Characterization and Multipotentiality of Human Fetal FemurCDerived Cells: Impli - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:07 |只看该作者 |倒序浏览 |打印

作者：Sayed-Hadi Mirmalek-Sania, Rahul S. Tarea, Suzanne M. Morgana, Helmtrud I. Roacha, David I. Wilsonb, Neil A. Hanleyb, Richard O.C. Oreffoa作者单位：a Bone and Joint Research Group, Developmental Origins of Health and Disease, University of Southampton, Southampton, United Kingdom;b Human Genetics Division, University of Southampton, Southampton, United Kingdom
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      【摘要】7 D2 ?7 N2 y. u7 `1 q. B, ~
   To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult-derived mesenchymal cell populations. Real-time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal-derived cells in comparison with unselected adult-derived and immunoselected STRO-1¨Cenriched adult populations. Multipotentiality was examined using cells derived from femurs and single-cell clones, culture-expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation-activated receptor- detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single-cell clonal analysis was used to demonstrate multipotentiality of the fetal-derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur¨Cderived cells in comparison with adult-derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue. ; W1 e6 }- d( c4 @. Y4 f
      【关键词】 Fetal mesenchymal stem cell Osteoprogenitor Differentiation Tissue regeneration. R3 w* U5 q9 w1 f! O
   INTRODUCTION/ I  ?7 B, ?% r+ t7 @( ~# d

Tissue loss as a result of injury or disease, in an increasing aging population, is responsible for a reduced quality of life for many and has led to the need for procedures to generate cartilage and bone for skeletal use . However, to date, the isolation and characterization of homogenous mesenchymal stem cells have been hampered by the lack of specifically reactive antibodies and the paucity of these cells.# n% L3 l) G4 o  W2 _( Q

Isolation and expansion of mesenchymal stem cells have been undertaken using a restricted panel of monoclonal antibodies, including SB-10 , which has opened up the potential of these cells in cartilage and bone repair and therefore obviating the need for autologous marrow although the donor age at which to select mesenchymal stem cell populations remains unclear.
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Alternative mesenchymal populations include cells isolated from cord blood and placenta , termed multipotent adult progenitor cells. These cells, CD34, CD44, CD45, c-Kit, and major histocompatibility complex class I and II negative, were derived from adult marrow by cell sorting and extended cell culture. Critically, these multipotent adult progenitor cells appear to engraft in vivo when transplanted into nondamaged recipients although, to date, only a few groups worldwide have cultured these cells.
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Understanding the differentiation of mesenchymal stem cells and skeletogenesis, and the effects of key differentiation agents such as vitamin A and related molecules, the retinoids, requires robust and clinically relevant cell models. In the absence of accepted facile strategies for the generation of sufficient mesenchymal stem/progenitor cells, the current study set out to examine the presence, characteristics, activity, and plasticity of cells derived from the cartilage anlage of fetal femurs and compared and contrasted their phenotypic properties with those of adult mesenchymal progenitor populations, which were isolated using STRO-1 immunoselection. To date, only one other study has reported on some of the properties of human fetal bone cells from 13¨C16 weeks . The current study has sought to characterize the multipotential properties and plasticity as well as further phenotypic characterization of human fetal femur¨Cderived cells from 8¨C11 weeks and their potential as a unique alternative cell source in skeletal tissue regeneration strategies and in fundamental research on human mesenchymal stem cell differentiation.
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MATERIALS AND METHODS
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Materials and Reagents

Tissue culture reagents, -minimum essential medium (-MEM), fetal calf serum (FCS), growth factors, including insulin transferrin selenium (ITS) solution, staining solutions, and all other biochemical reagents were obtained from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com) unless otherwise stated. Molecular biology reagents were purchased from Promega UK Ltd. (Southampton, U.K., http://www.promega.com) and Invitrogen Life Technologies (Paisley, U.K., http://www.invitrogen.com). Cell Tracker Green (CTG, 5-chloromethyl-fluorescein diacetate) and ethidium homodimer-1 (EH-1) were purchased from Molecular Probes (Leiden, The Netherlands, http://probes.invitrogen.com). Recombinant transforming growth factor (TGF)-ß-3 (PF073) and type II collagen polyclonal antibody (234187) were purchased from Calbiochem (Nottingham, U.K., http://www.emdbiosciences.com). Type I collagen polyclonal antibody was a gift from Dr. Larry Fisher, of the National Institutes of Health (NIH). Collagenase B was purchased from Roche (1088807; Manheim, Germany, http://www.roche.com) and 3D Calcium Phosphate Scaffold blocks came from Becton, Dickinson and Company (354617; Franklin Lakes, NJ, http://www.bd.com). Polyglycolic acid (PGA) fleece was purchased from Cellon (Bereldange, Luxembourg, http://www.cellon.lu), and the polylactic acid/hydroxyapatite (PLA/HA) composite scaffold was supplied by Kevin Shakesheff and Steven Howdle (University of Nottingham, U.K.) SOX9 polyclonal antibody (AB5535) was obtained from Chemicon International, Inc. (Temecula, CA, http://www.chemicon.com), and aP2 monologue fatty acid binding protein-3 (FABP-3) polyclonal antibody (AF1678) was purchased from R&D Systems (Abingdon, Oxon, U.K., http://www.rndsystems.com). TRIzol (15596¨C018) and Super-Script First-strand synthesis system for reverse transcription¨Cpolymerase chain reaction (RT-PCR) (11904¨C018) were purchased from Invitrogen Life Technologies, whereas the DNA-free RNA Kit (R1013) was procured from Zymo Research Corporation (Orange, CA, http://www.zymoresearch.com). Primers for quantifying glyser-aldehyde-3-phosphate dehydrogenase (GAPDH) expression and the SYBR green master-mix kit (RT-SN2X-03-075) were purchased from Eurogentec (Seraing, Belgium, http://www.eurogentec.com).
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Cell Culture: \7 J- z4 m: s% i% a; @/ U- d6 F$ T+ f

Human bone marrow samples were obtained from hematologically normal patients undergoing routine hip replacement surgery (seven patients . Cultures were maintained in basal medium (-MEM containing 10% FCS) at 37¡ãC, supplemented with 5% CO2. After 12¨C15 days, at confluence, samples were used for differentiation experiments or harvested for RNA./ T* f) a8 B: |: {* j; r6 H2 j( ~  _
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Stro-1  marrow stromal cells from human bone marrow were immunoselected with the antibody STRO-1, using a magnetically activated cell separation system as described previously . Passage 0 cultures were maintained in basal medium for 12 days to reach confluence and were then used for further studies.6 k% w" W) d6 Y) ?4 M- C% s# J

Human fetal femurs were obtained after termination of pregnancy according to guidelines issued by the Polkinghome Report and with ethical approval from the Southampton & South West Hampshire Local Research Ethics Committee (six samples; mean age 8.6 ¡À 1.2 weeks). Fetal age was determined by measuring fetal foot length and expressed in weeks postconception (WPC) (Table 1). The femurs were placed in sterile phosphate-buffered saline (PBS), and surrounding skeletal muscle was removed. Femurs were dissected and plated into T25 flasks in 2 ml basal medium, changed weekly. Cells were also maintained at 37¡ãC and supplemented with 5% CO2. Cells were cultured for 21 days from explants before passage and expanded for an additional 7 days before use in multipotential experiments. Samples were denoted by collection number. Fetal clonal populations were produced via a gated sort on a BD FACSAria Cell Sorter (Becton, Dickinson and Company) delivering single cells to a tissue culture well plate. Fetal cells were seeded into 96-well plates via a gated sort of events per well (20:1, 10:1, 8:1, 5:1, 2:1, and 1:1 with a BD FACSAria Cell Sorter). Viable single-cell clones were identified via microscopy and expanded in basal conditions. Populations derived from a single cell were then used for multipotential experiments, differentiating along all three (adipogenic, chondrogenic, and osteogenic) lineages. These studies were repeated with different femurs (n = 3, separate studies) to establish true multipotentiality.

Table 1. Human fetal tissue samples and ages (weeks post-conception)9 F' j! D1 V0 d  V

Sections of articular cartilage (2¨C4 mm3) were obtained from femoral heads of patients undergoing routine hip replacement surgery. The tissue samples were digested overnight in collagenase B (10 mg/ml). The cell suspension was then filtered using a 70-µm filter, and the resulting cells were pelleted by centrifugation. Cells were then resuspended in an appropriate volume of TRIzol for RNA extraction.
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Osteogenic, Chondrogenic, and Adipogenic Conditions0 L& x- W9 ~! {: j7 V
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After expansion in tissue culture flasks, cells were passaged to six-well plates for adipogenic and osteogenic differentiation or pelleted for chondrogenic conditions. Control cultures were refreshed with basal medium (-MEM containing 10% FCS) every 3 days. Adipogenic conditions: Confluent monolayers grown in basal conditions were transferred to adipogenic medium (-MEM supplemented with 10% FCS) with 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 1 µM dexamethasone, 10 µg/ml insulin (in the form of 100 x ITS Supplement Solution), and 100 µM indomethacin) as described in Pittenger et al. .+ \1 ~/ u8 }. l+ l# s; X. D+ J
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Fetal cells were seeded onto three-dimensional (3D) calcium phosphate scaffold blocks (diameter, 3 x 5 mm; average pore size, 200¨C400 µm), PGA fleece sections (5 x 5 x 2 mm), or PLA/HA scaffolds (4-mm cubes; average pore size, 100¨C300 µm). Cells were seeded dynamically at a density of 5 x 104 cells per calcium phosphate or PGA scaffold, and 3 x 105 for PLA/HA blocks. Cultures were maintained in basal conditions for 4 days before being transferred to osteogenic medium, as described above, with media changes every 3 days. Cultures were stopped after 7 days, then fixed in 95% ethanol, and analyzed histologically.$ l- q4 p0 w, n

Fetal femur¨Cderived cell seeded and unseeded PLA/HA blocks were cultured in osteogenic medium for 24 hours prior to subcutaneous implantation into female MFI-nu/nu severe combined immunodeficient mice (23¨C26 g; Harlan, Loughborough, U.K., http://www.harlan.com) . Control cultures of fetal cell¨Cseeded PLA/HA scaffolds were maintained in vitro in basal conditions. After 28 days, mice were killed and parallel cell cultures were stopped. Samples were explanted for fixing and histological analysis.
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Histological Analysis0 u6 L1 ]$ r+ y1 }) b( x  d- j5 g- i+ R
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Chondrogenic pellets and whole femurs were fixed in 95% ethanol and embedded in paraffin wax before sectioning at 6 µm. After explantation, PLA/HA samples were fixed in 4% paraformaldehyde, then decalcified in 5% EDTA in 0.1 M TRIS-HCL (pH 7.2) for 14 days, before wax embedding and sectioning. Day-21 fetal cell¨Cseeded scaffolds were fixed in 10% formalin and embedded in glycol methylacrylate (GMA) resin according to manufacturer¡¯s instructions (JB-4 Embedding Kit; Polysciences, Inc., Warrington, PA, http://www.polysciences.com). Sections were cut at 2 µm and allowed to dry overnight. All images were captured with Carl Zeiss Axio-vision software via AxioCam HR digital camera (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Staining Procedures
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To demonstrate adipogenesis, Oil Red O staining was used as previously described .1 K9 e, A* t2 j) x6 Y

Immunohistochemistry  S* i/ e1 r: m, |1 d' B
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After quenching endogenous peroxidase activity with 3% H2O2 and blocking with 1% bovine serum albumin (BSA) in PBS, sections were incubated with the relevant primary antiserum at 4¡ãC overnight, followed by 1-hour incubation with the appropriate biotinylated secondary antibody. Visualization of the immune complex involved the avidin-biotin method linked to peroxidase and 3-amino-9-ethylcarbazole (AEC), resulting in a reddish brown reaction product. Sections were mounted in glycerol jelly. Appropriate isotype controls were used with negative controls lacking the respective primary antibodies. No staining was observed in the control sections.3 W, B2 J1 S, G

aP2. Presence of aP2 was determined in adipogenic cultures. Plates were incubated with FABP-3 antibody (1:100) then with anti-sheep immunoglobulin G (IgG) biotin-conjugated secondary antibody (1:50 with 1% BSA in PBS)." |9 }! D- l8 L" |
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SOX9. The anti-SOX9 antibody was used at a dilution of 1:150, following the antigen retrieval procedure in the manufacturer¡¯s instructions. Briefly, it involved treating sections in 0.01 M citrate buffer in a microwave for 5 minutes before the routine immunocytochemistry procedure described above. Sections were incubated with anti-rabbit IgG biotin-conjugated secondary antibody (1:200 with 1% BSA in PBS; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com), and SOX9-stained sections were counterstained in Light Green for 2 minutes before mounting in glycerol jelly.! h! U1 Y+ ]9 W8 h2 k  s
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Type I & II collagen. Blocking and primary antibody incubation periods were also as described above for LF67 type I collagen antibody (1:300) or type II collagen (1:1,000) primary antibodies, with anti-rabbit IgG biotin-conjugated secondary antibody (1:200 with 1% BSA in PBS; DakoCytomation).
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Vimentin. Mouse monoclonal anti-vimentin antibody, specific for human tissue, (V6630; Sigma-Aldrich) was used in conjunction with the Animal Research Kit (ARK, K3954; DakoCytomation) according to manufacturer¡¯s instructions. Briefly, the primary antibody was incubated with a biotin-conjugated secondary antibody prior to application to tissue, then developed with 3,3'-diaminobenzidine (DAB) chromogen solution, counterstained with Light Green, and mounted in glycerol jelly.

STRO-1 Immunofluorescence5 D, u9 T4 z3 k7 g# I6 o) H  v
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After the blocking stage with 1% BSA in PBS, fetal cells fixed in 4% paraformaldehyde were incubated overnight with STRO-1 mouse monoclonal antibody (undiluted culture supernatant from the STRO-1 hydridoma provided by Dr. J. Beresford, University of Bath). A 1:50 dilution of fluorescein isothiocyanate (FITC)¨Cconjugated anti-mouse IgM secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, http://www.jacksonimmuno.com, and Stratech Scientific Ltd., Soham, Cambridgeshire, U.K., http://www.stratech.co.uk) was applied, and after three PBS washes, samples were incubated with 1:100 4',6-diamidino-2-phenylindole (DAPI, D3571; Molecular Probes) stain for 5 minutes. Cultures were again washed in PBS and visualized with appropriate fluorescent filters on a Carl Zeiss Axiovert 200.

Cell Viability
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Cells were incubated with 10 µg per ml CTG and 5 µg per ml EH-1 to label viable and necrotic cells, respectively. After incubation with CTG/EH-1 at 37¡ãC, cells were bathed in -MEM for 1 hour before fixing in 70% ethanol. Cells were transferred to PBS and visualized with a 20x water immersion lens on a Leica Leitz DM BRE confocal microscope (Heerbrugg, Switzerland, http://www.leica.com). Leica Confocal Software (version 2.5) was used to capture labeled cells on an image series at 5-µm stages.
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RNA Extraction and PCR Analysis

Total RNA was extracted using TRIzol reagent, subjected to DNAse treatment (DNA-free RNA kit), and reverse-transcribed using the Super-Script First-strand synthesis system for RT-PCR. Real-time quantitative PCR was performed using the BIORAD iCycler for quantifying the expression of activated leukocyte cell adhesion molecule (ALCAM/CD166), CD63, human stem cell factor (HSCF), and bone morphogenetic protein (BMP) receptor-type IA (BMPR-IA). Primer sequences for the genes are illustrated in Table 2. Values were calculated using the comparative threshold cycle (Ct) method and normalized to GAPDH expression. Values were expressed as the mean ¡À SD. Experiments were performed at least three times, and results of representative experiments have been presented. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey-Kramer Multiple Comparisons post-test using GraphPad Instant Software (GraphPad Software, Inc., San Diego, http://www.graphpad.com).! c5 l1 k1 f! l( L( E

Table 2. Primer sequences used for real-time quantitative PCR and RT-PCR

Conventional PCR was performed for analyzing GAPDH, type II collagen, ALP, type I collagen, osteopontin (OP), OC, peroxisome proliferator-activated receptor  (PPAR-, transcript encoding the full-length type 2 isoform), retinoic acid receptor (RAR)-, -ß-1, -ß-2, and -, and 9-cis RAR (RXR)-, -ß, and - expression in populations of fetal femur¨Cderived cells from three individual donor samples. Primer sequences for these genes are illustrated in Table 2.

RESULTS

Isolation of Fetal Femur¨CDerived Populations

The human fetal femurs at 8¨C11 WPC were predominantly a cartilaginous anlage with an emergent bone collar (as seen in Alcian Blue/Sirius red¨Cstained sections, Fig. 1A¨C1C). In the center of the diaphysis, cells displayed a well defined chondrogenic phenotype (Fig. 1B), and no vascular invasion had yet occurred. Presence of the transcription factor SOX9 (Fig. 1D, 1E) confirmed that the chondrocytes were at an early stage of differentiation. The femur was surrounded by a layer of fibroblastic cells staining for vimentin (Fig. 1F). Growth of human fetal cells after dissection and culture as minced 1-mm3 explants is shown in Figure 1G and 1H. Cells were observed to grow rapidly in culture and displayed, typically, a characteristic fibroblastic morphology (Fig. 1H, 1I). Cultured cells from fetal explant cultures closely resembled the fibroblastic morphology displayed in adult-derived bone marrow stromal cultures (Fig. 1I). Some fetal cells were observed to express the STRO-1 antigen after 48 hours of culture in basal conditions (Fig. 1J).
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Figure 1. Histology and explant cultures of 8-week postconception human fetal femurs. (A): Cartilaginous matrix highlighted by Alcian Blue staining in distal head. (B): Diaphysis centre showing further Alcian Blue staining with chondrocytic cells within lacunae and Sirius Red matrix stain for emerging bone collar. (C): Proximal (femoral) head displaying same staining characteristics as distal head. (D, E): SOX9 staining of chondrocytic populations throughout the femur. (F): Presence of fibroblastic marker vimentin in early perichondrium/periosteum surrounding femur. (G, H): Cellular outgrowth from fetal femur explants in basal media conditions (- minimum essential medium containing 10% fetal calf serum). (I): Human fetal femur-derived cells on tissue culture flask, maintained in basal conditions. (J): Positive STRO-1 staining of fetal cells, counterstained with DAPI nuclear stain. Bars = 200 µm. Abbreviation: DAPI, 4',6-diamidino-2-phenylindole.: K' s* `" x+ j  f* B; t6 V
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Expression of Stromal Antigens and Stem Cell Markers by Fetal Femur¨CDerived Cells

The expression of stem cell markers by the fetal femur¨Cderived cells, which were thought to include a form of primitive precursor cells, possibly mesenchymal, was examined by real-time quantitative PCR using accepted stromal antigens, namely ALCAM/CD166 and CD63, and putative stem cell markers like HSCF . HSCF expression was found to be higher in Stro-1  cells than in the fetal cells, followed by unselected bone marrow cells and articular chondrocytes. Fetal femur¨C derived cells expressed highest levels of BMPR-IA in comparison with the other cell populations, followed by Stro-1  cells, unselected bone marrow cells, and articular chondrocytes.
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Figure 2. Quantitative expression of ALCAM/CD166, CD63, HSCF, and BMPR-IA by real-time quantitative PCR in fetal femur¨Cderived cells, Stro-1  human bone marrow stromal cells, unselected human bone marrow cells and human articular chondrocytes. Transcript levels were determined using the comparative threshold cycle (Ct) method and normalized to GAPDH expression. Transcript levels of cell populations have been represented on the y-axis relative to those of Stro-1  cells set as 1. Results expressed as mean ¡À SD and n = 4¨C6 in the various cell populations. ***p 7 ~/ U! T+ m+ W" x  N" ~

Differentiation of Fetal Femur¨CDerived Populations6 Z8 L0 K$ w2 d+ s) X

The multipotentiality of the fetal femur¨Cderived populations was compared with adult-derived bone marrow stromal populations (Fig. 3). Fetal cells grown in adipogenic conditions for 21 days displayed extensive adipogenesis as demonstrated by Oil Red O staining for lipid vacuoles (Fig. 3A) and expression of the FABP-3 monologue aP2 (Fig. 3D, 3E). Cultures from fetal bones displayed enhanced adipogenesis in comparison with adult-derived marrow stromal populations (Fig. 3A compared with 3C). Culture of the adult-derived STRO-1¨Cisolated populations showed reduced lipid accumulation in adipogenic conditions in comparison with fetal cells (Fig. 3F). Fetal clones derived from single cells after fluorescence-activated cell sorting (FACS) were cultured. A total of 14 clones (from individual cells in individual wells) were confirmed by microscopy. Single cell¨Cderived clones with the greatest growth rate were used for multipotential experiments. Clonal studies using these FACS-sorted individual cells, expanded in basal media and subsequently differentiated in adipogenic media, confirmed the ability to generate adipocyte populations from individual fetal femur¨Cderived populations.) H+ `' [- R5 X$ ]9 w; [

Figure 3. Multipotential differentiation of cell populations. (A): Monolayer cultures grown in described adipogenic conditions for 21 days, fixed and stained for lipid presence with Oil Red O solution for 15 minutes, of whole fetal populations. (B): Oil Red O staining of 21 day fetal clonal populations in adipogenic conditions. (C): Adult marrow-derived cells cultured in adipogenic conditions for 24 days and stained for Oil Red O. (D): Cytoplasmic presence of aP2 (fatty acid binding protein-3) confirmed by immunocytochemistry in day 21 adipogenic fetal cell cultures, and (E) within clonal fetal populations in adipogenic conditions. (F): Oil Red O staining of adult-derived STRO-1 immunos-elected cells in adipogenic culture conditions for 22 days. (G): Alcian Blue & Sirius red (A/S) staining of sectioned pellets cultured in chondrogenic conditions showing the deposited matrix surrounding the fetal cells and the cells within lacunae appearance sectioned pellets for day 21 fetal and day 21 fetal clone (H) (Insets showing whole pellet). (I): Adult marrow-derived whole pellet culture, fixed on day 28 and stained with A/S (whole pellet shown in inset). (J¨CL): Type II collagen and (M¨CO) (SOX9) immunocytochemistry of day 21 fetal pellet, day 21 clonal fetal pellet, and day 28 adult-derived pellet cultures respectively, illustrating cartilaginous matrix deposition and formation. (P): Fetal cells in osteogenic monolayer conditions for 7 days, stained for alkaline phosphatase. (Q): Oil Red O staining of lipid accumulation within day 7 osteogenic cultures grown for a further 14 days in adipogenic differentiation conditions with day 21 control culture (inset). (R): Alkaline phosphatase staining of fetal-derived clonal culture after 7 days in osteogenic conditions. (S): Fetal cells in osteogenic monolayer conditions for 7 days, stained for type I collagen via immunocytochemistry. (T): Fetal clonal cultures in osteogenic cultures after 7 days displaying presence of type I collagen via immunocytochemistry. Bars = 200 µm (A¨CC, F, P¨CT), 50µm (D, E), 100 µm (G¨CO).
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After culture under chondrogenic conditions, using the pellet culture assay model, fetal femur¨Cderived and clonal fetal populations as well as adult mesenchymal cells gave rise to chondrogenic tissue, as evidenced by extensive deposition of a matrix containing proteoglycans (positive Alcian Blue staining; Fig. 3G¨C3I) and type II collagen (Fig. 3J¨C3L). Presence of SOX9 (Fig. 3M¨C3O) further confirmed the chondrogenic phenotype. Well defined chondrocyte morphology within the pellet culture was reached after 21 days in the fetal pellet cultures, demonstrated by more intense proteoglycan and type II collagen staining (Fig. 3G, 3J, 3M), compared with 28 days for adult-derived pellets (Fig. 3I, 3L, 3O).9 c0 J( v: ^# l- ^

Under osteogenic conditions, fetal femur¨Cderived cells expressed ALP (Fig. 3P) and type I collagen (Fig. 3S) within 7 days of culture, similar to the adult-derived mesenchymal stromal cell populations. Colonies derived from single cells confirmed the ability to differentiate fetal-derived populations along the osteogenic lineage (Fig. 3R, 3T). Cell growth was observed to be enhanced in fetal populations compared with adult bone marrow stromal cultures, at equivalent time points (data not shown) with extensive matrix deposition and clustering.
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Plasticity of Fetal Femur¨CDerived Populations
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The plasticity of fetal femur¨Cderived populations from the osteogenic to adipogenic phenotype was examined in extended culture. First-passage fetal cells were cultured in osteogenic conditions for 7 days with extensive ALP activity observed (Fig. 3P). Cultures were then transferred to adipogenic cultures conditions for an additional 14 days (Fig. 3Q). Oil Red O stain demonstrated extensive lipid accumulation within the culture. No lipid accumulation was observed within day 21 control cultures maintained in basal conditions (Fig. 3Q, inset).

Molecular Characterization; n6 e9 |: K7 p: Q# }0 m" b
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Expression of lineage-specific genes was analyzed to examine differentiation of three individual sets of fetal femur¨Cderived cell populations along the respective lineages (Fig. 4A). RNA from freshly isolated human articular chondrocytes served as a control for the gene expression analysis. GAPDH served as the housekeeping gene. Type I collagen, an early marker of osteoblasts, as well as of fibroblasts, was expressed by the cells under basal and osteogenic conditions. However, its expression under adipogenic conditions was probably indicative of a dedifferentiation of the fetal femur¨Cderived cells into fibroblasts. ALP, an early marker of osteogenic differentiation, was expressed prominently under basal and osteogenic conditions. It was possible to detect low-level ALP expression under adipogenic conditions as a consequence of initiation of adipogenic treatment after the cells had reached confluence in basal culture conditions. OP, a relatively late marker of osteogenic differentiation, was expressed only under basal and osteogenic conditions, whereas low-level expression of OC, a marker of terminally differentiated osteoblasts, could be detected only under osteogenic conditions. PPAR-, a distinguishing marker for adipocytes, was detected only under adipogenic culture conditions.
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Figure 4. Molecular characterization of differentiated fetal populations. (A): Analysis of lineage-specific gene expression in three sets of fetal femur¨Cderived cells cultured under basal, osteogenic, and adipogenic conditions. (B): Analysis of RAR and RXR gene expression in three sets of fetal femur¨Cderived cells cultured under basal, osteogenic, and adipogenic conditions. Freshly isolated human articular chondrocytes served as a positive control for the chondrogenic phenotype and expressed all isoforms of RAR and the  and ß isoforms of RXR. GAPDH served as the housekeeping gene. Type I collagen was detected not only under basal and osteogenic conditions but under adipogenic conditions; its expression under adipogenic conditions indicates a dedifferentiation of the fetal cells as fibroblasts. Although ALP was expressed predominantly under basal and osteogenic conditions, it is possible to detect low level ALP expression under adipogenic conditions. OP was expressed only under basal and osteogenic conditions, whereas low-level OC expression was detected only in osteogenic conditions. PPAR- was detected only under adipogenic conditions. Although RAR-, -ß-1, -ß-2, and - were expressed by the fetal cells under basal and osteogenic conditions, expression of these receptors was downregulated under adipogenic culture conditions. Expression of RXR- and -ß isoforms was detected in the fetal cells under basal, osteogenic, and adipogenic conditions. RXR- expression could not be detected in fetal cells or the human primary articular chondrocytes (data not shown). Abbreviations: ADI, adipogenic; ALP, alkaline phosphatase; BAS, basal; GAPDH, glyseraldehyde-3-phosphate dehydrogenase; OC, osteocalcin; OP, osteopontin; OST, osteogenic; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; RXR, 9-cis retinoic acid receptor.
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Expression of RARs and RXRs in Fetal Femur¨CDerived Cells Cultured Under Basal, Osteogenic, and Adipogenic Conditions
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The potential application of fetal-derived populations as a model system for skeletogenesis was examined by analyzing expression of retinoid receptors in three sets of fetal femur¨Cderived cells cultured under basal, osteogenic, and adipogenic conditions. RNA extracted from freshly isolated human articular chondrocytes was used as a positive control for retinoid receptor expression. We analyzed the expression patterns of , ß, and  isoforms of the RARs and RXRs in differentiating populations of cartilage-derived fetal cells (Fig. 4B). Freshly isolated human articular chondrocytes were found to express all isoforms of RAR and the  and ß isoforms of RXR. Although RAR-, -ß1, -ß2, and - were expressed by the fetal cells under basal and osteogenic conditions, adipogenic culture conditions were found to downregulate the expression of these receptors in adipocytes. Unlike the differential expression pattern observed for the RAR, expression of RXR- and -ß isoforms was detected in the fetal cells under all three culture conditions. RXR- expression was not detected in fetal cells or the human primary articular chondrocytes, because its expression is normally only observed in fetal cells undergoing myogenic condensations  (data not shown).5 |8 n$ v- R: a* d0 d
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Growth and Viability of Fetal Femur¨CDerived Cells on 3D Scaffolds In Vitro and In Vivo+ G: ~" J+ F% I- b9 s0 V
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The potential application of fetal femur¨Cderived populations for tissue engineering of bone was examined using culture and characterization on a variety of typical substrates, including calcium phosphate, PGA fleece, and PLA/HA scaffolds (Fig. 5). Fetal femur¨Cderived populations grew extensively within and on the calcium phosphate scaffolds as observed by ALP expression (Fig. 5A). Cell viability within the scaffolds was confirmed using CTG/EH-1 with confocal microscopy (Fig. 5B) as well as Toluidine blue, which demonstrated cell growth and deposition around the calcium phosphate scaffolds (Fig. 5C). Similarly, matrix deposition as evidenced by Sirius red and Alcian Blue staining was observed after culture of fetal-derived mesenchymal cells on PGA fleece substrates (Fig. 5D) and PLA/HA scaffolds (Fig. 5E) in in vitro cultures. Presence of the osteogenic and fibroblastic markers¡ªtype I collagen and vimentin, respectively¡ªwas confirmed via immunocytochemistry (Fig. 5F, 5G).

Figure 5. In vitro and in vivo demonstration of human fetal cell growth on three-dimensional scaffolds. (A): Fetal cells (day 7) seeded onto calcium phosphate scaffold growing within the macropores in osteogenic culture conditions, stained for alkaline phosphatase activity. (B): Cell viability demonstrated by live (green) and necrotic (red) cells labeled with Cell Tracker Green 5-chloromethyl-fluorescein diacetate and ethidium homodimer-1, respectively, captured by fluorescence confocal microscopy. (C): Fetal cells on calcium phosphate block after 21 days in basal conditions stained with Toluidine blue. (D): Fetal cells on PGA fleece after 7 days in osteogenic conditions stained with Alcian Blue and Sirius red. Fleece fibers (arrows) within matrix deposited by human fetal cells. (E): Alcian Blue/Sirius red staining of matrix deposition by fetal cells on PLA/HA composite scaffold after 28 days in vitro culture. (F): Immunocytochemistry of fetal cells after 28 days in vitro, demonstrating presence of type I collagen. (G): Immunocytochemistry of fetal cells after 28 day in vitro, demonstrating presence of vimentin (arrows). (H): Mouse-only tissue showing negative staining for anti-human vimentin. (I): Fetal cell growth on PLA/HA scaffold at day 28 in vivo, stained for presence of type I collagen, collagenous matrix stained for Sirius Red (J) with organized fibers shown by polarized light microscopy (K). (L): Anti-human vimentin staining (arrows) of human fetal¨Cderived fibroblasts within deposited matrix in in vivo culture on PLA/HA blocks. Bars = 500 µm (A), 100 µm (B¨CL). Abbreviations: PGA, polyglycolic acid; PLA/HA, poly-lactic acid/hydroxyapatite.2 ?, J8 [0 @4 D2 M
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After demonstration of the multipotential capability of fetal femur¨Cderived populations and the ability of these cells to colonize a variety of scaffolds, the potential of fetal-derived cells to differentiate and form new osteoid in vivo on 3D scaffold templates was examined. Primary fetal-derived cells were impregnated onto PLA/HA porous scaffolds and cell/scaffold constructs placed subcutaneously into athymic mice. Tissue from the mice was used as a negative control for the anti-human vimentin stain, used to determine human-derived cells, and no staining was observed in mice tissue (Fig. 5H). After 4 weeks, subcutaneous implants were removed from the mice and analyzed for new bone formation (Fig. 5I¨C5K). Cell differentiation and bone matrix synthesis on/within PLA/HA scaffolds with morphological evidence of new bone matrix synthesis was detected by type I collagen and Sirius red staining on all scaffolds pretreated with osteogenic induction medium (Fig. 5I and 5J, respectively). Evidence of organized new woven bone was confirmed by birefringence of collagen using polarized microscopy (Fig. 5K). Anti-human vimentin immunocytochemistry was used to confirm cell origin of the cells on the scaffolds (Fig. 5L).* u6 z) i) h. l3 Z- C2 V& h6 m

DISCUSSION

This study has examined the multipotentiality and phenotypic properties of cell populations derived from human fetal femurs collected at 8¨C11 WPC and compared these with adult-derived mesenchymal stem cell populations, including those isolated using STRO-1 immunoselection. In addition, we have examined the potential implications for skeletal tissue engineering after in vitro and in vivo culture of fetal-derived mesenchymal populations on scaffolds and the use of these cells as a model to study osteogenesis.

Critical to these studies is an evaluation of the starting material. The human fetal femurs used in these studies contained predominantly epiphyseal chondrocytes and were surrounded by a perichondrium/periosteum, which consisted of an outer fibroblastic layer and an inner layer of noncommitted mesenchymal stem cells, the latter capable of differentiating either along the chondrogenic or the osteogenic lineage. In addition, relatively small numbers of differentiated osteoblasts were present along the central diaphysis, as indicated by the presence of the first bone collar. The cells that grew out from the minced cut explants during culture could thus theoretically be derived from the cut edges of the epiphysis (early chondrocytes) or from the perichondrium (fibroblastic or mesenchymal stem cells). At present, it is not known whether any cell type preferentially grew out of the explants. Because epiphyseal chondrocytes were by far the most frequent cell type present in the fetal femurs and because most outgrowth was observed from the cut edges, it is probable that the starting cells were early chondroprogenitor and mesenchymal progenitor populations. Nevertheless, we have precluded labeling our populations fetal-derived mesenchymal populations, because it may well be that within the explant-derived whole populations, individual progenitors for the adipogenic, chondrogenic, and osteogenic lineages have been present.
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In the current study, culture of fetal femur¨Cderived populations in the presence of dexamethasone, IBMX, insulin, and indomethacin favored adipogenesis, whereas culture in presence of TGF-ß-3 favored chondrogenesis. Osteogenic differentiation, evidenced by enhanced ALP expression and matrix production, was observed in cells cultured in serum with dexamethasone and ascorbic acid, as reported by many other groups  showed the ability of osteogenic cells from clonal cell lines to form adipocytes in the presence of dexamethasone and IBMX.

Given the ability to modulate the phenotype of these cells along the osteogenic, chondrogenic and adipogenic lineage, it would appear the cells were still at an early stage in their differentiation pathway. In support of this, molecular analysis indicated that the fetal femur¨Cderived cells displayed an undifferentiated skeletal phenotype rather than cartilaginous phenotype with ALP, type I collagen, and OP expression in basal conditions, whereas culture in osteogenic conditions resulted in expression of OC. The undifferentiated phenotype is suggested by the expression of type I collagen observed in monolayer culture . There are at least two possible explanations for the observed multipotentiality of the fetal cartilage¨Cderived cells: The cells either dedifferentiated in culture followed by redifferentiation along mesenchymal lineages or the fetal cells were predominantly early chondroprogenitor or skeletal progenitor cells able to differentiate as a consequence of external cues and/or factors, as suggested by the molecular phenotyping./ N3 @% e* q# p& b- ?* A) |
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The donor age at which to select mesenchymal stem cell populations remains unclear. The enhanced proliferation and differentiation observed in these studies and also reported by Montjovent and coworkers  found that the number and proliferative capacity of osteogenic stem cells was maintained during aging and in patients with osteoporosis. The enhanced proliferation observed and earlier timeframe of differentiation for adipogenesis and chondrogenesis in these studies could have important implications for tissue regeneration strategies.$ ]' [! x9 H! F9 G1 C3 U; f7 V

However, extrapolation of in vitro findings on cell differentiation and multipotentiality must always be tempered with a need to demonstrate in vivo functionality and this is an important caveat in these studies. Therefore, a key aim of the work was the evaluation of fetal-derived cells as a model for tissue regeneration strategies. A variety of materials have been used for bone regeneration together with mesenchymal progenitors including ceramics or materials based on HA, ceramic forms of ß-tricalcium phosphate and composites of both HA and ß-tri-calcium phosphate . The current studies show the efficacy of these human fetal cell populations, both in vitro and in vivo, for the bone formation when seeded onto PLA/HA.' k. g, b* _( m1 W( E
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In addition, the use of a fetal cell source as a robust and facile model system may give insight not only into regeneration strategies for injury and disease but also into developmental mechanisms for bone formation. Vitamin A and related molecules, the retinoids, have profound effects on cellular differentiation, growth, and modulation of a number of cell lineages, including those of mesenchymal and chondrogenic origin. Vitamin A deficiency and excess have opposing effects on bone metabolism. In hypovitaminosis A, bone thickness is increased in a number of sites whereas hypervitaminosis A results in increased bone resorption . Similarly, in the current study, RAR-, -ß-1, -ß-2, and - were expressed by the fetal cells under basal and osteogenic conditions and no RXR- expression could be detected. In addition, in keeping with a key role in chondrogenesis, RAR-, -ß-1, -ß-2, and - were downregulated in fetal cartilage¨Cderived populations after culture in adipogenic culture conditions. In contrast, expression of RXR- and -ß isoforms was detected in the fetal cells in basal, osteogenic, and adipogenic conditions. These results indicate the potential of these cells as a model system to further delineate human skeletal differentiation and offer the potential to use these cells, given their clinical relevance, as a pharmaceutical screening paradigm.
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With an increasing aging population, developments in our understanding of mesenchymal stem cell and osteoprogenitor biology are paramount. One unachieved goal remains the ability to provide sufficient stem/progenitor cell populations for tissue regeneration. The ability to select, expand, and differentiate these fetal populations suggests the potential future use for orthopedic applications, although issues of immunorejection, ethical implications, and cell availability await resolution. Clearly, harnessing the homing potentials of such cells and channeling their development into the required human tissues for regeneration remain a significant challenge. Although development pathways are clearly delineated from in vitro observations, their relevance in vivo requires further investigation.

The current studies indicate that these human fetal mesenchymal populations will provide a useful model for the study of osteogenesis and mesenchymal cell differentiation as well as a robust model to examine regenerative/tissue engineering strategies for human skeletal repair. Given the complexity, technical challenges, and ethical issues surrounding current embryonic and embryonic germ cell research, human fetal cell populations may provide a unique half-way model and system to address stem cell differentiation and provide unique approaches to understanding musculoskeletal development in comparison with adult stem cell differentiation. In summary, the present studies indicate that fetal-derived mesenchymal populations warrant further examination for fundamental research and for determination of their immunoprivilege properties for clinical use which, ultimately, may improve the quality of life for many as a result of derived strategies to augment skeletal regeneration.
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ACKNOWLEDGMENTS

The polyclonal antibody to type I collagen was a generous gift from Dr. Larry Fisher (NIH). We thank the orthopedics surgeons at the Southampton General Hospital for their aid in facilitating the bone marrow samples and Dr. Rob Powell (Allergy and Inflammation Research, Southampton) for help with real-time primer design and set-up. We also thank Kevin Shakesheff and Steven Howdle (University of Nottingham, U.K.) for the PLA/HA scaffolds and the Biotechnology and Biological Sciences Research Council (BBSRC) for Ph.D. studentship funding (to S.-H.M.-S.) and research support (to R.S.T.). N.A.H. is a U.K. Department of Health Clinician Scientist. This work was supported by grants from the BBSRC and the Engineering and Physical Sciences Research Council.
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DISCLOSURES

The authors indicate no potential conflicts of interest.7 T/ K+ T. }: D5 A
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