossifi cation (Kronenberg, 2003; Goldring et al., 2006). However, to date, the cellular and molecular events that occur during initial membranous bone formation, in particular which events are obligatory (vs. associated) remain unclear. Caplan (Caplan, 1987) identifi ed a layer of 4-6 fl attened ‘stacked cells’ as the *Address for correspondence: cells that differentiate to the osteoblasts that produce Richard O.C. Oreffo the fi rst bone collar at the mid-diaphysis. Bianco et al. Bone & Joint Research Group (Bianco et al., 1998; Riminucci et al., 1998) demonstrated Institute of Developmental Sciences that both osteoblasts differentiating from the osteogenic University of Southampton School of Medicine, perichondrium and chondrocytes facing these osteoblasts Southampton, SO17 1BJ, U.K. (vis-à-vis chondrocytes) contribute to the pool of cells that E-mail: [email protected] form the initial bone collar. www.ecmjournal.org 558 AT El-Serafi et al. Self-assembly of human foetal skeletal cells into an osteo-chondrogenic shell We have previously reported on the phenotype, basal media (α-minimal essential medium (MEM), 10 % multipotentiality and osteogenic (bone forming capacity FCS and 100 U/mL penicillin and streptomycin), then of human foetal derived skeletal cells in monolayer culture frozen in 10 % dimethylsulphoxide (DMSO) in foetal calf (Mirmalek-Sani et al., 2006) as well as osteogenic matrix serum (FCS) and stored at -80 °C. In addition, femurs were formation by human bone marrow stromal cells in pellet micro-dissected into three parts: cartilaginous epiphyses, culture (El-Serafi et al., 2011). The current study has bone collar and hypertrophic cartilage and cells extracted examined the differentiation potential of human foetal as detailed above. femur derived cells in 3D pellet culture to determine if this will provide a new approach to examine and inform Cell culture bone formation and to delineate the mechanisms therein. Skeletal cells were expanded in monolayer in basal medium o 3D culture is known to enhance cell differentiation and under 5 % CO2 at 37 C in humidifi ed air. At confl uence, function, in comparison to human bone marrow stromal cells were washed twice with phosphate buffered saline cells. Matrix formation in the pellets derived from human (PBS) and incubated at 37 oC with collagenase IV (0.2 foetal femur derived skeletal cells was observed to be non- mg/mL) for 45 min and with trypsin for 5 min to allow uniform with a central core of cells within an indistinct isolation of individual cells. For pellet cultures typically matrix surrounded by a distinct shell of osteoid-like matrix 5x105 cells were transferred to 30 mL concave-end beneath an outer fi brous layer of fl attened cells reminiscent polystyrene universal tubes (Greiner Bio-One, Stonehouse, of the initial bone collar in the original foetal femur. These Gloucestershire, U.K.) in 1 mL of medium, centrifuged at fi ndings were not observed in human bone marrow stromal 400 g for 10 min and then incubated at 37 oC under 5 % cell pellets. CO2. The cells were observed to form a pellet within 24 The current approach provides a unique model to to 48 h and media in these studies changed three times i) examine, delineate and interrogate normal human per week. Pellets were cultured in either osteogenic or bone development including an approach to interrogate chondrogenic media. Osteogenic medium consisted of intramembranous bone formation in 3D, ii) bone tissue basal medium with ascorbate-2-phosphate (100 μM) and regeneration through intervention of the development dexamethasone (10 nM). Chondrogenic medium consisted process and mechanisms therein and, iii) a model to further of basal medium, ascorbate-2-phosphate (100 μM), examine the developmental plasticity of foetal skeletal dexamethasone (10 nM), TGF-β3 (10 ng/mL; VWR) and cells indicating the importance of the microenvironment ITS supplement (1.0 mg/mL insulin from bovine pancreas, in modulating lineage commitment and differentiation. 0.55 mg/mL human transferrin and 0.5 μg/mL sodium selenite) in the absence of FCS. Materials and Methods Histology At the end of the culture period, pellets were fi xed in 4 % Tissue culture reagents, α-Minimum Essential Medium paraformaldehyde, processed through graded ethanol and (α-MEM), dexamethasone, Insulin Transferrin Selenium chloroform and embedded in paraffi n wax. 7 μm sections (ITS) solution, staining solutions, and all biochemical were stained with Alcian blue/Sirius red to distinguish reagents were obtained from Sigma-Aldrich, Poole, Dorset, bone from cartilage matrix. Alcian blue (0.5 % in 1 % U.K., unless otherwise stated. Foetal Calf Serum (FCS) was acetic acid) stains proteoglycans, which typically stain purchased from Invitrogen Ltd (Paisley, U.K.). an indistinct light blue in connective tissues, whereas the aggrecan of cartilage matrix stains a typical turquoise blue. Cell isolation Sirius red (1 % in 33 % saturated picric acid) stains fi brous Human bone marrow stromal cells (HBMSCs) were collagens type I and II. In normal cartilage, the staining isolated from bone marrow samples obtained from patients sites for type II collagen are covered up by aggrecan and undergoing routine hip arthroplasty with appropriate only Alcian blue staining is observed.