Human stem cells from adult sources have been shown, in our laboratory and others, to promote tissue repair. Different populations of stem cells have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature, although the mechanisms by which they accomplish this are still not well understood. Stem cells are known, however, to secrete a variety of factors that have both paracrine and autocrine activities. One theory of tissue repair and regeneration by adult stem cells is that they home to hypoxic and/or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive factors suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, mitosis and differentiation of tissueresiding stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of stem cells into the tissue to be regenerated. We tested human umbilical cord blood (UCB) derived CD34+, ALDHhiLin− and ALDHloLin− cells following transplantation to NOD/SCID or NOD/SCID/B2M null mice with experimentally induced acute myocardial infarction (AMI). We used combined nanoparticle labeling and whole organ fluorescent imaging to detect homing of the cells to multiple organs 48 hours post transplantation. Long term engraftment and the regenerative effect of cell treatment was assessed four weeks post transplant. There was superior homing of ALDHhiLin− cells to the site of injury, as compared to CD34+ or ALDHloLin− cells at 48 hours post transplantation. At four weeks post transplantation, ALDHhiLin− cells engrafted multiple organs, including the heart, liver and kidney, at higher frequencies than ALDHloLin− cells. We found no donor derived cardiomyocytes and only few endothelial cells of donor origin. However, there was a significant increase in the density of large caliber vessels in the central infarct zone of ALDHhiLin− cell-transplanted mice, as compared to PBS and ALDHloLin− cell treated groups. Tissue staining in the damaged regions revealed that the transplanted human cells had undergone very few cell divisions after homing to the area of hypoxia or inflammation. It is important to note that, with over 300 mice now analyzed in tissue repair experiments in our group, no adult human stem cell-derived tumors have ever been observed, even though the mouse strains used have no capacity to reject the human cells. The paucity of human cells remaining in the tissue after repair suggest that the tissue improvements that were observed were not the result of generation of transplanted cell-derived endothelial cells or cardiac tissue, but suggest that cytokines secreted from transplanted cells potentiated angiogenic activity and tissue repair by the endogenous murine cells. Our data indicate that adult human stem cells do not become a significant part of the damaged tissue, but rapidly home to and persist only temporarily at a site of hypoxia or inflammation to exert significant trophic effects on tissue repair, and to enhance recovery of the vasculature.

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.

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.

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, d TcapQ. TcapQ has potential utility in detecting and monitoring single-cell apoptosis in glaucoma in vivo.

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.

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 dextrancoated 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 fluorophorelabeled iron oxide nanoparticles for fluorescence imaging in combination with flow cytometry allows evaluation of labeling efficiencies and homing capabilities of defined human HSC subsets.

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.

Novel stem cell-based therapies require new imaging techniques to enable the visualization and tracking of transplanted cells in vivo for evaluation of homing and engraftment parameters. Here we present in vitro and in vivo data on nanoparticle labeling of umbilical cord blood (UCB) CD34+ and lineage depleted HSC subsets, in addition to labeling of CD34+ human bone marrow, G-CSF and AMD-3100 M-PBSC. For these studies, we used a 24 hr. clinically applicable ex vivo labeling protocol including protamine complexed ferrumoxide nanoparticles conjugated to Alexa 647 dye or Alexa 750 (FE-PRO[647] or Fe-Pro[750]). Cell cultivation was carried out using serum free X-Vivo 15 defined medium with 10 ng/ml rhTPO, rhSCF, and Flt-3-ligand on retronectin. Transplantation of FACS sorted 97.5% pure FE-PRO [647] labeled human UCB-derived CD34+ cells into NOD/SCID/B2M null mice resulted in mean engraftment levels of 66.7%+/−1.0% CD45+ human cells, after 8 weeks, as compared to 41.8%+/−20.4% in control mice that received non-loaded cells. These data indicate that the FE-PRO [647] did not compromise the engraftment capacity of the human HSC (p>0.05). Moreover, transplantation of labeled human UCB-derived CD34+ cells into NOD/SCID/B2M null mice for in vivo tracking using flow cytometry and magnetic resonance imaging allowed visualization of the FE-PRO[647] labeled CD34+ cells in the spleen and marrow of the recipients, up to three weeks post transplantation. In spleens, human CD34+ FE-PRO [647]+ levels decreased from 20.6.0+/−13.4% (N=5) one week post transplantation to undetectable levels after three weeks (N=7). The total human CD45+ engraftment as evaluated in total murine marrow was 18.7+/−11.3% (N=7) after three weeks. All animals in the cohort were positive for CD34+ FE-PRO[647]+ engrafted human cells (0.8+/− 0.2 %, N=7). In vivo imaging of animals transplanted with 2–5 x 105 human CD34+ cells (16.8% Fe-Pro[750]+ labeled) was performed using the Kodak 4000 MM multimodal imaging unit in which the luminescence signal arising from the nano-labeled human cells can be precisely localized by overlaying the images with x-ray pictures of the animals. Surprisingly, asymmetric engraftment was repeatedly observed between right leg tibia-femur and left-leg tibia-femur in a cohort of 8 NOD/SCID mice at various timepoints over a total of 20 days after intravenous transplantation. Human engraftment was subsequently confirmed and correlated to the luminescence signal by flow cytometry of the bones and spleens of the imaged animals, at the same timepoints. These data demonstrate that nano-particles can be used to label repopulating human HSC for subsequent in vivo tracking, without toxicity to the engrafting cells. This technique offers new methods to dynamically image the homing and engraftment of purified human hematopoietic stem cells over the initial three weeks post-transplantation, in live animals.

The use of novel nano-sized iron particles and magnetic imaging techniques are ideal for studies of homing and trafficking after labeling and transplantation of long-term repopulating, pluripotent human hematopoietic stem cells (HSC). Whereas the use of luciferase as a reporter for in vivo imaging requires transfection or viral transduction of the target cells to generate a measurable signal, we present an in vivo imaging system based upon the measurement of deep tissue penetrating, near far-red Alexa 750 nm organic dye conjugated to nano-sized ferum oxide particles (FE [750]), transiently introduced into highly purified human hematopoietic stem/progenitor subsets through complexing to the cationic agent protamine sulphate (Pro). Previous results from our group demonstrate that we can track the FE-Pro [750] labeled cells for a minimum of 30 days post transplantation using flow cytometry, before the signal diminishes due to cell division. We used a Kodak 4000MM multimodal imaging unit, which allows a precise anatomical localization of the signal measured through overlaying of the high resolution luminescent profile with x-ray images. NOD/SCID Beta2M null mice were transplanted using intravenous (IV) or intra femural (IF) injection with 1 x 105 or 2 x 105 human cord blood CD34+ cells labeled with the FE-Pro[750] nano particles. The animals were imaged directly after the injections to confirm successful transplantation, and then were subsequently imaged over a period of 8 days (cohort 1), 20 days (cohort 2) or 30 days (cohort 3). At the end point of each time period, animals were sacrificed and flow cytometry was performed to assess and confirm the location of the human engraftment in right and left leg bones as well as in spleens. Our imaging data shows that the human stem cells transplanted IF reside in the injection site for up to 10 days post transplantation, before the dilution of the signal becomes evident, with migration to the spleen at that time point indicating active engraftment, but without noticeable spreading of labeled cells to the non-injected leg. IV injected animals showed an initial strong repopulation of the spleens, with subsequent however asymmetric homing to the femur-tibiae of the legs over 8 days post transplantation, indicating a delayed homing as compared to the more direct IF delivery of the transplantation dose. Flow cytometry results confirmed the asymmetric homing to the femur-tibia bones of IV transplanted animals with one mouse in particular showing a 0.6% CD45+/Fe-Pro[750]low engraftment in the left femur-tibia whereas the right femur-tibia showed a stronger 1.3% CD45+/Fe-Pro[750]low engraftment at day 8. In conclusion, we present a novel system for imaging of human hematopoietic stem cell homing and engraftment post transplantation using dye conjugated nano-particles. This system allow an unprecedented capacity to observe and assess the in vivo dynamics of the engraftment process with high resolution, following intravenous or intrafemoral injection of different purified human stem cell populations.

Top of pageAbstract Transient transduction of human hematopoietic stem cells with novel nano-sized iron particles offers a new approach to transient cell modulation in vitro and in vivo. Moreover, nano particles serve a dual purpose by enabling tracking of labeled cells in vivo by means of magnetic resonance imaging. However, defining ex vivo cultivation strategies for the transduction and/or labeling of human HSC using these particles has not yet been well investigated. Here we present in vitro and in vivo data on human cord blood CD34+ HSC subjected to a 24 hr. clinically applicable ex vivo transduction protocol including protamine complexed ferrumoxide nanoparticles conjugated to Alexa 647 dye (FE-PRO[647]). Cell cultivation were carried out in tissue culture-treated 6 well plates coated with the CH-296 fibronectin fragment. We used X-Vivo 15 defined serum free media supplemented with 10 ng/ml rhTPO, rhSCF, and Flt-3-ligand. Initial CFU-GEMM data indicated that the presence of the FE-PRO [647] did not compromise the human HSC ability to generate colonies in vitro. Subsequent transplantation of labeled human cord blood CD34+ into beta2-NOD/SCID mice for in vivo tracking using flow cytometry consistently showed FE-PRO[647] labeled CD34+ cells in the marrow of the recipients three weeks post transplantation. The total human CD45 engraftment as evaluated in total murine marrow was 18.7+/|[minus]|11.3% (N=7), with a CD34+ component of 10.2+/|[minus]|9.0% (N=7). All 7 animals in the cohort were positive for FE-PRO[647] positive engrafted human cells (0.9+/|[minus]| 0.2 %, N=7). Most interestingly, a striking correlation of the total FE-PRO[647] positive cells and the CD34+ HSC was found in the marrow of the recipients (FE-PRO[647]+/CD34+ was 0.8+/|[minus]|0.2, N=7). Though a subset of CD133+ cells was found in all engrafted mice, interestingly enough no correlation of FE-PRO[647] and CD133+ was found. Moreover, correlating the mean fluorescence activity of the initially FE-PRO [647] transduced CB CD34+ cells to the FE-PRO[647]+/ CD34+ cells found in the recipient marrows 3 weeks post transplantation (relative value: 90.5 versus 2.9+/|[minus]|0.7, N=7) indicated that these CD34+ cells had only divided a total of 5 times over the 3 weeks time period. We hypothesize that these CD34+ cells represent long term engrafting human HSC, whereas the majority of the resulting CD34+ engraftment represents committed progenitors, giving rise to short term engraftment. Overall, these data show that nano particles can indeed be used to transiently transduce repopulating human HSC. This has lead us to define conditions for in vivo modulation of human HSC by co-transduction of FE-PRO[647] and the HIVEF1-EGFP lenti viral vector, with the aim of improving the lenti viral transduction efficiency. In vitro and in vivo data will be presented.

The use of novel nano-sized iron particles and magnetic imaging techniques are ideal for studies of homing and trafficking of long-term repopulating, human hematopoietic stem cells (HSC). However, defining ex vivo cultivation strategies for the transduction of nano-particles into the HSC involves the use of strictly defined in vitro culture conditions with the aim of maintaining viability without inducing terminal differentiation. Here we present data on in vitro uptake and retainment of nanoparticles in defined human stem cell subsets. We have chosen to use protamine sulphate-complexed Ferridex nano-particles conjugated to an Alexa 647 dye (FE-PRO[647]) to load the cells ex vivo prior to analysis, as these particles are biodegradable and, eventually, will be metabolized by the cells. This makes these particles very well suited for loading of primary cells for transplantation and subsequent imaging of the transplanted cells by MRI. Whereas most cell line studies performed show a uniform 60-75% uptake of the FE-PRO within the first 24 hours in vitro, our study of primary human cord blood (CB) lineage depleted (lin-) cells shows that the overall load of Fe-Pro [647] particles declines from 61.4+/|[minus]|0.9 (n=2) after 24hrs to a level of 39.7+/|[minus]|0.4 (n=2) after 72 hours in vitro. This indicates that the cells ability to uptake the FE-Pro [647] is declining with a prolonged ex vivo cultivation. Evaluating the FE-Pro [647] uptake based on the primary stem cell markers CD34 and CD133, however, reveals a different picture. Most notably, after 72 hours in culture the two stem cell populations have a very uniform level of the G0 determinant p27kip1, indicating that the cells are highly quiescent, but only 8.3+/|[minus]|3.7% CD34 and 5.1+/|[minus]|4.5% (n=2) CD133 cells contain the FE-Pro [647]. This points towards the idea that quiescent stem cells after subjection to ex vivo cultivation might not easily uptake the particles, possibly due to their low metabolic activity. Based on these data we next reduced the ex vivo protocol to a single 24 hr. exposure of the cells to the FE-PRO [647] particles. Using this modified ex vivo protocol we have tested a number of defined stem cell subsets for their ability to uptake and retain FE-PRO [647]. FE-PRO [647] uptake in CB CD34 was 17%, with only 15.1% of these displaying the p27kip1 high quiescent phenotype, indicating that the majority of the transduced cells were actively cycling ex vivo. Currently in vivo data are being collected from cohorts of beta-2 NOD/SCID mice. Using the clinically interesting cell sources of AMD3100 and G-CSF mobilized CD34 cells, we found FE-PRO [647] uptake over 24 hrs. ex vivo to be 7% (G-CSF) and 10.3% (AMD3100), indicating that in these cell sources, targeting of the nanoparticles to the quiescent repopulating stem cells might be even more challenging. Overall our data shows that the use of nanoparticles to transiently transduce human HSC is indeed feasible, using a clinically applicable protocol.

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.

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.

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.

We report the rational design and development of nanostructured thin-film materials that are highly efficient for surface-enhanced optical processes such as surface-enhanced Raman scattering (SERS). These materials consist of fractionated colloidal Ag particles in the size ranges of 30−50, 50−80, 80−100, and >100 nm. The basic rationale comes from correlated optical and topographic studies of single nanoparticles, which reveal a strong relationship between particle size and the excitation wavelength for efficient optical enhancement. The enhancement efficiencies are dependent on the particle size, the excitation wavelength, and the presence of activating ions such as Cl- and Br-. These nanostructured films provide a reproducible SERS substrate for ultrasensitive detection and spectroscopy of native biological molecules.