Mesenchymal stem cells (MSC) are a promising tool for cell therapy, either through direct contribution to the repair of bone, tendon and cartilage or as an adjunct therapy through protein production and immune mediation. They are an attractive vehicle for cellular therapies due to a variety of cell intrinsic and environmentally responsive properties. Following transplantation, MSC are capable of systemic migration, are not prone to tumor formation, and appear to tolerize the immune response across donor mismatch. These attributes combine to allow MSC to reside in many different tissue types without disrupting the local microenvironment and, in some cases, responding to the local environment with appropriate protein secretion. We describe work done by our group and others in using human MSC for the sustained in vivo production of supraphysiological levels of cytokines for the support of cotransplanted hematopoietic stem cells and enzymes that are deficient in animal models of lysosomal storage disorders such as MPSVII. In addition, the use of MSC engineered to secrete protein products has been reviewed in several fields of tissue injury repair, including but not limited to revascularization after myocardial infarction, regeneration of intervertebral disc defects and spine therapy, repair of stroke, therapy for epilepsy, skeletal tissue repair, chondrogenesis/knee and joint repair, and neurodegenerative diseases. Genetically engineered MSC have thus proven safe and efficacious in numerous animal models of disease modification and tissue repair and are poised to be tested in human clinical trials. The potential for these interesting cells to secrete endogenous or transgene products in a sustained and long-term manner is highly promising and is discussed in the current review.

Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme beta-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+ CD133+ CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

Conventional 1H Magnetic Resonance Imaging (MRI) has been employed to define the migratory behavior of stem/progenitor cells noninvasively in vivo. In contrast, we propose fluorine (19F) MRI of liquid perfluorocarbon nanoparticles (PFC NP: 200 nm) comprising a phospholipid/surfactant monolayer surrounding a PFC core could provide a unique and sensitive cell marker distinct from tissue background signal. To determine the utility for cell tracking, stem/progenitor cells were harvested by density gradient centrifugation from human umbilical cord blood, grown under proendothelial conditions, and labeled with fluorescent PFC NP. Internalization of NP alone, without aid of any additional agents, was confirmed with confocal microscopy showing abundant NP uptake and distribution throughout the cytosol. Flow cytometry indicated ≥68% of cells contained NP and stained positive for CD34, CD133, and CD31 markers. Labeled cells were readily imaged using the 19F nuclear NP signal at 1.5T in vitro; and at 12T after injection of 106 cells into mouse skeletal muscle in situ. PFC NP provide an unequivocal signal for stem/progenitor cells, do not require adjunctive procedures to achieve cellular uptake, enable spatial cell localization with 19F MR imaging, and provide opportunity for quantification via 19F MR spectroscopy. This molecular imaging and tracking approach should facilitate longitudinal investigation of cellular events in vivo such as stem/progenitor cell localization, implantation, and differentiation.

The potential for human adipose-derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune-deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human beta-globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor-derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell-based therapies that involve a wide range of end tissue targets.

Top of pageAbstract Thoracic radiation is used to treat patients with malignancies to improve survival and decrease symptoms. Radiation induced lung toxicity limits thoracic radiation doses and volumes and can restrict therapeutic use of radiation. Clinical radiation pneumonitis can be life-threatening, despite aggressive steroid treatment, especially in those patients with pre-existing pulmonary disease. We hypothesize that bone marrow derived mesenchymal stem cells (BMSC) assist in repair of irradiated lung tissue, and will preferentially localize to tissue with radiation-induced DNA damage. We have developed a xenotransplant model of radiation-induced pulmonary pneumonitis that will allow transplantation of adult human bone marrow mesenchymal stem cells for support of endogenous repair mechanisms and attenuation of pro-fibrotic inflammatory cytokine cascades. We have previously shown that BMSC have functional CXCR4 and c-met receptors, two molecules involved in trafficking and homing of various cell populations to areas of injury. Ionizing radiation was delivered to NOD-SCID MPS7 in increasing amounts either as total body irradiation (TBI) or thoracic-limited irradiation. Dose rate was controlled as well as total radiation administered. BMSC retrovirally engineered with an MND-eGFP-SN retroviral vector were administered at the time of irradiation and post-radiation serum collections were performed at hourly intervals for 12 hours. Serum elevation of hepatocyte growth factor (HGF), transforming growth factor beta-1 (TGF-b1), and interferon-gamma (IFN-g) were measured by ELISA; HGF in particular was upregulated in a dose-response fashion by total radiation dose and rate of dose administration. In addition to serum collections, lungs of irradiated mice were excised, inflated, and sectioned for immunohistochemistry against phosphorylated gamma-H2Ax, and TUNEL, both early markers of DNA damage and predictive of apoptosis. Parallel cohorts were allowed to develop pulmonary pneumonitis and blinded histologic analysis was performed against non-transplanted controls for fibrosis. Finally, transplanted mice were screened by viral sequence specific PCR for persistence of BMSC at up to 75 days post-transplant, and explanted GFP+BMSC cold be explanted and cultured in G418 media at all timepoints. Total human cell engraftment was determined simultaneously with retroviral insertions per cell by multiplex QPCR specific for murine Rapsyn, human beta-Globin, and eGFP respectively. In summary, BMSC in this model of radiation-induced pneumonitis served to reduce total serum amounts of pro-inflammatory cytokines, as well as reduce overall fibrosis. The cells maintained transgene expression for greater than 75 days and were viable on explant. We conclude that mesenchymal stem cells are particularly well suited for this application due to their immune-privileged status and ability to extravasate in response to chemotactic injury signals, and may hold promise in an autologous transplant setting to alleviate radiation-induced lung toxicity.

Recent reports have suggested phenotypic similarities between an adherent umbilical cord blood-derived cell population (AdUCB) and traditional bone marrow derived mesenchymal stem cells (BMSC). Given the advantageous interactions demonstrated in HSC/MSC cultures, as well as AFT024 feeder systems, we reasoned that cord blood derived HSC would benefit from co-culture with their native stromal component.

Our group has previously reported the development of novel CD34-TK chimeric suicide genes for optimal T cell engraftment, graft versus leukemia effect and minimal graft versus host disease (GvHD) in murine BMT models (Rettig et al., Mol. Ther. 2003; Rettig et al., J. Immunol. 2004). These studies have demonstrated critical functional impairment of murine T cells after selected methods of activation, transduction and selection. Unfortunately no in-vivo models exist to consistently examine the impact of ex-vivo manipulation of human T cells (HuT) on T cell function in-vivo. NOD SCID2M null mice (2 mice) were conditioned with 250cGy TBI on day -1 (n=31), or 300cGy on day 0 (n=22). 107 naive HuT or CD3/28 bead activated (XcyteTMDynabeads®) with 50 U/ml IL-2 for 4 days (Act 4d) or 8 days (Act 8d) HuT were injected retroorbitaly (ro), intravenous tail vein injection (iv) or lower HuT doses resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood (PB) of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. (See Table 1)

Osteogenic growth peptide (OGP) is a bone anabolic protein synthesized by leaky ribosomal scanning through an imperfect initiator sequence within histone H4 to a perfect alternative site at codon 85. OGP has been shown previously to be upregulated following bone fracture or injury, as well as following massive blood loss. These observations prompted us to investigate the role of OGP in bone formation and endosteal vascular niche interactions.

In this study we assessed the transfer and expression of the gene for beta-glucuronidase (GUSB) into multiple tissues by infusing normal human mesenchymal stem cells (MSC) into unconditioned neonatal NOD/SCID/MPSVII (GUSB null) mice. In this study, pups were transplanted before 3 days of age using the facial vein injection strategy. We hypothesized that neonatal pups would provide a rapidly growing environment for engraftment and expansion of transplanted normal human MSC into different tissues. The Sands laboratory has used the MPSVII mouse for many years to study disease progression, including neural defects, and to study correction of the disease by gene replacement therapies. In order to study cellular therapies using normal human cells, the murine MPSVII mutation was backcrossed for ten generations onto the NOD/SCID strain. Human umbilical cord blood CD34+ cells engraft in the resultant NOD/SCID/GUSB null strain to levels of 86% marrow replacement, similar to the NOD/SCID parent (Hofling 2004). The tissues of the mice can be homogenized and subjected to a GUSB enzyme assay, and plotted against a standard curve composed of normal human and GUSB null mouse cells, to quantitate the percentage of human cells that had been residing in the tissue of interest at the time of harvest. There is also a FACS|[ndash]|based assay to detect levels of the GUSB protein, and to allow sorting of donor-derived cells from any tissue after transplantation. The aspect of the NOD/SCID/GUSB null strain that makes it so particularly well suited for the study of human stem cell therapy is the clear-cut enzymatic stain for normal, GUSB positive human cells transplanted into the mice. On tissue slides prepared from mice that had been transplanted at birth with human mesenchymal stem cells, the histochemical stain for GUSB revealed normal cells in multiple tissues. Almost all tissues surveyed (N=19) demonstrated the presence of human MSC by staining for GUSB. The stain is quite specific, and although the released enzyme can be taken up by neighboring cells, it appears to be in very low abundance or, more likely, in a processed form where it is no longer detectable by the histochemical analysis. Thus, the individual transplanted human cells stand out vividly against the background murine tissues. In summary, the use of the NOD/SCID/MPSVII neonatal injection model has allowed accurate tracking and quantitation of human MSC into multiple tissues of mice injected by facial vein within three days of birth. This study may set the stage for neonatal gene/cell therapy for newborns with lysosomal storage diseases.

This study was designed to examine the effect of hepatocyte growth factor (HGF) addition to traditional cytokine cocktails (Flt3, stem cell factor, and thrombopoietin), during human umbilical cord blood ex vivo transduction. Our hypothesis is that the HGF/c-met axis provides signaling cascades that promote symmetrical division of hematopoietic progenitors, thus allowing oncoretroviral gene transfer without the associated loss of potentiality that traditionally accompanies cell cycle progression.

Hematopoietic stem cells (HSC) are important targets for gene therapy. Most protocols involve ex vivo modification, in which HSC are transduced in vitro and injected into the recipient. An in vivo delivery method might simplify HSC gene therapy. We previously demonstrated that iv injection of an amphotropic retroviral vector (RV) into newborn mice resulted in long-term expression from hepatocytes. The goal of this study was to determine if HSC were also transduced. After neonatal administration of 1 × 1010 transducing units/kg of RV, peripheral blood cells had ∼0.1 copy of RV per cell for up to 22 months. At 18 months, RV sequences were detected in T, B, and myeloid cells from bone marrow (BM). Unfractionated BM was transplanted into naive recipients after total body irradiation. Recipients maintained similar levels of the RV in their blood cells for 10 months, at which time RV sequences were present at the same integration site in all lineages of cells from BM. We conclude that neonatal iv injection of RV results in transduction of HSC in mice, which might be used for BM-directed gene therapy. Transduction of blood cells after liver-directed neonatal gene therapy might have adverse effects in patients, although no leukemias developed here.

The survival, expansion and Graft versus Host Disease (GvHD) potential of human T cells in immunodeficient mouse models (SCID, SCID-Beige) have been limited by residual immune function and the need to inject large numbers of human T cells (>108–109) in conjunction with human antigen presenting cells into the peritoneal cavities of the animals. To overcome this problem, we used an immunodeficient mouse strain carrying the Beta 2 microglobulin knockout (NOD-SCID-β2M Null), which consistently permits survival, expansion, activation and GvHD potential of human T cells, even after intravenous (IV) injection of small numbers of enriched CD3/CD28 activated cells. In the current studies, T cells were isolated by Ficoll gradient centrifugation, and stimulated for 48 hours on immobilized antibodies to CD3 and CD28 in 24 well plates. Also, T cells transduced with a retroviral vector transferring a truncated CD34 receptor in conjunction with a Herpes Simplex Virus Thymidine Kinase (HSV TK) were prepared in the same way. FACS analyses of T cells pre-and post stimulation demonstrated up-regulation of stimulation markers (CD69 and CD25) after CD3/CD28 stimulation. Gene expressing human T cells were selected to a purity of over 90% using the Miltenyi magnetic bead separation system. A normal ratio of CD4+ and CD8+ cells and high viability was maintained during the culture period. Small numbers of cells (5–7×106 cells per mouse) were injected IV retro-orbitally, and the animals were followed for 4 weeks. Peripheral blood from the mice was analyzed on day 7, day 14, and day 30. The animals were sacrificed shortly after day 30 due to weight loss and clinical findings consistent with GvHD. Blood, spleen, liver, lymph nodes and other tissues were analyzed for the presence of human T cells. On day 7, human T cells (0.5–5%) could be detected in the peripheral blood of the mice. Human T cell numbers increased steadily over the follow up period, and high levels of human T cells (10–45%) could be measured on day 30. The T cell activation markers CD25 and CD69 were up-regulated. Gene transduced human T cells had also expanded in the same way in vivo and could be detected by FACS analysis via expression of the CD34 surface marker. Human CD3+ cells infiltrated the spleen, GI tract and other organs by day 30, coincident with the development of runting, weight loss and clinical findings consistent with GvHD. In summary, the NOD-SCID-β2M Null mouse provides a unique, efficient and consistent model to study gene transduced human T cell activation, expansion, survival and GvHD in a murine preclinical xenotransplantation model.