Raanan Margalit

Isolation and Characterization of Nontubular Sca-1+Lin− Multipotent Stem/Progenitor Cells from Adult Mouse Kidney

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific embryonic kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro differentiation assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-1 (Sca-1). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for beta1-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow-derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-beta/bone morphogenic protein, Wnt, or fibroblast growth factor, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer factor 2A, YY1-associated factor 2, and filamin-beta). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8(+) T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-1(+)Lin(-) cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney-derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.

Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas β2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

Direct imaging of immune rejection and memory induction by allogeneic mesenchymal stromal cells.

Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase‐labeled MSCs (Luc+ MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune‐deficient Balb‐Nude or non‐obese diabetic severe combined immunodeficiency (NOD‐SCID) recipients. Graft rejection in re‐challenge experiments infusing Luc+ Fib into mice, which had previously rejected Luc+ MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T‐cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re‐challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8+ or TEA CD4+ T cells exhibited a memory phenotype (CD122+, CD44+, and CD62Llow). Collectively, these results demonstrate that MSCs are not intrinsically immune‐privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (“off the shelf”) MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long‐term survival of allogeneic MSCs likely represents a major challenge. STEM CELLS 2009;27:2865–2874

Directed evolution of hydrolases for prevention of G-type nerve agent intoxication

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1s catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1s activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.

Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.

Dexanabinol (HU-211) effect on experimental autoimmune encephalomyelitis: implications for the treatment of acute relapses of multiple sclerosis

Dexanabinol (HU-211) is a synthetic non-psychotropic cannabinoid which suppresses TNF-alpha production in the brain and peripheral blood. The effects of dexanabinol in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses, modes of administration and time regimes. Dexanabinol, 5 mg/kg i.v. given once after disease onset (day 10), significantly reduced maximal EAE score. Increasing the dose or treatment duration resulted in further suppression of EAE. Drug administration at earlier phases during disease induction was not effective. Histological studies supported the clinical findings demonstrating reduction in the inflammatory response in the brain and spinal cord in animals treated with dexanabinol. The results suggest that dexanabinol may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis (MS).

Noninvasive Magnetic Resonance Imaging of Transport and Interstitial Fluid Pressure in Ectopic Human Lung Tumors

Tumor response to blood borne drugs is critically dependent on the efficiency of vascular delivery and transcapillary transfer. However, increased tumor interstitial fluid pressure (IFP) forms a barrier to transcapillary transfer, leading to resistance to drug delivery. We present here a new, noninvasive method which estimates IFP and its spatial distribution in vivo using contrast-enhanced magnetic resonance imaging (MRI). This method was tested in ectopic human non-small-cell lung cancer which exhibited a high IFP of approximately 28 mm Hg and, for comparison, in orthotopic MCF7 human breast tumors which exhibited a lower IFP of approximately 14 mm Hg, both implanted in nude mice. The MRI protocol consisted of slow infusion of the contrast agent [gadolinium-diethylenetriaminepentaacetic acid (GdDTPA)] into the blood for approximately 2 hours, sequential acquisition of images before and during the infusion, and measurements of T1 relaxation rates before infusion and after blood and tumor GdDTPA concentration reached a steady state. Image analysis yielded parametric images of steady-state tissue GdDTPA concentration with high values of this concentration outside the tumor boundaries, approximately 1 mmol/L, declining in the tumor periphery to approximately 0.5 mmol/L, and then steeply decreasing to low or null values. The distribution of steady-state tissue GdDTPA concentration reflected the distribution of IFP, showing an increase from the rim inward, with a high IFP plateau inside the tumor. The changes outside the borders of the tumors with high IFP were indicative of convective transport through the interstitium. This work presents a noninvasive method for assessing the spatial distribution of tumor IFP and mapping barriers to drug delivery and transport.

Heat Shock Protein 60 Activates Cytokine-Associated Negative Regulator Suppressor of Cytokine Signaling 3 in T Cells: Effects on Signaling, Chemotaxis, and Inflammation

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1α (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1α and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.

Transplantation of Human Hematopoietic Stem Cells into Ischemic and Growing Kidneys Suggests a Role in Vasculogenesis but Not Tubulogenesis

Transplantation of murine bone marrow‐derived stem cells has been reported recently to promote regeneration of the injured kidney. We investigated the potential of human adult CD34+ progenitor cells to undergo renal differentiation once xenotransplanted into ischemic and developing kidneys. Immunostaining with human‐specific antibodies for tubular cells (broad‐spectrum cytokeratin), endothelial cells (CD31, PECAM), stromal cells (vimentin), and hematopoietic cells (pan‐leukocyte CD45) demonstrated that although kidney ischemia enhanced engraftment of human cells, they were mostly hematopoietic cells (CD45+) residing in the interstitial spaces. Few other engrafted cells demonstrated an endothelial phenotype (human CD31+in morphologically appearing peritubular capillaries), but no evidence of tubular or stromal cells of human origin was found. Upregulation of SDF1 and HIF1 transcript levels in the ischemic kidneys might explain the diffuse engraftment of CD45+cells following injury. Similarly, when embryonic kidneys rudiments were co‐transplanted with human CD34+cells in mice, we found both human CD45+and CD31+cells in the periphery of the developing renal grafts, whereas parenchymal elements failed to stain. In addition, human CD34+cells had no effect on kidney growth and differentiation. This first demonstration of human CD34+stem cell transplantation into injured and developing kidneys indicates that these cells do not readily acquire a tubular phenotype and are restricted mainly to hematopoietic and, to a lesser extent, to endothelial lineages. Efforts should be made to identify additional stem cell sources applicable for kidney growth and regeneration.

Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis.