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Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients
Cytotherapy (2009) Vol. 11, No. 5, 584–594
Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients Patrick Coipeau1,2, Philippe Rosset1,2, Alain Langonné2,4, Julien Gaillard2,4, Bruno Delorme2, Angélique Rico2,4, Jorge Domenech2,3, Pierre Charbord2 and Luc Sensebé2,4 1Department of Orthopedic Surgery, University Hospital, Tours, France, 2Laboratory INSERM ESPRI-EA3855, Université François-Rabelais,
Faculté de Médecine, Tours, France, 3Laboratory of Hematology, University Hospital, Tours, France, and 4EFS Centre-Atlantique, Tours, France Background aims
whatever the starting material. However, the differentiation poten-
Advances in bone tissue engineering with mesenchymal stromal cells
tial of MSC obtained by bone washing was impaired compared with
(MSC) as an alternative to conventional orthopedic procedures has
aspiration; culture-amplified cells showed few Oil Red O-positive
opened new horizons for the treatment of large bone defects. Bone
adipocytes and few mineralized areas and formed inconsistent Alcian
marrow (BM) and trabecular bone are both sources of MSC. Regard-
blue-positive high-density micropellets after growth under adipo-
ing clinical use, we tested the potency of MSC from different sources.
genic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed
Methods
better differentiation potential.
We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and tro-
Conclusions
chanter, as well as BM aspirates of the iliac crest and trochanter. The
Trabecular bone MSC from elderly patients is not good starting
starting material was evaluated by bistologic analysis and assessment
material for use in cell therapy for bone repair and regeneration,
of colony-forming unit–fibroblasts (CFU-F). The MSC populations
unless cultured in the presence of FGF-2.
were compared for proliferation and differentiation potential, at RNA and morphologic levels.
Keywords Colony-forming unit–fibroblasts, differentiation potential, fibroblast
Results
growth factor-2, human mesenchymal stromal cells, trabecular bone.
MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar
Introduction The treatment of extensive bone defects (e.g. arthroplasty loosening, trauma and bone tumors) remains a challenge in skeletal surgery [1,2]. Trabecular bone fragments, either autologous (iliac crest and femoral head) or allogeneic (femoral head and long bones), are used for the treatment of large bone defects [3,4]. However, conventional treatments for loss of bone and non-union can fail, and cell therapy is an alternative. The transplantation of heterogeneous
marrow cell populations, usually harvested from the iliac crest [5], is routinely used in human clinical applications for bone repair [6]. In this context, the use of mesenchymal stromal cells (MSC), or multipotent MSC, seems to be promising [1,7–10]. MSC are present in virtually all adult tissues, including trabecular bone, but are usually cultivated from bone marrow (BM) [11]. The anatomical sites for cell harvest, i.e. the femoral head [12], iliac crest [13] and vertebral body [14], and
*These authors contributed equally. Correspondence to: Dr Luc Sensebé, Directeur Médical et Scientifique, Etablissement Français du Sang (EFS) Centre-Atlantique 2 and Inserm ESPRI-EA3855, 2 Boulevard Tonnellé, BP52009, 37020Tours Cedex 1, France. E-mail: [email protected] © 2009 ISCT
DOI: 10.1080/14653240903079385
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the age of the donor [15,16] are major factors influenc-
a heterogeneous population containing clonogenic cells,
ing both the incidence of colony-forming unit–fibroblasts
some multipotent, and mature stromal cells.
(CFU-F) and the proliferation rate of culture-amplified removed during surgical procedures can be an alternative
Methods Isolation and culture of human MSC
source of MSC for tissue repair and engineering. Before
We obtained trabecular bone and BM from 17 patients
using such cells in clinical trials, the potential of trabecular
with hip osteoarthritis who were undergoing total hip
bone MSC for bone repair must be tested. We evaluated
arthroplasty under general anesthesia. The study fol-
MSC obtained from the femoral head, trochanter region
lowed the guidelines of Tours University Hospital
cells. In terms of ease of procurement, trabecular bone
and iliac crest after bone washing or marrow aspiration in elderly patients in terms of proliferation and differentiation potential. Because fibroblast growth factor-2 (FGF-2) has shown good effects in population doubling and maintenance of differentiation potential [17–19], and is used in France for clinical-grade MSC culture, we tested the possible use of FGF-2 for trabecular bone MSC culture.
(Tours, France) and was approved by the local ethics committee. Patients gave their informed consent to be in the study. The characteristics of the patients are shown in Table I. Using two methods, extensive bone washing (W) and marrow aspiration (A), we obtained cells for MSC culture from three sites: the femoral head, trochanter and iliac crest. The femoral head and trochanter region are filled with yellow marrow, in
Recently, however, from in vivo experiments, it has been
contrast to the iliac crest, which is filled with red mar-
reported that FGF-2 can impair the stemness potential
row. Trabecular bone fragments were obtained from
of MSC [20].
the trochanter and femoral head. From the femoral
According to the position statement of the International
head, a cube (mean volume 6.2 cm3) of trabecular bone
Society for Cellular Therapy [21], we use the term MSC
(n 16) was obtained with the use of a surgical power
for the cultivated cells used in the present study. MSC
saw, and trabecular bone fragments were removed from
refers to cultivated BM mesenchymal stromal cells that are
the trochanteric region (metaphysis) by curettage (n 8).
Table I. Characteristics of patients and bone samples. Donor
Age (years)
Sex
Osteoarthritis etiology
Femoral head
Trochanter
Iliac crest
1
68
F
PA
W
W
ND
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
65 55 74 76 74 64 61 73 57 53 53 78 48 61 65 74
M F F M M F F F F F F F F M M M
AN CD PA PA PA PA CD PA PA PA CD PA PA PA PA PA
W W W W W W W W W W W W W ND W W
W W W W W W W A A A A A A A A A
ND ND ND ND ND ND ND ND ND ND ND ND ND A A A
F, female; M, male. Etiology of osteoarthritis: PA, primary arthritis; CD, congenital dysplasia; AN, avascular necrosis. ND, not done.
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The trabecular bone samples were fragmented, and each piece was meticulously washed by multiple injections of culture medium with a 19-gauge needle; erythrocytes were lysed (diluted 1:10 v/v with acetic acid 10%) then nucleated cells counted. Trochanter and iliac crest samples underwent BM aspiration. During surgery, using a Jamshidi biopsy needle, 10 mL BM were harvested with successive 2-mL aspirations from the trochanteric region (n 9) and iliac crest (n 3) (Table I). In order to avoid dilution by peripheral blood, after each aspiration the needle was repositioned. The BM samples were washed twice in complete culture medium, and nucleated cells were counted with a Cobas Argos automated hematology analyzer (Roche ABX, Montpellier, France). Referring to the method used (A or W), samples were named iliac crest A, trochanter A, trochanter W and femoral W. Nucleated cells were seeded at 5 104/cm2 in 75-cm2 tissue culture flasks (Becton Dickinson, Franklin Lake, NJ, USA) and cultured for 21 days (passage 0; P0) in complete culture medium: alpha-minimal essential medium (MEM; Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) screened fetal calf serum (FCS; Hyclone, Logan, UT, USA), 20 mm l-glutamine (Gibco Invitrogen), 100 U/mL penicillin–streptomycin (Gibco Invitrogen) and 0.25 mg/L amphotericin B (Fungizone, Bristol-Myers Squibb, New York, NY, USA). The medium was changed twice weekly. At day 21, cells were harvested from confluent or subconfluent layers with the use of trypsin (0.05% v/v solution containing 1 mm EDTA; Gibco Invitrogen). After washing twice in phosphate-buffered saline (PBS), viable cells were counted by trypan blue staining and replated at 103/cm2 (passage 1; P1). At day 42, cells were harvested and replated at the same density (passage 2; P2) and cultured until day 63. To evaluate the effects of FGF-2, cells from donors 15, 16 and 17 were cultured in complete culture medium with and without FGF-2 (1 ng/mL; R&D Systems, Minneapolis, MN, USA).
CFU-F assay A CFU-F assay was performed at day 0, P0 (day 21) and P1 (day 42). At day 0, nucleated cells were seeded in 25-cm2 flasks at two densities (2 103 and 4 103/cm2) for cells obtained by washing and three densities (2 103, 4 103 and 40 103/cm2) for cells from aspirates. At P0 and P1, 200 and 500 cells, respectively, were cultured in 25-cm2 flasks. Cultures were incubated for 10 days, then the
medium removed and, after two washes in PBS, cells fixed in methanol (5 min) and stained with Giemsa (10 min). Colonies of 50 or more cells were counted.
Cell phenotype analysis of cultured MSC Flow cytometry analysis was performed at P0, P1 and P2. Mouse anti-human monoclonal antibodies (MAb) were conjugated for CD13–phycoerythrin (PE), CD14–PE, CD29–PE, CD34–PE, CD49a–PE, CD73–PE, CD90–PE, CD105–PE, CD106–PE, CD164–PE, CD45–fluorescein isothiocyanate (FITC) (BD Biosciences, San Jose, CA, USA), CD146–PE (Biocytex, Marseille, France) and unconjugated for Stro-1 (R&D Systems). The following control MAb were included: mouse IgG1–PE, mouse IgG1–FITC and mouse IgM–PE for Stro-1 (BD Biosciences). Cells were incubated with MAb at 4°C for 30 min. Cells incubated with unconjugated Stro-1 were washed and incubated with PE-conjugated goat anti-mouse IgM IgG IgA (BD Biosciences). For each cell-surface marker, at least 104 events were analyzed with a BD FACSCalibur flow cytometer (BD Biosciences).
In vitro differentiation Osteogenic and adipogenic differentiation were evaluated at P0, P1 and P2 for MSC from six patients (patients 12–17) and chondrogenic differentiation at P1 for MSC from three patients (patients 15–17). To test FGF-2 for differentiation potential, we used MSC cultivated with or without FGF-2 in three patients (patients 15–17).
Osteogenic differentiation MSC were plated at 7 103 cells/cm2 in six-well culture plates (Corning, Corning, NY, USA) and cultured for 3 weeks in low-glucose Dulbecco’s modified essential medium (DMEM; Gibco Invitrogen) supplemented with 10% FCS, 0.15 mm ascorbate-2-phosphate, 2 mm -glycerophosphate and 10−8 m dexamethasone (Sigma-Aldrich, St Louis, MO, USA). Mineralized areas were revealed by Von Kossa staining. Cells were fixed with 4% paraformaldehyde (v/v) and stained for 30 min in 5% (v/v) silver nitrate solution (Sigma). After ultraviolet exposure for 45 min, cells were stained for 2 min in 5% (v/v) sodium thiosulfate solution (Sigma).
Adipogenic differentiation MSC were plated at 14103 cells/cm2 in Lab-Tek chamber slides (Nunc, Rochester, NY, USA) and cultured for 2 weeks
Impaired osteogenic potential of MSC
in low-glucose DMEM supplemented with 20% FCS, 0.5 mm isobutyl-methyl-xanthine IBMX, 60 μm indomethacin and 10−6 m dexamethasone (all Sigma). Lipid vacuoles were revealed by Oil Red O staining. Cells were fixed with 4% paraformaldehyde and stained for 30 min with 1Oil Red O (Sigma). Nuclear staining involved use of Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; AbCys, Paris, France). The percentage of differentiated cells was assessed according to the following scale: more than 75%, 50–75%, 25–50% and less than 25%.
Chondrogenic differentiation MSC differentiation was induced in aggregate cultures for 3 weeks. MSC, 2.5105, were spun down in 15-mL polypropylene conical tubes at 350 g for 5 min. The FCS-containing medium was then replaced with 1 mL defined medium consisting of high-glucose DMEM supplemented with 107 m dexamethasone, 103 m sodium pyruvate, 1.7104 ml-ascorbic acid-2-phosphate, 3.5104 m proline (all Sigma) and 1 insulin–transferring–selenium (ITS; Cambrex, East Rutherford, NJ, USA). After a 24-h incubation, the medium was changed and replaced with fresh medium 10 ng/mL transforming growth factor-1 (TGF-1; R&D Systems). The medium was changed every 3–4 days. The presence of glycosaminoglycans in cell pellets was revealed by Alcian blue staining and toluidine blue. At day 21, pellets were fixed for 48 h in 4% paraformaldehyde and embedded
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in paraffin. Sections 4 μm thick were rehydrated in 70%, 90% and 100% xylene and ethanol. Slides were stained for 10 min in a 1:1 mixture of 3% Alcian blue 8GX (Sigma) (v/v) solution in distilled water and 3% (v/v) methanol.
Reverse transcription–polymerase chain reaction analysis Reverse transcription–polymerase chain reaction (RT-PCR) involved use of SuperScript one-step RT-PCR with platinum Taq (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression of the following genes was studied: alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) for the osteogenic pathway, and peroxisome proliferator-activated receptor- (PPAR-), lipoprotein lipase (LPL) and fatty acid binding protein 4 (FABP4) for the adipogenic pathway. As a control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used. The sequences of the oligonucleotides are shown in Table II. All total cellular RNA samples were extracted with the use of an RNA easy kit (Qiagen SA, Courtaboeuf, France) from cells cultured in differentiation medium at the end of P1 (day 42); 50 ng total RNA were used for specific genes and 5 ng for the GAPDH gene. RT-PCR products were analyzed on agarose gel electrophoresis.
Histology Bone samples were obtained from two donors (16 and 17) by BM biopsy of the iliac crest and trochanter region with
Table II. Sequences of the primers used in RT PCR study. Target mRNA Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Sequence (forward) 5 -GGTGAAGGTCGGTGTGAACG-3
(reverse) 5 -CATCATACTTGGCAGGTTTCTCC-3 (forward) 5 -CTGGACCTCGTTGACACCTG-3 (reverse) 5 -GAC ATTCTCTCGTTC ACCGC- Osteopontin (forward) 5 -CAGGCTGATTCTGGAAGTTCTGAG-3 (reverse) 5 -GGTGATGTCCTCGTCTGTAGCATC-3 Bone sialoprotein (forward) 5 -TTTCCAGTTCAGGGCAGTAGTGAC (reverse) 5 -CTTCCCCTTCTTCTCCATTGTCTC Peroxisome proliferator-activated (forward) 5 -GGAGAAGCTGTTGGCGGAGA-3 receptor- (PPAR-) (reverse) 5 -TCAAGGAGGCCAGCATTGTG-3 Fatty acid binding protein 4 (forward) 5 -GTACCTGGAAACTTGTCTCC-3 (FABP4) (reverse) 5 -GTTCAATGCGAACTTCAGTCC-3 Lipoprotein lipase (forward) 5 -AAAGCCCTGCTCGTGCTGAC-3 (reverse) 5 -TAAACCGGGCCACATCCTGT-3
Alkaline phosphatase
Product length Gene accession number 761pb
NM_002046.3
545pb
NM_000478
509pb
NM_001040058
436pb
NM 004967 3
430pb
NM_015869.4
418pb
NM_001442.1
405pb
NM_000237.2
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a Jamshidi biopsy needle and from the femoral head cube after fragmentation. Tissue samples were fixed in Bouin solution (VWR Prolabo, Fontenay sous bois, France), decalcified for 30 min in rapid bone decalcification media (Eurobio, Courtaboeuf, France) and embedded in paraffin. Sections 4 μm thick were rehydrated in xylene and 50%, 80% and 100% ethanol. Slides were then stained in Mayer’s Hemalun for 5 min (VWR Merck Eurolab, Leuven, Belgium) and Eosin for 3 min (Merck Eurolab). The presence and ratio of compact bone, trabeculae, adipocytes and myeloid elements were assessed.
Statistical analysis Statistical analyzes involved the Student’s t-test with StatView software v.5 (SAS Institute, Cary, NC, USA). Values are means standard error of the mean (SEM). A P-value of 0.05 was considered statistically significant.
Results Cell composition of starting materials In our 17 elderly patients (mean age 64 years, range 48–78 years), histology showed femoral head tissue and trochanteric regions with no hematopoietic tissue but normal levels in the iliac crest (Figure 1A–C). The femoral head biopsy showed necrotic trabecular bone fragments, and trochanter tissue biopsy showed adipocytes and compact bone, with no hematopoiesis. Iliac crest biopsy showed hematopoiesis in cords located within bone trabeculae and some adipocytes within the cords.
CFU-F differences The frequency and size of CFU-F did not differ with age of donor (53–78 years) or starting material (iliac crest, BM or trabecular bone). CFU-F frequency was significantly lower with trochanter A than with trochanter W (0.04 0.02 versus 0.21 0.07/103 cells; P 0.027) (Figure 2A). In all MSC cultures, CFU-F frequency decreased from cell passage P0 to P2 (P1 versus P0, P 0.05; P2 versus P1, P 0.05; n 17; Figure 2B, C). Cell proliferation was identical with cell passage, whatever the starting material. Only trochanter MSC at P2 showed a significant difference in cell proliferation when comparing washing and aspiration (Figure 2D–F). Finally, whatever the starting material (femoral head A, n 16; trochanter W, n 8; trochanter A, n 9; iliac crest, W; n 3), the calculated mean of population doublings was 15 1.
Phenotypes of MSC As early as P0, cells were positive for CD13, CD29, CD49a, CD73, CD90, CD105, CD106, CD164 and CD146 (Figure 3). CD146 expression was low at the end of P0 (ratio of signal to noise, S:N, 8.14 1.44) and decreased continuously in subsequent passages. Stro-1 expression was low at P0 and P1 and non-existent by the end of P2, as reported previously [22]. In our culture conditions, hematopoietic cells disappeared rapidly: at P0, a few CD45-positive cells were sometimes detected (less than 1% in three experiments and less than 5% in two); at P1, all cultivated cells were negative for CD45, CD34 and CD14.
Figure 1. Histologic analysis of BM biopsies. Samples from BM biopsies (patients 1 and 17) were fixed, decalcified and embedded in paraffin, and then sections stained with hematoxylin & eosin (H&E). The figure shows representative images (patient 17). (A) Femoral head: avascular and necrotic trabecular bone fragments. (B) Trochanter: aplastic-like, with adipocytes, compact bone and lack of hematopoietic tissue. (C) Iliac crest: large proportion of hematopoietic tissue (original magnification 100).
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Figure 2. CFU-F and cell proliferation in culture. (A,B,C) Variation in CFU-F number with cell passage. P1 42 days; P2 63 days; W, cells obtained by washing; A, obtained by aspiration. *P 0.05. (D,E,F) Variation in proliferation with cell passage. Cell proliferation total number of cells at the end of passage/total number of cells seeded. *P 0.05.
Differentiation potential The osteogenic differentiation potential, assessed by evaluation of mineralization with von Kossa staining, and adipogenic differentiation potential, assessed by the presence of cells with Oil Red O-positive vesicles ( 5 μm), was evaluated at P0, P1 and P2 in MSC from patients 12–17. Because of the number of cells needed, the chondrogenic differentiation potential was evaluated at only P1 in three patients (patients 15–17). MSC from material obtained by A and W at P0–P2 (n 3, patients 15–17) underwent RT-PCR to test mRNA expression of genes specific for bone (ALP, OPN and BSP) and adipose (PPAR-, FABP4 and LPL). When cultivated in basic culture conditions, MSC from the femoral head and trochanter region (after washing) showed impaired osteogenic differentiation potential, with negative or very low von Kossa staining. Mineralized areas were present in only P1 and P2 MSC from the femoral head and trochanter region grown in the presence of FGF-2 (Figure 4A). At P2, no
mineralized areas were present (Figure 4A). At P0 and P1, iliac crest A and trochanter A MSC generated many mineralized areas; however, on cultivation without FGF-2, at P2, iliac crest A and trochanter A MSC inconsistently generated mineralized areas (n 2/3 without FGF-2 versus n 3/3 with FGF-2). In all MSC (A and W) from the three patients analyzed, RT-PCR analysis revealed the expression of bone-specific genes (ALP and OPN) in all differentiated MSC (Figure 4D). Over time, the adipogenic differentiation potential was rapidly impaired in MSC from material obtained by washing (n 6) (Table III). The adipogenic potential was present at P0 and preserved only in MSC cultured with FGF-2 (n 3; Figure 4B). For iliac crest A MSC, lipid vacuoles were observed in more than 25% of cells at P0 and adipogenic differentiation decreased with time, with lessthan 25% differentiation at P1 and no differentiation at P2. However, at P1, after culture in adipogenic differentiation medium, RT-PCR analysis showed the
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Figure 3. Cell phenotype. Data from one representative experiment: patient 16, femoral head W MSC at P1. MSC were cultured in normal medium supplemented with 10% FCS without FGF-2.
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expression of adipose-specific genes (PPAR-γ, FABP4 and LPL) in all MSC, whatever the starting material (Figure 4D). Chondrogenic differentiation, assessed by the appearance of Alcian blue-positive glycosaminoglycans in high-density pellets, was impaired in P1 MSC and was inconsistently
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preserved (n 1/3) even on culture with FGF-2, whatever the starting material (Figure 4C).
Discussion We cultured MSC from trabecular bone using a simple method of extensive washing followed by a classic
Figure 4. (Continued).
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culture procedure. The MSC showed proliferation potential, a proportion of clonogenic cells and a phenotypic profile similar to that of BM MSC. However, in our elderly donors, the trabecular bone MSC showed impaired potential for differentiation to osteogenic and chondrogenic pathways, as well as the adipogenic pathway, over time. The washing method we used to obtain MSC is simple, safe and not time-consuming. It enables a sufficient number of cells to be obtained with a good yield of CFU-F that are representative of the stem cell/progenitor population. Compared with other methods, bone washing prevents the use of an exogenous enzyme such as collagenase [11], which might be a safety concern. In contrast to culture of entire bone fragments [23], bone washing allows entire qualification (e.g. cell counting and flow cytometry) of the starting material. In terms of safety and regulatory issues, all these aspects have an advantage for clinical use. MSC from material obtained by washing, as well as those obtained by aspiration, expressed the classic mesenchymal markers CD90, CD73, CD105 and CD49a at high levels and CD146 and Stro-1 at low levels. With increasing cell passage, cell number and CFU-F incidence did not differ significantly, whatever the starting material. These results are in agreement with previous data for trabecular bone MSC [11]. In contrast with unmodified proliferation potential, CFU-F cloning efficiency and immunophenotype in MSC whatever the method, MSC from material obtained by washing showed a significant decrease in osteogenic and adipogenic differentiation potential. Beyond the scope of this study, in vivo analyzes of the osteogenic differentiation potential must be performed to confirm the results obtained in vitro. Our results are different to those of Tuli et al. [23]
and Sakaguchi et al. [11], who showed trabecular bone MSC correctly differentiated following the three canonical pathways even in elderly patients and after long-term culture. Although the phenotypes of MSC were identical in the studies, the impaired in vitro differentiation in our study might be linked to a difference in the procedures for cell collection: in our case extensive washing, compared with collagenase treatment [11] or culture explants [12,24] without washing steps. These previously reported procedures might release or allow the expression of more potent MSC and some mature osteoblastic progenitors. Moreover, as shown previously, the matrix elasticity can drive MSC into specific differentiation pathways [25]. With bone explant cultures, the presence of bone matrix with a specific stiffness might explain the better osteoblastic differentiation. In contrast to previous series, our series contained a predominance of female donors (11/17). Previously it has been shown that MSC cultivated from post-menopausal osteoporotic women have high phosphorylation of extracellular signalregulated kinase (ERK) levels [26], which plays an important role in the phosphorylation of RUNX2 [27]. We did not test the ERK-MAPK pathway in our patients, but the predominance of elderly female donors could play a role in our finding of impaired differentiation of trabecular bone MSC. Finally, we cannot exclude the possibility that the difference could be related to the arthritis in patients included in the study. The positive effect of FGF-2 on proliferative capacity was demonstrated at concentrations as low as 1 ng/mL [17]; in France, FGF-2 is used at this concentration for producing BM MSC in clinically approved protocols [28]. In our study, adding low-dose FGF-2 (1 ng/mL) to culture medium during the proliferation phase allowed better preservation of the differentiation potential of MSC.
Figure 4. In vitro differentiation of MSC. (A) Osteogenic differentiation: von Kossa staining was used to determine osteogenic differentiation in all MSC from six patients (patients 12–17). All data shown are from patient 16: femoral head W MSC (P1), trochanter W MSC (P1) and iliac crest A MSC (P2) (original magnification 50). (B) Adipogenic differentiation: Oil Red O staining was used to determine adipogenic differentiation in all MSC from six patients (patients 12–17). All data shown are from patient 17: femoral head W MSC (P0), trochanter W MSC (P0) and iliac crest A MSC (P0) cultivated without or with FGF-2 (1 ng/mL) (original magnification 400). (C) Chondrogenic differentiation: micropellets and Toluidine blue staining were used to determine chondrogenic differentiation in all MSC from three patients (patients 15–17). All data shown are from patient 17: femoral head W MSC (P1), trochanter W MSC (P1) and iliac crest A MSC (P1) (original magnification 40). (D) RT-PCR analysis: at P1, total mRNA was obtained from iliac crest A MSC, femoral head W MSC, trochanter W and A MSC after culture in either osteogenic or adipocytic differentiation media. Results are from a representative experiment (patient 17) of three (patients 15–17) with () or without () FGF-2 (1 ng/mL): ALP, OPN and BSP gene expressions were tested for osteogenic differentiation, and LPL, FABP4 and PPAR- gene expressions were tested for adipogenic differentiation. NC, negative control without mRNA.
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Table III. Adipocytic differentiation of MSC from different sites. Site
Femoral head
Trochanter
Iliac crest
W
W
A
Without FGF-2 With FGF-2
10% (3/3) 10– 25% (3/3)
10% (3/3) 10– 25% (3/3)
25– 50% (3/3) 25– 50% (3/3)
Without FGF-2 With FGF-2
10% (3/3) 10– 25% (2/3)
10% (3/3) 10– 25% (2/3)
25% (3/3) 25% (3/3)
Method P0
P1
Adipocytic differentiation was evaluated in MSC cultivated or not in the presence of FGF-2. The numbers represent the percentage of cells having lipid vacuoles revealed by Oil Red O staining. The starting cells were obtained by washing of femoral head and trochanter (W) and aspiration of BM of iliac crest (A).
The effect of FGF-2 on maintaining the osteoblastic differentiation potential of MSC has been reported elsewhere [18,19]. This cytokine might select for MSC with preserved osteogenic potential that are still present in these elderly patients but overwhelmed by senescent cells that have lost most of their differentiation capacity. In elderly patients, BM aspiration and trabecular bone washing enabled the generation of MSC. With both techniques, the rate of proliferation and CFU-F content were equivalent, but MSC cultivated from BM aspiration showed better in vitro differentiation potential. Although these results for MSC must be confirmed in vivo, this difference may have implications for clinical applicability.
Acknowledgements This research was supported by the integrated project Genostem of the European Commission FP6 research funding program and by grant number 2004-08 from the Etablissement Français du Sang. The authors thank Jean-Louis Brémond for histologic analysis of bone marrow biopsies and James E. Dennis for a critical review of the manuscript. Declaration of interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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14. Risbud MV, Shapiro IM, Guttapalli A, Seghatoleslami R, Denaro V, Vaccaro AR, et al. Osteogenic potential of adult human stem cells of the lumbar vertebral body and the iliac crest. Spine 2006;31:83–89. 15. Stenderup K, Justensen J, Clausen C, Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone 2003; 33:919–26. 16. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 2004;22:675–82. 17. Sensebe L, Li J, Lilly M, Crittenden C, Hervé P, Charbord P, et al. Non transformed colony-derived stromal cell lines from normal human marrows. I. Growth requirement and myelopoiesis supportive ability. Exp Hematol 1995;23:507–13. 18. Bianchi G, Banfi A, Mastrogiacomo M, Notaro R, Luzzatto L, Cancedda R, et al. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 2003;287:98–105. 19. Martin I, Muraglia A, Campanile G, Cancedda R, Quarto R. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology 1997;138:4456–62. 20. Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, et al. Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment. Cell 2007;131:324–36.
21. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marchi FC, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 2005;7:393–99. 22. Gronthos S, Zannettino AC, Hay SJ, Shi S, Graves SE, Kortesidis A, et al. Molecular and cellular characterization of highly purified stromal stem cells derived from human bone marrow. J Cell Science 2003;116:1827–35. 23. Tuli R, Tuli S, Nandi S, Wang ML, Alexander PG, Haleem-Smith H, et al. Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone. Stem Cells 2003;21:681–93. 24. Gundle R, Beresford JN. The isolation and culture of cells from explants of human trabecular bone. Calcif Tissue Int 1995;56:S8–10. 25. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–89. 26. Rodriguez JP, Rios S, Fernandez M, Santibanez JF. Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women. J Cell Biochem 2004;92:745–54. 27. Ge C, Xiao G, Jiang D, Franceschi RT. Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. J Cell Biology 2007;176:709–18. 28. Sensebé L. Clinical grade production of mesenchymal stem cells. Bio-Med Mater Eng 2008;18:S3–10.
Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients Patrick Coipeau1,2, Philippe Rosset1,2, Alain Langonné2,4, Julien Gaillard2,4, Bruno Delorme2, Angélique Rico2,4, Jorge Domenech2,3, Pierre Charbord2 and Luc Sensebé2,4 1Department of Orthopedic Surgery, University Hospital, Tours, France, 2Laboratory INSERM ESPRI-EA3855, Université François-Rabelais,
Faculté de Médecine, Tours, France, 3Laboratory of Hematology, University Hospital, Tours, France, and 4EFS Centre-Atlantique, Tours, France Background aims
whatever the starting material. However, the differentiation poten-
Advances in bone tissue engineering with mesenchymal stromal cells
tial of MSC obtained by bone washing was impaired compared with
(MSC) as an alternative to conventional orthopedic procedures has
aspiration; culture-amplified cells showed few Oil Red O-positive
opened new horizons for the treatment of large bone defects. Bone
adipocytes and few mineralized areas and formed inconsistent Alcian
marrow (BM) and trabecular bone are both sources of MSC. Regard-
blue-positive high-density micropellets after growth under adipo-
ing clinical use, we tested the potency of MSC from different sources.
genic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed
Methods
better differentiation potential.
We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and tro-
Conclusions
chanter, as well as BM aspirates of the iliac crest and trochanter. The
Trabecular bone MSC from elderly patients is not good starting
starting material was evaluated by bistologic analysis and assessment
material for use in cell therapy for bone repair and regeneration,
of colony-forming unit–fibroblasts (CFU-F). The MSC populations
unless cultured in the presence of FGF-2.
were compared for proliferation and differentiation potential, at RNA and morphologic levels.
Keywords Colony-forming unit–fibroblasts, differentiation potential, fibroblast
Results
growth factor-2, human mesenchymal stromal cells, trabecular bone.
MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar
Introduction The treatment of extensive bone defects (e.g. arthroplasty loosening, trauma and bone tumors) remains a challenge in skeletal surgery [1,2]. Trabecular bone fragments, either autologous (iliac crest and femoral head) or allogeneic (femoral head and long bones), are used for the treatment of large bone defects [3,4]. However, conventional treatments for loss of bone and non-union can fail, and cell therapy is an alternative. The transplantation of heterogeneous
marrow cell populations, usually harvested from the iliac crest [5], is routinely used in human clinical applications for bone repair [6]. In this context, the use of mesenchymal stromal cells (MSC), or multipotent MSC, seems to be promising [1,7–10]. MSC are present in virtually all adult tissues, including trabecular bone, but are usually cultivated from bone marrow (BM) [11]. The anatomical sites for cell harvest, i.e. the femoral head [12], iliac crest [13] and vertebral body [14], and
*These authors contributed equally. Correspondence to: Dr Luc Sensebé, Directeur Médical et Scientifique, Etablissement Français du Sang (EFS) Centre-Atlantique 2 and Inserm ESPRI-EA3855, 2 Boulevard Tonnellé, BP52009, 37020Tours Cedex 1, France. E-mail: [email protected] © 2009 ISCT
DOI: 10.1080/14653240903079385
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the age of the donor [15,16] are major factors influenc-
a heterogeneous population containing clonogenic cells,
ing both the incidence of colony-forming unit–fibroblasts
some multipotent, and mature stromal cells.
(CFU-F) and the proliferation rate of culture-amplified removed during surgical procedures can be an alternative
Methods Isolation and culture of human MSC
source of MSC for tissue repair and engineering. Before
We obtained trabecular bone and BM from 17 patients
using such cells in clinical trials, the potential of trabecular
with hip osteoarthritis who were undergoing total hip
bone MSC for bone repair must be tested. We evaluated
arthroplasty under general anesthesia. The study fol-
MSC obtained from the femoral head, trochanter region
lowed the guidelines of Tours University Hospital
cells. In terms of ease of procurement, trabecular bone
and iliac crest after bone washing or marrow aspiration in elderly patients in terms of proliferation and differentiation potential. Because fibroblast growth factor-2 (FGF-2) has shown good effects in population doubling and maintenance of differentiation potential [17–19], and is used in France for clinical-grade MSC culture, we tested the possible use of FGF-2 for trabecular bone MSC culture.
(Tours, France) and was approved by the local ethics committee. Patients gave their informed consent to be in the study. The characteristics of the patients are shown in Table I. Using two methods, extensive bone washing (W) and marrow aspiration (A), we obtained cells for MSC culture from three sites: the femoral head, trochanter and iliac crest. The femoral head and trochanter region are filled with yellow marrow, in
Recently, however, from in vivo experiments, it has been
contrast to the iliac crest, which is filled with red mar-
reported that FGF-2 can impair the stemness potential
row. Trabecular bone fragments were obtained from
of MSC [20].
the trochanter and femoral head. From the femoral
According to the position statement of the International
head, a cube (mean volume 6.2 cm3) of trabecular bone
Society for Cellular Therapy [21], we use the term MSC
(n 16) was obtained with the use of a surgical power
for the cultivated cells used in the present study. MSC
saw, and trabecular bone fragments were removed from
refers to cultivated BM mesenchymal stromal cells that are
the trochanteric region (metaphysis) by curettage (n 8).
Table I. Characteristics of patients and bone samples. Donor
Age (years)
Sex
Osteoarthritis etiology
Femoral head
Trochanter
Iliac crest
1
68
F
PA
W
W
ND
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
65 55 74 76 74 64 61 73 57 53 53 78 48 61 65 74
M F F M M F F F F F F F F M M M
AN CD PA PA PA PA CD PA PA PA CD PA PA PA PA PA
W W W W W W W W W W W W W ND W W
W W W W W W W A A A A A A A A A
ND ND ND ND ND ND ND ND ND ND ND ND ND A A A
F, female; M, male. Etiology of osteoarthritis: PA, primary arthritis; CD, congenital dysplasia; AN, avascular necrosis. ND, not done.
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The trabecular bone samples were fragmented, and each piece was meticulously washed by multiple injections of culture medium with a 19-gauge needle; erythrocytes were lysed (diluted 1:10 v/v with acetic acid 10%) then nucleated cells counted. Trochanter and iliac crest samples underwent BM aspiration. During surgery, using a Jamshidi biopsy needle, 10 mL BM were harvested with successive 2-mL aspirations from the trochanteric region (n 9) and iliac crest (n 3) (Table I). In order to avoid dilution by peripheral blood, after each aspiration the needle was repositioned. The BM samples were washed twice in complete culture medium, and nucleated cells were counted with a Cobas Argos automated hematology analyzer (Roche ABX, Montpellier, France). Referring to the method used (A or W), samples were named iliac crest A, trochanter A, trochanter W and femoral W. Nucleated cells were seeded at 5 104/cm2 in 75-cm2 tissue culture flasks (Becton Dickinson, Franklin Lake, NJ, USA) and cultured for 21 days (passage 0; P0) in complete culture medium: alpha-minimal essential medium (MEM; Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) screened fetal calf serum (FCS; Hyclone, Logan, UT, USA), 20 mm l-glutamine (Gibco Invitrogen), 100 U/mL penicillin–streptomycin (Gibco Invitrogen) and 0.25 mg/L amphotericin B (Fungizone, Bristol-Myers Squibb, New York, NY, USA). The medium was changed twice weekly. At day 21, cells were harvested from confluent or subconfluent layers with the use of trypsin (0.05% v/v solution containing 1 mm EDTA; Gibco Invitrogen). After washing twice in phosphate-buffered saline (PBS), viable cells were counted by trypan blue staining and replated at 103/cm2 (passage 1; P1). At day 42, cells were harvested and replated at the same density (passage 2; P2) and cultured until day 63. To evaluate the effects of FGF-2, cells from donors 15, 16 and 17 were cultured in complete culture medium with and without FGF-2 (1 ng/mL; R&D Systems, Minneapolis, MN, USA).
CFU-F assay A CFU-F assay was performed at day 0, P0 (day 21) and P1 (day 42). At day 0, nucleated cells were seeded in 25-cm2 flasks at two densities (2 103 and 4 103/cm2) for cells obtained by washing and three densities (2 103, 4 103 and 40 103/cm2) for cells from aspirates. At P0 and P1, 200 and 500 cells, respectively, were cultured in 25-cm2 flasks. Cultures were incubated for 10 days, then the
medium removed and, after two washes in PBS, cells fixed in methanol (5 min) and stained with Giemsa (10 min). Colonies of 50 or more cells were counted.
Cell phenotype analysis of cultured MSC Flow cytometry analysis was performed at P0, P1 and P2. Mouse anti-human monoclonal antibodies (MAb) were conjugated for CD13–phycoerythrin (PE), CD14–PE, CD29–PE, CD34–PE, CD49a–PE, CD73–PE, CD90–PE, CD105–PE, CD106–PE, CD164–PE, CD45–fluorescein isothiocyanate (FITC) (BD Biosciences, San Jose, CA, USA), CD146–PE (Biocytex, Marseille, France) and unconjugated for Stro-1 (R&D Systems). The following control MAb were included: mouse IgG1–PE, mouse IgG1–FITC and mouse IgM–PE for Stro-1 (BD Biosciences). Cells were incubated with MAb at 4°C for 30 min. Cells incubated with unconjugated Stro-1 were washed and incubated with PE-conjugated goat anti-mouse IgM IgG IgA (BD Biosciences). For each cell-surface marker, at least 104 events were analyzed with a BD FACSCalibur flow cytometer (BD Biosciences).
In vitro differentiation Osteogenic and adipogenic differentiation were evaluated at P0, P1 and P2 for MSC from six patients (patients 12–17) and chondrogenic differentiation at P1 for MSC from three patients (patients 15–17). To test FGF-2 for differentiation potential, we used MSC cultivated with or without FGF-2 in three patients (patients 15–17).
Osteogenic differentiation MSC were plated at 7 103 cells/cm2 in six-well culture plates (Corning, Corning, NY, USA) and cultured for 3 weeks in low-glucose Dulbecco’s modified essential medium (DMEM; Gibco Invitrogen) supplemented with 10% FCS, 0.15 mm ascorbate-2-phosphate, 2 mm -glycerophosphate and 10−8 m dexamethasone (Sigma-Aldrich, St Louis, MO, USA). Mineralized areas were revealed by Von Kossa staining. Cells were fixed with 4% paraformaldehyde (v/v) and stained for 30 min in 5% (v/v) silver nitrate solution (Sigma). After ultraviolet exposure for 45 min, cells were stained for 2 min in 5% (v/v) sodium thiosulfate solution (Sigma).
Adipogenic differentiation MSC were plated at 14103 cells/cm2 in Lab-Tek chamber slides (Nunc, Rochester, NY, USA) and cultured for 2 weeks
Impaired osteogenic potential of MSC
in low-glucose DMEM supplemented with 20% FCS, 0.5 mm isobutyl-methyl-xanthine IBMX, 60 μm indomethacin and 10−6 m dexamethasone (all Sigma). Lipid vacuoles were revealed by Oil Red O staining. Cells were fixed with 4% paraformaldehyde and stained for 30 min with 1Oil Red O (Sigma). Nuclear staining involved use of Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; AbCys, Paris, France). The percentage of differentiated cells was assessed according to the following scale: more than 75%, 50–75%, 25–50% and less than 25%.
Chondrogenic differentiation MSC differentiation was induced in aggregate cultures for 3 weeks. MSC, 2.5105, were spun down in 15-mL polypropylene conical tubes at 350 g for 5 min. The FCS-containing medium was then replaced with 1 mL defined medium consisting of high-glucose DMEM supplemented with 107 m dexamethasone, 103 m sodium pyruvate, 1.7104 ml-ascorbic acid-2-phosphate, 3.5104 m proline (all Sigma) and 1 insulin–transferring–selenium (ITS; Cambrex, East Rutherford, NJ, USA). After a 24-h incubation, the medium was changed and replaced with fresh medium 10 ng/mL transforming growth factor-1 (TGF-1; R&D Systems). The medium was changed every 3–4 days. The presence of glycosaminoglycans in cell pellets was revealed by Alcian blue staining and toluidine blue. At day 21, pellets were fixed for 48 h in 4% paraformaldehyde and embedded
587
in paraffin. Sections 4 μm thick were rehydrated in 70%, 90% and 100% xylene and ethanol. Slides were stained for 10 min in a 1:1 mixture of 3% Alcian blue 8GX (Sigma) (v/v) solution in distilled water and 3% (v/v) methanol.
Reverse transcription–polymerase chain reaction analysis Reverse transcription–polymerase chain reaction (RT-PCR) involved use of SuperScript one-step RT-PCR with platinum Taq (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression of the following genes was studied: alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) for the osteogenic pathway, and peroxisome proliferator-activated receptor- (PPAR-), lipoprotein lipase (LPL) and fatty acid binding protein 4 (FABP4) for the adipogenic pathway. As a control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used. The sequences of the oligonucleotides are shown in Table II. All total cellular RNA samples were extracted with the use of an RNA easy kit (Qiagen SA, Courtaboeuf, France) from cells cultured in differentiation medium at the end of P1 (day 42); 50 ng total RNA were used for specific genes and 5 ng for the GAPDH gene. RT-PCR products were analyzed on agarose gel electrophoresis.
Histology Bone samples were obtained from two donors (16 and 17) by BM biopsy of the iliac crest and trochanter region with
Table II. Sequences of the primers used in RT PCR study. Target mRNA Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Sequence (forward) 5 -GGTGAAGGTCGGTGTGAACG-3
(reverse) 5 -CATCATACTTGGCAGGTTTCTCC-3 (forward) 5 -CTGGACCTCGTTGACACCTG-3 (reverse) 5 -GAC ATTCTCTCGTTC ACCGC- Osteopontin (forward) 5 -CAGGCTGATTCTGGAAGTTCTGAG-3 (reverse) 5 -GGTGATGTCCTCGTCTGTAGCATC-3 Bone sialoprotein (forward) 5 -TTTCCAGTTCAGGGCAGTAGTGAC (reverse) 5 -CTTCCCCTTCTTCTCCATTGTCTC Peroxisome proliferator-activated (forward) 5 -GGAGAAGCTGTTGGCGGAGA-3 receptor- (PPAR-) (reverse) 5 -TCAAGGAGGCCAGCATTGTG-3 Fatty acid binding protein 4 (forward) 5 -GTACCTGGAAACTTGTCTCC-3 (FABP4) (reverse) 5 -GTTCAATGCGAACTTCAGTCC-3 Lipoprotein lipase (forward) 5 -AAAGCCCTGCTCGTGCTGAC-3 (reverse) 5 -TAAACCGGGCCACATCCTGT-3
Alkaline phosphatase
Product length Gene accession number 761pb
NM_002046.3
545pb
NM_000478
509pb
NM_001040058
436pb
NM 004967 3
430pb
NM_015869.4
418pb
NM_001442.1
405pb
NM_000237.2
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a Jamshidi biopsy needle and from the femoral head cube after fragmentation. Tissue samples were fixed in Bouin solution (VWR Prolabo, Fontenay sous bois, France), decalcified for 30 min in rapid bone decalcification media (Eurobio, Courtaboeuf, France) and embedded in paraffin. Sections 4 μm thick were rehydrated in xylene and 50%, 80% and 100% ethanol. Slides were then stained in Mayer’s Hemalun for 5 min (VWR Merck Eurolab, Leuven, Belgium) and Eosin for 3 min (Merck Eurolab). The presence and ratio of compact bone, trabeculae, adipocytes and myeloid elements were assessed.
Statistical analysis Statistical analyzes involved the Student’s t-test with StatView software v.5 (SAS Institute, Cary, NC, USA). Values are means standard error of the mean (SEM). A P-value of 0.05 was considered statistically significant.
Results Cell composition of starting materials In our 17 elderly patients (mean age 64 years, range 48–78 years), histology showed femoral head tissue and trochanteric regions with no hematopoietic tissue but normal levels in the iliac crest (Figure 1A–C). The femoral head biopsy showed necrotic trabecular bone fragments, and trochanter tissue biopsy showed adipocytes and compact bone, with no hematopoiesis. Iliac crest biopsy showed hematopoiesis in cords located within bone trabeculae and some adipocytes within the cords.
CFU-F differences The frequency and size of CFU-F did not differ with age of donor (53–78 years) or starting material (iliac crest, BM or trabecular bone). CFU-F frequency was significantly lower with trochanter A than with trochanter W (0.04 0.02 versus 0.21 0.07/103 cells; P 0.027) (Figure 2A). In all MSC cultures, CFU-F frequency decreased from cell passage P0 to P2 (P1 versus P0, P 0.05; P2 versus P1, P 0.05; n 17; Figure 2B, C). Cell proliferation was identical with cell passage, whatever the starting material. Only trochanter MSC at P2 showed a significant difference in cell proliferation when comparing washing and aspiration (Figure 2D–F). Finally, whatever the starting material (femoral head A, n 16; trochanter W, n 8; trochanter A, n 9; iliac crest, W; n 3), the calculated mean of population doublings was 15 1.
Phenotypes of MSC As early as P0, cells were positive for CD13, CD29, CD49a, CD73, CD90, CD105, CD106, CD164 and CD146 (Figure 3). CD146 expression was low at the end of P0 (ratio of signal to noise, S:N, 8.14 1.44) and decreased continuously in subsequent passages. Stro-1 expression was low at P0 and P1 and non-existent by the end of P2, as reported previously [22]. In our culture conditions, hematopoietic cells disappeared rapidly: at P0, a few CD45-positive cells were sometimes detected (less than 1% in three experiments and less than 5% in two); at P1, all cultivated cells were negative for CD45, CD34 and CD14.
Figure 1. Histologic analysis of BM biopsies. Samples from BM biopsies (patients 1 and 17) were fixed, decalcified and embedded in paraffin, and then sections stained with hematoxylin & eosin (H&E). The figure shows representative images (patient 17). (A) Femoral head: avascular and necrotic trabecular bone fragments. (B) Trochanter: aplastic-like, with adipocytes, compact bone and lack of hematopoietic tissue. (C) Iliac crest: large proportion of hematopoietic tissue (original magnification 100).
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Figure 2. CFU-F and cell proliferation in culture. (A,B,C) Variation in CFU-F number with cell passage. P1 42 days; P2 63 days; W, cells obtained by washing; A, obtained by aspiration. *P 0.05. (D,E,F) Variation in proliferation with cell passage. Cell proliferation total number of cells at the end of passage/total number of cells seeded. *P 0.05.
Differentiation potential The osteogenic differentiation potential, assessed by evaluation of mineralization with von Kossa staining, and adipogenic differentiation potential, assessed by the presence of cells with Oil Red O-positive vesicles ( 5 μm), was evaluated at P0, P1 and P2 in MSC from patients 12–17. Because of the number of cells needed, the chondrogenic differentiation potential was evaluated at only P1 in three patients (patients 15–17). MSC from material obtained by A and W at P0–P2 (n 3, patients 15–17) underwent RT-PCR to test mRNA expression of genes specific for bone (ALP, OPN and BSP) and adipose (PPAR-, FABP4 and LPL). When cultivated in basic culture conditions, MSC from the femoral head and trochanter region (after washing) showed impaired osteogenic differentiation potential, with negative or very low von Kossa staining. Mineralized areas were present in only P1 and P2 MSC from the femoral head and trochanter region grown in the presence of FGF-2 (Figure 4A). At P2, no
mineralized areas were present (Figure 4A). At P0 and P1, iliac crest A and trochanter A MSC generated many mineralized areas; however, on cultivation without FGF-2, at P2, iliac crest A and trochanter A MSC inconsistently generated mineralized areas (n 2/3 without FGF-2 versus n 3/3 with FGF-2). In all MSC (A and W) from the three patients analyzed, RT-PCR analysis revealed the expression of bone-specific genes (ALP and OPN) in all differentiated MSC (Figure 4D). Over time, the adipogenic differentiation potential was rapidly impaired in MSC from material obtained by washing (n 6) (Table III). The adipogenic potential was present at P0 and preserved only in MSC cultured with FGF-2 (n 3; Figure 4B). For iliac crest A MSC, lipid vacuoles were observed in more than 25% of cells at P0 and adipogenic differentiation decreased with time, with lessthan 25% differentiation at P1 and no differentiation at P2. However, at P1, after culture in adipogenic differentiation medium, RT-PCR analysis showed the
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Figure 3. Cell phenotype. Data from one representative experiment: patient 16, femoral head W MSC at P1. MSC were cultured in normal medium supplemented with 10% FCS without FGF-2.
Impaired osteogenic potential of MSC
expression of adipose-specific genes (PPAR-γ, FABP4 and LPL) in all MSC, whatever the starting material (Figure 4D). Chondrogenic differentiation, assessed by the appearance of Alcian blue-positive glycosaminoglycans in high-density pellets, was impaired in P1 MSC and was inconsistently
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preserved (n 1/3) even on culture with FGF-2, whatever the starting material (Figure 4C).
Discussion We cultured MSC from trabecular bone using a simple method of extensive washing followed by a classic
Figure 4. (Continued).
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culture procedure. The MSC showed proliferation potential, a proportion of clonogenic cells and a phenotypic profile similar to that of BM MSC. However, in our elderly donors, the trabecular bone MSC showed impaired potential for differentiation to osteogenic and chondrogenic pathways, as well as the adipogenic pathway, over time. The washing method we used to obtain MSC is simple, safe and not time-consuming. It enables a sufficient number of cells to be obtained with a good yield of CFU-F that are representative of the stem cell/progenitor population. Compared with other methods, bone washing prevents the use of an exogenous enzyme such as collagenase [11], which might be a safety concern. In contrast to culture of entire bone fragments [23], bone washing allows entire qualification (e.g. cell counting and flow cytometry) of the starting material. In terms of safety and regulatory issues, all these aspects have an advantage for clinical use. MSC from material obtained by washing, as well as those obtained by aspiration, expressed the classic mesenchymal markers CD90, CD73, CD105 and CD49a at high levels and CD146 and Stro-1 at low levels. With increasing cell passage, cell number and CFU-F incidence did not differ significantly, whatever the starting material. These results are in agreement with previous data for trabecular bone MSC [11]. In contrast with unmodified proliferation potential, CFU-F cloning efficiency and immunophenotype in MSC whatever the method, MSC from material obtained by washing showed a significant decrease in osteogenic and adipogenic differentiation potential. Beyond the scope of this study, in vivo analyzes of the osteogenic differentiation potential must be performed to confirm the results obtained in vitro. Our results are different to those of Tuli et al. [23]
and Sakaguchi et al. [11], who showed trabecular bone MSC correctly differentiated following the three canonical pathways even in elderly patients and after long-term culture. Although the phenotypes of MSC were identical in the studies, the impaired in vitro differentiation in our study might be linked to a difference in the procedures for cell collection: in our case extensive washing, compared with collagenase treatment [11] or culture explants [12,24] without washing steps. These previously reported procedures might release or allow the expression of more potent MSC and some mature osteoblastic progenitors. Moreover, as shown previously, the matrix elasticity can drive MSC into specific differentiation pathways [25]. With bone explant cultures, the presence of bone matrix with a specific stiffness might explain the better osteoblastic differentiation. In contrast to previous series, our series contained a predominance of female donors (11/17). Previously it has been shown that MSC cultivated from post-menopausal osteoporotic women have high phosphorylation of extracellular signalregulated kinase (ERK) levels [26], which plays an important role in the phosphorylation of RUNX2 [27]. We did not test the ERK-MAPK pathway in our patients, but the predominance of elderly female donors could play a role in our finding of impaired differentiation of trabecular bone MSC. Finally, we cannot exclude the possibility that the difference could be related to the arthritis in patients included in the study. The positive effect of FGF-2 on proliferative capacity was demonstrated at concentrations as low as 1 ng/mL [17]; in France, FGF-2 is used at this concentration for producing BM MSC in clinically approved protocols [28]. In our study, adding low-dose FGF-2 (1 ng/mL) to culture medium during the proliferation phase allowed better preservation of the differentiation potential of MSC.
Figure 4. In vitro differentiation of MSC. (A) Osteogenic differentiation: von Kossa staining was used to determine osteogenic differentiation in all MSC from six patients (patients 12–17). All data shown are from patient 16: femoral head W MSC (P1), trochanter W MSC (P1) and iliac crest A MSC (P2) (original magnification 50). (B) Adipogenic differentiation: Oil Red O staining was used to determine adipogenic differentiation in all MSC from six patients (patients 12–17). All data shown are from patient 17: femoral head W MSC (P0), trochanter W MSC (P0) and iliac crest A MSC (P0) cultivated without or with FGF-2 (1 ng/mL) (original magnification 400). (C) Chondrogenic differentiation: micropellets and Toluidine blue staining were used to determine chondrogenic differentiation in all MSC from three patients (patients 15–17). All data shown are from patient 17: femoral head W MSC (P1), trochanter W MSC (P1) and iliac crest A MSC (P1) (original magnification 40). (D) RT-PCR analysis: at P1, total mRNA was obtained from iliac crest A MSC, femoral head W MSC, trochanter W and A MSC after culture in either osteogenic or adipocytic differentiation media. Results are from a representative experiment (patient 17) of three (patients 15–17) with () or without () FGF-2 (1 ng/mL): ALP, OPN and BSP gene expressions were tested for osteogenic differentiation, and LPL, FABP4 and PPAR- gene expressions were tested for adipogenic differentiation. NC, negative control without mRNA.
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Table III. Adipocytic differentiation of MSC from different sites. Site
Femoral head
Trochanter
Iliac crest
W
W
A
Without FGF-2 With FGF-2
10% (3/3) 10– 25% (3/3)
10% (3/3) 10– 25% (3/3)
25– 50% (3/3) 25– 50% (3/3)
Without FGF-2 With FGF-2
10% (3/3) 10– 25% (2/3)
10% (3/3) 10– 25% (2/3)
25% (3/3) 25% (3/3)
Method P0
P1
Adipocytic differentiation was evaluated in MSC cultivated or not in the presence of FGF-2. The numbers represent the percentage of cells having lipid vacuoles revealed by Oil Red O staining. The starting cells were obtained by washing of femoral head and trochanter (W) and aspiration of BM of iliac crest (A).
The effect of FGF-2 on maintaining the osteoblastic differentiation potential of MSC has been reported elsewhere [18,19]. This cytokine might select for MSC with preserved osteogenic potential that are still present in these elderly patients but overwhelmed by senescent cells that have lost most of their differentiation capacity. In elderly patients, BM aspiration and trabecular bone washing enabled the generation of MSC. With both techniques, the rate of proliferation and CFU-F content were equivalent, but MSC cultivated from BM aspiration showed better in vitro differentiation potential. Although these results for MSC must be confirmed in vivo, this difference may have implications for clinical applicability.
Acknowledgements This research was supported by the integrated project Genostem of the European Commission FP6 research funding program and by grant number 2004-08 from the Etablissement Français du Sang. The authors thank Jean-Louis Brémond for histologic analysis of bone marrow biopsies and James E. Dennis for a critical review of the manuscript. Declaration of interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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