Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders.

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM-derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

The role of human decay-accelerating factor in the pathogenesis of preterm labor.

Complement activation is thought to contribute to the pathogenesis of preterm labor (PTL). Decay-accelerating factor (DAF) is a natural complement pathway inhibitor. Our hypothesis was that DAF expression on maternal white blood cells (WBCs) in women with preterm labor is elevated compared with women with no preterm labor. Our secondary objective was to determine if differences in upregulation of DAF levels correlated with clinical outcomes. Serial blood samples were obtained from 30 patients with a clinical diagnosis of PTL and a control group of 30 pregnant individuals (same gestational age range) to determine DAF expression in peripheral WBCs in both groups. DAF expression was higher in women with PTL (less than 37 weeks) compared with the control group without PTL. Subjects with PTL who delivered before 34 weeks had less DAF expression and different kinetics of expression compared with those carrying pregnancies beyond 34 weeks. These data suggest that women with a clinical diagnosis of preterm labor have increased DAF expression on peripheral WBCs. Furthermore, it appears that failure to elevate DAF expression is associated with a risk of early premature delivery.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (P = 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. CONCLUSIONS: 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.

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.

In Vivo Cellular Imaging for Translational Medical Research.

Personalized treatment using stem, modified or genetically engineered, cells is becoming a reality in the field of medicine, in which allogenic or autologous cells can be used for treatment and possibly for early diagnosis of diseases. Hematopoietic, stromal and organ specific stem cells are under evaluation for cell-based therapies for cardiac, neurological, autoimmune and other disorders. Cytotoxic or genetically altered T-cells are under clinical trial for the treatment of hematopoietic or other malignant diseases. Before using stem cells in clinical trials, translational research in experimental animal models are essential, with a critical emphasis on developing noninvasive methods for tracking the temporal and spatial homing of these cells to target tissues. Moreover, it is necessary to determine the transplanted cell's engraftment efficiency and functional capability. Various in vivo imaging modalities are in use to track the movement and incorporation of administered cells. Tagging cells with reporter genes, fluorescent dyes or different contrast agents transforms them into cellular probes or imaging agents. Recent reports have shown that magnetically labeled cells can be used as cellular magnetic resonance imaging (MRI) probes, demonstrating the cell trafficking to target tissues. In this review, we will discuss the methods to transform cells into probes for in vivo imaging, along with their advantages and disadvantages as well as the future clinical applicability of cellular imaging method and corresponding imaging modality.

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.

Preterm labor: CD55 in maternal blood leukocytes.

PROBLEM: Intrauterine inflammation is a frequent and significant factor associated with the pathogenesis of preterm labor/birth (PTL/PTB). However, it remains unclear whether the intrauterine inflammatory responses activate the maternal peripheral circulation. We explored the association between PTL/PTB and the 'activation' of the peripheral circulatory system by determining whether CD55 mRNA expression within peripheral WBCs differed between PTL and control patients not in labor. METHOD OF STUDY: RNA was purified from white blood cells collected from pregnant women with preterm labor (n = 45), and from pregnant (n = 30) control women. CD55 gene expression was evaluated by quantitative PCR. RESULTS: The mean CD55 mRNA level within the PTL group (0.77 +/- 0.03) was 1.48-fold higher than that observed (0.52 +/- 0.02) within the control group (P < 0.0001); 71% of PTL patients and only 6.7% of control subjects expressed elevated CD55 mRNA. The receiver operating characteristics (with 95% CI) of CD55 as a marker for PTL were as follows: Sensitivity, 69% (53-82%); Specificity, 93% (78-99%); Positive Predictive Value, 94% (80-99%); and Negative Predictive Value, 67% (51-80%). In the patient population that delivered prematurely (before 37 weeks), 81% expressed elevated CD55 mRNA levels with a mean of 0.78 +/- 0.03 and 95% CI of 0.71-0.84. The receiver operating characteristics were as follows: Sensitivity, 73% (54-88%); Specificity, 86% (71-95%); Positive Predictive Value, 81.5% (62-94%); and Negative Predictive Value, 80% (64-91%). CONCLUSION: Here we report for the first time that CD55 mRNA expression was elevated in the peripheral WBCs of subjects with preterm labor compared with control gestationally-matched pregnant woman and that elevated leukocyte CD55 may be a useful predictor of subsequent PTB.

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.

In vitro model of bromodeoxyuridine or iron oxide nanoparticle uptake by activated macrophages from labeled stem cells: implications for cellular therapy.

There is increasing interest in using exogenous labels such as bromodeoxyuridine (BrdU) or superparamagnetic iron oxide nanoparticles (SPION) to label cells to identify transplanted cells and monitor their migration by fluorescent microscopy or in vivo magnetic resonance imaging (MRI), respectively. Direct implantation of cells into target tissue can result in >80% cell death due to trauma or apoptosis. Bystander uptake of labeled cells by activated macrophages (AM) can confound the interpretation of results. This study investigated the frequency of BrdU or SPION uptake by AM using the Boyden chamber model of inflammation. SPION/BrdU-labeled bone marrow stromal cells or HeLa cells, AM, and mouse fibroblasts (MF) or human fibroblasts (HF) were mixed in various ratios in Matrigel in the upper chamber and incubated for up to 96 hours. The AM were chemotactically induced to migrate to the lower chamber. Fluorescence-activated cell sorting analysis of AM from lower and upper chambers, in the presence of either MF or HF using anti-CD68, anti-BrdU, anti-dextran antibodies, revealed 10%-20% dextran-positive or 10% BrdU-positive AM after 96 hours of incubation. Transfer of iron to AM accounted for <10% of the total iron in labeled cells. The uptake of BrdU and SPION was dependent on the ratio of labeled cells to inflammatory cells and microenvironmental conditions. Direct implantation of BrdU/SPION-labeled cells into target tissue can result in uptake of label by AM; therefore, care should be taken to validate by histology transplanted cells for bystander cell markers and correlation with MRI results.

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 samples.

Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging.

Ferumoxides-protamine sulfate (FE-Pro) complexes are used for intracellular magnetic labeling of cells to non-invasively monitor cell trafficking by in vivo MRI. FE-Pro labeling is non-toxic to cells; however, the effects of FE-Pro labeling on cellular expression of transferrin receptor (TfR-1) and ferritin, proteins involved in iron transport and storage, has not been reported. FE-Pro-labeled human mesenchymal stem cells (MSCs), HeLa cells and primary macrophages were cultured from 1 week to 2 months and evaluated for TfR-1 and ferritin gene expression by RT-PCR and protein levels were determined using Western blots. MTT (proliferation assay) and reactive oxygen species (ROS) analysis were performed. FE-Pro labeling of HeLa and MSCs resulted in a transient decrease in TfR-1 mRNA and protein levels. In contrast, Fe-Pro labeling of primary macrophages resulted in an increase in TfR-1 mRNA but not in TfR-1 protein levels. Ferritin mRNA and protein levels increased transiently in labeled HeLa and macrophages but were sustained in MSCs. No changes in MTT and ROS analysis were noted. In conclusion, FE-Pro labeling elicited physiological changes of iron metabolism or storage, validating the safety of this procedure for cellular tracking by MRI.

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 approximately 20 A apart.