Dustin J. Maxwell

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.

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.

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

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 ALDHhiLin-, and ALDHloLin- cells following transplantation to NOD/SCID or NOD/SCID β2m 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 ALDHhiLin- stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDHloLin- 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 ALDHhiLin- cell-treated mice, as compared to PBS and ALDHloLin- cell-treated mice.ConclusionsOur 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.

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.

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model (Proceedings of the National Academy of Sciences of the United States of America (2009) 106, 23, (9391-9396) DOI: 10.1073/pnas.0812884106)

Correction for ‘‘Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model,’’ by Edward M. Barnett, Xu Zhang, Dustin Maxwell, Qing Chang, and David Piwnica-Worms, which appeared in issue 23, June 9, 2009, of Proc Natl Acad Sci USA (106:9391–9396; first published May 20, 2009; 10.1073/ pnas.0812884106). The authors note that author Xu Zhang contributed equally with author Edward M. Barnett, and should have been among those credited with writing the paper. The corrected author line, author contribution footnote, and related footnotes appear below.

783. Transient Transduction of Repopulating Human CD34+ Cells Using Nanoparticles in a Clinically Applicable Ex Vivo Protocol

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.