Superparamagnetic Iron Oxide Nanoparticles Labeling of Bone Marrow Stromal (Mesenchymal) Cells Does Not Affect Their “Stemness”

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called “mesenchymal stem cells”) to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the “stemness” of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the “gold standard” of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs

Spontaneous preterm labor is associated with an increase in the proinflammatory signal transducer TLR4 receptor on maternal blood monocytes

<p>Abstract</p> <p>Background</p> <p>Localized inflammation and increased expression of TLR4 receptors within the uterus has been implicated in the pathogenesis of preterm labor. It remains unclear whether intrauterine inflammatory responses activate the maternal peripheral circulatory system. Therefore we determined whether increased TLR4 expression is present in the peripheral maternal white blood cells of women with spontaneous preterm labor.</p> <p>Methods</p> <p>This is a cross-sectional study of 41 preterm labor cases and 41 non-preterm controls. For each case and control sample, RNA was purified from white blood cells and TLR4 mRNA pool size was evaluated by quantitative PCR. Protein expression levels were determined by flow cytometry. Statistical evaluation using multiple linear regressions was used to determine any significant differences between the cases and controls. The purpose was to determine association prevalence of TLR4 levels and preterm labor.</p> <p>Results</p> <p>Adjusted mean TLR4 mRNA levels of 0.788 ± 0.037 (standard error) for preterm labor and 0.348 ± 0.038 for the corresponding pregnant control women were statistically significantly different <it>(P </it>= 0.002). Using the lower 95% confidence interval of the mean expression level in PTL subjects (0.7) as a cutoff value for elevated TLR4 mRNA levels, 25/41 (60.9%) of PTL patients expressed elevated TLR4 mRNA as compared to 0/41 (0%) in control subjects. The TLR4 receptor levels in the granulocyte fraction of white blood cells from preterm labor and pregnant controls were similar. However, TLR4⁺/CD14⁺monocytes were 2.3 times more frequent (70% vs. 30%) and TLR4 also had a 2.6-fold higher density (750 vs. 280 molecules per cell) in preterm labor women compared with pregnant controls. There was no difference in the levels of TLR4 in patients at term.</p> <p>Conclusions</p> <p>Patients with preterm labor exhibited elevated levels of CD14⁺maternal blood monocytes each bearing enhanced expression of TLR4, indicating that the peripheral circulatory system is activated in patients with preterm labor. Elevated leukocyte TLR4 levels may be a useful biomarker associated with preterm labor.</p

Color Transformation and Fluorescence of Prussian Blue–Positive Cells: Implications for Histologic Verification of Cells Labeled with Superparamagnetic Iron Oxide Nanoparticles

Superparamagnetic iron oxide (SPIO) nanoparticles, either modified or in combination with other macromolecules, are being used for magnetic labeling of stem cells and other cells to monitor cell trafficking by magnetic resonance imaging (MRI) in experimental models. The correlation of histology to MRI depends on the ability to detect SPIO-labeled cells using Prussian blue (PB) stain and fluorescent tags to cell surface markers. Exposure of PB-positive sections to ultraviolet light at a wavelength of 365 nm commonly used fluorescence microscopy can result in color transformation of PB-positive material from blue to brown. Although the PB color transformation is primarily an artifact that may occur during fluorescence microscopy, the transformation can be manipulated using imaging process software for the detection of low levels of iron labeled cells in tissues sample

In vivo transfer of intracellular labels from locally implanted bone marrow stromal cells to resident tissue macrophages.

Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION), bromodeoxyuridine (BrdU) or green fluorescent protein (GFP) are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM) can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs) that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU(+), 5-10% of GFP(+) and 5-15% of dextran(+) macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells

Structure-Function Analysis of Decay-Accelerating Factor: Identification of Residues Important for Binding of the Escherichia coli Dr Adhesin and Complement Regulation

Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a(−)); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids—predominantly those in close proximity to Ser165 to alanine—and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart

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-1α (HIF-1α), 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-1α 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-1α 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.—Arbab, A. S., Janic, B., Knight, R. A., Anderson, S. A., Pawelczyk, E., Rad, A. M., Read, E. J., Pandit, S. D., Frank, J. A. Detection of migration of locally implanted AC133+ stem cells by cellular magnetic resonance imaging with histological findings

Prussian blue staining of ossicles derived from FePro or unlabeled BMSCs.

<p>Prussian blue (PB) staining of a representative ossicle derived from BMSCs labeled with FePro (A) and control unlabeled BMSCs (B). PB staining of a representative ossicle derived from BMSCs labeled with FePro showing PB⁺ adipocytes (C). PB staining of a representative ossicle derived from labeled BMSCs showing PB⁺ pericytes (D).</p

Global gene expression and multidimensional scaling analysis of FePro labeled BMSCs.

<p>BMSC samples from 3 donors (FePro-labeled, gold nanoparticle-labeled and unlabeled control) and control cells (3 samples from human embryonic stem cells and 3 samples of adult cells) were analyzed by an oligonucleotide microarray. The multidimensional scaling plot similarly grouped the hES cells together, the adult cells other than BMSCs together in another group, and all the BMSC samples into a third group. The BMSCs did not cluster according to the type of labeling method. hES- human embryonic stem cell; adult indicated the adult cells: Fb-fibroblasts, EC endothelial cells, SMC-smooth muscle cells; BMSC-FePro: bone marrow stromal cellslabeled with FePro; BMSC-Gold: bone marrow stromal cells labeled with gold nanoparticle; BMSC-control: unlabeled BMSC control; D1: donor 1; D2-donor 2; D3 donor 3.</p