Stem Cells 2003;21:681-693
www.StemCells.com
© 2003 AlphaMed Press
Characterization of Multipotential Mesenchymal Progenitor Cells Derived from Human Trabecular Bone
Richard Tulia,b,
Suraj Tulia,
Sumon Nandia,
Mark L. Wanga,b,
Peter G. Alexandera,
Hana Haleem-Smitha,
William J. Hozackb,
Paul A. Mannera,c,
Keith G. Danielsonb,
Rocky S. Tuana,b,c
a Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA;
b Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, USA;
c Department of Orthopaedic Surgery, George Washington University, Washington, D.C., USA
Key Words. Mesenchymal progenitor cell • Chondrogenesis • Osteogenesis • Adipogenesis • Tissue engineering
Rocky S. Tuan, Ph.D., Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Building 50, Room 1503, 50 South Drive, MSC 8022, Bethesda, Maryland 20892-8022. Telephone: 301-451-6854; Fax: 301-435-8017; e-mail: tuanr{at}mail.nih.gov
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ABSTRACT
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The in vitro culture of human trabecular bone-derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase-treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone-derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73+, STRO-1+, CD105+, CD34-, CD45-, CD144- cell population resident within collagenase-treated, culture-processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. When cultured as described, the trabecular bone-derived cells display stem cell-like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long-term in vitro culture.
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INTRODUCTION
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The in vitro culture of human trabecular bone-derived cells has served as a useful system for the investigation of the biology of osteoblasts and has also provided insights into the interactions between bone and biomaterial for orthopedic and dental implants, their response to different growth factors and hormones, cell-matrix interactions, and osteoblast differentiation and maturation [1–9]. Our recent discovery of the multilineage mesenchymal differentiation potential of primary cell cultures derived from collagenase-treated human trabecular bone fragments [10] has prompted further interest in these cells in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma [11, 12]. Moreover, the identification and maintenance of MPCs in the undifferentiated phenotype depend on efficient methods of isolation as well as optimal conditions for subsequent culture in vitro, such as the tissue culture substrate, supplementation with proliferative and differentiation factors, and specific culture media. As such, establishing an optimal, standardized cell culture system is of critical importance, given the inherent complications associated with comparing studies that use a variety of different methodologies to obtain results. We have previously reported a simple, high-yield procedure that allows the isolation and production of a significant number of these MPCs from human trabecular bone, with improved yields over MPCs aspirated from bone marrow [13]. However, the origin, identification, characterization, and behavior of these trabecular bone-derived cells during primary and extensive subcultivation have yet to be provided.
As is the case for bone marrow-derived human MPCs, an understanding of the variability associated with the isolation of these cells is crucial for optimizing the experimental design of studies that utilize them. For example, techniques have been established to standardize the quantity of bone marrow aspirate of MPCs, the site and methodology of aspiration itself, the yield of progenitor cells identified by cell surface immunophenotyping, and their in vitro self-renewing capacity and multilineage differentiation potential following extensive subcultivation [14–17]. These known properties have permitted the utilization of MPCs in cell-matrix composites for various connective tissue engineering applications involving bone, adipose, cartilage, tendon, and muscle [12].
With this in mind, we have sought to characterize the native and culture-expanded trabecular bone-derived cell population and describe and optimize the proliferative and differentiative capacity of these cells during long-term in vitro culture. Specifically, we have compared the performance of two basal media, Dulbecco’s modified Eagle’s medium (DMEM), widely used for the culture of various cell types including bone marrow-derived MPCs [14, 18, 19], and DMEM with Kaighn’s modification of Ham’s F12 (F12K) supplemented with ascorbic acid and L-glutamine, which has been routinely used in studies involving cells derived from trabecular bone [9, 20, 21].
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MATERIALS AND METHODS
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Isolation of Human Trabecular Bone-Derived Cells
Otherwise normal trabecular bone was obtained from the femoral heads of five patients (ages 51–78 years) undergoing total hip arthroplasty as a result of primary osteoarthritis, and processed using a rapid, high-yield protocol recently established in our laboratory [13] and approved by the Institutional Review Board of Thomas Jefferson University. Culture-processed trabecular bone fragments were subsequently plated in either A) DMEM (high-glucose and L-glutamine; Mediatech, Inc.; Herndon, VA; http://gomediatech.com) supplemented with 10% fetal bovine serum (FBS, Premium Select, Atlanta Biologicals; Atlanta, GA; http://www.atlantabio.com) from selected lots [22] and 50 µg/ml penicillin-streptomycin, or B) calcium-free DMEM-F12K (Specialty Media; Phillipsburg, NJ; http://www.cmt-inc.net) supplemented with 10% FBS from the same selected lots, 50 µg/ml ascorbate, 2 mM L-glutamine, and 50 µg/ml penicillin-streptomycin. Medium was changed every 3–4 days. Subconfluent monolayers of cells were removed with 0.25% trypsin containing 1 mM EDTA (GIBCO/BRL, Life Technologies; Grand Island, NY; http://www.invitrogen.com) and utilized for study or serially passaged at a ratio of 1:3. Primary cells that were initially cultured in calcium-free DMEM-F12K were switched to medium with normal amounts of calcium upon subculture.
Immunofluorescence Analysis of Human Trabecular Bone Chips and Cell Monolayers
Collagenase-treated human trabecular bone chips were harvested following 0, 7, and 14 days of culture; decalcified for 14 days using 0.38 M EDTA; fixed overnight at 4°C in 4% phosphate-buffered paraformaldehyde (FD Neuro Technologies, Inc.; Baltimore, MD; http://www.fdneurotech.com); embedded in paraffin; and sectioned at a thickness of 8 µm. STRO-1 was detected using a mouse IgM primary antibody (Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA; Dr. B. Torok-Storb). CD105 was detected using a mouse IgG1 primary antibody (BD Transduction Laboratories; San Diego, CA; http://www.bdbiosciences.com); CD73 was detected using an R-phycoerythrin-conjugated mouse primary antibody (PharMingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen). CD45 was detected using a PerCP-Cy5.5-conjugated mouse IgG1 primary antibody (BD Biosciences; San Jose, CA; http://www.bdbiosciences.com). CD34 was detected using a monoclonal mouse IgG1 primary antibody and CD144 (VE-cadherin) was detected using a polyclonal rabbit primary antibody (Zymed Laboratories, Inc.; San Francisco, CA; http://www.zymed.com). The sections were incubated in the following secondary antibodies: for STRO-1, fluorescein-conjugated goat anti-mouse IgM (Vector Laboratories, Inc.; Burlingame, CA; http://www.vectorlabs.com); for CD105, tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse IgG was used; for CD34, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse was used; for CD144, FITC-conjugated goat anti-rabbit was used. All sections were Hoechst Dye 33342 (Molecular Probes; Eugene, OR; http://www.probes.com) stained. Primary cultures of cell monolayers, fixed in 2% phosphate-buffered paraformaldehyde, were treated as described above.
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Determination of Colony-Forming Unit-Fibroblast (CFU-F) Frequency
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The initial cell population that exited the culture-processed trabecular bone fragments was trypsinized as described above and replated as single cells at a density of 100 cells per well of a 24-well plate in either DMEM or DMEM-F12K. The total number of cells that replicated to form colonies was subsequently determined following 7 days of culture (n = 416 and 400, respectively).
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Differentiation of Cell Cultures along Mesenchymal Lineages
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Osteogenic induction of confluent monolayer cultures was accomplished using DMEM supplemented with 10% FBS, 50 µg/ml ascorbate, 10 mM ß-glycerophosphate, and 0.1 µM dexamethasone for 21 days, with medium changes every 3–4 days [17]. Adipogenic differentiation was induced using DMEM supplemented with 10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 µg/ml insulin for 14 days [17]. Control cultures were maintained without osteogenic or adipogenic supplements, respectively. Chondrogenesis of high-density pellet cultures (2 x 105 cells/pellet, 500 x g for 5 minutes) was induced using serum-free DMEM supplemented with 50 µg/ml ascorbate, 0.1 µM dexamethasone, 40 µg/ml L-proline, 100 µg/ml sodium pyruvate, and ITS-plus (Collaborative Biomedical Products; Cambridge, MA; http://www.bioscience.org/company/hbecton.htm) [18, 19, 23]. Recombinant human transforming growth factor-ß1 (TGF-ß1; R&D Systems; Minneapolis, MN; http://www.rndsystems.com) was also added to the chondrogenic induction medium at a final concentration of 10 ng/ml. Pellet cultures were maintained for 21 days with medium changes every 3–4 days. Control pellet cultures were maintained without the addition of TGF-ß1.
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Cell Proliferation Assay
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Undifferentiated cells were added to a 96-well plate (1 x 103 cells/well) and assessed for cell viability and proliferation at days 1, 2, 4, 8, 16, and 24 using the CellTiter 96 AQueous One Solution MTS Cell Proliferation Assay (Promega; Madison, WI; http://www.promega.com) according to the manufacturer’s protocol.
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Population-Doubling Potential
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For primary cultures, the initial number of cells that migrated from the bone chips and replicated to form colonies was counted, averaged from a total of five donors, and used to ascertain the number of population doublings based upon the total number of cells obtained at 80% confluency. For each subsequent passage, cells were seeded at a density of 10,000 cells/cm2, and the population doublings were also calculated based upon the total number of cells obtained when 80% of the tissue culture polystyrene was covered with cells (approximately 4 weeks).
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Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
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Total cellular RNA was extracted using Trizol Reagent (GIBCO/BRL) according to the manufacturer’s protocol. RNA samples were reverse transcribed using random hexamers and the SuperScript First Strand Synthesis System (GIBCO/BRL). PCR amplification of cDNA was carried out using AmpliTaq DNA Polymerase (Perkin Elmer; Norwalk, CT; http://www.perkinelmer.com) and the gene-specific primer sets listed in Table 1
. These genes included the bone-specific genes, alkaline phosphatase (ALP), bone sialoprotein (BSP), collagen type I
2 (Col I2), and osteocalcin (OC); the adipose-specific genes, lipoprotein lipase (LPL) and peroxisome proliferator-activator receptor-
2 (PPAR2); and the cartilage-specific genes, collagen types II (Col II), IX (Col IX), and XI (Col XI), aggrecan (AGN), and Sox 9. Thirty-two cycles were used for all genes and consisted of a 1-minute denaturation at 95°C, a 1-minute annealing at 57°C (Col II, Col IX, Col XI, AGN, and Sox 9) or 51°C (all remaining genes); a 1-minute polymerization at 72°C; and a final 10-minute extension at 72°C. The housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used as a control for RNA loading of samples. PCR products were analyzed electrophoretically using an ethidium bromide 2% MetaPhor agarose gel (BioWhittaker Molecular Applications; Rockland, ME; http://www.cambrex.com).
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Table 1. RT-PCR primers for differentiation-specific gene expression analysis: sequence and expected product size
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Alkaline Phosphatase Histochemistry
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Osteogenic monolayer cultures were stained histochemically for alkaline phosphatase (Sigma Cat. No. 86-C; St. Louis, MO; http://www.sigmaaldrich.com) according to the manufacturer’s protocol.
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Oil Red O Histochemistry
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Control and adipogenic monolayer cultures were fixed for 15 minutes using 2% phosphate-buffered paraformaldehyde (FD NeuroTechnologies), incubated in 60% isopropanol for 5 minutes, stained with Oil Red O for 5 minutes, rinsed with tap water, and counterstained with hematoxylin for 1 minute.
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Alcian Blue Histochemistry
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Cell pellet cultures, rinsed twice with phosphate-buffered saline (PBS), were fixed for 2 hours in 2% PBS-buffered paraformaldehyde, dehydrated through a graded series of ethanol, infiltrated with isoamyl alcohol, embedded in paraffin, and sectioned at an 8 µm thickness for histological staining with alcian blue (pH 1.0) [24].
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RESULTS
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Immunofluorescence Analysis of Human Trabecular Bone Chips and Monolayer Cultures
Collagenase-treated, culture-processed trabecular bone fragments harbor cells distributed throughout the confines of the mineralized matrix tissue (Fig. 1). As a function of culture time, these cells, appearing mitotically active, migrated outward where they lined the periphery of the fragments (Fig. 1C, 1F, 1I, 1L, 1O, 1R). This migration time apparently coincided with the lag period of 1 week, as described below, required before cellular outgrowth from the explanted bone began. Additionally, all cells resident within the fragments detected by Hoechst stain also stained positively for CD73, STRO-1, and CD105, individually, but negatively for CD34, CD45, and CD144. Following approximately 3 weeks of culture, cells that had successfully migrated out from within the trabecular bone chips retained the CD73, STRO-1, and CD105 cell surface antigens, while remaining CD34, CD45, and CD144 negative. There were no cells in monolayer culture that did not stain positively for the CD73, STRO-1, or CD105 antigens or negatively for CD34, CD45, or CD144 (Fig. 2).
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Figure 1. Collagenase-treated human trabecular bone chips harvested following 0, 7, and 14 days of culture and immunostained as described in Materials and Methods reveal cells that express the cell surface antigens CD73 (A-C, orange-red), STRO-1 (D-F, green), and CD105 (G-I, red), and fail to express CD34 (J-L), CD45 (M-O), and CD144 (P-R). Between days 7 and 14 of culture, most of the cells have migrated to the periphery of the bone fragments. Cell nuclei are counterstained with Hoechst Dye 33342. Bar = 20 µm.
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Figure 2. Cell monolayers derived from collagenase-treated trabecular bone chips uniformly express the CD73 (A, orange-red), STRO-1 (B, green), and CD105 (C, red) cell surface antigens. No cells expressing CD34 (D), CD45 (E), or CD144 (F) are present within the culture. Cell nuclei are counterstained with Hoechst Dye 33342. Bar = 20 µm.
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Cell Viability and Proliferation
Phase contrast microscopy of the trabecular bone fragments seeded in DMEM and DMEM-F12K culture media initially showed fragments devoid of soft tissue residue and contaminating cells (Fig. 3A, 3B). Upon culture, an initial lag phase of 4–6 days was seen in DMEM cultures, after which cells entered the log phase of growth and began to actively migrate from the bone chips and proliferate. This was in sharp contrast to the 10- to 12-day lag phase seen in DMEM-F12K cell cultures, followed by the slow, gradual cellular migration from the bone fragments (Fig. 3C, 3D). This difference was more apparent when cultures were observed microscopically on days 14 (Fig. 3E, 3F) and 21 (Fig. 3G, 3H), revealing the ability of DMEM cultures to attain a higher limiting cell density. Cellular morphology during the initial primary culture was similar in both populations, revealing homogeneous spindle-shaped cells.
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Figure 3. Morphology of reamed trabecular bone explants cultured in DMEM-F12K (A, C, E, G) and DMEM (B, D, F, H). Approximately 8 hours after plating, no attached cells can be seen contaminating the culture, and all bone fragments appear completely devoid of soft-tissue components (A, B). After 7 and 14 days, cells began to migrate and proliferate more rapidly from bone chips cultured in DMEM (C, D and E, F). Differences in the number of cells and colonies became more apparent following 21 days in culture (G, H). Bar = 25 µm.
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The CFU-F assay was used to ascertain the colony-forming potential of trabecular bone-derived cells cultured in the two media, which were in the process of exiting the bone fragments. Following 3 days of culture in DMEM, approximately 73% of individual cells established small (4–8 cells) colonies, all of which proliferated and grew rapidly, forming large (100–200 cells) colonies by day 7. In contrast, only 48% of single isolated cells cultured in DMEM-F12K followed the same pattern of colony formation. Following increased culture time, a small percentage of the cells cultured in either medium that failed to yield colony formation during the first 7 days of culture began to proliferate and established latent colonies.
Cells serially passaged and grown in the two media followed patterns of growth similar to their respective primary cultures (Fig. 4). DMEM-F12K cell cultures experienced an initial lag phase of 1–3 days, followed by a log phase of 10–14 days, and a plateau phase of 4–6 days, after which mitotic division slowed dramatically (Fig. 4A–4C). In comparison, the growth curves of cells cultured in DMEM depict a minimal lag phase, followed by steeper log and plateau phases. As passage number increased, growth rates for DMEM-F12K cultures decreased, resulting in a smaller total number of cells, i.e., the log phase cell growth rate diminished more rapidly with passaging for the cells cultured in DMEM-F12K.
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Figure 4. Viability and proliferation of cells grown in DMEM-F12K and DMEM, obtained from the first (A), third (B), and fifth (C) serial subculture. Cultures were established from the same patient and analyzed using the MTS assay (see Materials and Methods). Compared with DMEM cultures, DMEM-F12K cultures show a decrease in the slope of the log phase as a function of increasing passage. A similar pattern of cell viability and proliferation was seen among the three arthroplasty specimens (data not shown). The results represent the mean cell number (absorbance at 490 nm) ± standard deviation (n = 3). * p <0.05.
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Moreover, cells in DMEM-F12K attained a broad, flattened morphological appearance, a cessation of mitotic activity, and an increased accumulation of cellular debris after an average of 6 passages, thereby signifying replicative senescence. On average, the cumulative population doublings for DMEM-F12K cultured cells were 21.9 ± 1.8, with 10.1 doublings taking place during the primary culture, accounting for 46.1% of the total, and two doublings occurring at each subsequent passage (Fig. 5). In comparison, the mean cumulative population doublings for cells cultured in DMEM was 43.6 ± 2.2 through an average of 13 passages. Analysis of primary DMEM cultures revealed an average of 12.4 population doublings, with 2.4 doublings per subsequent passage (Fig. 5); this difference can be partially attributed to the higher limiting density reached by the cell population cultured in DMEM.
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Figure 5. Population-doubling potential of trabecular bone-derived cells in two different culture media. The cumulative number of doublings was calculated from the initial establishment of colonies in primary culture through each subsequent passage until replicative senescence occurred. DMEM primary cultures (passage 0) underwent a total of 12.4 population doublings, as compared with 10.1 for DMEM-F12K cultures. Serially subcultured cells passaged at known densities underwent an average of 2.4 and 1.8 population doublings per passage in DMEM and DMEM-F12K cultures, respectively. Replicative senescence occurred after an average of five passages for DMEM-F12K cultures, while DMEM cultures continued to replicate until an average of 13 passages. The results represent the mean number of doublings ± SD of cells obtained from three donors.
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Analysis of Osteogenic Differentiation
Confluent first and fifth passage cells maintained in monolayer and cultured in basal DMEM and DMEM-F12K medium served as controls for the analysis of osteogenic gene expression (Fig. 6A, 6C). While these control cultures expressed Col I mRNA at similar levels, none of the other osteogenesis-specific marker genes were expressed. On the other hand, upon culture in the presence of osteogenic supplements (OS) for 21 days, RNA expression of ALP, BSP, Col I, and OC was readily detectable in first and fifth passage cells. In the first passage, OS-treated DMEM cultures exhibited a significantly higher level of alkaline phosphatase gene expression as compared with similarly treated DMEM-F12K cultures, whereas the expression levels of BSP, Col I, and OC were equivalent (Fig. 6B). After serial subculture to passage five, significantly higher levels of ALP, BSP, Col I, and OC gene expression were seen in OS-treated DMEM cultures compared with similarly treated DMEM-F12K cultures (Fig. 6C, 6D). In all cases, the ALP enzyme activity levels were in direct agreement with the RT-PCR analysis of mRNA levels after 21 days of OS treatment (Fig. 7).
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Figure 6. Osteogenic nature of serially passaged trabecular bone-derived cells cultured in DMEM and DMEM-F12K. The mRNA was obtained from untreated control cells (-OS) and from cells treated with osteogenic supplements (+OS) for 21 days, and analyzed by RT-PCR to compare osteogenic gene expression (ALP, BSP, Col I, OC). All RT-PCR products were fractionated by electrophoresis, and ethidium bromide intensities were quantified densitometrically. (A, B) Passage 1. (C, D) Passage 5. Although both cell cultures, DMEM and DMEM-F12K, responded osteogenically to the supplements during the first passage (A), the osteogenic capacity of DMEM-F12K cultures diminished significantly by passage 5. Moreover, DMEM cultures were more responsive to the OS supplements, indicated by the significant upregulation of ALP gene expression in the first passage (B), and of ALP, BSP, Col I, and OC in the fifth passage (D) upon OS treatment, compared with DMEM-F12K cultures.
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Figure 7. ALP activity of serially passaged cells grown in DMEM and DMEM-F12K, and then treated with (+OS) or without (-OS) osteogenic supplements for 10 days. (A-D) Passage 1; (E-H) Passage 5. Bar = 500 µm. DMEM cells (C, D and G, H) demonstrated a significant increase in ALP activity upon osteogenic supplementation for both Passage 1 and Passage 5 cultures, as did Passage 1 DMEM-F12K cultures (A, B). However, Passage 5 DMEM-F12K cultures (E, F) failed to respond to OS supplementation, as evidenced by unchanged staining intensity. Additionally, DMEM cultures displayed a comparatively higher level of ALP activity during each passage.
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Analysis of Adipogenic Differentiation
Subconfluent monolayer cultures of first passage cells maintained in either DMEM or DMEM-F12K and cultured in media permissive for adipogenic differentiation for 14 days exhibited similar levels of LPL and PPAR2 gene expression, whereas control cells treated without adipogenic supplements expressed significantly lower levels of LPL and failed to express PPAR2 (Fig. 8A, 8B). Serial subculture appeared not to effect cells cultured in DMEM, as adipogenically induced cells of the fifth passage continued to express levels of LPL and PPAR2 comparable to those of first passage cells (Fig. 8C, 8D). However, serial subculture appeared to significantly affect the adipogenic capacity of cells cultured in basal DMEM-F12K, as evidenced by fifth passage cells treated with adipogenic supplements, which expressed minimal amounts of LPL and failed to express PPAR2, similar to the untreated controls. Oil Red O staining of similarly treated cultures was in accordance with adipogenic gene expression results (Fig. 9). As expected, control cultures maintained without adipogenic supplements did not stain positively for lipid droplets.
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Figure 9. Oil Red O staining of serially passaged trabecular bone-derived MPCs cultured in DMEM or DMEM-F12K basal medium and treated with (+AS) or without (-AS) adipogenic supplements for 14 days. Similar to RT-PCR results, passage 1 cells cultured in either basal medium responded to AS, as evidenced by the accumulation of cytoplasmic lipid droplets (A-D). Passage 5 DMEM cultures responded similarly when induced (H). However, serially subcultured DMEM-F12K cultures could not be adipogenically induced beginning at passage 5, as evidenced by absence of stain (F). Bar = 20 µm.
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Analysis of Chondrogenic Differentiation
In the absence of TGF-ß1, RT-PCR analysis showed constitutive expression of GAPDH in the first and fifth passage cells, but no cartilage-specific genes were expressed (Fig. 10). On the other hand, all cell pellets maintained in chondrogenic media and treated with TGF-ß1 significantly upregulated the cartilage-specific genes, i.e., Col II, Col IX, Col XI, AGN, and Sox 9. These mRNA transcripts were readily detectable in both first and fifth passage cells treated with TGF-ß1. First passage DMEM cultured cells maintained in high-density chondrogenic cultures with TGF-ß1 exhibited a significantly higher level of Col II gene expression as compared with similarly treated DMEM-F12K cells, whereas expression of Col IX, Col XI, AGN, and Sox 9 was not significantly different between the two groups of cells (Fig. 10B). TGF-ß1-treated cell pellets derived from fifth passage DMEM cultures expressed significantly higher levels of Col IX, AGN, and Sox 9, in addition to Col II as compared with similar passage DMEM-F12K medium cultures (Fig. 10C, 10D).
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Figure 10. Chondrogenic differentiation of serially passaged cells grown in DMEM and DMEM-F12K (A, B) Passage 1; (C, D) Passage 5. Cells were initially cultured in either DMEM or DMEM-F12K medium, followed by chondrogenic induction. The mRNA was obtained from pellet cultures maintained with (+) or without (-) TGF-ß1 for 21 days, and analyzed by RT-PCR to compare chondrogenic gene expression (Col II, IX, and XI, aggrecan, and Sox 9). RT-PCR products were fractionated by electrophoresis, and ethidium bromide intensities were quantified densitometrically. All treated cultures responded chondrogenically; however, the DMEM cultures were more responsive to chondrogenic induction as indicated by significant upregulation of Col II during the first passage (B), and Col II and XI, aggrecan, and Sox 9 during the fifth passage (D) compared with similarly treated DMEM-F12K cultures.
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As shown in Figure 11, 21-day TGF-ß1-treated pellets derived from DMEM cultures of the first (D-F) and fifth (J-L) passage exhibited a dense, extensive, and organized sulfated proteoglycan-rich extracellular matrix surrounding chondrocyte-like cells, as compared with control pellets maintained without TGF-ß1 (data not shown). The alcian blue staining intensity was higher than similarly treated pellets derived from DMEM-F12K cultured cells (A-C, G-I), which displayed a disorganized extracellular matrix devoid of the proteoglycan-rich characteristic of the in vitro chondrogenic phenotype. The pattern of the alcian blue histochemical staining was consistent with the mRNA gene expression of 21-day TGF-ß1 pellet cultures.
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Figure 11. Alcian blue staining of serially passaged cells grown in DMEM and DMEM-F12K media, subsequently maintained as pellet cultures in the presence of TGF-ß1 for 21 days. (A-F) Passage 1; (G-L) Passage 5. DMEM cultures from both passages exhibit larger pellets with a more abundant, alcian blue-stained sulfated proteoglycan matrix as compared with DMEM-F12K cultures. Cells also have a more organized appearance in DMEM cultures. Successively higher magnifications are shown: Bar = 300 µm (A, D, G, J); 80 µm (B, E, H, K); or 40 µm (C, F, I, L).
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DISCUSSION
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In this study, we have characterized in detail a population of cells resident within culture-processed human trabecular bone fragments that expressed STRO-1, CD73, and CD105, initially identified as antigens specific for nonhematopoietic bone marrow progenitor cells, and were negative for CD34, CD45, and CD144, common markers of hematopoietic and endothelial cells. The antibodies SH-2 and SH-3, and SH-4 [25], which recognize epitopes on CD105 [26] and CD73 [27], respectively, have also been shown to be nonreactive with the cell surfaces of osteoblasts and osteocytes, suggesting the presence of a population of multipotential cells distinct from other cell types normally associated with and present within trabecular bone. It is noteworthy that not all cells resident within the bone fragments display these cell surface epitopes, i.e., the multipotential cells are a subset of cells of the trabecular bone. Following migration from within the explants, these cells, which retain the same cell surface profile, proceeded to establish colonies. Analysis of the cell surface antigens of confluent monolayer cultures revealed a highly homogenous population of cells expressing CD73, STRO-1, and CD105, all previously shown to be associated with cells capable of differentiating along multiple mesenchymal lineages [26–29]. It is noteworthy that, in comparison, bone marrow-derived MPCs contain considerably fewer cells expressing these particular antigens than trabecular bone-derived cells, suggesting the presence of a significantly larger population of MPCs isolated from trabecular bone. Additionally, no cells expressing CD34, CD45, or CD144 were found within or associated with the trabecular bone fragments, indicating lack of any hematopoietic or endothelial cell contamination.
Analysis of cultures from all patients revealed that DMEM was significantly more effective in stimulating cellular outgrowth from the bone chips and promoting the establishment of colonies as compared with DMEM-F12K cultures. However, the growth of individual colonies throughout primary culture in either medium appeared to occur at different rates. Some cells appeared to begin dividing immediately upon migration from the bone fragment, and others failed to yield colonies at all or did so only after several days in culture. Previous studies have shown that fibroblasts, which are capable of quickly adhering and establishing rapidly growing colonies, undergo an increased number of total population doublings as compared with cells that slowly divide and form smaller colonies [30]. Our data support previous research into the aging characteristics of bone marrow stromal cells, where the confirmed presence of two types of adherent cells in primary culture, a rapidly dividing spindle-shaped cell type and a much slower dividing broad and flattened cell type, was observed [14, 31]. A much larger proportion of the former cell type was present in DMEM primary cultures as compared with DMEM-F12K cultures. Moreover, the proportion of spindle-shaped cells to flattened cells began to decrease as a function of increasing passage number, indicating a general progression of cells into a state of replicative senescence [14]. Since serially subcultured DMEM cultured cells maintained under control conditions, i.e., without osteogenic, adipogenic, or chondrogenic stimuli, did not express markers of terminal differentiation after long-term culture and also retained the ability to differentiate along mesenchymal lineages upon subsequent stimulation, it is reasonable to conclude that these cells did not senesce by entering into a state of terminal differentiation [14, 32, 33]. This transition to a cell population with diminished replicative capacity occurred much more rapidly for DMEM-F12K cultures.
Primary cell cultures followed normal, predictable growth curves with lag, exponential, and plateau growth phases; however, the characteristics of each phase varied between DMEM and DMEM-F12K cell populations through long-term culture. DMEM cultures consistently exhibited a shorter lag phase as well as a steeper exponential growth phase, resulting in a higher cellular density before proliferation was inhibited by cell-cell contact. These cultures were able to maintain a linear increase in population doubling through an average of 12 passages before the rate of population growth began to decline. Passaged cultures proceeded through the same stages; however, the rate of growth in the log phase and the final number of cells after a fixed period in culture gradually diminished as a function of continued passaging. This decrease in the rate of growth, as well as the total number of cells as a function of increasing passage number, could be attributed to a small population of cells in each passage developing the broad, flattened morphology characteristic of cells entering G0, thus resulting in a smaller number of actively dividing cells remaining in the population [14, 30, 34]. Therefore, loss of doubling potential was accelerated in the DMEM-F12K cultures by the onset of cellular senescence.
Additionally, the two culture media were found to differentially affect expression of the osteoblastic, adipogenic, and chondrogenic phenotypes. Besides being more potent in stimulating cell growth, culturing in DMEM elicited a stronger differentiation-inducing effect as determined by RT-PCR mRNA phenotyping and histology. Serially passaged cells grown in DMEM and treated with OS consistently expressed several bone-specific markers, such as ALP, BSP, Col 1, and OC, as well as elevated ALPase activity, thus confirming the retention of osteogenic potential as a function of passage. The loss of osteogenic differentiation potential of DMEM cell cultures was concurrent with the onset of cellular replicative senescence, which occurred between passages 10 and 12. In contrast, cells serially cultured in DMEM-F12K medium showed diminished osteogenic potential as early as passage 5 upon OS treatment. This was indicated by a reduced level of ALPase activity, as well as by lower mRNA levels of OC and ALP. Since DMEM and DMEM-F12K cultures in the absence of OS treatment maintained low basal levels of ALPase activity and failed to express any osteogenesis-, adipogenesis-, and chondrogenesis-specific genes besides limited levels of Col I, the cells are likely to have become senescent during extensive passage and lost the ability to become osteogenic upon treatment, thereby failing to make a lineage commitment when induced. This early onset of senescence in DMEM-F12K cultures may also have prevented them from responding to the mitogenic effects of the osteoinductive medium, thereby contributing to the lower levels of OS-specific mRNA and ALPase activity, as compared with the highly OS-responsive DMEM cultures.
Trabecular bone-derived cells cultured in DMEM were also capable of chondrogenic differentiation upon serial subculture up to an average of passage 10 before their ability to produce a proteoglycan-rich extracellular matrix began to diminish. The increased pellet culture size, extracellular matrix production, and morphology and orientation of cells derived from DMEM cultures compared with those derived from DMEM-F12K medium cultures can be attributed to the presence of a higher percentage of multipotent cells that are able to respond to chondrogenic induction upon serial subculture, similar to osteogenically and adipogenically induced DMEM cell cultures. Thus, the onset of senescence in DMEM and DMEM-F12K medium cultures, which began with passage 10 and 5 cultures, respectively, can be directly correlated to the loss of multipotentiality, the onset of which was induced more rapidly in the latter cultures.
In conclusion, we have identified the presence of a CD73+, STRO-1+, CD105+, CD34-, CD45-, CD144- cell population resident within collagenase-treated bone fragments, which upon migration established a homogenous population of cells with stem cell-like capabilities. We have also introduced a system of culturing trabecular bone-derived MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. Similar to human bone marrow-derived MPCs, these cells, when cultured as described, have the characteristics of mesenchymal progenitors, displaying a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate exclusively along the osteogenic, adipogenic, and chondrogenic lineages, even when maintained in long-term in vitro culture [17, 35–37]. It is also noteworthy that several other connective tissues, including adipose and striated muscle [38–40], harbor MPCs with multilineage differentiation potential. It would be of interest to assess the developmental relationship of MPCs obtained from these different sources. Studies using such an in vitro model should provide insights into the molecular and cellular events that occur during lineage-specific mesenchymal differentiation and demonstrate their potential application for the repair and regeneration of damaged or diseased tissues.
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ACKNOWLEDGMENT
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We would like to thank Dr. David Hall and Dr. Ulrich Nöth for their invaluable advice and technical assistance. Richard Tuli was supported in part by a Percival E. and Ethel Brown Foerderer Foundation fellowship from Thomas Jefferson University.
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Abstract
Introduction
