Filgrastim for the treatment of hematopoietic acute radiation syndrome.

The U.S. Food and Drug Administration (FDA) recently approved Neupogen(®) (filgrastim) for the treatment of patients with radiation-induced myelosuppression following a radiological/nuclear incident. It is the first medical countermeasure currently approved by the FDA for this indication under the criteria of the FDA "animal rule". This article summarizes the consequences of high-dose radiation exposure, a description of the hematopoietic acute radiation syndrome (H-ARS), the use of hematopoietic growth factors in radiation accident victims and current available treatments for H-ARS with an emphasis on the use of Neupogen in this scenario.

Imaging Macrophage and Hematopoietic Progenitor Proliferation in Atherosclerosis.

RATIONALE: Local plaque macrophage proliferation and monocyte production in hematopoietic organs promote progression of atherosclerosis. Therefore, noninvasive imaging of proliferation could serve as a biomarker and monitor therapeutic intervention. OBJECTIVE: To explore (18)F-FLT positron emission tomography-computed tomography imaging of cell proliferation in atherosclerosis. METHODS AND RESULTS: (18)F-FLT positron emission tomography-computed tomography was performed in mice, rabbits, and humans with atherosclerosis. In apolipoprotein E knock out mice, increased (18)F-FLT signal was observed in atherosclerotic lesions, spleen, and bone marrow (standardized uptake values wild-type versus apolipoprotein E knock out mice, 0.05 ± 0.01 versus 0.17 ± 0.01, P<0.05 in aorta; 0.13 ± 0.01 versus 0.28 ± 0.02, P<0.05 in bone marrow; 0.06 ± 0.01 versus 0.22 ± 0.01, P<0.05 in spleen), corroborated by ex vivo scintillation counting and autoradiography. Flow cytometry confirmed significantly higher proliferation of macrophages in aortic lesions and hematopoietic stem and progenitor cells in the spleen and bone marrow in these mice. In addition, (18)F-FLT plaque signal correlated with the duration of high cholesterol diet (r(2)=0.33, P<0.05). Aortic (18)F-FLT uptake was reduced when cell proliferation was suppressed with fluorouracil in apolipoprotein E knock out mice (P<0.05). In rabbits, inflamed atherosclerotic vasculature with the highest (18)F-fluorodeoxyglucose uptake enriched (18)F-FLT. In patients with atherosclerosis, (18)F-FLT signal significantly increased in the inflamed carotid artery and in the aorta. CONCLUSIONS: (18)F-FLT positron emission tomography imaging may serve as an imaging biomarker for cell proliferation in plaque and hematopoietic activity in individuals with atherosclerosis.

HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice.

Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 5173-83. ©2014 AACR.

Trafficking and homing of systemically administered stem cells: the need for appropriate analysis tools of radionuclide images.

AIM: Despite its enormous relevance, homing of hematopoietic stem cells (SCs) remains relatively uncertain due to the limitations of measuring small number of systemically administered cells in the different organs. Despite its high sensitivity, radionuclide detection has been relatively underutilized to this purpose since it cannot differentiate hematopietic SCs recruited by target tissues from those circulating in the blood pool. Our study aims to verify the potential of tracer kinetic approaches in estimating the recruitment of labeled SCs after their systemic administration. METHODS: Twenty-four Lewis rats underwent administration of 2 millions cells labeled with 37 MBq of 99mTc-exametazime. Animals were divided into 2 groups according to administered cells: hematopoietic SCs or cells obtained from a line of rat hepatoma. Cell injection was performed during a planar dynamic acquisition. Regions of interest were positioned to plot time activity curves on heart, lungs, liver and spleen. Blood cell clearance was evaluated according to common stochastic analysis approach. Either fraction of dose in each organ at the end of the experiment or computing the slope of regression line provided by Patlak or Logan graphical approach estimated cell recruitment. At the end of the study, animals were sacrificed and the number of cells retained in the same organs was estimated by in vitro counting. RESULTS: Cell number, documented by the dose fraction retained in each organ at imaging was consistently higher with respect to the "gold standard" in vitro counting in all experiments. An inverse correlation was observed between degree of overestimation and blood clearance of labeled cells (r=-0.56, P<0.05). Logan plot analysis consistently provided identifiable lines, whose slope values closely agreed with the "in vitro" estimation of hepatic and splenic cell recruitment. CONCLUSION: The simple evaluation of organ radioactivity concentration does not provide reliable estimates of local recruitment of systemically administered cells. Yet, the combined analysis of temporal trends of tracer (cell) tissue accumulation and blood clearance can provide quantitative estimations of cell homing in the different organs.

Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma.

BACKGROUND: A major challenge in the development of cell based therapies for glioma is to deliver optimal number of cells (therapeutic dose) to the tumor. Imaging tools such as magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) has been used in cell tracking and/or biodistribution studies. In this study, we evaluate the dynamic biodistribution of systemic injected labeled cells [human cord blood derived endothelial progenitor cells (EPCs) and cytotoxic T-cells (CTLs)] in rat glioma model with in vivo SPECT imaging. METHODS: Human cord blood EPCs, T-cells and CD14⁺ cells (monocytes/dendritic cells) were isolated using the MidiMACS system. CD14⁺ cells were converted to dendritic cells (DC) and also primed with U251 tumor cell line lysate. T-cells were co-cultured with irradiated primed DCs at 10:1 ratio to make CTLs. Both EPCs and CTLs were labeled with In-111-oxine at 37°C in serum free DMEM media. Glioma bearing animals were randomly assigned into three groups. In-111 labeled cells or In-111 oxine alone were injected through tail vein and SPECT imaging was performed on day 0, 1, and 3. In-111 oxine activity in various organs and tumor area was determined. Histochemical analysis was performed to further confirm the migration and homing of injected cells at the tumor site. RESULTS: EPCs and CTLs showed an In-111 labeling efficiency of 87.06 ± 7.75% and 70.8 ± 12.9% respectively. Initially cell migration was observed in lung following inravenous administration of In-111 labeled cells and decreased on day 1 and 3, which indicate re-distribution of labeled cells from lung to other organs. Relatively higher In-111 oxine activity was observed in tumor areas at 24 hours in animals received In-111 labeled cells (EPCs or CTLs). Histiological analysis revealed iron positive cells in and around the tumor area in animals that received labeled cells (CTLs and EPCs). CONCLUSION: We observed differential biodistribution of In-111-oxine labeled EPCs and CTLs in different organs and intracranial glioma. This study indicates In-111 oxine based SPECT imaging is an effective tool to study the biodistribution of therapeutically important cells.

Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene.

Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.

Combined reporter gene PET and iron oxide MRI for monitoring survival and localization of transplanted cells in the rat heart.

UNLABELLED: There is a need for in vivo monitoring of cell engraftment and survival after cardiac cell transplantation therapy. This study assessed the feasibility and usefulness of combined PET and MRI for monitoring cell engraftment and survival after cell transplantation. METHODS: Human endothelial progenitor cells (HEPCs), derived from CD34+ mononuclear cells of umbilical cord blood, were retrovirally transduced with the sodium iodide symporter (NIS) gene for reporter gene imaging by (124)I-PET and labeled with iron oxides for visualization by MRI. Imaging and histologic analysis were performed on 3 groups of nude rats on days 1, 3, and 7 after intramyocardial injection of 4 million HEPCs. RESULTS: In vitro studies demonstrated stable expression of functional NIS protein and normal viability of HEPCs after transduction. On day 1, after intramyocardial transplantation, iron- and NIS-labeled HEPCs were visualized successfully on MRI as a regional signal void in the healthy myocardium and on PET as (124)I accumulation. The (124)I uptake decreased on day 3 and was undetectable on day 7, and the MRI signal remained unchanged throughout the follow-up period. Histologic analysis with CD31 and CD68 antibodies confirmed the presence of either labeled or nonlabeled control transplanted HEPCs at the site of injection on day 1 but not on day 7, when only iron-loaded macrophages were seen. Furthermore, deoxyuride-5'-triphosphate biotin nick end labeling showed extensive apoptosis at the site of transplantation. CONCLUSION: The combination of MRI and PET allows imaging of localization and survival of transplanted HEPCs together with morphologic information about the heart. Although iron labeling rapidly loses specificity for cell viability because of phagocytosis of iron particles released from dead cells, reporter gene expression provided specific information on the number of surviving cells. This multimodality approach allows complementary analysis of cell localization and viability.

Administration of granulocyte colony-stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair.

The administration of granulocyte colony-stimulating factor (G-CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)-derived c-kit(+) cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM-derived hematopoietic cells mobilized by G-CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)-transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G-CSF for 10 days. In the acute phase, a number of GFP(+) cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and alpha-smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G-CSF administration, and the enhanced myofibroblast-rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G-CSF-mobilized monocytes were harvested from the peripheral blood of GFP-transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP(+) myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G-CSF promotes this recruitment and enhances cardiac protection.

In vivo visualization of 111In labeled CD133+ peripheral blood stem cells after intracoronary administration in patients with chronic ischemic heart disease.

AIM: Stem cell homing to injured tissue is necessary for local tissue repair. But homing of stem cells in chronic ischemic heart disease (CIHD) is poorly understood. This study investigated homing of peripheral blood stem cells (PBSC) expressing the CD133 antigen. After intracoronary injection. The cells were (111)In labeled for in vivo visualization. METHODS: PBSC were mobilized with granulocyte-colony stimulating factor and collected by apheresis on d-1. On d0, CD133+ cells were enriched up to a median purity of 89% (range: 79-97%) with an immunomagnetic separation device (CliniMACS, Miltenyi). A fraction of the cells was radiolabeled with [(111)In]oxine in 0.1 M TRIS at pH 7.4 for 45-60 min. Cell viability after labeling was assessed using trypan-blue. The cells were injected at a radioactive concentration of 0.9 MBq/10(6) cells into the target open coronary vessel through a balloon catheter. During balloon inflation [(99m)Tc]sestamibi was injected intravenously to identify the myocardium and the target vascular territory. Eight patients (mean age: 53 years; range: 50-72 years) with stable CIHD and reduced left ventricular function (NYHA class I-II) after acute myocardial infarction (>12 months) were studied. After a first cohort of 3 patients received an injectate of 5-10 x 10(6) cells, our final protocol was applied in 5 patients in whom an average of 34.4 x 10(6) (range: 18.6-49.4) CD133+ cells was injected. Whole body and single photon emission computed tomography (SPECT) scans were acquired at different time points after injection (energy windows set at 140, 171 and 245 keV). Residual activity in the heart was assessed by drawing a region of interest around the heart on the anterior whole body views. RESULTS: Mean labeling efficiency of [111In]oxine labeling was 51.2% and cell viability after labeling averaged 88%. In the 5 patients receiving the higher amount of labeled cells, a clear (111)In-signal was observed in the heart region up to 3 days after administration. Fused [(99m)Tc]sestamibi/(111)In SPECT images demonstrated that the regional distribution of the transplanted cells within the target zone, as delineated by the flow tracer, remained unchanged over time. A biodistribution study in 2 patients showed a residual activity in the heart, liver and spleen of 6.9-8%, 23.1-26.8%, 3.1-3.7%, respectively, after 1-2 h and 2.3-3.2% 23.8-28.3%, 3.5-3.8%, respectively, after 12 h (decay corrected and expressed as a percentage of total body initial activity). No adverse events were observed during the procedure and up to 3 months follow-up. CONCLUSIONS: Radiolabeling with [(111)In]oxine is a suitable method for follow-up of cell distribution during the first days after transplantation. A significant amount of CD133+ PBSC home to the heart after intracoronary injection in patients with CIHD. The results of this study are useful for the design of trials that evaluate the tissue repair potential of CD133+ PBSC in the setting of CIHD.

Indium-111 oxine labelling affects the cellular integrity of haematopoietic progenitor cells.

PURPOSE: Cell-based therapy by transplantation of progenitor cells has emerged as a promising development for organ repair, but non-invasive imaging approaches are required to monitor the fate of transplanted cells. Radioactive labelling with (111)In-oxine has been used in preclinical trials. This study aimed to validate (111)In-oxine labelling and subsequent in vivo and ex vivo detection of haematopoietic progenitor cells. METHODS: Murine haematopoietic progenitor cells (10(6), FDCPmix) were labelled with 0.1 MBq (low dose) or 1.0 MBq (high dose) (111)In-oxine and compared with unlabelled controls. Cellular retention of (111)In, viability and proliferation were determined up to 48 h after labelling. Labelled cells were injected into the cavity of the left or right cardiac ventricle in mice. Scintigraphic images were acquired 24 h later. Organ samples were harvested to determine the tissue-specific activity. RESULTS: Labelling efficiency was 75 +/- 14%. Cellular retention of incorporated (111)In after 48 h was 18 +/- 4%. Percentage viability after 48 h was 90 +/- 1% (control), 58 +/- 7% (low dose) and 48 +/- 8% (high dose) (p<0.0001). Numbers of viable cells after 48 h (normalised to 0 h) were 249 +/- 51% (control), 42 +/- 8% (low dose) and 32 +/- 5% (high dose) (p<0.0001). Cells accumulated in the spleen (86.6 +/- 27.0% ID/g), bone marrow (59.1 +/- 16.1% ID/g) and liver (30.3 +/- 9.5% ID/g) after left ventricular injection, whereas most of the cells were detected in the lungs (42.4 +/- 21.8% ID/g) after right ventricular injection. CONCLUSION: Radiolabelling of haematopoietic progenitor cells with (111)In-oxine is feasible, with high labelling efficiency but restricted stability. The integrity of labelled cells is significantly affected, with substantially reduced viability and proliferation and limited migration after systemic transfusion.

Bone marrow stem cell imaging after intracoronary administration.

Although feasibility and safety of autologous stem cells administration to the post-infarction heart has been proven it is not known what proportion of cells effectively do home at the damaged site. Therefore, we have labeled autologous bone marrow cells (ABMC's) by radioactive Indium and single photon emission computed tomography (SPECT) tissue distribution has been analyzed. It was detected that up to 10% of the cells were retained within the myocardium while their majority migrated or has been anchored at the spleen and liver. Comparing the number of homed cells to the total number of cells delivered one may postulate the indirect role for few hundred thousands ABMC's at heart regeneration.

Improved expression of gamma-aminobutyric acid receptor in mice with cerebral infarct and transplanted bone marrow stromal cells: an autoradiographic and histologic analysis.

UNLABELLED: Recent studies have indicated that bone marrow stromal cells (BMSC) have the potential to improve neurologic function when transplanted into animal models of central nervous system disorders. However, how the transplanted BMSC restore the lost neurologic function is not clear. In the present study, therefore, we aimed to elucidate whether BMSC express the neuron-specific gamma-aminobutyric acid (GABA) receptor when transplanted into brain that has been subjected to cerebral infarction. METHODS: The BMSC were harvested from green fluorescent protein-transgenic mice and were cultured. The mice were subjected to permanent middle cerebral artery occlusion. The BMSC or vehicle was transplanted into the ipsilateral striatum 7 d after the insult. Using autoradiography and fluorescence immunohistochemistry, we evaluated the binding of 125I-iomazenil and the expression of GABA receptor protein in and around the cerebral infarct 4 wk after transplantation. RESULTS: Binding of 125I-iomazenil was significantly higher in the periinfarct neocortex in the BMSC-transplanted animals than in the vehicle-transplanted animals. Likewise, the number of the GABAA receptor-positive cells was significantly higher in the periinfarct neocortex in the BMSC-transplanted animals than in the vehicle-transplanted animals. A certain subpopulation of the transplanted BMSC expressed a neuron-specific marker, microtubule-associated protein 2, and the marker protein specific for GABAA receptor in the periinfarct area. CONCLUSION: These findings suggest that BMSC may contribute to neural tissue regeneration through migrating toward the periinfarct area and acquiring the neuron-specific receptor function.

Visualization of a primary anti-tumor immune response by positron emission tomography.

Current methodologies that monitor immune responses rely on invasive techniques that sample tissues at a given point in time. New technologies are needed to elucidate the temporal patterns of immune responses and the spatial distribution of immune cells on a whole-body scale. We describe a noninvasive, quantitative, and tomographic approach to visualize a primary anti-tumor immune response by using positron emission tomography (PET). Bone marrow chimeric mice were generated by engraftment of hematopoietic stem and progenitor cells transduced with a trifusion reporter gene encoding synthetic Renilla luciferase (hRluc), EGFP, and Herpes virus thymidine kinase (sr39TK). Mice were challenged with the Moloney murine sarcoma and leukemia virus complex (M-MSV/M-MuLV), and the induced immune response was monitored by using PET. Hematopoietic cells were visualized by using 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG), a radioactive substrate specific for the sr39TK PET reporter protein. Immune cell localization and expansion were seen at the tumor and draining lymph nodes (DLNs). 2-[(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG), which is sequestered in metabolically active cells, was used to follow tumor growth and regression. Elevated glucose metabolism was also seen in activated lymphocytes in the DLNs by using the [(18)F]FDG probe. When M-MSV/M-MuLV-challenged mice were treated with the immunosuppressive drug dexamethasone, activation and expansion of immune cell populations in the DLNs could no longer be detected with PET imaging. The method we describe can be used to kinetically measure the induction and therapeutic modulations of cell-mediated immune responses.

111In-labeled CD34+ hematopoietic progenitor cells in a rat myocardial infarction model.

UNLABELLED: Transplantation of progenitor cells (PCs) has been shown to improve neovascularization and left ventricular function after myocardial ischemia. The fate of transplanted PCs has been monitored by fluorescence labeling or by genetic modifications introducing reporter genes. However, these techniques are limited by the need to kill the experimental animal. The aim of this study was to radiolabel CD34(+) hematopoietic PCs (HPCs) with (111)In-oxine and to evaluate the feasibility of this in vivo method for monitoring myocardial homing of transplanted cells in a rat myocardial infarction model. METHODS: Human HPCs were isolated from mobilized peripheral blood and labeled with (111)In-oxine. Labeled HPCs were injected into the cavity of the left ventricle in nude rats 24 h after induction of myocardial infarction (n = 4) or sham operation (n = 4). Scintigraphic images were acquired up to 96 h after HPC injection. After animals were killed, tissue samples of various organs were harvested to calculate tissue-specific activity and for immunostaining. RESULTS: Labeling efficiency of HPCs was 32% +/- 11%. According to trypan-blue staining, viability of radiolabeled HPCs was impaired by 30% after 48 and 96 h in comparison with unlabeled cells, whereas proliferation and differentiation of HPCs was nullified after 7 d, as assessed by colony-forming assays. After injection of HPCs, the specific activity ratio of heart to peripheral muscle tissue increased from 1.10 +/- 0.32 in sham-operated rats to 2.47 +/- 0.92 (P = 0.020) in infarcted rats. However, the overall radioactivity detected in the heart was only about 1%. A transient high lung uptake of 17% +/- 6% was observed within the first hour after infusion of HPCs. At 24 h after injection, the initial lung activity had shifted toward liver, kidneys, and spleen, resulting in an increase of radioactivity in these organs from 37% +/- 6% to 57% +/- 5%. CONCLUSION: Radiolabeling with (111)In-oxine is a feasible in vivo method for monitoring transplanted HPCs in a rat myocardial infarction model. The potential to detect differences in myocardial homing between infarcted and normal hearts suggests that this method may provide a noninvasive imaging approach for clinical trials using transplanted HPCs in patients. Our findings, however, also demonstrated a negative effect of (111)In-oxine on cellular function, which resulted in complete impairment of HPC proliferation and differentiation. For future trials in stem cell imaging with (111)In-oxine, therefore, it will be mandatory to carefully check for radiation-induced cell damage.

Telomere length in subpopulations of human hematopoietic cells.

In order to test the hypothesis that the telomere length in human hematopoietic cells correlates with their proliferative potential, we analyzed the telomere length in highly purified subpopulations of bone marrow cells. Cells were sorted on the basis of CD34 and CD38 cell surface markers, and two samples were additionally sorted on the basis of Hoechst 33342 dye efflux allowing isolation of side population (SP) cells. The telomere length in limiting numbers of sorted cells was analyzed using a newly developed fluorescence in situ hybridization (flow-FISH) method in which hybridization of telomere probe in cells of interest is measured relative to control cells in the same tube. In all seven bone marrow samples analyzed, the telomere length in CD34(+)CD38(-) cells was longer than in CD34(+)CD38(+) cells from the same donor (p < 0.02). Results with sorted SP cells were less clear: the telomere fluorescence in these cells was very heterogeneous, and a reproducible difference in telomere length relative to CD34(+)CD38(-) cells could not be observed. We conclude that the telomere length in subpopulations of hematopoietic cells does appear to be correlated with the known proliferative potential of such cells and that further characterization of cells on the basis of telomere length is warranted for enrichment of very rare precursors of hematopoietic and other tissues.

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate along different mesenchymal lineages including those forming bone, cartilage, tendon, fat, muscle and marrow stroma that supports hematopoiesis. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries and for hematopoietic disorders by both local and systemic application. In the present study, rat marrow-derived MSCs were ex vivo culture-expanded, labeled with (111)In-oxine, and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in excised organs, including liver, lungs, kidneys, spleen and long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected primarily in the lungs and then secondarily in the liver and other organs. When sodium nitroprusside was used, more labeled MSCs cleared the lungs resulting in a larger proportion detected in the liver. Most importantly, the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator.

Pattern of localization of primitive hematopoietic cells in vivo using a novel mouse model.

Bone marrow transplantation is increasingly used as a treatment for numerous immunologic, hematologic, and malignant disorders. However, the mechanism by which transplanted hematopoietic stem cells are engrafted is not completely understood. Many traditional techniques have been used to study the engraftment of transplanted stem cells. Most of these methods are ex vivo and, in some cases, donor cells must be modified to enable detection. We describe a novel alternative for identifying unmodified primitive donor cells in a murine host. This mouse model is based on the differential capacity of adenine phosphoribosyltransferase (APRT)-positive and APRT-negative cells to sequester and incorporate radiolabeled adenine. Aprt is the gene encoding the adenine phosphoribosyltransferase purine salvage enzyme and has been ablated in 129sv mice. Following the injection of APRT-positive c-kit-positive enriched hematopoietic cells into syngeneic, sublethally irradiated APRT-deficient mice, engrafted cells and their presumptive progeny were successfully tracked by polymerase chain reaction. Their presence also was visualized by autoradiography of paraffin-embedded tissue sections. APRT-positive c-kit-positive enriched cells were detected in the bone marrow, spleen, lung, and thymus of nonirradiated mice. Donor cells and their progeny were more widely distributed in tissues of sublethally irradiated mice than of their nonirradiated counterparts, demonstrating that the pattern of localization of c-kit-positive enriched cells differs between nonirradiated and sublethally irradiated syngeneic recipients. The Aprt mouse model provides a sensitive method for further studying the mechanism of engraftment of unmodified donor hematopoietic cells in relation to the tissue architecture of the recipient.