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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

To test the hypothesis that CD45lowCD271+ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional “fitness” in health and osteoarthritis (OA).

Methods

Following enzymatic extraction, MSC release was evaluated using colony-forming unit–fibroblast (CFU-F) and colony-forming unit–osteoblast assays, flow cytometry, and confocal microscopy. CD45lowCD271+ cells isolated by fluorescence-activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45lowCD271+ cells. In vitro and in vivo MSC “aging” was measured using quantitative polymerase chain reaction–based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality.

Results

Cellular isolates from trabecular bone cavities contained ∼65-fold more CD45lowCD271+ cells compared with aspirates (P < 0.0001) (median 1.89% [n = 39] and 0.029% [n = 46], respectively), concordant with increased CFU-F release. Aspirated and enzymatically released CD45lowCD271+ cells had identical MSC phenotypes (∼100% CD73+CD105+CD13+, ∼50–60% CD146+CD106+CD166+) and contained large proportions of highly clonogenic multipotential cells. In vitro osteogenic potency of freshly isolated CD45lowCD271+ cells was comparable with, and often above, that of early-passage MSCs (8–14%). Their frequency and in vivo telomere status in OA bone were similar to those in bone from age-matched controls.

Conclusion

Our findings show that CD45lowCD271+ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45lowCD271+ MSCs. In OA they display aging-related loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.

Bone marrow (BM)–derived cultured multipotential stromal cells (MSCs), which are also known as mesenchymal stem cells, are starting to be used clinically as cellular therapies for tissue repair and regeneration in orthopedic and related musculoskeletal disorders (1). However, clinical outcomes are often inconsistent and unpredictable, due at least in part to an absence of standardized consensus protocols for MSC isolation and expansion. Industrial practices in which purity and potency of MSC preparations are controlled from the beginning and at each stage of manufacture could be critical for improved treatment development (2). Poor understanding of the biology of MSCs in vivo and the inability to purify sufficient numbers of MSCs to characterize and control early stages of cultivation hinder progress in this area.

The dogma that BM MSCs are exceptionally rare has dominated the MSC field for several decades (3, 4). Hence, in vitro cultivation of MSCs was deemed necessary, not only for therapeutic exploitation, but also for basic molecular studies in health and disease. However, the assumption that BM aspiration procures all BM MSCs ignores the fact that their recovery from virtually all other tissue types requires an initial breakdown of extracellular matrix (5–9). Several recent investigations have shown that large numbers of colony-forming unit–fibroblasts (CFU-Fs) giving rise to cultures “virtually identical” to MSCs could be obtained by enzymatic extraction from trabecular bone cavities and/or endosteal surfaces (10–12). However, the phenotype of the extracted cells and their relationship to aspirated BM MSCs remain unknown.

We and others have shown that aspirated in vivo BM MSCs reside within the CD45−/lowCD271+ stromal cell fraction (13–18), denoted here as CD45lowCD271+. Fluorescence-activated cell sorter–purified CD45lowCD271+ cells resemble adventitial reticular cells (ARCs) (19), which are bona fide specialized pericytes of venous sinusoids in the marrow (20–22). Paradoxically, while MSCs are deemed to be rare, ARCs are known to be quite abundant in situ (23). Based on the above reasoning, we hypothesized that not all CD45lowCD271+ cells are easily aspirated, and therefore their complete extraction from local niches requires a breakdown of neighboring extracellular matrix.

We further speculated that the prospect of extracting large numbers of native noncultured MSCs could open up new avenues for the study of in vivo MSCs in health and osteoarthritis (OA). In OA, pathologic formation of bony structures (osteophytes) and subchondral bone thickening suggest increased osteogenic activity of MSCs, at least in the areas adjacent to cartilage damage. To date, results of analyses of culture-expanded MSCs derived from femoral heads of OA patients have been conflicting, revealing either no abnormality (24) or skewed differentiation capacities (25). We reasoned that reported discrepancies were most likely due to artifacts from variable culture manipulations, and hence we aimed to compare the frequencies and proliferative potentials of CD45lowCD271+ cells directly extracted from OA and normal bone. This knowledge could be very important for future bone repair strategies, since large amounts of bone, which represent a potentially useful source of MSCs, are routinely available and often discarded at the time of joint replacement surgery in OA.

In the current study, we found an ∼65-fold greater abundance of CD45lowCD271+ cells in fractions enzymatically extracted from bone fragments compared with aspirates. By extended phenotypic analysis using common MSC markers and by assessment of proliferative and differentiation capacities at the clonogenic cell level, we established that the aspirated and enzymatically extracted CD45lowCD271+ cell population had the same cellular identity. In OA hip joints, the frequency of CD45lowCD271+ cells was within the range of that in normal trabecular bone, and their telomere status, indicative of native in vivo age, was similar to that in aged healthy individuals. These findings showed that marrow occupying trabecular bone cavities is a rich source of MSCs. Their large-scale extraction based on the CD45lowCD271+ phenotype, short-cutting or minimizing culture manipulations, offers an excellent tool for studying these cells in health and disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patients and cells.

Ethics permission for specimen collection was obtained from the Leeds National Health Service Trust Ethics Committee. BM aspirates were obtained from 42 normal donors (median age 15 years [range 2–61]), and mononuclear cells (MNCs) were isolated using Lymphoprep (Axis-Shield). Additionally, frozen MNCs from 22 normal subjects (median age 22 years [range 19–39]) were purchased from Lonza. The average cellularity of BM aspirates was 107 MNCs/ml. Trabecular bone fragments were collected from the pelvic area of 20 trauma patients (median age 52 years [range 17–86]); none had underlying disease, and the samples were considered normal. Matched BM aspirates and bone samples were collected from the pelvic area of 6 trauma patients (median age 47 years [range 25–61]). For comparisons between health and OA, femoral heads were obtained at the time of joint replacement surgery, from 52 patients with hip OA (median age 64 years [range 43–91]).

Enzymatic digestion to release increased numbers of MSCs.

Small bone fragments (median 0.1 gm [range 0.05–0.3]) were produced manually using a 4-mm punch biopsy tool, or by mincing large fragments using a bone mill (DePuy International). Fragments were washed in 5 ml phosphate buffered saline (PBS) and placed, for 5 hours at 37oC, in 0.5 ml of 0.25% collagenase (Stem Cell Technologies). This corresponded to ∼250–375 units of enzyme per fragment. A rotator was used to ensure continuous mixing of sample with collagenase. At the end of digestion, collagenase solution was diluted with a >20-fold (volume/volume) excess of PBS, filtered through a 70-μm filter (BD), and the liquid fraction containing the cells was centrifuged. The cell pellet was resuspended in standard freezing medium containing 10% DMSO (Sigma), and cells were stored in liquid nitrogen prior to use. The average cellularity of enzymatically released fractions was 6.5 × 107 cells/gm of bone.

Confocal microscopy and scanning electron microscopy (SEM) of enzymatically treated trabecular bone fragments.

To monitor the completeness of cell release from trabecular bone fragments after 5-hour enzymatic treatment, fragments were fixed with 10% formalin, stained for 30 minutes with 0.5 mM calcein (Sigma) to visualize bone surfaces (26), washed twice with PBS, and further treated with 5 mM ToPro-3 (Invitrogen) to stain for cell nuclei. Images were acquired and analyzed using a TCS SP2 CLSM confocal microscope (Leica Microsystems). Residual DNA was measured using a commercial PicoGreen reagent (Invitrogen).

For SEM studies, biopsy samples were dehydrated in 50%, 70%, 90%, and 100% series of ethyl alcohol (30 minutes each), dried overnight in a vacuum dessicator, and mounted on aluminium stubs. Gold coat (20 nm thick) was applied using an Agar Auto sputter coater (Agar Scientific), and samples were analyzed using a Jeol JSM35 SEM with Genie 3000 operating software.

CD45lowCD271+ cell isolation from BM aspirates and trabecular bone digests and MSC expansion.

Aspirated BM MNCs or enzymatically released cell fractions were thawed in media containing DNase (20 units/ml). The MSC-enriched fraction was isolated following positive selection with Anti-Fibroblast Microbeads (Miltenyi Biotec) and subsequent sorting for the CD45lowCD271+ cell population (4, 13), using fluorescein isothiocyanate (FITC)–conjugated CD45 (Dako), phycoerythrin-conjugated CD271 (BD), and propidium iodide (Sigma) (to eliminate dead/dying cells) on a FACSVantage cell sorter (Becton Dickinson). For analysis of clonogenicity, 500 CD45lowCD271+ cells were seeded in 35-mm plastic dishes, grown in NH expansion medium (Miltenyi Biotec), and their CFU-F potential was assessed on day 14 using Crystal Violet staining, as described previously (10). Additionally, single CD45lowCD271+ cells were seeded at limiting-dilution density into 96-well plates (27, 28) to generate single cell–derived clones, which were grown for a minimum of 25 population doublings (PDs). Clones were expanded upon reaching 80% confluence, using a plating density of 104 cells/ml.

Polyclonal cultures were grown from 103 sorted CD45lowCD271+ cells that were initially seeded into 25-cm2 flasks and similarly expanded for a minimum of 25 PDs. Accrued PDs for polyclonal cultures at any time point were calculated according to the formula log2(n total cells/total CFU-F on day 0).

Osteoprogenitor capacity of CD45lowCD271+ cells in BM aspirates and enzymatic digests of normal and OA bone.

For analysis of the osteoprogenitor content of noncultured CD45lowCD271+ cells, a clonal osteogenesis assay adapted from the one described by Sakaguchi et al (29) was used. This assay allowed the assessment of osteogenesis from rare aspirated CD45lowCD271+ cells (500 cells/10 cm2). Seeded cells were grown for 8 days in NH medium followed by 14 days under osteoinductive conditions. At the end of culture, CFU-osteoblasts (CFU-Os) were scored following standard immunochemical staining for alkaline phosphatase (28). The same clonal osteogenesis assay was used to monitor CFU-F/CFU-O dynamics in standard MSC cultures derived from CD45lowCD271+ cells from matched donors. Additionally, standard in vitro trilineage differentiation assays on polyclonal and clonal cultures were performed as previously described (28).

Extended phenotyping of aspirated and enzymatically extracted CD45lowCD271+ cells and resulting MSC cultures.

Marker phenotyping of CD45lowCD271+ cells was performed on a BD LSRII flow cytometer, using 4-color flow cytometry. Due to their extreme rarity in BM aspirates (3), CD45lowCD271+ cells were pre-enriched with D7-FIB MicroBeads. FITC-conjugated CD45/allophycocyanin-conjugated CD271 (Miltenyi Biotec) was used as the gating combination. Bone digests contained higher proportions of CD45lowCD271+ cells, eliminating the need for MicroBead pre-enrichment. Test antibodies were CD73, CD106, CD146, CD166 (all from BD), CD105, and CD13 (both from Serotec). Extended phenotyping of polyclonal and clonal cultures was performed as previously described (28). Each culture was analyzed at different passages, using an average of 6 time points (3 before reaching 20 PDs and 3 after reaching 20 PDs). This was done to monitor marker expression as a function of the in vitro “age” of the cultures (30).

Relative telomere length assay using quantitative real-time polymerase chain reaction (PCR).

Telomere length measurements were used to assess MSC aging status, both in vitro (31) and “in vivo” (from noncultured CD45lowCD271+ cells). Genomic DNA from experimental samples and reference cell lines (Jurkat and K562) was purified using a Norgen RNA/DNA/Protein Purification Kit (Geneflow) and stored at −20oC until analysis. Relative telomere lengths were measured by quantitative real-time PCR amplification of telomere repeats (T) in relation to a single-copy gene, 36B4 (S) (32). The method was adapted from those described by Cawthon (32) and Guillot et al (33). T and S standard curves were generated using serial dilutions of DNA (50 ng to 6.25 ng) from the Jurkat and K562 cells. Relative telomere length was calculated as the T:S ratio, which is proportional to the mean restriction fragment telomere length, according to the formula (2Ct[telomere]/2Ct[36B4])−1 (32). The telomere and 36B4 quantitative PCRs were run on separate plates, consecutively, on the ABI 7900HT Real Time System (Applied Biosystems); standard curves were included in each run to allow relative quantification between plates. Samples were run in triplicate using 20 ng DNA. The 25-μl reactions were carried out using 2× SYBR Green PCR Master Mix (Applied Biosystems) and primers Tel1 (270 nM), Tel2 (900 nM), 36B4u (300 nM), and 36B4d (500 nM) (MWG Biotech). Thermal cycling conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 54°C for 2 minutes for telomere quantitative PCR, and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 58°C for 1 minute for 36B4 quantitative PCR.

Statistical analysis.

The Mann-Whitney U test for 2 independent samples was used to compare groups. Donor-matched samples were compared by Wilcoxon's signed rank test. Spearman's rank correlation coefficient was used to correlate variables. P values less than 0.05 and r values greater than 0.6 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Proliferative and osteogenic potencies of cultured MSCs and fresh CD45lowCD271+ cells.

Consistent with the results of previous studies (31, 34, 35), prolonged (>20 PDs) MSC expansion under standard conditions led to a significant decline in the CFU-F/CFU-O content of cultures, from an average of 12% CFU-F/9% CFU-O to 2 CFU-F/1% CFU-O (Figure 1A). A significant decline in the proportions of cells expressing self-renewing osteoprogenitor/MSC markers CD146 and CD106 (22, 27, 36) was also observed (Figure 1B). In contrast, CD73 expression remained stable (Figure 1B) and did not reflect loss of CFU-F/CFU-O potency. Similarly, stable expression of CD105 was observed (data not shown). An observed trend of telomere erosion during extended MSC cultivation (Figure 1B) further reflected the “aging” processes in cultured MSCs.

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Figure 1. Proliferative and osteogenic potencies of cultured multipotential stromal cells (MSCs) and fresh CD45lowCD271+ cells. A, Decline in colony-forming unit–fibroblasts (CFU-Fs) and CFU-osteoblasts (CFU-Os) upon prolonged MSC cultivation. Assays were established from 500 cultured MSCs (cultured for <20 population doublings [PDs] or >20 PDs). Values are the mean and SD. ∗ = P < 0.05. B, Decline in expression of CD146 and CD106 with increased accrued PDs (upper panels), and stability of CD73 expression despite evident telomere erosion with increased accrued PDs (lower panels). Telomore erosion was evidenced by the ratio of telomere repeats (T) to a single-copy gene 36B4 (S). The T:S ratios for the control cell lines Jurkat and K562 were 1.02 and 0.91, respectively. C, Osteogenic (purple) and nonosteogenic (brown) colonies, grown under osteoinductive conditions, from 500 CD45lowCD271+ cells (far left, accrued PDs = 0) or from 500 cultured MSCs at “ages” 12, 17, 22, and 27 PDs (representative bone marrow [BM] donor, age 9 years). Note the superior proliferative potential of freshly isolated CD45lowCD271+ cells compared with their significantly expanded progeny. MSCs and CD45lowCD271+ cells were obtained from iliac crest BM aspirates.

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Next, we tested the CFU-F/CFU-O potencies of CD45lowCD271+ cells freshly isolated from BM aspirates. We found that their CFU-F and CFU-O frequencies were above those of early-passage cultures (median 16% [range 14–19%] and 12% [range 8–14%], respectively; n = 4 donors). As seen in the representative experiment from a single donor depicted in Figure 1C, freshly isolated CD45lowCD271+ cells (denoted as accrued PDs = 0) had the longest innate proliferative potential, which in cultured MSCs is inversely related to their division history in vitro. Based on these data we concluded that freshly isolated CD45lowCD271+ cells could be suitable candidates for direct therapeutic exploitation. However, their low numbers in aspirates preclude such use, since relatively large seeding MSC numbers are required for current protocols (37).

Increased numbers of CD45lowCD271+ cells in enzymatic digests of bone.

In accordance with previous findings (10, 12), cell fractions released from bone cavities after enzymatic treatment contained more CFU-Fs than did BM aspirates (∼15-fold) (Figure 2A). The frequency of CD45lowCD271+ stromal cells was correspondingly increased (∼65-fold, from a median of 0.029% [n = 46] to a median of 1.89% [n = 39]), reaching as high as ∼6% of total released cells (Figures 2A and B). The liberation of CD45lowCD271+ cells occurred in a time-dependent manner and reached a maximum after 5 hours of incubation (data not shown). After 5 hours of treatment, marrow pockets were completely removed from bone cavities and the majority of bone-lining cells were liberated (Figure 2C). Only occasional lining cells were seen remaining on the bone, corresponding to a minor fraction of total released DNA (median 3.2% [range 0.3–4.0%]; n = 10). Using this procedure, 0.1 gm of trabecular bone biopsy material yielded ∼1.3 × 105 CD45lowCD271+cells, equivalent to ∼43 ml of marrow aspirate.

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Figure 2. Enzymatic extraction of CD45lowCD271+ cells from trabecular bone cavity. A, Massively increased recovery of CFU-Fs and CD45lowCD271+ cells following enzymatic digestion from trabecular bone. Bars show the median. ∗∗∗ = P < 0.0001. B, Representative flow cytometry plots both from the same normal subject, showing the high frequency of CD45lowCD271+ cells in an enzymatic digest of iliac crest bone (right) compared with BM aspirate (left). C, Confocal microscopy images (upper panels) and scanning electron microscopy (SEM) images (lower panels) of trabecular bone surfaces, illustrating the complete removal of marrow pockets and bone-lining cells after enzymatic digestion (right panels) versus before enzymatic digestion (left panels). Calcein staining of bone appears in green; 4′,6-diamidino-2-phenylindole staining of cell nuclei appears in blue. The SEM images illustrate matrix-rich, smooth, cell-lined bone surfaces before enzymatic digestion, as opposed to bare bone after enzymatic digestion. Bars = 100 μm. A = aspiration; ER = enzymatic release (see Figure 1 for other definitions).

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Growth kinetics and differentiation capacity of CD45lowCD271+ cells obtained by aspiration or enzymatic digestion.

Theoretically, enzymatically released CD45lowCD271+ cells could be functionally inferior to aspirated CD45lowCD271+ cells. Therefore, we compared their proliferative potential and growth characteristics upon expansion under standard conditions. The initial CFU-F content of aspirated and enzymatically released CD45lowCD271+ cells was similar (median 16% [range 14–19%] and median 16% [range 9–25%] for aspirated and enzymatically released cells, respectively; n = 4 in each group).

Culture growth rates were measured as days/PD. No statistically significant differences were found between cultures established with aspirated versus enzymatically released CD45lowCD271+ cells (median 3.5 days and 3.8 days, respectively); however, we observed a direct positive correlation between PD time and donor age (Figure 3A). Consequently, growth rates were further validated using age- and donor-matched samples. No differences in the growth rates of age-matched samples were found (Figure 3A). Furthermore, when clonal MSC cultures (capable of at least 20 PDs) were established from these samples, both the frequencies of highly clonogenic cells and the growth rates of resulting clonal cultures were similar (Figure 3B). Assessment of the growth rates of donor-matched samples further confirmed the lack of differences between aspirated and enzymatically released CD45lowCD271+ cells (Figure 3C).

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Figure 3. Growth kinetics of enzymatically released CD45lowCD271+ cells. A, Growth kinetics of polyclonal cultures derived from aspirated CD45lowCD271+ cells (solid circles) and enzymatically released CD45lowCD271+ cells (open circles). Relationship with donor age is shown in the left panel; results obtained with age-matched samples (28 years old) are shown in the right panel. B, Growth rates of MSC clones derived from age-matched aspirated (A) and enzymatically released (ER) CD45lowCD271+ cells. The frequencies of highly clonogenic cells were similar (22 and 20 per 500 seeded cells, respectively), as were the distributions of fast- and slow-growing clones. C, Growth rates of 6 donor-matched aspirated and enzymatically released CD45lowCD271+ cells. See Figure 1 for other definitions.

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The MSC nature of polyclonal and clonal cultures derived from enzymatically released CD45lowCD271+ cells was confirmed by standard assays of tripotentiality. Representative photomicrographs are shown in Figures 4A–C. Clonal MSC cultures were slightly more potent than polyclonal cultures; however, the differences in differentiation levels (measured as sulfated glycosaminoglycan, Ca++, and adipocyte production) were not significant (Figure 4D). Taken together, these data showed that enzymatically released CD45lowCD271+ cells were clonogenic and tripotential and that their proliferative capacities were similar to those of age-matched aspirated CD45lowCD271+ cells.

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Figure 4. A–C, Tripotentiality of standard polyclonal (B) and clonal (C) cultures derived from enzymatically released CD45lowCD271+ cells from a healthy 28-year-old donor compared with polyclonal MSCs derived from aspirated CD45lowCD271+ cells from an age-matched control (A). Left panels show chondrogenesis (toluidine blue staining), middle panels show osteogenesis (alkaline phosphatase staining), and right panels show adipogenesis (oil red O staining). Photomicrographs were taken on day 21 postinduction. Polyclonal and clonal cultures were tested at the time of 12–15 PDs and 20 PDs, respectively (original magnification × 100). D, Quantitative analysis of tripotentiality of cultures derived from enzymatically released CD45lowCD271+ cells. At least 4 donor-derived cultures were used for each test. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. sGAG = sulfated glycosaminoglycan (see Figure 1 for other definitions).

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Phenotypic comparison between CD45lowCD271+ cells obtained by aspiration and those obtained by enzymatic release.

In the next set of experiments, 4-color flow cytometry was used to compare the expression of a panel of MSC markers (3, 13–15, 22, 36, 38, 39) on noncultured CD45lowCD271+ cells (aspirated and enzymatically released), to further explore their extended phenotype. No significant differences in expression patterns were found (Figure 5A). The majority of cells were positive for MSC markers CD73 and CD105, and ∼50–60% of cells were positive for CD146, CD106, and CD166.

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Figure 5. MSC marker phenotype of enzymatically released CD45lowCD271+ cells (A) and their polyclonal (B) and clonal (C) progeny in comparison with aspirated CD45lowCD271+ cells. Solid and open bars represent aspirated and enzymatically released cells, respectively. Values are the mean and SD from a minimum of 4 donors. Bottom panels show marker histograms and marker positivity values for enzymatically released CD45lowCD271+ cells from a representative donor. Polyclonal and clonal cultures were tested at the time of 12–15 PDs and 20 PDs, respectively. See Figure 1 for definitions.

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We then investigated the phenotype of polyclonal and clonal MSC cultures derived from aspirated or enzymatically released CD45lowCD271+ cells (tested at 12–15 PDs and 20 PDs, respectively) (Figure 5B and C). No significant differences in the extended phenotype of any cultures were found. Therefore, with the panel of markers tested, CD45lowCD271+ cells (and their progeny) were phenotypically identical, regardless of the method of extraction used (aspiration or enzymatic release).

Frequencies and osteoprogenitor capacities of CD45lowCD271+ cells in healthy and OA trabecular bone.

Having established that large numbers of CD45lowCD271+ cells containing MSCs are present in normal trabecular bone, we next explored whether OA bone could represent a potentially abundant source of autologous MSCs, since the MSC yield could be substantially greater given the large volume of bone removed at joint replacement. The frequency of CD45lowCD271+ cells released from OA femoral heads was within the range of that found in normal bone (range 0.31–8% and 0.33–5.6%, respectively), and was not related to donor age in the age group studied (Figure 6A). Concordantly, no deficiency in released CFU-Fs was found in OA bone (range 120–2,580/106 cells and 250–2,158/106 cells in OA and normal controls, respectively; n = 10 donors in each group).

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Figure 6. Frequencies and functional fitness of CD45lowCD271+ stromal cells in osteoarthritic (OA) and normal (N) aged bone. A, Similar frequency of CD45lowCD271+ stromal cells, in relation to donor age, in enzymatic digests from OA femoral heads and normal trabecular bone. B, Osteogenic (purple) and nonosteogenic (brown) colonies, grown under osteoinductive conditions, from 500 CD45lowCD271+ cells (top) or their progeny at “ages” 12 and 16 PDs (representative age-matched donors, 67 years old). C, In vitro longevity of CD45lowCD271+ cells grown under standard MSC conditions, in relation to donor age. D, In vivo telomere status of CD45lowCD271+ cells in relation to donor age. Note the parallel trends depicted in C and D. A = aspirated; ER = enzymatically released (see Figure 1 for other definitions).

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Similar to findings in age-matched controls, osteogenic progenitors (CFU-O) were present within the CD45lowCD271+ cell population from OA femoral heads (freshly isolated) (Figure 6B). Following standard cultivation of normal “aged” or OA-derived CD45lowCD271+ cells, the CFU-O frequency declined at a similar rate (expanded for 12 PDs and 16 PDs) (Figure 6B), which was more rapid compared with the rate in samples from normal younger donors (see Figure 1C). Concordantly, MSCs derived from older and OA donors approached senescence more rapidly than MSCs from younger donors (Figure 6C).

In accordance with the functional data from normal and OA samples, similar age-related trends were observed following measurements of telomere lengths in native noncultured CD45lowCD271+ cells (Figure 6D). CD45lowCD271+ cells from younger individuals (<40 years old) had longer telomeres, consistent with the longevity results from the cultures (Figures 6B and C), whereas telomeres in CD45lowCD271+ cells extracted from OA or aged normal bone were shorter (Figure 6D). These findings suggested that in terms of innate osteogenic and proliferative capacities, BM MSCs from patients with OA were functionally similar to MSCs from aged normal subjects.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

This study addressed the hypothesis that complete extraction of MSCs from their intrabone niches requires enzymatic breakdown of extracellular matrix. The results show, for the first time, that marrow aspiration from an iliac crest anatomic niche procures only a minority of resident MSCs. Enhanced CFU-F release was described previously (10); however, it was not linked to a specific cell phenotype. We enumerated and comprehensively assessed CD45lowCD271+ MSCs from BM aspirates and trabecular bone digests and confirmed that they had the same cellular identity. We also demonstrated, for the first time, that freshly isolated noncultured CD45lowCD271+ cells were at least as proliferative, if not more proliferative, and as osteogenic as early-passage MSCs and that they were significantly more potent than late-passage MSCs. These data provide scientific rationale for the use of noncultured or minimally expanded MSCs for bone repair applications, and explain the good therapeutic outcomes that have been observed in previous investigations using fresh MSCs (34, 40, 41).

To our knowledge, this is also the first study to explore in vivo aging in native MSCs both in health and in disease. Our findings suggest that the loss of proliferative potency in aging and OA is likely to be related to the “in vivo age” of native MSCs, and not to a reduction in their in vivo numbers. Furthermore, given the numeric, functional, and telomeric data, which were virtually identical between samples from aged healthy individuals and those from OA patients, our findings help to explain why prominent new bone formation may be a feature of OA, as no gross defect in MSC osteogenic differentiation was evident.

In this study we used a broadly accepted MSC/ARC marker, CD271 (13, 15, 17, 18, 38), to identify the multipotential marrow stromal cell population in vivo. We acknowledge that CD271 is down-regulated in culture (13) and that ∼10% of CD45lowCD271+ cells are truly colony-forming under the conditions used; however, all of these cells expressed MSC markers CD73 and CD105, and ∼50% strongly expressed CD146 and CD106 (22, 27). Their ability to adhere to plastic, another MSC criterion, has been shown in our previous work (4, 13).

In the present work we demonstrated, for the first time, a decline in CD146 and CD106 expression as MSC cultures “aged” in vitro, suggesting a possible enrichment of the most clonogenic/self-renewing in vivo MSCs within a narrower CD271+CD146+CD106+ phenotype. Preliminary sorting experiments, however, have revealed no gross differences in the clonogenicity of CD146+ and CD146− subsets within the CD45lowCD271+ population (42), and further elucidation of cell heterogeneity in vivo was outside the scope of this study. Overall, the findings of our comprehensive cytometric analysis are fully consistent with recent evidence that MSCs and hematopoietic niche-forming stromal cells have overlapping in vivo phenotypes (22, 43). The “niche” for MSCs themselves is currently viewed as being perivascular (21, 22). However, given the abundance of MSCs isolated in the present study, our findings raise the possibility that the MSC niche may also extend to the bone surfaces, where MSCs may be involved in both the support of hematopoietic stem cells and the production of resident osteoblasts.

Our findings offer new possibilities for large-scale extraction of native MSCs as an “instant” cell therapy for bone fractures (44). One recent study showed that as few as 5 × 104 autologous CFU-Fs were needed to induce successful repair of nonunion fractures (41). According to our calculations, this equates to ∼5 × 105 CD45lowCD271+ cells, which could be extracted from ∼150–300 ml of marrow aspirate or from as little as ∼0.3–0.6 gm of trabecular bone. Marrow volume reduction techniques and implanting of cell concentrates into fracture sites have become popular in orthopedic surgery for trauma (44, 45), and harvesting and processing by-product bone fragments, which are normally discarded, may prove beneficial. Simple measurement of the proportion of CD45lowCD271+ cells in fresh cell isolates could be used for quality control of their MSC content for therapy (46). In contrast to retrospective CFU-F assay, almost instantaneous cytometric assessment would ensure that a therapeutic dose of “active” cells could be adjusted before implantation, which is currently not possible.

Repairing larger bone defects requires tissue engineering approaches based on scaffolds and much higher numbers of MSCs (47). In these settings, the large-scale extraction of MSCs described herein would allow therapeutically relevant numbers of MSCs to be obtained within a much shorter time frame. Moreover, such “minimal expansion” is likely to yield higher-potency MSCs, since their combined proliferative and osteogenic capacities clearly relate to the number of accrued PDs (35, 48, 49), as was fully confirmed in this study. Similarly, minimally passaged MSCs ought to cope better with oxidative stress and have higher multilineage differentiation capacity (30). New approaches based on noncultured or minimally expanded MSCs appear to be practical and are likely to be cost-effective; however, we acknowledge that the availability of safe, good manufacturing practice–compatible enzymes would be a critical factor in enabling them to enter the clinic.

Hematologists have now begun to study freshly extracted MSCs for molecular and cytogenetic analysis of potential abnormalities in disease (50). Widespread utilization of this approach is as yet precluded by the complexity of the techniques required to capture and analyze rare cells. The present results provide a much simpler “toolbox” for investigating native MSCs ex vivo and, using the OA disease model, demonstrate the feasibility of studying MSCs without the requirement for in vitro culture. Using a quantitative PCR–based method for measuring telomere lengths in native CD45lowCD271+ cells, we found a trend toward age-related telomere erosion in vivo. However, no further decline was found in OA bone, and the proliferative span of OA MSCs was similar to that of normal aged control bone. Based on our findings we envisage a technology whereby prospectively enriched autologous MSC isolates from removed femoral heads of OA patients are banked for potential future use in revision surgery, either to strengthen the bone–implant interface or to build up new osteochondral tissue. Theoretically, as many as 108 autologous CD45lowCD271+ cells could be obtained from an average-sized femoral head (median 76 gm). We acknowledge that allogeneic MSCs from younger donors could be more potent; however, autologous isolates would be safer from an immunologic and regulatory perspective. Similarly, the present findings enable assessment of potential MSC dysfunctions in osteoporosis and other bone diseases, avoiding artifacts that are inherent to in vitro culture–based procedures.

In summary, we have shown that CD45lowCD271+ MSCs are abundant in trabecular bone cavities, opening new avenues for direct study of these cells in musculoskeletal diseases. Investigations to determine their abundance in human cortical bone shaft and fatty marrow are ongoing. Furthermore, our study results provide a rationale for the use of native and minimally expanded MSCs as cell therapy. This strategy, which avoids the loss of potency associated with extended culture and involves significantly shorter in vitro manipulations, is likely to be more efficient and cost-effective. Future clinical trials and in vivo animal studies will determine the efficacy of such therapies in comparison with standard expanded MSCs.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Jones had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Jones, English, Kinsey, Emery, McGonagle.

Acquisition of data. Jones, English, Churchman, Kouroupis, Boxall, Kinsey, Giannoudis, McGonagle.

Analysis and interpretation of data. Jones, English, Churchman, Kouroupis, McGonagle.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Drs. Brian Flood, David Macdonald, Geoff Shenton, Ippokratis Pountos, Argyris Papathanasopoulos, and George Cox for providing valuable clinical samples, Mrs. Jackie Hudson for help with confocal microscopy and SEM, and Dr. Mostafa Raif for DNA analysis of bone chips. We are also very grateful to Dr. Ilaria Bellantuono for helpful discussion on the topic of telomere and provision of control cell lines for telomere length analysis.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES