Glenn P. Niemeyer

Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow

OBJECTIVE Although several types of stem cells have been isolated from rodent and human tissues, very few data exist on stem cell isolation from nonrodent animals, which seriously limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Domestic cats are used frequently in biomedical research and are the preferred species for studies of normal physiology and disease, particularly in neuroscience. Therefore, the objective of this study was to characterize mesenchymal stem cells (MSC) from feline bone marrow for use in research on the application of stem cells to human health problems for which cats are the preferred model. METHODS Mesenchymal stem cells from feline bone marrow were isolated by standard methodology developed for other species and characterized according to morphology, growth traits, cell-surface antigen profile, and differentiation repertoire in vitro. RESULTS Feline mesenchymal stem cells exhibit a fibroblast-like morphology with bipolar or polygonal cell bodies and possess a cell-surface antigen profile similar to their rodent and human counterparts. Feline MSC exist at a frequency of 1 in 3.8 x 10(5) bone marrow mononuclear cells and are capable of differentiation to adipocytic, osteocytic, and neuronal phenotypes when exposed to appropriate induction media. CONCLUSIONS Mesenchymal stem cells isolated from feline bone marrow possess several traits typical of MSC from other species. Characterization of feline mesenchymal stem cells will facilitate future studies of stem cell biology and therapeutics for which the domestic cat is an indispensable model.

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) β-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

Neural stem/progenitor cells modulate immune responses by suppressing T lymphocytes with nitric oxide and prostaglandin E2

We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T-cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and non-activated T-cells were co-cultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when co-cultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in co-culture with T-cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by co-culture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T-cells, at least in part, by NO and PGE2 production which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.

Isolation and characterization of canine hematopoietic progenitor cells

The purpose of this study was to purify and characterize canine hematopoietic progenitor cells for surface antigen phenotype and reconstitution ability. Canine hematopoietic progenitor cells were isolated by density gradient sedimentation, lineage depletion with monoclonal antibodies, and fluorescence-activated cell sorting (FACS) for selection of cells with low-forward and right-angle scatter that were rhodamine 123 (Rh-123)(dull). Isolated cells were characterized for expression of CD34, c-kit, and Flt-3. A canine/murine xenograft model and a mixed-chimerism assay were used to examine the in vivo proliferative potential of isolated cells. The lineage-positive (Lin(+)) cells represented 80 +/- 11% (n = 22) of the input mononuclear cells. Lineage depletion resulted in a fourfold increase in colony-forming unit granulocyte/monocyte (CFU-GM), a 2.5-fold increase in burst-forming unit-erythroid (BFU-E), and a twofold increase in the number of Rh-123(dull) cells over nonlineage-depleted bone marrow mononuclear cells (BMMCs). Lineage depletion led to a 2.7-fold enrichment of CD34 cells, a 10.4-fold enrichment of c-kit cells, and a 10.8-fold enrichment of CD34/c-kit(+1) cells over total BMMCs. Nineteen percent of lineage-negative (Lin(-)) cells were positive for Flt-3. Injection of canine cells into irradiated (400 rads) NOD/SCID mice resulted in the detection of canine CD45(+) cells with BMMCs, Lin(-) cells, or Rh-123(dull) cells. Transplantation of purified Lin(-) cells in dog leukocyte antigen-matched littermates resulted in low-level engraftment for at least 10 weeks. The development of methods for purification and characterization of canine hematopoietic progenitor cells should enhance the utilization of the canine model for a variety of experimental and therapeutic purposes.

Neutrophil elastase-processing defect in cyclic hematopoietic dogs

OBJECTIVE Canine cyclic hematopoiesis (CH), a model of human cyclic neutropenia and severe congenital neutropenia, is characterized by a periodic reduced neutrophil count and decreased neutrophil elastase (NE) enzymatic activity. Canine CH is caused by a mutation of AP3B1 encoding the beta3A subunit of adaptor protein complex-3 (AP-3). It has been proposed that trafficking of elastase is affected by AP-3. The aim of this study was to study intracellular sorting/trafficking of NE in CH dogs using antibodies specific to canine NE. MATERIALS AND METHODS Polyclonal and monoclonal antibodies were generated to immunogenic epitopes in the middle (aa85-98) and C-terminal (aa269-282) regions of NE. The antibodies to canine NE were characterized by Western immunoblotting and immunocytochemistry. RESULTS Antibody ELA85 (antibody to canine NE aa 85-98) specifically recognized mature 28-kD NE. Immunocytochemical analysis using ELA85 and an antibody to myeloperoxidase demonstrated colocalizaton of NE and myeloperoxidase in primary granules of normal dogs. Antibody ELA269 (antibody to canine NE aa 269-282) reacted exclusively with the 33-kD NE presumptive precursor form. Immunocytochemical analysis demonstrated that the NE precursor was not colocalized with myeloperoxidase in the primary granules of normal or CH dogs. Western immunoblotting using these antibodies demonstrated that CH dogs contained reduced mature NE, but accumulated a large amount of the NE precursor protein that was not enzymatically active. CONCLUSION Antibodies ELA85 and ELA269 were found to be useful reagents for studying the biosynthesis, processing, and trafficking of NE during normal myelopoiesis. Neutrophils from CH dogs accumulated large amounts of higher molecular weight elastase precursors compared to normal dogs.

962. Treatment of Feline GM1 Gangliosidosis with Mesenchymal Stem Cells and Lentiviral Gene Therapy

Deficiency of lysosomal |[beta]|-galactosidase causes GM1 gangliosidosis, an inherited, progressive neurological disorder in which GM1 ganglioside accumulates in all tissues, including thymus, liver and brain. In addition to abnormal neuronal histology and function, gangliosidosis pathology involves a significant inflammatory component and therefore is similar to other neurodegenerative disorders such as Alzheimer and Parkinson disease. GM1 gangliosidosis occurs in humans, mice and domesticated animals, including cats, and the feline gangliosidosis model has been used previously by our laboratory to test several therapeutic strategies. In this study, stem cell transplantation and gene therapy have been evaluated separately and in combination as therapeutic approaches for feline GM1 gangliosidosis.