Stem cells and burns: review and therapeutic implications.

Despite significant advances in burn resuscitation and wound care over the past 30 years, morbidity and mortality from thermal injury remain high. Limited donor skin in severely burned patients hinders effective wound excision and closure, leading to infectious complications and prolonged hospitalizations. Even with large-volume fluid resuscitation, the systemic inflammatory response syndrome compromises end-organ perfusion in burn patients, with resultant multiorgan failure. Stem cells, which enhance wound healing and counteract systemic inflammation, now offer potential therapies for these challenges. Through a review of the literature, this article seeks to illustrate applications of stem cell therapy to burn care and to highlight promising areas of research.

Optimization and validation of FePro cell labeling method.

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

AC133+ progenitor cells as gene delivery vehicle and cellular probe in subcutaneous tumor models: a preliminary study.

BACKGROUND: Despite enormous progress in gene therapy for breast cancer, an optimal systemic vehicle for delivering gene products to the target tissue is still lacking. The purpose of this study was to determine whether AC133+ progenitor cells (APC) can be used as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI). In this study, we used superparamagentic iron oxide (SPIO)-labeled APCs to carry the human sodium iodide symporter (hNIS) gene to the sites of implanted breast cancer in mouse model. In vivo real time tracking of these cells was performed by MRI and expression of hNIS was determined by Tc-99m pertechnetate (Tc-99m) scan. RESULTS: Three million human breast cancer (MDA-MB-231) cells were subcutaneously implanted in the right flank of nude mice. APCs, isolated from fresh human cord blood, were genetically transformed to carry the hNIS gene using adenoviral vectors and magnetically labeled with ferumoxides-protamine sulfate (FePro) complexes. Magnetically labeled genetically transformed cells were administered intravenously in tumor bearing mice when tumors reached 0.5 cm in the largest dimension. MRI and single photon emission computed tomography (SPECT) images were acquired 3 and 7 days after cell injection, with a 7 Tesla animal MRI system and a custom built micro-SPECT using Tc-99m, respectively. Expression of hNIS in accumulated cells was determined by staining with anti-hNIS antibody. APCs were efficiently labeled with ferumoxide-protamine sulfate (FePro) complexes and transduced with hNIS gene. Our study showed not only the accumulation of intravenously administered genetically transformed, magnetically labeled APCs in the implanted breast cancer, but also the expression of hNIS gene at the tumor site. Tc-99m activity ratio (tumor/non-tumor) was significantly different between animals that received non-transduced and transduced cells (P < 0.001). CONCLUSION: This study indicates that genetically transformed, magnetically labeled APCs can be used both as delivery vehicles and cellular probes for detecting in vivo migration and homing of cells. Furthermore, they can potentially be used as a gene carrier system for the treatment of tumor or other diseases.

Investigation of relationships between transverse relaxation rate, diffusion coefficient, and labeled cell concentration in ischemic rat brain using MRI.

MRI has been used to evaluate labeled cell migration and distribution. However, quantitative determination of labeled cell concentration using MRI has not been systematically investigated. In the current study, we investigated the relationships between labeled cell concentration and MRI parameters of transverse relaxation rate, R(2), and apparent diffusion coefficient (ADC), in vitro in phantoms and in vivo in rats after stroke. Significant correlations were detected between iron concentration or labeled cell concentration and MRI measurements of R(2), ADC, and ADC x R(2) in vitro. In contrast, in vivo labeled cell concentration did not significantly correlate with R(2), ADC, and ADC x R(2). A major factor for the absence of a significant correlation between labeled cell concentration and MRI measurements in vivo may be attributed to background effects of ischemic tissue. By correcting the background effects caused by ischemic damage, DeltaR(2) (difference in R(2) values in the ischemic tissue with and without labeled cells) exhibited a significant correlation to labeled cell concentration. Our study suggests that MRI parameters have the potential to quantitatively determine labeled cell concentration in vivo.

Detection of migration of locally implanted AC133+ stem cells by cellular magnetic resonance imaging with histological findings.

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB), platelet-derived growth factor (PDGF), hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1 (SDF-1), matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells, both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1, MMP-2, and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells.

Effects of ferumoxides-protamine sulfate labeling on immunomodulatory characteristics of macrophage-like THP-1 cells.

Superparamagnetic Iron Oxide (SPIO) complexed with cationic transfection agent is used to label various mammalian cells. Labeled cells can then be utilized as an in vivo magnetic resonance imaging (MRI) probes. However, certain number of in vivo administered labeled cells may be cleared from tissues by the host's macrophages. For successful translation to routine clinical application of SPIO labeling method it is important that this mode of in vivo clearance of iron does not elicit any diverse immunological effects. The purpose of this study was to demonstrate that SPIO agent ferumoxides-protamine sulfate (FePro) incorporation into macrophages does not alter immunological properties of these cells with regard to differentiation, chemotaxis, and ability to respond to the activation stimuli and to modulate T cell response. We used THP-1 cell line as a model for studying macrophage cell type. THP-1 cells were magnetically labeled with FePro, differentiated with 100 nM of phorbol ester, 12-Myristate-13-acetate (TPA) and stimulated with 100 ng/ml of LPS. The results showed 1) FePro labeling had no effect on the changes in morphology and expression of cell surface proteins associated with TPA induced differentiation; 2) FePro labeled cells responded to LPS with slightly higher levels of NFkappaB pathway activation, as shown by immunobloting; TNF-alpha secretion and cell surface expression levels of CD54 and CD83 activation markers, under these conditions, were still comparable to the levels observed in non-labeled cells; 3) FePro labeling exhibited differential, chemokine dependent, effect on THP-1 chemotaxis with a decrease in cell directional migration to MCP-1; 4) FePro labeling did not affect the ability of THP-1 cells to down-regulate T cell expression of CD4 and CD8 and to induce T cell proliferation. Our study demonstrated that intracellular incorporation of FePro complexes does not alter overall immunological properties of THP-1 cells. The described experiments provide the model for studying the effects of in vivo clearance of iron particles via incorporation into the host's macrophages that may follow after in vivo application of any type of magnetically labeled mammalian cells. To better mimic the complex in vivo scenario, this model may be further exploited by introducing additional cellular and biological, immunologically relevant, components.

Measurement of quantity of iron in magnetically labeled cells: comparison among different UV/VIS spectrometric methods.

Cell labeling with superparamagnetic iron oxides (SPIO) is becoming a routine procedure in cellular magnetic resonance imaging (MRI). Quantifying the intracellular iron in labeled cells is a prerequisite for determining the number of accumulated cells by quantitative MRI studies. To establish the most sensitive and reproducible method for measuring iron concentration in magnetically labeled cells, we investigated and compared four different methods using an ultraviolet-visible (UV/VIS) spectrophotometer. Background spectra were obtained for 5 and 10 M hydrochloric acids, a mixture of 100 mM citric acid plus ascorbic acid and bathophenanthroline sulphonate (BPS), and a mixture of 5 M hydrochloric acid plus 5% ferrocyanide. Spectra of the same solutions containing either 10 or 5 microg/mL iron oxides were also created to determine the peak absorbance wavelengths for the dissolved iron. In addition, different known iron concentrations were used to obtain calibration lines for each method. Based on the calibration factors, iron was measured in samples with a known amount of iron and in labeled cells. Methods based on the use of 10 M hydrochloric acid underestimated iron concentration in all experiments; for this method to give an accurate measurement, iron concentration in sample needs to be at least 3 microg/mL.

Magnetically-labeled sensitized splenocytes to identify glioma by MRI: a preliminary study.

This study investigated the feasibility of imaging the migration and incorporation of magnetically-labeled sensitized splenocytes in an experimental 9L glioma brain tumor model. Splenocytes collected from tumor-bearing (sensitized splenocytes) or control (nonsensitized splenocytes) host rats were analyzed to determine the population of different cells, labeled with ferumoxides-protamine sulfate (FePro) and injected intravenously to recipient rats (N=4, for each group) bearing intracranial 9L tumors. Day 3 postinjection of splenocytes multiecho T2*-weighted and three-dimensional (3D) gradient echo MRI were obtained using a 7 Tesla MR system. R2* (1/T2*) maps were created from the T2*-weighted images. Signal intensities (SIs) and R2* values in the tumors and contralateral brain were determined by hand drawn regions of interest (ROIs). Brain sections were stained for the evidence of administered cells. Both 3D and T2*-weighted MRI showed low signal intensity areas in and around the tumors in rats that received labeled sensitized splenocytes. Prussian blue (PB), CD45- and CD8-positive cells were present in areas at the corresponding sites of low signal intensities seen on MRI. Rats that received labeled nonsensitized splenocytes did not show low signal intensity areas or PB positive cells in or around the implanted tumors. In conclusion, the immunogenic reaction can be exploited to delineate recurrent glioma using MRI following systemically delivered magnetically labeled sensitized splenocytes or T-cells.

Quantification of superparamagnetic iron oxide (SPIO)-labeled cells using MRI.

PURPOSE: To show the feasibility of using magnetic resonance imaging (MRI) to quantify superparamagnetic iron oxide (SPIO)-labeled cells. MATERIALS AND METHODS: Lymphocytes and 9L rat gliosarcoma cells were labeled with ferumoxides-protamine sulfate complex (FE-PRO). The cells were labeled efficiently (more than 95%) and the iron concentration inside each cell was measured by spectrophotometry (4.77-30.21 pg). Phantom tubes containing different numbers of labeled or unlabeled cells, as well as different concentrations of FE-PRO, were made. In addition, labeled and unlabeled cells were injected into fresh and fixed rat brains. RESULTS: Cellular viability and proliferation of labeled and unlabeled cells were shown to be similar. T2-weighted images were acquired using 7T and 3T MRI systems, and R2 maps of the tubes containing cells, free FE-PRO, and brains were made. There was a strong linear correlation between R2 values and labeled cell numbers, but the regression lines were different for the lymphocytes and gliosarcoma cells. Similarly, there was strong correlation between R2 values and free iron. However, free iron had higher R2 values than the labeled cells for the same concentration of iron. CONCLUSION: Our data indicate that in vivo quantification of labeled cells can be done by careful consideration of different factors and specific control groups.

The role of vascular cell adhesion molecule 1/ very late activation antigen 4 in endothelial progenitor cell recruitment to rheumatoid arthritis synovium.

OBJECTIVE: Marrow-derived endothelial progenitor cells (EPCs) are important in the neovascularization that occurs in diverse conditions such as cardiovascular disorders, inflammatory diseases, and neoplasms. In rheumatoid arthritis (RA), synovial neovascularization propels disease by nourishing the inflamed and hyperproliferative synovium. This study was undertaken to investigate the hypothesis that EPCs selectively home to inflamed joint tissue and may perpetuate synovial neovascularization. METHODS: In a collagen-induced arthritis (CIA) model, neovascularization and EPC accumulation in mouse ankle synovium was measured. In an antibody-induced arthritis model, EPC recruitment to inflamed synovium was evaluated. In a chimeric SCID mouse/human synovial tissue (ST) model, mice were engrafted subcutaneously with human ST, and EPC homing to grafts was assessed 2 days later. EPC adhesion to RA fibroblasts and RA ST was evaluated in vitro. RESULTS: In mice with CIA, cells bearing EPC markers were significantly increased in peripheral blood and accumulated in inflamed synovial pannus. EPCs were 4-fold more numerous in inflamed synovium from mice with anti-type II collagen antibody-induced arthritis versus controls. In SCID mice, EPC homing to RA ST was 3-fold greater than to normal synovium. Antibody neutralization of vascular cell adhesion molecule 1 (VCAM-1) and its ligand component alpha4 integrin potently inhibited EPC adhesion to RA fibroblasts and RA ST cryosections. CONCLUSION: These data demonstrate the selective recruitment of EPCs to inflamed joint tissue. The VCAM-1/very late activation antigen 4 adhesive system critically mediates EPC adhesion to cultured RA fibroblasts and to RA ST cryosections. These findings provide evidence of a possible role of EPCs in the synovial neovascularization that is critical to RA pathogenesis.

Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells.

Two FDA-approved agents, ferumoxides (Feridex), a suspension of superparamagnetic iron oxide (SPIO) nanoparticles and protamine sulfate, a drug used to reverse heparin anticoagulation, can be complexed and used to label cells magnetically ex vivo. Labeling stem cells with ferumoxides-protamine sulfate (FePro) complexes allows for non-invasive monitoring by MRI. However, in order for stem cell trials or therapies to be effective, this labeling technique must not inhibit the ability of cells to differentiate. In this study, we examined the effect of FePro labeling on stem cell differentiation. Viability, phenotypic expression and differential capacity of FePro labeled CD34 + hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) were compared with unlabeled control cells. Colony-forming unit (CFU) assays showed that the capacity to differentiate was equivalent for labeled and unlabeled HSC. Furthermore, labeling did not alter expression of surface phenotypic markers (CD34, CD31, CXCR4, CD20, CD3 and CD14) on HSC, as measured by flow cytometry. SDF-1-induced HSC migration and HSC differentiation to dendritic cells were also unaffected by FePro labeling. Both FePro-labeled and unlabeled MSC were cultured in chondrogenesis-inducing conditions. Alcian blue staining for proteoglycans revealed similar chondrogenic differentiation for both FePro-labeled and unlabeled cells. Furthermore, collagen X proteins, indicators of cartilage formation, were detected at similar levels in both labeled and unlabeled cell pellets. Prussian blue staining confirmed that cells in labeled pellets contained iron oxide, whereas cells in unlabeled pellets did not. It is concluded that FePro labeling does not alter the function or differentiation capacity of HSC and MSC. These data increase confidence that MRI studies of FePro-labeled HSC or MSC will provide an accurate representation of in vivo trafficking of unlabeled cells.