Review

Gene Therapy (2012) 19, 1–7; doi:10.1038/gt.2011.68; published online 12 May 2011

Non-hematopoietic stem cells as factories for in vivo therapeutic protein production

L Sanz1, M Compte1, I Guijarro-Muñoz1 and L Álvarez-Vallina1

1Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain

Correspondence: Dr L Sanz, Unidad de Inmunología Molecular, Hospital Universitario Puerta de Hierro, Joaquín Rodrigo 2, Majadahonda, Madrid 28222, Spain. E-mail: lsanz.hpth@salud.madrid.org

Received 2 December 2010; Accepted 16 February 2011; Published online 12 May 2011.

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Abstract

As an alternative to recombinant protein administration, ex vivo gene-modified cells may provide a novel strategy for systemic delivery of therapeutic proteins. This approach has been used in preclinical and clinical studies of a plethora of pathological conditions, including anemia, hemophilia and cancer for the production of erythropoietin, coagulation factors, immunostimulatory cytokines, recombinant antibodies and angiogenesis inhibitors. Cell delivery vehicles may also be varied: autologous or allogeneic, precursor or terminally differentiated cells, with targeting properties or immobilized in immunoprotective devices. This field did not meet the expectation raised initially, mainly because of difficulties with obtaining therapeutic plasma levels and the short lifespan of producer cells that hampered clinical application. Different non-hematopoietic stem/progenitor cells have emerged as potential delivery vehicles, since they are easy to obtain, expand and transduce, and they exhibit prolonged lifespans (with mesenchymal stem cells probably being the most popular cell type, but not the only one). Special emphasis is placed on the different routes used to deliver these cellular vehicles and the controversies about their targeting abilities.

Keywords:

mesenchymal stem cell; neural stem cell; endothelial progenitor cell; cell-based gene delivery; cell factory

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Introduction

Systemic administration of recombinant proteins (cytokines, antibodies and coagulation factors) has been widely used in the clinical setting for decades. As an alternative, gene therapy may provide a novel means for in vivo delivery of therapeutic proteins, resulting in effective and persistent levels of protein with a syngenic glycosylation pattern and without any additional formulation or manufacturing, that could make the protein less immunogenic and better tolerated. At the same time, this approach could circumvent problems related to large-scale production and high cost of recombinant proteins.

The two main gene therapy approaches are based on direct gene delivery (using viral or non-viral vectors) or on inoculation of ex vivo genetically modified cells (autologous or allogeneic). Viral vectors are highly efficient as gene delivery vehicles, and have been tested in numerous clinical trials, but raise concerns about safety risks1 and limitation of the effect due to immune responses against viral antigens.2, 3 On the other hand, use of non-viral vectors has been hampered by their low transduction efficiency.

The use of cells as delivery vehicles for therapeutic proteins4 (Figure 1) offers several conveniences: after ex vivo cell transduction, remaining viral particles are eliminated, reducing the risk of unwanted virus dissemination; levels of expression by transduced cells can be quantified in vitro and serum levels can be predicted; high-expression clones can be selected and expanded prior to administration; less likelihood of immune responses against autologous cells; cell vehicles can be endowed (naturally or artificially) with targeting capabilities5 and cells can be retrieved once the therapeutic effect is fulfilled if administered in certain formats.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Schematic diagram of SCDV generation with viral vectors. Ex vivo manipulation (a) of autologous or allogeneic stem/progenitor cells (collection and isolation, expansion and lentiviral transduction) to generate a therapeutic SCDV (b). Producer cells can be injected directly (c), confined to s.c. scaffolds (d) or microencapsulated (e).

Full figure and legend (160K)

Non-hematopoietic stem/progenitor cells have been successfully used as vehicles for suicide genes and oncolytic virus in cancer treatment strategies6, 7 or trophic factors in regenerative medicine. However, we intend here to focus on the potential of these cells as ‘cell factories’ for the in vivo production of therapeutic proteins in a variety of pathological conditions, including anemia, hemophilia and cancer.

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Cells of choice

Terminally differentiated cells possess a short lifespan, and this implies an obvious limitation to their application in gene therapy strategies. In contrast, stem/progenitor cells are endowed, at least theoretically, with a great expansion capacity and constitute a more appropriate cellular source than senescence-susceptible cells.

In contrast with hematopoietic stem cells, mesenchymal stem cells (MSCs) are easily transduced and exhibit a unique in vitro proliferative capacity using a simple media formulation. Several other advantages have been attributed to MSCs as cell vehicles, being availability, tumor tropism and low immunogenicity the most appealing.6

Not only MSCs, but also other human adult stem cells, such as neural stem cells (NSCs) and endothelial progenitor cells (EPCs), have emerged as promising delivery vehicles of therapeutic proteins. NSCs were initially devised as potential tools for the treatment of neurodegenerative diseases or central nervous system injuries, but in 2000, their capacity to migrate throughout normal brain tissue to central nervous system tumors8, 9 and deliver a therapeutic payload was demonstrated. Unfortunately, there are serious limitations to the obtention of primary NSCs, and most studies in preclinical models have used immortalized NSCs, with the safety concerns that it implies.10 Circulating EPCs incorporate to growing tumor vasculature and, therefore, might represent a particularly well-suited delivery system for anti-angiogenic therapy of cancer. Blood late outgrowth endothelial cells (BOECs) obtained from peripheral blood are easy to isolate and expand, have extended longevity and represent true EPCs.11 However, only a fraction of systemically administered EPCs seems to incorporate into tumor vessels.

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Local administration: intratumoral injection of stem cell delivery vehicles

One of the most interesting properties of MSCs, NSCs and EPCs, at least in the context of cancer therapy, is their tropism to primary/metastatic tumors that open the opportunity of systemic administration for the targeted production of the therapeutic protein. This tropism has been widely documented in the literature; however, a brief look at Table 1 reveals an intriguing fact. Most studies showing therapeutic effect of these stem cell delivery vehicles (SCDV) employ one of these two strategies: either coinjection or intratumoral (i.t.) injection for localized tumors (even if the authors have demonstrated previously specific homing after systemic administration), or intravenous (i.v.) administration for disseminated lung metastasis (Figure 1).


The first modality has been used extensively for the experimental treatment of intracranial malignant glioma. Several studies demonstrated that MSCs and NSCs, inoculated intracerebrally, could migrate to gliomas and exert a therapeutic effect. In a seminal work, Aboody et al. demonstrated that not only NSCs implanted intracranially at distant sites from the tumor (for example, into the contralateral hemisphere) could migrate through normal tissue targeting the tumor cells, but also after i.v. injection.8 Moreover, the administration of genetically modified NSCs-expressing cytosine deaminase resulted in a reduction in tumor cell burden. But in most of the reports using SCDV for therapeutic protein production cells are administered i.t., and only a few works use a peritumoral delivery route. The i.t. inoculation of MSCs has been validated for the delivery of interleukin (IL)-2,12 IL-7,13 IL-18,14 interferon (IFN)-β,15 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)16, 17 and NSCs for the delivery of IL-4,9 IL-12,18 PEX (hemopexin-like protein)19 and TRAIL.20 Local production of the therapeutic payload decreased tumor growth in every case.

Interestingly, TRAIL-expressing MSCs injected ipsilaterally to an established intracranial glioma xenograft model effectively inhibited tumor growth.21 On the contrary, IL-12-secreting MSCs (MSCIL-12) administered in the peritumoral region extended animal survival but did not result in a statistically significant difference in comparison to control groups.22 Another work showed migration of peritumorally injected lacZ-expressing NSCs into the tumor mass; however, the therapeutic effect of NSCs cells expressing IL-23 was demonstrated inoculating the cells into the tumor.23

In contrast with previous works, Bexell et al.24 found no evidence of MSC homing to established gliomas following i.v. injections and concluded that MSC in glioma therapy should be administered by i.t. implantation rather than by i.v. injections.

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Systemic administration: homing, preferential engraftment or physical trapping?

In fact, accumulating evidence suggests that a great proportion of i.v.-injected MSC are trapped within the lungs of mice, rats and pigs, as assessed by different techniques: nuclear imaging of 99Tc- or 111In-labeled MSCs, tissue iridium content for nanoparticle-labeled MSCs, in vivo bioluminescence imaging, ex vivo infrared imaging and real time PCR.25, 26, 27, 28, 29, 30 This ‘pulmonary first-pass effect’ is attributed to the combined effect of cell size and adhesion molecule expression pattern: NSC pulmonary passage was twofold and bone marrow mononuclear cells passage was 30-fold increased as compared with MSC.26 In two unrelated studies, <0.3% from injected rat MSCs could pass the lungs and reach the carotid artery.26, 28 In a recent work with human MSCs inoculated in mice, 99% of the cells were cleared from the circulation within 5min as assessed by real time PCR, and cells trapped in lungs disappeared with a half-life of around 24h, being undetectable in other organs.29 Using bioluminescence imaging, Wang et al.24 detected MSCs from firefly luciferase transgenic mice primarily into the lungs of healthy mice 1 day after their i.v. administration, as the majority of cells were trapped within the pulmonary capillaries. The number of cells that were able to reach other target organs or tumors in different publications is controversial, but generally very low. Preferential location in lungs have also been documented after systemic administration of EPCs.31 These facts pose a challenging conundrum: how do MSCs exert their proved systemic therapeutic effects after i.v. administration, if they are mainly retained in lungs?

These observations have also interesting implications for the interpretation of SCDV tumor tropism in mouse lung metastasis models. If we assume that most of the systemically administered MSC, or at least an important proportion of them, will be physically retained in the pulmonary filter (as tumor cells themselves have been previously trapped after i.v. inoculation to generate the metastasis model), detection of MSCs close to or in contact with tumor cells in lungs perhaps should not be considered strictly as tumor homing. Anyway, these studies have given important clues about the ability of MSCs to engraft, secrete therapeutic proteins and exert anti-tumoral effects. The extent at which these considerations might apply to NSCs and BOECs remains to be elucidated.

Studeny et al.32 investigated the fate of MSCs injected i.v. into mice with human melanoma lung metastasis. MSCs were found randomly distributed 1 day after in healthy lung and tumor nodules by immunohistochemistry, but after 8 days MSCs persisted mainly in tumors, suggesting that tumor microenvironment is more permissive for their engraftment as compared with normal lung tissue. These results are in accordance with those by Wang et al.,30 where firefly luciferase-expressing MSCs could be detected by bioluminescence imaging 11 days after i.v. inoculation in the lungs of mice carrying murine breast cancer metastasis, but not in control mice.

In the work by Studeny et al.,33 i.v.-injected IFN-β-expressing MSCs significantly decreased tumor growth rate and prolonged animal survival. In another study by the same group, systemically administered IFN-β-expressing MSC inhibited the growth of pre-established melanoma and breast cancer lung metastasis. These results were supported by Ren et al.,34 who reported the therapeutic effect of systemically administered murine IFN-β-expressing MSC in a model of murine prostate cancer lung metastasis, with no detectable increase in serum levels of the cytokine. They also demonstrated the therapeutic effect of IFN-α-expressing MSC in a melanoma lung metastasis model.35

The systemic delivery of NK4 (an antagonist of hepatocyte growth factor) was compared using MSCs as a cell vehicle or adenoviral vectors (Ad) in mice with different types of lung metastasis. MSCNK4 inhibited development of lung metastasis and prolonged survival without the severe liver damage associated with AdNK4 administration.36 The same group explored the use of MSCs for the expression of the immunostimulatory chemokine CX3CL1. After systemic administration in a model of colon carcinoma lung metastasis, expression of CX3CL1 increased in the lung with metastasis, but not in the normal lungs. The numbers of lung metastatic nodules decreased significantly in the MSCCX3CL1-treated group, but not in mice that received fibroblasts expressing the chemokine.37 As an alternative route of systemic delivery, MSCIL-12 were administered intraperitoneally (i.p.) prior to an i.v. challenge with melanoma cells.38 Treatment led to a marked decrease in the number of lung metastasis, but unfortunately no data about potential MSC trafficking or elevated IL-12 mouse serum levels are provided.

An altogether different setting is the use of systemically administered SCDVs for the targeting of subcutaneous (s.c.) tumors, assuming that a number of cells high enough pass the pulmonary filter as to exert a therapeutic effect. Frank et al.39 tested the ability of NSCs to deliver intact anti-Human Epidermal growth factor Receptor 2 antibodies to human breast cancer foci inoculated in the mammary fat pad. Four days later, mice were euthanized and tumors were harvested. NSCs were detected within the tumor mass of each treated animal by immunohistochemistry and the local production of the antibody was demonstrated. Unfortunately, the anti-tumor effect of anti-Human Epidermal growth factor Receptor 2 antibodies was not assessed in an extended follow-up.

A therapeutic effect was shown by Chen et al.,40 who inoculated s.c. different types of tumor cells into the footpad of syngenic mice. After i.v. administration of MSCIL-12, the authors reported long-lasting inhibition on local tumor growth and reduction on spontaneous metastasis numbers. MSCs could be detected into the tumor foci 5 weeks after administration, but interestingly they were absent from normal tissues, such as lung and liver.40 In a related work, i.v.-injected fluorescent-labeled MSCs were not only found in s.c. Ewing's sarcoma tumors 10 days after, but also detected in lung, liver and spleen. In this model, i.v.-inoculated MSCIL-12 decreased tumor growth, and local expression of IL-12 was detected in tumors of mice receiving MSCIL-12, but not untransfected MSCs.41 Recently, it was reported that adipose-derived MSCs expressing TRAIL, administered i.t. or i.v. in mice bearing s.c. HeLa tumors, caused a reduction in tumor burden in both models.42 Presence of transduced MSCs in tumors after systemic administration was demonstrated by green fluorescent protein (GFP) amplification, but no data on MSC potential localization in normal tissues were provided. Using radiolabeled BOECs systemically administered, Dudek et al.43 observed preferential accumulation in lung and to a lower extent in spleen and liver, with only a small fraction localized in s.c. tumors after 4h. At 72h post-administration, BOEC concentration remained the same in the spleen, liver and tumor, but decreased in lungs. The i.v. injection of endostatin-expressing human BOECs into mice bearing s.c. Lewis lung carcinoma resulted in decreased tumor growth.

Taking into account that it is difficult to estimate the percentage of SCDVs effectively homing to s.c. tumors in these models, and given the anti-tumoral effect observed in most of them, it cannot be ruled out that these results could be attributed, at least in part, to therapeutic protein production in locations other than tumors. Alternatively, the observed effects may be due to the small number of cells that escape lung trapping.44 Systematic data about plasma levels and local production of the protein could help to clarify this point.

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Matrix-embedded SCDVs as retrievable s.c. depots

As seen above, tumor-homing capacities of different types of SCDVs are somehow a controversial issue. Moreover, recent evidence also suggests that MSCs display immunomodulatory and pro-angiogenic properties45, 46, 47 and could have a role in tumor growth and metastasis,48 implying a potential risk in the use of MSCs in cancer-targeting approaches.

In fact, for strategies where stem cells are used as therapeutic factories, their ability to disseminate throughout the body may be simply not required (for example, therapeutic levels of factor VIII (fVIII) have been achieved by intrasplenic injection of genetically modified MSC).49 As an alternative to systemic administration or implantation into organs, producer cells can be confined to s.c. scaffolds that would improve engraftment and keep cells at the implantation site (Figure 1d), with the therapeutic protein acting at distance after being secreted into circulation. The s.c. delivery of MSC-loaded scaffolds would provide an easily accessible implant that could be retrieved once the therapeutic effect is fulfilled or in the event of an unexpected adverse reaction. A seminal work by Eliopoulos et al.50 reported that erythropoietin (Epo)-secreting MSCs (MSCEpo), when administered as ‘free’ cells by s.c or i.p. injection, led to a temporary hematocrit increase. In contrast, s.c. implantation of the same cell dose of Matrigel-embedded MSCEpo led to a more significant and prolonged therapeutic effect. Moreover, MSCs participated in blood vessel formation to give rise to a neovascularized organoid that supported the release of Epo directly into the bloodstream.

Matrigel is an injectable, rapid gelling murine basement membrane preparation constituted by a mixture of extracellular matrix proteins, widely used in angiogenesis studies in vitro and in vivo, but probably not best suited for MSC immobilization in a clinical setting. Similar results were observed when MSCEpo were embedded within the human-compatible, food and drug administration approved, bovine collagen-based matrix Contigen.51 Upon retrieval of implants of matrix-embedded MSCEpo, the effect on the hematocrit was reversed. The authors also demonstrated that implantation of embedded MSCEpo can correct anemia in a murine model of chronic renal failure.52

As a proof of principle, we explored the production in vivo of a recombinant antibody by lentiviral-transduced MSC. In search of an alternative to Matrigel, we tested different hydrogel formulations, commercially available, that offer several advantages: they are synthetic, potentially less immunogenic and their composition is the same batch to batch. Human bone marrow-derived MSCs were engineered for the expression of a recombinant T-cell activating bispecific antibody, embedded in hydrogel and inoculated in the ventral s.c. space of nude mice. The antibody was released into the bloodstream at detectable levels for at least 7 weeks and inhibited the growth of human colon carcinoma cells s.c. inoculated in the dorsal region in the presence of i.v. administered human T cells.53 Recently, stem cells have been proposed as an emerging platform for antibody therapy of cancer.54 The systemic effect of a locally produced protein was also reported in the context of cancer therapy by Wang et al.55 Autologous MSCs, matrix embedded and s.c. implanted provided sustained delivery of the decoy soluble IGF-1 receptor for at least 3–4 weeks post-implantation. The protein could access the systemic circulation and achieved therapeutically effective plasma concentrations, inhibiting the development of experimental hepatic metastases of colon and lung carcinoma cells after administration of tumor cells via the intrasplenic/portal route 9–14 days later.

In contrast, the therapeutic effect of MSCIL-2 and MSCIL-12 seems to be due to a local action of the secreted cytokine. Stagg et al.56 observed that matrix-embedded MSCIL-12 injected in the vicinity of pre-established B16 melanoma tumors led to the absence of tumor growth in 90% of treated mice. Similarly, MSCIL-12 implanted peritumorally in a model of breast cancer led to a significant slowing of cancer growth and to increased survival.57 Although MSCIL-12 scaffolds supported an increase in plasma levels of the cytokine, the observed therapeutic benefit was not due to a systemic effect, since MSCIL-2 and MSCIL-12 implanted contralaterally did not inhibit significantly tumor growth. The enhanced properties of matrix-embedded MSCs were further demonstrated by the fact that peritumoral injection of the same number of ‘free’ MSCIL-12 did not exhibit any therapeutic effect.

Another important field of application of SCDV-seeded scaffolds is hemophilia. Van Damme et al.58 transduced MSCs for the expression of fVIII and monitored production over a 5-month period in vitro. After 3 weeks in culture, expression started to decline gradually, but remained detectable for at least 15 weeks. MSCGFP implanted in collagen scaffolds into immunodeficient mice resulted in efficient engraftment of gene-modified cells, with GFP fluorescence detectable by whole-body transdermal imaging for at least 2 months post-implantation. Lin et al.59 had demonstrated that i.v. administration of genetically modified BOECs (mainly located afterwards in marrow and spleen) resulted in sustained therapeutic levels of fVIII. In a related study, and to avoid concerns about cell dissemination throughout the body, BOECfVIII were implanted s.c. in Matrigel scaffolds in non-obese diabetic-severe combined immunodeficiency or in immunocompetent hemophilic mice, showing therapeutic fVIII expression for several months before the eventual return to baseline levels.60 Using an MSC-based strategy for the treatment of hemophilia B, autologous factor IX-producing MSCs were loaded into sophisticated porous scaffolds specifically designed to maximize cell capacity and provide MSC with the appropriate adhesion cues. When implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic factor IX delivery by MSCs that corrected the hemophilic phenotype of most animals for up to 12 weeks.61

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Allogeneic MSCs: not so invisible to the host immune system

It is obvious that the use of autologous cells as SCDVs would reduce the risk of an immune response against the vehicle. However, the only potential cost-effective method to bring this approach to the clinical setting would imply the use of ‘off-the-shelf’ stocks of genetically modified cells ready to be applied in a series of patients. The low immunogenicity and the immunomodulatory properties of MSCs would make them ideal candidates for this strategy, but some reports cast doubts on the administration of genetically engineered allogeneic MSCs to immunocompetent recipients.62

Campeau et al.63 explored whether MSCs from C57BL/6 mice would sustain erythropoietin production in BALB/c allorecipients. Implantation of MSCEpo led to increases in hematocrit in syngeneic and allogeneic mice, but the latter eventually developed severe anemia due to the production of neutralizing anti-erythropoietin antibodies.63 Interestingly, while plasma soluble IGF-1 receptor levels declined progressively in immunocompetent mice in the study by Wang et al.,55 they were more stable in athymic mice, suggesting host immunity implication in levels and duration of soluble IGF-1 receptor production by MSCs. Given that the MSCs used were syngenic to the injected mice, the authors suggest that the immune response could be directed at the product of the GFP reporter gene.

Not only the transgene, but also the same MSC may trigger an immune response under certain circumstances (and not only in immunocompetent mice), contrary to the expected of their immunomodulatory properties. In the work by Elzaouk et al.,38 human MSCIL-12 was detected for only 7 days after i.t. injection in immunocompetent mice that generated antibodies against these cells. Insulin-producing human MSCs transplanted into the liver normalized glucose levels in diabetic non-obese diabetic-severe combined immunodeficiency mice, but hyperglucemia recurred 7 days post-implantation, probably due to an innate immune response against the xenogeneic cells that was somehow ameliorated after i.p. administration.64 In contrast, human MSCs producing the enzyme β-glucuronidase and inoculated i.p. in a non-obese diabetic-severe combined immunodeficiency model of mucopolysaccharidosis type VII, expressed therapeutic levels of protein and persisted for at least 4 months, with no apparent immune response.65

If immune tolerance elicited by MSCs in vivo is not completely reliable, an option is to enclose gene-modified MSC into devices (Figure 1e) that protect them from the host immune system and at the same time allow entry of nutrients and oxygen and exit of the therapeutic protein.66 Goren et al.67 designed alginate-poly-L-lysine microcapsules that can encapsulate human MSCs for extended periods. As a proof of principle, encapsulated MSCPEX were injected adjacent to glioblastoma tumors in nude mice. Live imaging and tumor measurements showed a significant reduction in tumor volume. The authors suggest that MSCs are the cell of choice for microencapsulation cell-based therapy, thus driving this technology closer to clinical application.67

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Concluding remarks

Stem cell-based in vivo therapeutic protein production shares challenges common to any cell-based strategy, as obtaining optimal therapeutic levels, loss of transgene expression over time (due to the lifespan of producer cells, immune responses against the cell or the gene product, or transcriptional repression in vivo) and regulated gene expression. With respect to this point, an MSC glucose-responsive promoter able to drive insulin production in diabetic mice by intrahepatic or i.p. administration with near-physiological characteristics has been recently identified.64

A different question is how to cope with the contradictory results found in the literature concerning tumor-homing capacities and immunomodulatory properties of these cells, especially when trying to apply them to a clinical setting. Possible explanations for these discrepancies are that perhaps we all are not using the same cells, even if we name them similarly (isolation and expansion protocols may influence radically in their properties) and the realization that cultures in vitro are not homogeneous and different subpopulations may have different therapeutic potentials.7, 44 The development of unequivocal, widely accepted markers for different types and subtypes of stem cell populations of different sources (bone marrow, adipose tissue and umbilical cord) and species will be crucial to compare data on a solid ground.68 Standardization of methods used to assess homing efficiencies would also help to clarify MSC trafficking after systemic administration.69

Regardless of these issues, MSCs are emerging as the best option for the generation of long-lasting cell factories. Given the doubts about the capacity of MSCs to target ‘extrapulmonary’ tumors and the evidences suggesting the potential role of MSCs in tumor biology, we believe that the safest approach in cancer therapy might be the use of scaffolds that keep genetically modified MSCs at the implantation site. On the other hand, in approaches aimed to the treatment of inherited protein deficiencies, such as hemophilia, producer cells do not require tissue specificity, but long-term systemic protein delivery. And recent advances in the field of biomaterials have allowed the design of scaffolds that considerably improve the engraftment of ex vivo transduced cells.70 Finally, the use of encapsulation systems to protect SCDV from the host immune system would be highly desirable in an allogenic context.

Probably, the only consensus that can be extracted from all the studies commented in this review is the shared hope in the therapeutic potential of SCDV. How to manage to fulfill this potential is still an open question.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This study was supported by grants from the Ministerio de Ciencia e Innovación (BIO2008-03233 and PSE-01000-2009-11), the Comunidad de Madrid (S-BIO-0236-2006) and the European Union (SUDOE-FEDER. IMMUNONET-SOE1/P1/E014) to LA-V; and from the Fondo de Investigación Sanitaria/ Instituto de Salud Carlos III (PI08/90856 and PS09/00227) and Fundación Investigación Biomédica Hospital Puerta de Hierro to LS. MC was supported by Instituto de Salud Carlos III (Contrato Rio Hortega, CM06/00055).

AUTHOR CONTRIBUTIONS

LS, MC, IG-M and LA-V wrote the manuscript; LS approved the final draft of the manuscript.