AbstractLife-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271brightCD146− and CD271brightCD146+ subsets correlates with donor age. The main subset in adults was CD271brightCD146−, whereas the CD271brightCD146+ population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271−CD146+ cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.
Mesenchymal stromal cells (MSC) cultured from adult and fetal tissues constitute a heterogeneous cell population. Although a panel of markers, including CD105 (Endoglin) and CD90 (Thy-1), was introduced to define cultured MSC,1 the cells initiating the culture remain unidentified. Recently, the low-affinity nerve growth factor receptor CD2712–4 and melanoma cell adhesion molecule CD1465,6 were described for prospective isolation of MSC with colony forming unit-fibroblast capacity. The combination of these markers revealed that two different MSC subsets are present in adult BM: CD271CD146 and CD271CD146 These subsets had a similar capacity to differentiate and to support hematopoiesis, but in human BM they were found at different sites; CD271/CD146 cells were bone-lining, while CD271CD146 had a perivascular localization.7 It is still not clear whether the contribution of these subsets to the MSC population changes during development and aging, two phenomena known to influence BM cellular composition.8–10 Here, we report that MSC subset distribution, characterized by CD271 and CD146, correlates with donor age. CD271CD146 cells were most common in adults, whereas CD271CD146 cells are dominant in children. In addition, fetal CD271CD146 cells represent a third MSC subset containing CFU-F.
Design and Methods
Bone marrow was aspirated from 53 adults (mean age 65 years, range 19–86 years). Ten were healthy donors and 43 were patients undergoing cardiac surgery; 27 pediatric patients (mean age 5 years, range 0–16 years) had malignancies (solid n=17, hematologic n=10). Patients’ characteristics are listed in the Online Supplementary Table S1. Because we experienced a large variation in BM aspirate quality, we decided to analyze the relative distribution of MSC subsets. Fetal bones (n=6) were obtained from legally terminated pregnancies (15–20 weeks gestation). Informed consent was obtained for all samples according to the protocol approved by the Medical Research Ethics Committee of the AMC and the Erasmus MC.
Mononuclear cells (MNC) were isolated by Ficoll density gradient centrifugation (GE Health Care Bio-Sciences AB, Uppsala, Sweden) and erythrocyte lysis using NH4Cl. Fetal bones were flushed with IMDM (Lonza, Verviers, Belgium) containing 10% FCS (Bodinco, Alkmaar, The Netherlands), 1% penicillin-streptomycin and erythrocytes were lysed.
MNC were analyzed for expression of: CD34 (clone 8G12), CD73 (AD2), CD90 (5E10), CD45 (HI30), CD140b (PDGFRβ, 28D4), CD146 (P1H12), CXCR4 (12G5), (all from BD, San Jose CA), CD36 (CLB-IVC7, Sanquin, Amsterdam, The Netherlands), CD271 (ME20.4-1.H4), MSCA1 (W8B2) (Miltenyi Biotec, Gladbach, Germany), CXCR7 (358426), NG2 (LHM-2) (R&D systems, Abingdon, UK), CD56 (C5.9), CD235a (JC159) (Dako Cambridgeshire, UK), CD105 (SN6, Ancell, Bayport, MN, USA), isotype matched controls (Sanquin, BD, USA). At least 1,000,000 events were recorded using an LSR II flow cytometer (BD, USA). For six-color sorting (FACS Aria, BD, USA), MNC were stained for CD45, CD34, CD271, CD146, CD105, CD90.
For CFU-F assays, 500 sorted cells/cm were plated in M199 (Gibco, Paisley, UK) containing 10% FCS, 1% penicillin-streptomycin, 20 μg/mL Endothelial Cell Growth factor (Roche Diagnostics, Indianapolis, IN, USA) and 8IU/ml heparin (Leo Pharma, Breda, The Netherlands). For CFU-F with MNC, 20,000–80,000 adult or pediatric cells/cm or 2,500–20,000 fetal cells/cm were plated. For depletion experiments, 80,000 cells/cm were seeded. CFU-F were counted after 14 days.
RNA was isolated using Trizol (Invitrogen, Breda, The Netherlands). cDNA was synthesized using Sensiscript (Qiagen, Venlo, The Netherlands). Primer- and probe-sequences: Nestin (5′>3′) F-GCTGCGGGCTACTGAAAAGT, R-TCTGTAGGCC-CTGTTTCTCCTG, Probe AGCTGGCTGTGGAGGCCCTGG. ABL sequences were published previously.11
For MSC expansion, CFU-F were trypsinized and replated at 2,500 cells/cm in M199. The cells were passaged at 70–80% confluency.
Passage three MSC were used for adipogenic and osteogenic differentiation, as previously described,12 and imaged on a Leica DC300 microscope (IM500 software, 20X magnification, Leica, Wetzlar, Germany). Chondrocyte differentiation was performed using NH ChondroDiff medium (Miltenyi), detected by aggrecan-staining (MAB19310, Millipore, Amsterdam, The Netherlands) and imaged on an LSM 510 META confocal microscope (Zeiss, Jena, Germany; ZEN 2007 software, 10X magnification).
Statistical significance was determined by Mann-Whitney’s U test, Wilcoxon’s signed rank test or Spearman’s correlation (SPSS 15.0; SPSS Inc, Chicago, IL, USA). Results were considered significant at P≤0.05.
Results and Discussion
We used the differential distribution of CD271 and CD146 to analyze MSC subsets in human BM at distinct phases during development and aging. We first analyzed the distribution of MSC populations in CD45CD34cells in adult BM (n=53, mean age 65 years, range 19–86 years) and identified three subpopulations: CD271CD146, CD271CD146 and CD271CD146 (Figure 1A and B). Only the CD271 populations contained CFU-F, indicative for functional MSC, with a tendency towards more CFU-F in CD271CD146 cells (P<0.072; Figure 1C). CFU-F were absent in CD271-CD146 and CD271CD146 fractions, except for one sample (Figure 1C) in accordance with Tormin et al.7 Depletion of CD271CD146 did not reduce CFU-F from the remaining BM cells, while CFU-F were absent in CD271 depleted MNC cells (Figure 1D and E). Thus CFU-F are only present in CD271 cells and clonogenic MSC do not require support from other BM-derived cells.
Expanded CD271 subsets showed trilineage differentiation potential in vitro (Figure 1F) and maintained expression of MSC markers CD73, CD90 and CD105 (data not shown) fulfilling the ISCT criteria.1 Next, the expression of recently described MSC markers, including Nestin,13 CD140b (PDGFRβ),3 CD562 and MSCA-12 was analyzed. High levels of Nestin mRNA were detected in CD271CD146 and CD271CD146 cells. Far less Nestin mRNA was detected in culture-expanded MSC (Figure 1G). Both subsets contained CD56- and CD140b-positive subpopulations. Uniform expression was observed for MSCA-1 and chemokine receptors CXCR4 and CXCR7; however, only 15–40% of the cells expressed CXCR4 and CXCR7 above background (Figure 1H).
Cellular composition of BM changes during development9 and aging.8,10,14 The frequency of CFU-F declines with age,8 but the effect on the composition of the MSC compartment is unknown. Considering all adult BM samples, the largest subpopulation that co-expressed CD90 and CD105 was CD271CD146 (Online Supplementary Figure S1A; n=53, 60.4±3.0%; 0.011±0.001% of nucleated cells), whereas CD271CD146 and CD271CD146 populations accounted for 30.6±2.9% (0.006±0.001%) and 9.4±2.1% (0.003±0.001%), respectively (Online Supplementary Figure S1A).
When separating samples from younger adults (19–55 years, i.e. the eligible age for stem cell donors; www.europ-donor.nl) and elderly adult donors (>55 years), it was observed that CD271CD146 and CD271CD146 occurred at a similar frequency in younger adults whereas the CD271CD146 fraction was significantly reduced in elderly adults (Online Supplementary Figure S1B and C). This suggests that CD146 expression on CD271 cells decreases with aging, as illustrated by representative donor samples of distinct ages (Online Supplementary Figure S1D-G).
To further investigate the age-related distribution of MSC subsets, we studied pediatric and fetal BM. In contrast to adults, CD271CD146CD90CD105 cells were dominant in children (Figure 2A and B; n=27, 67.7±4.2%; 0.010±0.002% MNC count). This fraction also contained most CFU-F (Figure 2C; n=3). The CD271CD146CD90CD105 (0.003±0.001% from MNC) and CD271CD146 CD90CD105 (0.0006±0.0002%) populations represented 25.6 and 6.6% of the MSC, respectively.
A relatively large subpopulation of fetal CD271CD146 cells co-expressed CD105 and CD90. They accounted for 35.5±8.8% (Figure 2D and E; n=6, 0.107±0.004% from MNC) of all MSC. Interestingly, fetal CD271CD146 cells contained CFU-F (Figure 2F). CD271CD146 cells represented 60.8±8.2% (0.174±0.025%) and also formed CFU-F (Figure 2D and F). CD271CD146 cells were nearly absent (0.012±0.004%) and they did not contain CFU-F (Figure 2D-F). The newly defined fetal CD271CD146 subset had trilineage differentiation potential (Figure 2G). The fetal subsets had a comparable proliferation rate (data not shown). These data show that fetal BM contains a third MSC subset, identified as CD271CD146CD105CD90 that unlike their adult counterparts do generate CFU-F. In addition, these results indicate that CFU-F content and the distribution of MSC subsets differs between adult, pediatric and fetal BM.
The proportion of CD271CD146 cells in BM was significantly correlated to donor age (n=86, R=0.36, P<0.0001, Figure 2H) while the distribution of CD271CD146 cells was inversely correlated (n=86, R=0.26, P<0.0001, Figure 2I). Therefore, the previously reported age-related decrease in BM CFU-F8,15,16 may now be explained by the loss of CD271CD146 cells that tend to generate more CFU-F. The aspiration site may influence CFU-F content and stromal cell phenotype.17 In our study, BM from cardiac patients was sternum-derived; fetal femurs were flushed, whereas BM from other patients was iliac crest-derived. Excluding cardiac patients and fetal samples, thereby correcting for aspiration site-related effects, did not affect the age-related correlation (n=37, age 0–86, CD271CD146 cells, R=0.13; P<0.031; CD271CD146 cells, R=0.12; P<0.036).
In conclusion, we have shown for the first time that the distribution of defined MSC subsets significantly correlates with donor age, and we identified a novel MSC subset specific for fetal BM. Tormin et al. reported that CD271CD146 and CD271CD146, respectively, localize to endosteal or perivascular niches in vivo,7 while we report an age-related distribution. This suggests that the relative size of specialized BM niches is dynamic, and that distinct phases in life require different MSC subtypes. The increase in CD271CD146 MSC in aged BM may, therefore, correspond to the increase in long-term hematopoietic stem cells (HSC) in aged murine BM.18,19 These quiescent HSC predominantly localize to the endosteal niche, where the human CD271CD146 also reside.7 Accordingly, our preliminary co-culture data suggest that adult CD271CD146cells provide better long-term hematopoietic support than CD271CD146 cells. Although the ontogeny and the relationship between the subsets remain unclear, MSC seem to comprise a dynamic system during human life, in which the subpopulations could have different functions during bone marrow development, homeostasis and regeneration.
We would like to thank Dr BJ Biemond, A van der Laan, Dr SE Dohmen, K. de Heer and the Dept. of Cardiothoracic Surgery, Academic Medical Center Amsterdam for collection of bone marrow aspirates and Ms N Papazian and Dr T Cupedo for collection of fetal bone marrow. Furthermore, the authors would like to thank Ms T Schaap and Dr D Hamman for technical assistance, and Dr M von Lindern for critically reading the manuscript and helpful discussions.
- Funding: this work was supported by DPTE grant n. 06728 and Sanquin PPO-C.
- The online version of this article has a Supplementary Appendix.
- Authorship and Disclosures The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
- Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.
- Received May 16, 2011.
- Revision received October 6, 2011.
- Accepted October 10, 2011.
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