Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction.

UNLABELLED: Human stem cells from adult sources have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature. The mechanisms by which this is accomplished are, however, still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDH(hi)Lin(-), and ALDH(lo)Lin(-) cells following transplantation to NOD/SCID or NOD/SCID beta2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDH(hi)Lin(-) stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDH(lo)Lin(-) committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was, however, a significant increase in vascular density in the central infarct zone of ALDH(hi)Lin(-) cell-treated mice, as compared to PBS and ALDH(lo)Lin(-) cell-treated mice. CONCLUSIONS: Our data indicate that adult human stem cells do not become a significant part of the regenerating tissue, but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery.

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model.

Molecular imaging probes have potential for in vivo identification of apoptosis and other intracellular processes. TcapQ, a cell-penetrating, near-infrared fluorescent peptide probe designed to be optically silent through intramolecular fluorescence quenching and activated by effector caspases, has been previously described and validated in vitro. Herein, using NMDA-induced apoptosis of retinal ganglion cells (RGCs), representing an in vivo rat model of glaucoma, we assessed the ability of TcapQ to image single-cell apoptosis through effector caspase activity. Following intravitreal injection, intracellular TcapQ activation occurred specifically in RGCs, identified individual apoptotic cells, showed a clear dose-response relationship with NMDA, and colocalized with TUNEL labeling in the retina. There was a significant diminution of probe activation following pretreatment with a specific inhibitor of caspase-3. Stereospecificity was also exhibited by the lack of intracellular fluorescence upon administration of the noncleavable isomer, dTcapQ. TcapQ has potential utility in detecting and monitoring single-cell apoptosis in glaucoma in vivo.

An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging.

Apoptosis is required for normal cellular homeostasis, and deregulation of the apoptotic process is implicated in various diseases. Previously, we developed a cell-penetrating near-infrared fluorescence (NIRF) probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an effector recognition sequence (DEVD) that was flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Once exposed to effector caspases, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. Herein, we present biochemical analysis of a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV). This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed an unexpectedly higher quenching efficiency between the fluorophore-quencher pair, and was potentially less toxic to cells. Assays using recombinant caspase enzymes revealed that the probe was specific for effector caspases (caspase 3 > 7 > 6). Analysis of apoptosis in HeLa cells treated with doxorubicin showed probe activation specific to apoptotic cells. In a rat model of retinal neuronal excitotoxicity, intravitreal injection of N-methyl-d-aspartate (NMDA) induced apoptosis of retinal ganglion cells (RGCs). Eyecup and retinal flat-mount images of NMDA-pretreated animals injected intravitreally with KcapQ using a clinically applicable protocol showed specific and widely distributed cell-associated fluorescence signals compared to untreated control animals. Fluorescence microscopy images of vertical retinal sections from NMDA-pretreated animals confirmed that activated probe was predominantly localized to RGCs and colocalized with TUNEL labeling. Thus, KcapQ represents an improved effector caspase-activatable NIRF probe for enhanced noninvasive analysis of apoptosis in whole cells and live animals.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity.

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Fluorophore-conjugated iron oxide nanoparticle labeling and analysis of engrafting human hematopoietic stem cells.

The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enables dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores to the dextran coat for fluorescence-activated cell sorting purification eliminated spurious signals from nonsequestered nanoparticle contaminants. A short-term defined incubation strategy was developed that allowed efficient labeling of both quiescent and cycling HSC, with no discernable toxicity in vitro or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34(+) cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles and to follow their fate post-transplantation. Flow cytometry endpoint analysis confirmed the presence of nanoparticle-labeled human stem cells in the marrow. The use of fluorophore-labeled iron oxide nanoparticles for fluorescence imaging in combination with flow cytometry allows evaluation of labeling efficiencies and homing capabilities of defined human HSC subsets.

Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis.

Apoptosis is an important process involved in diverse developmental pathways, homeostasis, and response to therapy for a variety of diseases. Thus, noninvasive methods to study regulation and to monitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, a novel cell-permeable activatable caspase substrate, TcapQ647, was synthesized and Km, kcat, and Ki values were biochemically characterized. Specific cleavage of TcapQ647 by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ647 with a kcat 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ647 cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C6-ceramide, TcapQ647 detected apoptosis in individual- and population-based fluorescent cell assays in an effector caspase inhibitor-specific manner. Further, only background fluorescence was observed in cells incubated with dTcapQ647, a noncleavable all d-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ647 to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ647 represents a sensitive, effector caspase-specific far-red "smart" probe to noninvasively monitor apoptosis in vivo.

Rationally designed peptides for controlled release of nerve growth factor from fibrin matrices.

The purpose of this research was to identify peptide sequences with varying affinity for nerve growth factor (NGF) and use them in the rational design of affinity-based drug delivery systems. A phage display library (12 amino acid random peptide sequence) was screened against NGF-conjugated chromatography resin three times and fractions containing phage of varying affinity were eluted by decreasing the pH of the eluent. These phages were isolated, amplified; then their DNA was purified and sequenced to determine the identity of the random peptide domain. Consensus peptides based on these sequences were synthesized and screened for their ability to bind NGF and release it at different rates from fibrin matrices. The ability of fibrin matrices containing these peptides and NGF to deliver to biologically active NGF was tested using a chick dorsal root ganglia model. A mathematical model was developed to further understand how the affinity of a peptide can modulate release of NGF and to aid in design optimization for the delivery system. The peptides identified in this study were determined to have varying affinities for NGF suggesting that this approach can serve as a model for tailoring the affinity of a drug delivery system for a target protein drug.

Optical imaging of bacterial infection in living mice using a fluorescent near-infrared molecular probe.

An optical imaging probe was synthesized by attaching a near-infrared carbocyanine fluorophore to an affinity group containing two zinc(II) dipicolylamine (Zn-DPA) units. The probe has a strong and selective affinity for the surfaces of bacteria, and it was used to image infections of Gram-positive S. aureus and Gram-negative E. coli bacteria in living nude mice. After intravenous injection, the probe selectively accumulates at the sites of localized bacterial infections in the thigh muscles of the mice.

Development of rationally designed affinity-based drug delivery systems.

Many drug delivery systems have been developed to provide sustained release of proteins in vivo. However, the ability to predict and control the rate of release from delivery systems is still a challenge. Toward this goal, we screened a random drug-binding peptide library (12 amino acids) to identify peptides of varying (i.e. low, moderate, and high) affinity for a model polysaccharide drug (heparin). Peptide domains of varying affinity for heparin identified from the library were synthesized using standard solid phase chemistry. A mathematical model of drug release from a biomaterial scaffold containing drug-binding peptide domains identified from the library was developed. This model describes the binding kinetics of drugs to the peptides, the diffusion of free drug, and the kinetics of enzymatic matrix degradation. The effect of the ratio of binding sites to drug, the effect of varying the binding kinetics and the rate of enzymatic matrix degradation on the rate of drug release was examined. The in vitro release of the model drug from scaffold containing the peptide drug-binding domains was measured. The ability of this system to deliver and modulate the biological activity of protein drugs was also assessed using nerve growth factor (NGF) in a chick dorsal root ganglia (DRG) neurite extension model. These studies demonstrate that our rational approach to drug delivery system design can be used to control drug release from tissue-engineered scaffolds and may be useful for promoting tissue regeneration in vivo.

Self-assembled nanoparticle probes for recognition and detection of biomolecules.

Colloidal gold nanocrystals have been used to develop a new class of nanobiosensors that is able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. At the core of this biosensor is a 2.5-nm gold nanoparticle that functions as both a nano-scaffold and a nano-quencher (efficient energy acceptor). Attached to this core are oligonucleotide molecules labeled with a thiol group at one end and a fluorophore at the other. This hybrid bio/inorganic construct is found to spontaneously assemble into a constrained arch-like conformation on the particle surface. Binding of target molecules results in a conformational change, which restores the fluorescence of the quenched fluorophore. Unlike conventional molecular beacons with a stem-and-loop structure, the nanoparticle probes do not require a stem, and their background fluorescence increases little with temperature. In comparison with the organic quencher Dabcyl (4,4'-dimethylaminophenyl azo benzoic acid), metal nanoparticles have unique structural and optical properties for new applications in biosensing and molecular engineering.

Luminescent quantum dots for multiplexed biological detection and imaging.

Recent advances in nanomaterials have produced a new class of fluorescent labels by conjugating semiconductor quantum dots with biorecognition molecules. These nanometer-sized conjugates are water-soluble and biocompatible, and provide important advantages over organic dyes and lanthanide probes. In particular, the emission wavelength of quantum-dot nanocrystals can be continuously tuned by changing the particle size, and a single light source can be used for simultaneous excitation of all different-sized dots. High-quality dots are also highly stable against photobleaching and have narrow, symmetric emission spectra. These novel optical properties render quantum dots ideal fluorophores for ultrasensitive, multicolor, and multiplexing applications in molecular biotechnology and bioengineering.