Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well-differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies.
Disclosure of potential conflicts of interest is found at the end of this article.
MSCs (also known as marrow stromal cells) are bone marrow-derived stem cells with the potential to differentiate into bone, cartilage, and fat [1, 2]. Recent studies suggest that they may have further potential, developing into muscle cells , neuronal progenitors , and lung and kidney epithelium [5, 6]. This raises the exciting possibility that MSCs may have therapeutic potential. MSCs can be quickly expanded in vitro and could be used to engraft into diseased tissues expressing missing or damaged genes, as has been reported for the correction of CFTR gene in cystic fibrosis in humans .
In vivo experiments have confirmed expectations of traditional MSC differentiation by showing repair of bone and cartilage after local injection using injury models [5, 7, –9]. Intraperitoneal injection of human MSCs in sheep strongly suggested that MSCs have the capacity to differentiate into multiple cell types , whereas systemic infusion of these cells leads to an even wider distribution into multiple organs, with evidence of differentiation into epithelial cells of local tissues [5, 9, 11].
After venous injection, bone marrow-derived cells (BMDC) must pass through the lungs. Crucial to the regeneration of lung epithelium or replacement of genes in genetic disorders, BMDC must engraft and differentiate into an epithelial phenotype. Previous studies suggest higher engraftment of BMDC into the lungs compared with other organs [9, 12], and evidence exists that tissue damage may be important for this engraftment after systemic delivery of both hematopoietic stem cells (HSCs) and MSCs [5, 12, 13]. For example, the engraftment of enhanced green fluorescent protein (eGFP)-labeled BMDC 1 week after endotoxin inhalation  or 3 weeks after inhalation of elastase  was seen only after a pulmonary insult but not in undamaged controls.
Murine MSC transplantation has led to both lung engraftment as type 2 pneumocytes and attenuation of lung damage . However, others have documented that 80% of collagen 1-producing fibroblasts found at sites of bleomycin-induced fibrosis were of donor origin . One reason for this may be the preselection of cells before transplant. We previously described a culture system for enrichment of murine MSCs that removes contaminating hematopoietic cells and leaves cells capable of in vitro differentiation into bone, cartilage, fat, muscle, and neuronal cells . These cells were transduced with a lentiviral construct carrying eGFP, allowing the tracking of engrafted MSC progeny. Although we saw some evidence for differentiation of these cells into multiple cell types, including airway epithelial cells, the mice became short of breath and had to be sacrificed after 28 days. This study examines in detail the kinetics of MSC engraftment in the lung parenchyma and their subsequent differentiation potential.
We found that after systemic delivery of MSCs, cells embolized in lung parenchyma capillaries. These cells both transmigrated and divided over the next 24 hours. However, far from showing the expected local differentiation pattern, the majority of cells expanded to form tumors of immature disorganized bone resembling well-differentiated osteosarcoma or exaggerated callus. However, there were no definitive features of neoplastic transformation, such as atypical nuclei, tumor necrosis, or areas of undifferentiated sarcoma. The lesions expanded rapidly, destroying the lung parenchyma, and in several cases led to recipient death by 28 days. Importantly, we found that the formation of these tumors appears specific to murine MSCs. The same type of experiments using MSCs derived from human fetal blood underwent similar initial engraftment but subsequent clearance from the lungs. The precise mechanism of the tumor development is not clear; however, we demonstrated that murine MSCs acquired significant chromosomal abnormalities after four in vitro passages, whereas we did not detect any abnormalities in human MSCs after six or more passages in vitro. Our results show that an enriched population of murine MSCs can be expanded in vitro for cell therapy studies, but the cells are chromosomally unstable, and this may contribute, in part, to tumor formation after systemic injection.
Materials and Methods
Murine MSC Isolation, Purification, and Expansion
MSCs from NOD/SCID bone marrow were used for initial experiments because of the ease of culturing MSCs without the presence of contaminating hematopoietic cells in this mouse strain . Later experiments used MSCs from Rosa26-LacZ mice (background C57Bl/6J × 129S2). These cells were transplanted into both NOD/SCID mice and syngenic B6/129S2F1 ([C57Bl/6J × 129S2] F1) mice. Bone marrow cells were collected by flushing the femurs, tibias, and iliac crests from 8–12-week-old mice with phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS) (Gibco, Paisley, U.K., http://www.invitrogen.com). Red blood cell-depleted bone marrow mononuclear cells were plated at a density of 106 cells per cm2 in murine mesenchymal medium with murine mesenchymal supplements (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco). Half the culture medium was changed at day 3 to remove some nonadherent cells. Whole medium was subsequently replaced weekly. The cells were grown for 2–3 weeks until almost confluent. Adherent cells were then detached by 0.25% trypsin-EDTA and replated using a 1:3 dilution factor. Subsequent passaging and seeding of the cells was performed at a density of 5,000 cells per cm2. MSCs were enriched at passages 2 and 3 by elimination of cells stained with rat anti-mouse CD45-CyChrome and CD11b-PE (BD Biosciences, Oxford, U.K., http://www.bdbiosciences.com). The negative fraction (35%–40% of adherent cells) was sorted using the FACSVantage (Becton, Dickinson and Company, Oxford, U.K., http://www.bd.com) and expanded before transduction and injection as described below. At injection, MSCs were resuspended in PBS, 2% fetal calf serum with 1 mM EDTA and filtered to avoid cell aggregates.
Human Adult and Fetal MSC Sample Collection
Human adult bone marrow cells were purchased from Stem Cell Technologies. Fetal blood collection was approved by the Research Ethics Committee (Hammersmith and Queen Charlotte's Hospitals) in compliance with national guidelines regarding the use of fetal tissue for research purposes (Polkinghorne Commission Recommendations). All women gave written informed consent for collection and use of human tissues. Murine experiments were carried out under full ethical approval from the host institutions and the appropriate Home Office license.
Fetal blood (100 μl) was obtained by ultrasound-guided cardiac aspiration (10 weeks of gestation) before a clinically indicated termination of pregnancy. Fetal gestational age was determined by crown-rump length measurement on ultrasound.
Culture of Adult and Fetal Blood MSCs
Fetal blood was plated in 100-mm dishes at 105 nucleated cells per milliliter, and bone marrow mononuclear cells were plated at a density of 106 cells per cm2. Both were cultured in MSC growth medium consisting of 10% FBS (Stem Cell Technologies) in Dulbecco's modified Eagle's medium (Sigma-Aldrich, Poole, Dorset, U.K., http://www.sigmaaldrich.com) supplemented with 2 mM l-glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) at 37°C in 5% CO2. After 3 days, nonadherent cells were removed, and the medium was replaced. After 10 days, adherent cells were then detached by 0.25% trypsin-EDTA (Stem Cell Technologies) and replated at a density of 5,000 cells per cm2, expanded, and cultured to confluence in 75-cm2 flasks. Adult BM-derived MSCs and fetal blood-derived MSCs underwent 4 and 5 passages, respectively, before being transduced.
In Vitro Lentivirus-Mediated Gene (eGFP) Transfer into Murine and Human MSCs
The HIV-1-based self-inactivating lentiviral vector (pHRSINcPPT-SEW), carrying the eGFP reporter gene under the control of the spleen focus-forming virus long terminal repeat, was used to transduce murine and human adult MSCs. For transduction, 1 × 104 purified MSCs from passage 4 were seeded into individual wells of a 12-well plate. The following day, virus particles were added at a multiplicity of infection (MOI) of 50 to murine MSCs and at an MOI of 10 to human adult MSCs, and transductions were performed for 20 hours.
Fetal MSCs were transduced with a lentivirus encoding the eGFP reporter gene driven by hPGK at an MOI of 12.5 as previously described .
Transduced MSCs were washed several times after virus removal with culture medium to avoid viral contamination before infusion into mice. Flow cytometry assessment of the transduced cells at day 5 showed expression of eGFP expression >95% for murine MSCs , 93% for human adult MSCs, and 98% for fetal MSCs .
A total of 39 NOD/SCID mice ages 8–12 weeks old received donor NOD/SCID MSCs in four independent in vivo studies. eGFP-MSCs (2 × 106), obtained 4 days post-virus removal to minimize further expansion, were delivered intravenously by tail vein injection into each sublethally irradiated mouse (375 cGy using a 137Cs source). Pairs of mice were then sacrificed on days 1, 2, 7, 14, and 28 postinfusion, and lungs were collected in three experiments. In the fourth experiment, nine NOD/SCID mice were used; five of them were injected with eGFP-MSCs (2 × 106), and four were injected with nontransduced MSCs (2 × 106). The fourth experiment also included nine NOD/SCID mice that were injected with MSCs from Rosa26-LacZ mice (0.35 × 106 and 2 × 106 cells) after sublethal irradiation (375 cGy). MSCs from Rosa26-LacZ mice were also injected (0.5 × 106 and 2 × 106 cells) into syngenic mice after sublethal irradiation of 500 cGy or no irradiation. For the human fetal MSCs, a total of 16 NOD/SCID mice were injected with 2 × 106 cells after sublethal irradiation (375 cGy). Human adult MSCs were injected (1 × 106 and 2 × 106 cells) into 12 NOD/SCID mice after sublethal irradiation of 375 cGy and into 5 NOD/SCID mice with no irradiation.
Tissue Processing and Immunohistochemistry
Tissues were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin. Sections (4 μm thick) were stained with H&E or Alizarin Red or immunostained using the avidin-biotin-peroxidase or alkaline phosphatase technique.
For all the mice used in the fourth experiment, lungs were inflated with 1% paraformaldehyde, fixed in NBF, and embedded in paraffin. For human cells and the MSCs from Rosa-LacZ mice, the left main bronchus was ligated after lung inflation, and the left lung was excised and embedded in optimal tissue compound for frozen sectioning.
The sections were immunostained with anti-eGFP antibody (rabbit polyclonal; 1:500; Molecular Probes Inc., Leiden, The Netherlands, http://probes.invitrogen.com), anti-Ki67 (rat monoclonal; 1:25; DAKO, Cambridgeshire, U.K., http://www.dako.com), anti-AE1/AE3 (mouse monoclonal; 1:100; DAKO), anti-endomucin (V.7C7 rat IgG2a polyclonal; 1:500; generously provided by Dr. Dieter Vestweber, Institute of Cell Biology, University of Munster, Munster, Germany), anti-TTF-1 (mouse monoclonal; 1:50; Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), anti-osteocalcin (goat polyclonal; 1:100; Santa Cruz Biotechnology Inc., Heidelberg, Germany, http://www.scbt.com), and anti-collagen type II (mouse monoclonal; 1:25; Biocarta, San Diego, http://www.biocarta.com).
The endomucin antibody required no antigen retrieval. For Ki67, eGFP, TTF-1, and osteocalcin, sections required microwave antigen retrieval (sodium citrate, pH 6, for 10 minutes), and sections were quenched for endogenous peroxidase (with 1.6% H2O2) and when necessary for endogenous alkaline phosphatase (2 mM levamisole). For AE1/AE3, sections were treated with protease (Streptomyces griseus) antigen retrieval (0.04% in PBS at 37°C for 10 minutes; Sigma-Aldrich), and for osteocalcin, sections were treated with pepsin antigen retrieval (1 mg/ml in 50 mM Tris-HCl, pH 2, for 15 minutes). Biotinylated secondary antibodies (1:250) were used, and immunoreactivity was detected using the ABC peroxidase-based system in combination with 3,3′-diaminobenzidine, the Vector Blue alkaline phosphatase-based system, or a combination of both (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), following the manufacturer's protocol. Absent and/or nonspecific primary negative controls were included. For secondary antibodies conjugated with fluorochromes, the sections were incubated with Sudan Black (0.1% in 70% ethanol for 30 minutes) to lower autofluorescence.
For the Alizarin Red S stain for calcium, sections were dewaxed and taken to 95% alcohol. The sections were then air-dried before being placed in Alizarin solution for 1–5 minutes (1% aqueous red S [Sigma-Aldrich], pH 6.3) until the desired intensity was seen, and they were then counterstained with methyl green (Vector Laboratories) for 2 minutes, rinsed in water, blotted, and then rinsed in acetone for 30 seconds.
All microscopy was performed on a Nikon Eclipse E1000 microscope, and all images were captured using a Nikon DXM1200F digital camera (Tokyo, http://www.nikon.com).
Metaphase analysis was performed by using 80% confluent flasks of MSCs that were subsequently incubated with 0.1 μg/ml colcemid (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) overnight for passage 1 and for 5 hours for later passages and human fetal MSCs. Mitotic cells were washed in PBS and trypsinized, and the cells were incubated in hypotonic solution (75 mM KCl) for 15 minutes at 37°C and fixed in methanol/acetic acid (3:1). Fixed chromosomes spreads were stained with 200 ng/ml 4,6-diamidino-2-phenylindole in 2× standard saline citrate and examined by immunofluorescence microscope. Forty metaphases of each passage and cell type were counted.
Isolation and Characterization of Murine MSCs
The murine MSCs used in these experiments have been extensively characterized, as previously described . Previous studies have found the isolation and expansion of these cells to be strain-dependent [19, 20], and we previously found that MSCs from the NOD/SCID mouse strain exhibited higher cell expansion and survival potential after removal of the hematopoietic cells in early passages . The initial experiments were conducted using NOD/SCID bone marrow because of this and the availability of this strain in the laboratory. As previously reported , we observed that MSC cultures were contaminated with hematopoietic cells co-expressing CD45 and CD11b. Contaminating cells consisted of 0.5%–2% B cells (CD19+), T cells (CD3+), NK cells (NK1.1+), and granulocytes (Ly-6Gr+) . After purification, the MSCs were transduced with eGFP-lentivirus vector without losing their in vitro differentiation capacities into adipocytes, chondrocytes, and osteoblasts (as described previously ).
MSC Engraftment Occurs Immediately Post-Transplantation by Embolization
We performed systemic injections of NOD/SCID-derived MSCs into sublethally irradiated NOD/SCID mice. Two mice per time point were killed on days 1, 2, 7, 14, and 28 in three separate experiments. A fourth experiment consisted of nine mice, all sacrificed at day 28. Only two mice (both killed on day 2, from the second experiment) showed no eGFP-positive cell engraftment. All other mice demonstrated engraftment. On day 1 (six mice), engraftment of eGFP-positive cells was between 0.45% and 3.2% of the total cells counted per section (Fig. 1A, 1B). Histopathological examination showed that murine MSCs embolize in pulmonary capillaries. Figure 1C shows an ectatic capillary with embolized MSCs. After impaction, MSCs transmigrate into air spaces (Fig. 1E), where they proliferate or cluster to form aggregates (Fig. 1F).
MSC Plasticity Is Rare in the Lung Despite Expansion of Engrafted Donor-Derived Cells
We and others have previously reported that bone marrow-derived cells have the ability to differentiate into lung epithelial cells [5, 13, 16, 17]. Examination of donor murine MSC-derived tissue (eGFP-positive) revealed rare cells (<1 in 1,000) that both were eGFP-positive and expressed markers specific to lung epithelium, such as TTF-1 or AE1/AE3 (Fig. 2A–2D). Although we used Sudan Black to reduce autofluorescence, we cannot rule out the possibility that these double-stained cells are the result of cell overlay or apoptosis, as has been described recently [21, 22]. However, the majority of eGFP-positive tissue derived from donor MSCs did not costain with either a lung epithelial cell nuclear marker (TTF-1) or an epithelial cell membrane marker (AE1/AE3) (Fig. 2E, 2F). Indeed, the lung epithelial architecture within the eGFP-positive tissue was grossly distorted by proliferating eGFP-positive cells.
Donor-Derived Murine MSCs Form Tumors Within the Lung Consisting of Bone and Cartilage
Postengraftment, donor-derived NOD/SCID MSCs rapidly expanded, forming large areas of tissue within the lungs of NOD/SCID mice by day 7 (Fig. 3A, 3B). The majority of this tissue had the morphological appearance of cartilage or bone, and by day 14, areas of lung stained clearly for cartilage-specific marker collagen 2 (Fig. 3C) and bone markers Alizarin Red (Fig. 3F) and osteocalcin (Fig. 3G, 3H). Large areas had the characteristic collagen deposition of bone on polarized light microscopy (Fig. 3I, 3J). The tumors continued to expand obliterating normal lung tissue until between 40% and 60% of the lung parenchyma contained eGFP-positive cells in 6 of 12 mice sacrificed at 28 days (six of six mice from the first three experiments). The other nine mice (five transduced with lentivirus-eGFP and four untransduced) had multiple nodules, but they were of smaller size, occupying <5% of the lung parenchymal area on tissue sections (all injected in the fourth experiment). These mice were injected over 2–3 minutes, in contrast to the bolus injections given previously to minimize the embolization of the cells within the capillaries. Detailed histopathological examination revealed osteochondroblastic proliferation that resembled the exaggerated healing response of fracture callus or well-differentiated osteosarcoma. (Fig. 3K, 3L).
Although other organs were analyzed in the first three experiments, no tumor formation was detected in kidney, liver, or heart (data not shown).
Murine MSCs Grew Within Vessel Lumen, Infiltrating and Tracking Along Vessel Walls, but Did Not Costain with Endothelial Markers
Reports of MSC differentiation into endothelial cells led us to search for donor-derived cells that costained with endothelial markers. MSCs were frequently seen to expand within vessels differentiating into bone or cartilage (Fig. 4A). eGFP-positive cells also invaded the wall of the vessel in an apparently organized manner (Fig. 4B, 4C) but did not costain for the endothelial markers CD31 or endomucin. Within the vessel wall, cells positive for endothelial markers were not donor-derived (Fig. 4C). eGFP-positive cells “pagetoid spread” along the vascular basement membrane, undermining and displacing the endothelium (Fig. 4E, 4F), but again, these donor-derived cells did not stain for endothelial markers.
Murine MSCs Rapidly Proliferated Following Systemic Injection
Lung epithelium divides irregularly in the steady state, with rare cells staining positively for proliferative markers. Using immunohistochemistry for Ki67, a marker of cellular proliferation, we determined that injected MSCs were proliferating following embolization within lung parenchymal capillaries within 24 hours (Fig. 4G) and continued to proliferate within the lung, explaining the rapid growth of the lesions formed (Fig. 4H).
Lentiviral Transduction of Donor Cells Does Not Cause the Tumor Formation
The cells injected in the first three experiments had been transduced with a lentivirus expressing eGFP, allowing easy identification of donor cells. To examine whether the lentiviral transduction led to the tumor formation, we performed a fourth experiment, in which we injected four NOD/SCID mice with nontransduced NOD/SCID cells and five NOD/SCID mice with cells transduced with the lentivirus. All nine mice developed equivalent tumors (Fig. 5A–5C). As noted above, the tumors were smaller than those seen in the first three experiments but had the same morphological and differentiation characteristics, indicating that the formation of tumor was not related to virus integration.
Murine MSCs from a Nonimmunocompromised Mouse Strain Show Less Tumor Formation Following Systemic Administration
To examine whether the formation of tumors only occurred in immunodeficient mice, we repeated our experiments in immunocompetent Bl6/129 mice. Donor cells from Rosa26-LacZ (Bl6/129) mice were cultured under the same conditions and infused into 13 sublethally irradiated Bl6/129 mice using two different cell doses (eight mice with 2 × 106 cells; five mice with 0.35 × 106 cells). The mice were sacrificed 28 days after infusion. The lungs were sectioned at different levels separated by 50 μm. Between 8 and 12 H&E sections (through at least two lobes each) per mouse were examined for evidence of tumor formation. None of the 13 mice analyzed had tumors (Table 1), suggesting that tumor formation may be dependent on mouse strain. To examine whether the absence of tumors in the Bl6/129 mice can be explained by recipient strain differences, we performed the same experiments injecting Rosa26-LacZ-derived donor cells into sublethally irradiated NOD/SCID recipient mice. Histopathological analysis showed a single tumor in one mouse (Fig. 5D; Table 1) but no tumors in the other eight mice (Fig. 5E; Table 1). This low frequency of tumor formation suggests that strain differences in the donor cells are important.
Table Table 1.. Summary of tumor nodule formation
Lentivirally Transduced Human Adult and Fetal MSCs Did Not Form Tumors
We next wished to determine whether human MSCs cultured in similar circumstances formed tumors after transplantation into mice. We used both human adult and fetal blood MSCs (passages 6 and 7, respectively). Human fetal MSCs have been extensively characterized by us previously and demonstrated in vitro differentiation into adipocytes, chondrocytes, and osteocytes [18, 23]. NOD/SCID mice were infused intravenously with 2 million fetal MSCs or 1 or 2 million adult MSCs after identical sublethal irradiation of 375 cGy to the murine experiments. For fetal MSC experiments, two mice were killed on day 1, two on day 14, two on day 21, six on day 28, and four at 8 weeks. No tumors were detected at any time point (Fig. 5F; Table 2). Indeed, the number of cells detected with anti-green fluorescent protein (GFP) staining reduced from approximately 1 in 350 at day 1 to <1 in 5,000 at day 28 (Table 2), suggesting clearance of these cells. In the case of human adult MSCs, we observed identical results with no engrafted cells and no tumors detected at 4 and 8 weeks postinjection of eGFP-transduced MSCs. Similarly, no tumors were found in mice injected with nontransduced or nonirradiated adult MSCs (Table 3).
Table Table 2.. Summary of lung engraftment and tumor formation in bone marrow transplantation of human fetal enhanced green fluorescent protein-transduced MSCs by histology
Table Table 3.. Summary of lung engraftment and tumor formation in bone marrow transplantation of human adult enhanced green fluorescent protein-transduced MSCs by histology
Murine MSCs Have an Abnormal Karyotype, but Human MSCs Do Not
Chromosomal abnormalities are thought to be critical in tumor formation, and recent studies have identified abnormalities in murine MSCs after high passage numbers [24, –26]. To check whether the donor MSCs used in our study had normal chromosomes, we performed a karyotype analysis on all the cell types injected, counting at least 40 metaphases per cell type and passage. We found that in both NOD/SCID- and Rosa26-LacZ (Bl6/129)-derived MSCs, the majority of metaphases had the expected number of chromosomes (n = 40) at passage 1 (data not shown). Unexpectedly, at the time of injection (passage 4 or 5), both strains of MSCs showed numerical chromosomal abnormalities, with a modal chromosome number of 69 for NOD/SCID-derived MSCs and 76 for LacZ-derived cells (Fig. 6A, 6B). These changes in cell karyotype may be responsible, in part, for the tumor formation observed. However, almost all the human fetal MSCs (35 of 40 cells) showed the normal number of chromosomes (n = 46) at passage 7 (Fig. 6A, 6B).
We systemically injected an enriched population of murine MSCs into mice following low-dose irradiation. The aim of our study was to evaluate the extent of lung parenchyma engraftment as epithelial cells under these conditions. Although we saw rare evidence of differentiation of MSCs into cells with staining characteristics of lung epithelium, we saw widespread engraftment and expansion of MSCs into large tumors, showing differentiation into bone and cartilage cells with destruction of normal lung architecture.
We have demonstrated that not only do murine MSCs cultured under our conditions have high in vitro capacity for osteogenic and chondrogenic differentiation  but this potential is retained in vivo and occurs outside of a normal biological microenvironment. On engraftment into the lungs, most likely at the first pass following venous injection, MSCs begin proliferating immediately, as indicated by immunopositivity with anti-Ki67 antibody, and then continue to proliferate and differentiate into bone and cartilage lineages. The lesions are multiple, throughout the lungs, and expand rapidly, involving between 40% and 60% of cross-sectional area of lung parenchyma at 28 days, leading to recipient sacrifice due to breathlessness. The lesions grew destructively both within alveolar spaces and vessels. The majority of lesions have osteoblastic predominance, but occasional tumors consist predominantly of chondrocytes and chondroblasts. Morphologically, these tumors are typical of fracture callus exuberance, resembling the well-differentiated osteosarcoma previously recognized in osteogenesis imperfecta .
Additional experiments using a different mouse strain, Bl6/129, revealed no tumor formation (0 of 13 mice analyzed). Hence, there does appear to be some strain dependence. To establish whether this was due to the genetic background of the recipient, we administered Rosa26-LacZ (Bl6/129)-derived MSCs into NOD/SCID mice and found that only one of nine mice displayed a small tumor. These results suggest that tumor formation is dependent on both recipient strain and the strain from which donor cells are derived. Although both donor murine strains had numerical abnormalities in karyotype, it is possible that differences in the cytogenetic events occurring between the two strains could explain the differences in the tumorigenesis capacity. This hypothesis is supported by a report that shows a variety of cytogenetic abnormalities in MSCs derived from C57/Bl6 mice, some capable of forming sarcomas in C57/Bl6 mice but others not . Moreover, another possible explanation could be differences in the expression of cell adhesion molecules that may favor the higher engraftment in the lung parenchyma of NOD/SCID-derived MSCs. Of note, all four separate cultures of NOD/SCID MSCs produced lung tumors upon injection in our experiments.
Murine MSCs, although less well studied than human MSCs, have similar in vitro differentiation potential but differ in a number of other attributes, including cell surface molecule expression [5, 29]. This may alter cell homing and engraftment after systemic injection. To assess whether human MSCs would also form tumors in our model, we injected both human fetal and adult MSCs carrying a lentivirally expressed eGFP marker. Human cells engrafted into the lung parenchyma, but at a lower level. Importantly, we found no evidence of tumor formation after 2 months, and the levels of GFP-positive cells decreased over time, with no evidence of proliferation.
Few studies have examined the effects of systemic delivery of MSCs. Ortiz et al. reported an attenuation of bleomycin-induced inflammation and fibrosis after transplanting murine MSCs, resulting in engraftment of donor-derived type 2 pneumocytes, although at lower levels than those of HSC transplants . A separate group reported engraftment of a plastic-adherent murine bone marrow population of cells as type 1 pneumocytes . Two recent studies, however, demonstrated that long-term cultures of MSCs (4–5 months) contain a population of transformed cells capable of tumor formation after systemic injection [24, 30]. In one study, there was no apparent differentiation of human MSC-derived tumors within the lung parenchyma , whereas in the other, murine MSC-derived tumors appeared to undergo fibroblastic differentiation . In vitro, both these cells had an abnormal karyotype and high telomerase activity. Similarly, in long-term culture, telomerase transduced human MSCs evolved spontaneous genetic changes leading to tumorigenicity . More recently still, a study demonstrated sarcoma-like tumors developing after injection of MSCs from C57/Bl6 mice. These cells showed cytogenetic abnormalities after nine in vitro passages that were capable of tumorigenesis. Nevertheless, the authors were unable to reproduce the results with subsequently derived MSCs despite demonstrating cytogenetic abnormalities in all their MSC cultures from two mouse strains , indicating that the nature of the chromosomal aberrations and/or other factors might influence the capacity of MSCs to form tumors. Importantly, the culture systems in these different studies and our own used standard culture conditions and thus are unlikely to be responsible for the tumor formation.
Why these enriched MSCs form tumors is intriguing. We have found that our highly selected murine MSCs rapidly accrue genetic changes, as demonstrated by their abnormal karyotype. It is likely that this has led to abnormal expression of genes or genetic/epigenetic changes responsible for tumor formation. It would be interesting to examine this short-term-cultured MSC population for the genetic abnormalities frequently found in osteosarcoma tumors and cell lines. The irradiation dose we administrated in our experiments was not likely critical for the tumor formation, as previous reports showed that this sublethal dose is not harmful to the lung [32, 33]. Supporting these data, the transplanted human MSCs were also given a sublethal dose of irradiation and no tumors were seen.
Our data provide further evidence for the rapid acquisition of chromosomal abnormalities and potential adverse effects of systemic injection of murine MSCs after a minimal in vitro culture necessary to obtain a quantity of cells required for cell therapy studies. These results stress the importance of careful analysis of cells used for cell therapy both before and after transplantation.
Disclosures of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
We thank Fernando Anjos-Afonso for help with these experiments, George Elias and his team in the Cancer Research UK histopathology laboratories, Karen Groot for critical reading of the manuscript, and Denise Sheer for help with karyotyping analysis. This work was supported by Cancer Research UK. S.M.J. is a Medical Research Council Clinician Scientist. D.B. and S.M.J. are joint last authors.