Open access journal of the Ferrata-Storti Foundation, a non-profit organization
Articles

Factors that influence short-term homing of human bone marrow-derived mesenchymal stem cells in a xenogeneic animal model

Vol. 93 No. 10 (2008): October, 2008 https://doi.org/10.3324/haematol.12553

Abstract

Background Human mesenchymal stem cells are potential agents for tissue regeneration, enhancing hematopoietic stem cell transplantation and delivering genes of therapeutic interest. To implement any of these strategies successfully, we need a better understanding of factors that influence the tissue distribution of systemically administered mesenchymal stem cells.Design and Methods The present study was designed to investigate the short-term tissue homing of mesenchymal stem cells in immunodeficient mouse models, exploring the effects of animal age, duration of ex vivo expansion of mesenchymal stem cells, lentiviral transduction and CXCR4 over-expression. Dye-labeled mesenchymal stem cells (1.5–2.0×106/animal) were injected via the tail vein into unconditioned β2m/NOD/SCID animals. Animals were sacrificed 20–24 hours later and cell suspensions from tissues were examined by flow cytometry for the presence of PKH-positive cells.Results PKH-positive cells were readily detected in the bone marrow, spleen, liver and lungs at 20–24 hours after infusion. The homing of systemically infused mesenchymal stem cells to the bone marrow and spleen of unconditioned β2m/NOD/SCID animals was significantly (>2-fold, p<0.001) higher in younger (<10 weeks) animals, and was reduced with increasing passage number. Despite low surface CXCR4 expression, human mesenchymal stem cells migrated to SDF-1 in vitro, and this was enhanced by over-expression of CXCR4 using lentiviral transduction. Over-expression of CXCR4 by lentiviral transduction (>80%) did not alter the bone marrow homing of mesenchymal stem cells in unconditioned animals, but caused a significant (p<0.05) increase in homing to bone marrow and spleen of animals that had received prior irradiation.Conclusions Tissue homing of systemically administered mesenchymal stem cells is influenced by host factors such as age, is diminished by prolonged in vitro culture, and can be increased by enforced expression of CXCR4, at least in irradiated hosts.

Introduction

Bone marrow-derived human mesenchymal stem cells (MSC) are an accessible population of progenitor cells that can differentiate into mesenchymal tissues including cartilage and bone1 are easily expanded ex vivo and readily transduced by viral vectors.2,3 Human MSC may prove useful for enhancing hematopoietic stem cell transplantation,4 modulating graft-versus-host disease,5 treating disorders of bone, cartilage6 and muscle7 and delivering therapeutic genes.8 The successful employment of human MSC for these in vivo strategies relies upon the efficient localization and retention of cells within appropriate tissues. Site-directed administration of MSC can result in successful engraftment and integration of MSC under specific circumstances, most commonly in injured tissue.9–11 It remains unresolved to what degree such effects are due to the local production of growth factors rather than direct participation of injected MSC in cellular regeneration.

Site-directed administration is practicable only for a limited number of applications, hence the localization of systemically administered MSC in appropriate tissues, for example bone marrow, is crucial to the success of many therapies. The initial localization of circulating cells to specific organs is referred to as homing. Homing of hematopoietic stem and progenitor cells to the bone marrow is a multistep process mediated by adhesion molecules and chemokines.12–15 Far less is known about the in vivo homing of human MSC, partly because in vivo studies using systemic administration have employed different models and read-out systems. Evidence for tissue localization of systemically infused MSC is most convincing in models employing tissue injury.16–18 Current evidence suggests that in the absence of tissue damage systemically administered MSC seed to the bone marrow only at low levels,19–21 with large numbers of MSC lodging in the pulmonary vascular bed. Although MSC can engraft in non-pulmonary tissues, levels are at the limit of detection and of clinical use only in disorders such as osteogenesis imperfecta.22

The initial localization of exogenously administered MSC to target tissues or organs is critical, and clearly we need a better understanding of the mechanisms that regulate the migration and homing of MSC to the bone marrow and other tissues in vivo. The chemokine stromal derived factor-1 (SDF-1) has a major role in the homing and engraftment of hematopoietic stem and progenitor cells to the bone marrow,15 and together with its receptor, CXCR4, functions in stem cell seeding of the bone marrow during embryonic development.24 Enforced expression of CXCR4 on CD34 cells increased migration and engraftment in immunodeficient animals.25 Levels of CXCR4 and SDF-1 are upregulated in stressed or injured tissues,26,27 hence this receptor/ligand pair may mediate metastatic invasion,28 or the migration of cells into damaged tissues.29,30 Human MSC express low levels of CXCR4,31,32 which may account for the low levels of seeding to the bone marrow following systemic infusion. Despite low levels of CXCR4, human MSC are reported to migrate to SDF-1 in vitro,31,33,34 suggesting that, like hematopoietic stem and progenitor cells, human MSC may upregulate functional CXCR4 molecules to the cell surface. Other factors important in MSC homing and migration in vivo include vascular endothelial growth factor, fibroblast growth factor-2, interleukin-6, hepatocyte growth factor and monocyte chemotactic protein-1.34–36 Irradiation or tissue damage may increase MSC migration by inducing such factors.37,38

The present study was designed to investigate the short-term homing of bone marrow-derived human MSC using unconditioned B6.CB17-Prkdc-SzJ SCID (NOD/SCID) and β2m/NOD/SCID animals. The aim was to evaluate factors, including animal age and CXCR4 expression, which influence the short-term biodistribution of human MSC in this unconditioned xenogeneic animal model.

Design and Methods

Human mesenchymal stem cells isolation and culture

Human MSC were obtained from the bone marrow of healthy donors undergoing bone marrow harvest after informed consent under a protocol approved by the local ethics committee. Human MSC were cultured from the adherent fraction of bone marrow mononuclear cells, and characterized for phenotype and differentiation function according to established protocols,39,2 (see the Online Supplementary Methods).

Mice

B6.CB17-Prkdc-SzJ severe combined immunodeficiency (NOD/SCID) and β2-microglobulin non-obese diabetic/severe combined immunodeficiency (β2m NOD/SCID) mice were obtained from Jackson Laboratories and bred and maintained under defined flora conditions in individually ventilated sterile microisolater cages in accordance with the Animals Scientific Procedures Act 1986 approved by the Institutional Animal Care and Use and licensed by the Home Office. Mice underwent transplantation with MSC by lateral tail vein injection and were killed by CO2 inhalation 20–24 hours after transplantation. For irradiation experiments mice were sublethally irradiated (325 cGy from a cesium 137 source).

Labeling human mesenchymal stem cells with PKH-26 and injection into animals

Cultured human MSC (passages 6–15) were labeled with PKH-26 according to manufacturer’s (Sigma) instructions (see the Online Supplementary Methods). Labeled cells were then injected into the lateral tail vein of NOD/SCID or β2m NOD/SCID mice (1.5–2.0×10 cells per animal).

Analysis of murine tissues for human mesenchymal stem cells homing

Animals were sacrificed 20–24 hours after transplantation. Mononuclear cells obtained from bone marrow and other tissues were analyzed for PKH-positive cells (see the Online Supplementary Methods).

Fluorescence in situ hybridization assay

FISH was performed using probes specific for the human X and Y chromosomes. Human MSC of male origin were transplanted into female mice. FISH analysis was performed on mononuclear cell suspensions from bone marrow and other organs using standard techniques. (see the Online Supplementary Methods).

Viral vector construction and transduction of human mesenchymal stem cells with CXCR4

Details of the bicistronic lentiviral vector containing cDNA for CXCR4 (Figure 1) are given in the Online Supplementary Methods. Human MSC at early passage (<3) were transduced using a multi-hit protocol as previously described2 (see the Online Supplementary Methods).

Analysis of surface and intracellular CXCR4 expression

Human MSC were incubated with phycoerythrin-conjugated anti-human CXCR4 (BD PharMingen) for 30 minutes on ice, washed and analyzed by flow cytometry. For intracellular CXCR4 staining, surface CXCR4 molecules were blocked with non-conjugated antihuman CXCR4 monoclonal antibody (10 μg/mL) for 1 hour on ice. Cells were washed, fixed, permeabilized (BD Cytofix/Cytoperm, BD Biosciences), and subsequently incubated with anti-human CXCR4-phycoerythrin monoclonal antibody. After further washing, cells were analyzed on an EPICS Elite flow cytometer (Beckman-Coulter).

In vitro migration assay

Recombinant human SDF-1 (0-100 ng/mL, Peprotech) was placed in the lower chamber of fibronectin-coated Transwell filters (8.0 μm Pore Size, Corning Inc). CXCR4-transduced, or control hMSC were plated in the upper chamber and allowed to migrate over 4 hours at 37°C. Filters were removed, fixed using 100% ice cold methanol (−20° C for 10 minutes) and washed once in phosphate-buffered saline. Filters were stained with hematoxylin and eosin, washed and air dried. Cells on the upper surface of each filter were carefully removed using a damp cotton bud, filters were detached, placed on a glass slide and mounted using DPX mounting medium. Migrated cells were visualized by light microscopy, and counted by two independent observers (20 fields per slide, x100 power).

Statistical analysis

Differences between groups were analyzed using the Mann-Whitney U test; p values less than 0.05 were considered statistically significant (GraphPad Prism Version 4.0).

Results

In vivo homing of human mesenchymal stem cells in unconditioned immunodeficient mice

PKH-26 (PKH)-labeled human MSC (1.5–2.0×10 cells/animal; for optimization of cell doses, see the Online Supplementary Methods) were administered via the tail vein into unconditioned β2m/NOD/SCID animals. Animals were sacrificed 20–24 hours later, and single cell suspensions from bone marrow, spleen, lungs and liver were analyzed for PKH-positive cells by flow cytometry. The highest levels of homing were consistently found in the lungs, while there was considerable variation of PKH-positive cells in the bone marrow and spleen between animals (Figure 2A, Table 1). PKH-positive cells were also detected in the liver. In two experiments, homing was also analyzed at 48 hours, with similar results to those at 24 hours. There was some donor-to-donor variation; this did not, however, correlate with donor age or sex. These findings were supported by FISH analysis using human X and Y specific probes (Online Supplementary Figure S1). PKH-positive cells identified in murine tissues also stained positive with a human-specific anti-HLA class I monoclonal antibody (data not shown). We also used unconditioned NOD/SCID animals as recipients. There was a generally lower level of homing in these animals than in β2m/NOD/SCID recipients (Table 1).

Figure 1.Schematic representation of the lentiviral vector constructs The pCL10.1 MSCV promoter is used to drive expression of either GFP cDNA in the control vector (bottom panel) or the CXCR4-IRES-GFP bisistronic cassette of the experimental vector (top panel).

Extended culture of cells is usually required to obtain sufficient numbers of human MSC for therapeutic applications. We studied the effect of passage number on homing behavior. The numbers of PKH-positive cells in the bone marrow of β2mNOD/SCID mice were lower in mice transplanted with advanced passage human MSC than in animals that had received cells from earlier passages (p<0.001, Figure 2B). A similar effect of passage number was seen in the spleen (p<0.01).

Efficiency of short-term homing alters with the animals’ age

The numbers of PKH-positive cells detected in the bone marrow of younger (<10 weeks) animals were significantly higher (median 433/10; range, 294–521) than the numbers in older (>10 weeks) animals (median, 166/10; range, 120–306; p<0.001, Figure 2C). To control for any age-related changes in bone marrow cellularity we determined the numbers of PKH-positive cells per bone. This analysis confirmed that bone marrow homing was greater in younger (<10 weeks) animals (median, 8836/bone; range, 4600–15977, vs median, 2725/bone; range, 1750–7933 in older animals; p<0.001). This effect of the animals’ age was also observed for human MSC homing to the spleen (p<0.001, Figure 2C), but not to lungs. (p=0.1457).

Figure 2.Short-term in vivo homing of human mesenchymal stem cells (MSC). (A) Flow cytometric identification of PKH-stained human MSC 20 hours after infusion. Cell suspensions from each organ were analyzed by flow cytometry for the presence of PKH-bright cells, using as negative control an animal infused with saline only. One representative animal. Control: animal infused with saline; test: animal infused with PKH-labeled human MSC. Gates indicate PKH-positive events. (B) Effect of passage number on homing to the bone marrow (left panel) and spleen (right panel). Data are given as number of PKH-positive events/105 cells analyzed, and are pooled from two different MSC donors, 5–7 animals per data point, *p<0.01, **p<0.001, compared with homing of MSC at passage 8 or less. (C) Effect of age on the homing efficiency of human MSC. Data presented as in (B), from 8–12 animals per data point, 4 experiments.

Table 1.Short-term homing of PKH-labeled human mesenchymal stem cells in two strains of unconditioned immunodeficient animals.

CXCR4 expression and homing of human mesenchymal stem cells

The consistent, albeit low, levels of homing to the bone marrow displayed by human MSC led us to investigate the possible contribution of CXCR4, as the bone marrow is reported to express high levels of SDF-1. We found that the surface expression of CXCR4 was low (2.3±1.8%, n=10) or absent (n=5) on all human MSC tested (Figure 3A). In contrast, human MSC harbored significant amounts of intracellular CXCR4 (63±12%, n=3, Figure 3A), as previously reported.31 Next we investigated whether CXCR4 receptors, despite their low expression on infused human MSC, did contribute to bone marrow homing. To do this, we used a phycoerythrin-cyanin 5 (PE-Cy5)-conjugated anti-CXCR4 monoclonal antibody to label human MSC that had homed to the bone marrow. We confirmed that the PE-Cy5-conjugated monoclonal antibody was able to stain surface CXCR4 molecules on PKH-labeled human MSC (Online Supplementary Figure S2). Bone marrow cells recovered from animals 24 hours after infusion of unmanipulated human MSC were incubated with PE-Cy5-conjugated anti-CXCR4 monoclonal antibody or the isotype control, washed and analyzed by flow cytometry. We found that bone marrow-homed human MSC, identified as PKH-bright cells (gated in Panel A, Figure 3B) expressed significant levels of CXCR4 (28.8±8.1%, n=5), compared with levels on pre-infusion human MSC (panel B, Figure 3B). This suggests that either human MSC upregulate intracellular CXCR4 in vivo, or there is preferential homing of a small population of human MSC expressing higher levels of CXCR4.

CXCR4 expression and in vitro migration of human mesenchymal stem cells

Human MSC were transduced with a bicistronic vector encoding CXCR4 and eGFP (MSC), or the control vector encoding eGFP (MSC). Significant levels of transgene expression were achieved (43–96% CXCR4, 50–93% eGFP positive, Figure 4A) and the expression was stable for up to 20 passages. CXCR4 expression on human MSC from five different donors was 41.8±9.8 (MCF) and 73.2±12.7 (% positive). Both human MSC and human MSC expressed intra-cellular CXCR4 (Figure 4A). In vitro, unmanipulated human MSC migrated to SDF-1, as previously reported.31 Human MSC (>80% CXCR4-positive) displayed significantly higher levels of migration to SDF-1 (Figure 4B). No further increase in migration occurred at SDF-1 concentrations >100 ng/mL.

Enforced CXCR4 expression does not alter homing in unconditioned recipients

We found no significant differences in bone marrow homing between eGFP-transduced (median 422/10 cells; range, 267–556), and unmanipulated human MSC (median 376/10 cells; range, 247–539), suggesting that lentiviral transduction did not affect homing. The presence of eGFP-expressing human MSC in murine tissues was confirmed by direct visualization of fluorescent cells in bone marrow trephine rolls (data not shown).

We next tested the in vivo homing of gene-modified human MSC expressing high (>85%) levels of CXCR4 (MSC), using young animals (6–8 weeks), and unmanipulated or MSC from the same donor as controls. To our surprise, enforced expression of CXCR4 had no effect on homing of human MSC to the bone marrow, spleen, liver or lungs of unconditioned β2m/NOD/SCID animals (Figure 5A). When we repeated these experiments in NOD/SCID animals, we found a small increase in homing of MSC to the spleen (Figure 5B), but no effect on bone marrow homing.

Figure 3.(A) Unmanipulated human mesenchymal stem cells (MSC) were stained for surface (top panel) or intracellular (bottom panel) CXCR4. Empty histograms indicate negative controls incubated with istoype-matched monoclonal antibody. (B) CXCR4 expression on MSC which had homed to bone marrow PKH+ cells present in the bone marrow at 20 hours after infusion (shown gated in a) were analyzed for CXCR4 expression by staining with anti-CXCR4-PE-Cy5 monoclonal antibody (c). For comparison, the profile of CXCR4 expression on MSC prior to infusion into animals is shown (b). Empty histograms represent negative controls, gray hatched histograms positive samples stained with CXCR4 monoclonal antibody. One representative experiment.

CXCR4 over-expression enhances in vivo homing in irradiated animals

It is possible that an effect of CXCR4 over-expression on homing would only be revealed in the context of tissue injury. We, therefore, repeated our experiments in sub-lethally irradiated hosts. Irradiation leads to loss of bone marrow cellularity (of up to 80%), so that the percentage of PKH-positive cells detected by flow cytometry is artefactually increased. The absolute numbers of PKH-positive human MSC detected per bone, however, remained unchanged (data not shown). To investigate the homing of human human MSC in irradiated hosts, β2m/NOD/SCID animals were treated with 325 cGy 24 hours prior to infusion of human MSC or hMSC. Under these conditions, human MSC demonstrated significantly higher levels of homing to the bone marrow (median, 260/10 cells; range, 219–283) compared with hMSC (median, 177/10 cells; range, 28–220; p<0.05, Figure 6A). Because irradiation reduces the total number of bone marrow cells, we also determined the number of PKH-positive cells/bone. These results confirmed the superior homing of CXCR4-expressing human MSC (Figure 6B). Similar effects of CXCR4 over-expression were also demonstrated for human MSC homing to the spleen (Figure 6A). The absolute number of human MSC homed to the spleen was also significantly higher for human MSC (p<0.05, Figure 6B).

Figure 4.Enforced CXCR4 expression on human mesenchymal stem cells (MSC) enhances in vitro migration (A) CXCR4-transduced human MSC (MSCCXCR4) or eGFP transduced (MSCGFP) human MSC were stained for surface or intracellular CXCR4 as indicated. Panels a, c, e, and g represent negative controls stained with IgG-PE, while panels b, d, f and h are positive samples stained with anti-CXCR4-PE. GFP fluorescence is shown on the X-axis to indicate transduction efficiency. Note that in panel f only GFP-positive MSC are positive for surface CXCR4, while in panel h, all MSC are positive for intracellular CXCR4. (B) Enhanced migration of MSCCX-CR4 (MSC/CXCR4) compared with that of MSCGFP (control MSC). Migration to SDF-1 (0–100 ng/mL) over 4 hours. Mean±SEM of three experiments, with duplicate filters for each condition, and ten high power fields/filter counted by each of two observers. The right panel shows representative filters stained with hematoxylin and eosin (x100 magnification).

Figure 5.In vivo homing of human mesenchymal stem cells (MSC) expressing CXCR4 by lentiviral transduction (A). Comparison of in vivo homing to the bone marrow (left panel) and spleen (right panel) between human MSCCXCR4 and control e-GFP-transduced human MSC from the same donor (MSC) in unconditioned β2m/NOD/SCID animals. Ten to 15 animals per data point, three experiments. (B) In vivo homing of MSCCXCR4 and control MSC to the bone marrow (left panel) and spleen (right panel) in unconditioned NOD/SCID animals. Ten to 12 animals per data point, two experiments.

Discussion

In this study we used a sensitive dye-labeling method to quantify the tissue biodistribution of human MSC after systemic injection in a xenograft model, and showed that a proportion of transplanted cells was capable of localizing into the bone marrow, spleen and lungs of unconditioned animals. A key observation reported here is that the efficiency of homing is critically influenced by the age of the recipient animal, and also declines with increased duration of cell culture. The adverse effect of the recipient’s age on short-term homing in this model remains to be confirmed in other models, and in human recipients. It may, however, have important implications for clinical transplantation in elderly recipients. A decline in homing may be related to the down-regulation of stromal cell-derived chemokines/cytokines during the aging process. The decline in homing efficiency with prolonged ex vivo culture of human bone marrow-derived MSC in this study using an unconditioned immunodeficient xenogeneic model is reminiscent of a similar previous report using murine MSC in a syngeneic model.40 Many clinical applications require high doses of MSC, achievable only with considerable ex vivo expansion. A clearer under-standing of the effect of extended in vitro culture on adhesion and chemokine receptor expression, and on migratory capacity is needed.

Figure 6.Effect of CXCR4 over-expression on in vivo homing of human mesenchymal stem cells (MSC) in irradiated recipients. Effect of enforced CXCR4 expression on homing of human MSC to the bone marrow and spleens of irradiated β2m/NOD/SCID animals. MSCCXCR4, CXCR4-transduced human MSC, MSC, control e-GFP-transduced human MSC from the same donor. Results expressed as number of PKH-26 positive events/105 cells (A) or number of PKH-26-positive events/organ (B), five to seven animals per data point, two experiments.

Importantly, our observations confirm a potential role for the CXCR4 receptor in the short-term homing behavior of systemically administered human MSC. While unmanipulated human MSC express low levels of surface CXCR4, cells that had localized to the bone marrow at 20 hours post-transplantation showed significant levels of expression. We demonstrate for the first time that enforced surface expression of CXCR4 by lentiviral gene transfer was able to enhance in vivo short-term homing, but only in the context of an irradiated model. The reported surface expression of CXCR4 on human MSC varies from almost negligible to 20–40%.33,34 Such variation may be related to differences in culture conditions. For example, CXCR4 is known to be upregulated when human MSC are exposed to cytokines, including insulin-like growth factor-1,41 which is present in cell culture serum. Several studies now report that human MSC migrate in vitro in response to SDF-1,31,34 perhaps mediated by upregulation of intracellular CXCR4 molecules. Similarly, the localization of human MSC to the bone marrow, despite low surface expression of CXCR4, may be explained by upregulation of intracellular CXCR4 molecules to the cell surface in vivo, in response to cytokines, sheer forces,42 or exposure to hypoxia. Similarly, upregulation of CXCR4 by cytokines in vitro may enhance short-term localization of MSC to the bone marrow of irradiated NOD/SCID animals.44

In the present study we used a lentiviral system to stably over-express a functional CXCR4 receptor on human MSC and examined the effects on SDF-1-directed migration in vitro and on homing in vivo. Lentiviral vectors are well suited to these studies because their safety profile is better than that of onco-retroviral vectors.45 Over-expression of CXCR4 on human MSC significantly promoted their migration in vitro, in confirmation of previous reports,31,46 but did not influence short-term homing to the bone marrow following systemic administration in unconditioned mice. CXCR4 expression is dynamic and regulated by cytokines, adhesion molecules, ligand-binding and proteolytic enzymes.15,28 Functional modulation by receptor internalization and trafficking, incorporation into lipid rafts, cleavage of both CXCR4 and SDF-1 by serine proteases and matrix metalloproteinases also occurs.28,47 It is not clear if, and how, lentivirally expressed surface molecules are subject to these physiological regulatory mechanisms.

Irradiation, and consequent tissue injury, leads to increased secretion of cytokines and chemokines such as SDF-1, and, in some models, to increased homing/engraftment of MSC.48,49 It is not surprising, therefore, that CXCR4–overexpressing human MSC demonstrate superior homing (compared with control human MSC) under these circumstances. What is perhaps surprising is that while more than 80% of infused cells express surface CXCR4 molecules, levels of homing remain below 1%. This may reflect the limited capacity of the system, or alternatively CXCR4-expressing human MSC may display increased localization to tissues other than the bone marrow and spleen. Lentivirally expressed receptors may not undergo physiological trafficking necessary for optimal function, as discussed above. Increased CXCR4 receptors on genetically modified human MSC may also affect the migratory response to other inflammatory or chemokinetic agents released following irradiation by receptor cross-talk.50

In conclusion we report that multiple factors influence the in vivo homing of human MSC in this xenogeneic model. In vivo homing to the bone marrow and spleen is greater in younger animals. Enforced surface expression of CXCR4 by lentiviral gene transfer leads to increased in vitro migration to SDF-1, and enhanced homing in vivo but this effect is only marked in irradiated hosts. These factors should be taken into account when using in vivo models for pre-clinical evaluation of MSC-based therapies.

Acknowledgments

we are grateful to Mike Blundell and Meera Ulganathan (Molecular Immunology Unit, Institute of Child Health) for help with tail vein injections, and to Professor David Linch for helpful comments on the manuscript.

Footnotes

  • Authorship and Disclosures CK designed and performed the research, analyzed data and wrote the paper. NR performed the research. KY designed the research, analyzed data and wrote the paper. AN designed the research, AP performed the research. The authors reported no potential conflicts of interest.
  • Fundings: this research was supported by the Comprehensive Biomedical Research Centre, University College London.
  • Received November 27, 2007.
  • Revision received May 21, 2008.
  • Accepted June 9, 2008.

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Vol. 93 No. 10 (2008): October, 2008 : Articles
 
DOI
https://doi.org/10.3324/haematol.12553
Pubmed
18728032
 
Published
2008-10-01
Published By
Ferrata Storti Foundation, Pavia, Italy
Print ISSN
0390-6078
Online ISSN
1592-8721
 

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