Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

Direct evaluation of myocardial viability and stem cell engraftment demonstrates salvage of the injured myocardium.

RATIONALE: The mechanism of functional restoration by stem cell therapy remains poorly understood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and precardiac properties. In this study, the restorative effects of 3 AMC-derived subpopulations were examined in a murine myocardial injury model: (1) unselected AMCs, (2) ckit(+)AMCs, and (3) AMC-derived induced pluripotent stem cells (MiPSCs). OBJECTIVE: To determine the differential restorative effects of the AMC-derived subpopulations in the murine myocardial injury model using multimodality imaging. METHODS AND RESULTS: SCID (severe combined immunodeficiency) mice underwent left anterior descending artery ligation and were divided into 4 treatment arms: (1) normal saline control (n=14), (2) unselected AMCs (n=10), (3) ckit(+)AMCs (n=13), and (4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular function, whereas bioluminescence imaging assessed stem cell engraftment during a 4-week period. Immunohistological labeling and reverse transcriptase polymerase chain reaction of the explanted myocardium were performed. The unselected AMC and ckit(+)AMC-treated mice demonstrated transient left ventricular functional improvement. However, the MiPSCs exhibited a significantly greater increase in left ventricular function compared with all the other groups during the entire 4-week period. Left ventricular functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSC-treated animals lacked any evidence of de novo cardiac differentiation. CONCLUSION: The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium.

High-resolution, serial intravital microscopic imaging of nanoparticle delivery and targeting in a small animal tumor model.

Nanoparticles are under active investigation for the detection and treatment of cancer. Yet our understanding of nanoparticle delivery to tumors is limited by our ability to observe the uptake process on its own scale in living subjects. We chose to study single-walled carbon nanotubes (SWNTs) because they exhibit among the highest levels of tumor uptake across the wide variety of available nanoparticles. We target them using RGD (arginine-glycine-aspartic acid) peptide which directs them to integrins overexpressed on tumor vasculature and on the surface of some tumor cells (e.g., U87MG as used here). We employ intravital microscopy (IVM) to quantitatively examine the spatiotemporal framework of targeted SWNT uptake in a murine tumor model. IVM provided a dynamic microscale window into nanoparticle circulation, binding to tumor blood vessels, extravasation, binding to tumor cells, and tumor retention. RGD-SWNTs bound to tumor vasculature significantly more than controls (P<0.0001). RGD-SWNTs extravasated similarly compared to control RAD-SWNTs, but post-extravasation we observed as RGD-SWNTs eventually bound to individual tumor cells significantly more than RAD-SWNTs (p<0.0001) over time. RGD-SWNTs and RAD-SWNTs displayed similar signal in tumor for a week, but over time their curves significantly diverged (p<0.001) showing increasing RGD-SWNTs relative to untargeted SWNTs. We uncovered the complex spatiotemporal interplay between these competing uptake mechanisms. Specific uptake was delimited to early (1-6 hours) and late (1-4 weeks) time-points, while non-specific uptake dominated from 6 hours to 1 week. Our analysis revealed critical, quantitative insights into the dynamic, multifaceted mechanisms implicated in ligand-targeted SWNT accumulation in tumor using real-time observation.

Real-time fluorescence tracking of dynamic transgene variegation in stem cells.

Transgene variegation is caused by epigenetic switching between expressing and silent states. gamma-retrovirus vectors can be variegated in stem cells, but the dynamics of epigenetic remodeling during transgene variegation are unknown. Here, we measured variegated enhanced green fluorescent protein gamma-retrovirus expression over 4 days in individual embryonic stem cells while tracking cells in order to create expression lineage trees: 56 colony founder cells and their progeny were tracked over seven generations. Nineteen lineages produced synchronized inheritable trajectories of transgene silencing or reactivation, indicative of epigenetic remodeling with long-term stable inheritance. Short-term fluctuations in fluorescence intensity were also observed, which contributed low-amplitude variation to transgene expression level. These two processes have different frequencies and inheritability, but together contribute to variegated transgene expression. Inhibition of DNA methylation with 5-azacytidine eliminated long-term transgene silencing over 4 days, but short-term fluctuations continued. Our approach applies real-time imaging technology to track the long-term dynamics of transgene expression to investigate the timing and expression patterns leading to variegation.

Design and analysis of a long-term live-cell imaging chamber for tracking cellular dynamics within cultured human islets of Langerhans.

A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.

High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal.

To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (>1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.

Probabilistic model-based cell tracking.

The study of cell behavior is of crucial importance in drug and disease research. The fields of bioinformatics and biotechnology rely on the collection, processing, and analysis of huge numbers of biocellular images, including cell features such as cell size, shape, and motility. However manual methods of inferring these values are so onerous that automated methods of cell tracking and segmentation are in high demand. In this paper, a novel model-based cell tracker is designed to locate and track individual cells. The proposed cell tracker has been successfully applied to track hematopoietic stem cells (HSCs) based on identified cell locations and probabilistic data association.