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I. Objective Isolate and characterize mesenchymal cells (MSC) from the bone marrow of a normal domestic cat (for future application to human health problems with cats as preferred model) II. Overview A. Methodology - Characterization of MSC based on: morphology, growth traits, cell-surface antigen profile, differentiation repertoire in vitro B. Results 1. Morphology: fibroblast-like, w/ bipolar or polyglonal cell bodies 2. Cell-surface Antigen Profile: similar to rodent and human counterparts 3. Differentiation: adipocytic, osteocytic and neuronal phenotypes (given: MSC’s are exposed to appropriate induction media) 4. Frequency: C. Conclusion 1. Feline MSC’s possess several traits similar from other species’ MSC’s. 2. Domestic cat can be used as an indispensable model for future studies on stem cell biology and therapeutics. III. Introduction A. Stem Cells 1. Characteristics: a. self-renewal capability b. ability to differentiate into cells of other tissues 2. Two Major Classifications of Stem Cells a. Embryonic Stem Cells Source: inner cell mass of fertilized ova Differentiation: into any cell type b. Tissue-specific Stem Cells Source: specific organs Differentiation: into cells of other tissues e.g. mesenchymal stem cells (MSC) B. MSC 1. Source: bone marrow stromal cells 2. Characteristic: can differentiate into other cells a. In-vitro: to marrow and non-marrow cell types (adipocytes, chondrocytes, osteocytes, myocytes, astrocytes and neurons) b. Invivo: astrocytes and neurons 3. Frequency: a. present in very small nos. in bone marrow 4. Characteristic of Undifferentiated MSC w/c qualifies it as a prototypic mesenchymal stem cell phenotype a. Morphology: fibroblast-like b. Ability to differentiate into many type of cells 1
Background 1. Differentiation reagents 4. species related to hematopoietic & immune system . Antibodies to CD9 & CD45 6. adult) and bone marrow transplantation in domestic cats b.Importance: provide opportunities for experimental assessment of the differentiation fate of transplanted stem cells . .g. CD9 (myeloid) b. MHC (almost all nucleated cells) IV. neonatal. Progress: Stem cells from nonrodent like mammals like cats 3. platelet) c.Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow c. Heterologous for histocompatibility loci (more closely mimic the challenges & limitations encountered in human patients receiving allogeneic cell transplants) c. Materials 1. on cell markers e. Domestic Cats as Source Advantages: a. Gives more info. derived from rodent and human tissues.4 to 6 progeny/breeding . Capable of substantial proliferation and expansion in culture d. rat. Percoll 5. postnatal). Antibodies to neuronal proteins: a. f.Multiple breeding cycles/year with short gestation period of 63±1 days g. erythrocyte. dog and human counterpart C.acquired immunodeficiency diseases e. Materials and Methods A. Characteristics consistent with those of mouse. Cells usu. Common Cell Surface Antigen in Feline & Rodent/Human MSC’s Cell surface antigen (expression) a. sampling of tissue and body fluids. immunology (fetal. h. CD44 (brain. Tissue culture media 2. Cats are large enough to permit freq.60 inherited human diseases e. diabetes & retinal atropy . Cats respond well to intrauterine surgery. 4. Stem Cell Research. mouse anti-pig neurofilament M (160 kDa) 2 . leukocyte.Several feline viral diseases adversely affect bone marrow and other organ systems. Cats were used as models for: . Supplements 3.g. 2. Studies about nervous system (stem cell transplantation therapy of neurological disorders) d. Cats can be easily maintained. Substantial knowledge: hematopoiesis (prenatal.
3 . Iscove’s Modified Dulbecco’s Medium (IMDM) w/ 200 units/ ml heparin Filter @ 100µm Pelletize: centrifuged @ 900 g Rinse w/ phosphate-buffered salive (PBS) 2X Load 10^8 cells in 12. General Steps/Overview of the Procedure 1. Procedure 1. flow cytometric determination of cell-surface antigen profile 3. isolation of mesenchymal stem cells (MSC) from feline marrow 2.073 g/ml in 0. enumeration of MSC in feline bone marrow C. mouse anti-human BIII-tubulin B. immunochemistry study of neuronal differentiated MSC 5. isolation of mesenchymal stem cells (MSC) from feline marrow Domestic cat Antemortem Source: greater trochanter of femur or greater tubercle of the humerus Necropsy Source: flushing of the shaft of a femur Bone marrow 1-5 ml vol.15 M NaCl) Centrifuge @ 1100 g for 30 mins.Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow b.5 ml Percoll (1. rabbit anti-human trkA (nerve growth factor receptor) c. induced differentiation 4.
05 % trypsin-EDTA) Replate at 8000/cm^2 4 . osteocytic and neuronal Get MSC from selected FBS Incubate in appropriate induction media* Get the lot of FBS that was optimal for proliferation of induced neuronal phenotype Remove the nonadherent cells .Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow Collect contaminating mononuclear cells @ Percoll interface Rinse w/ PBS 2X Initial Plating (3 Trials/setup) Plate crude extracted MSC at 2x10^5 /cm^2 Of Dulbecco’s Modified Eagle’s Medium (DMEM) (1g/L glucose) w/ 10 % Fetal Bovine Serum (FBS) Screen 3 lots of FBS For their ability to support feline MSC proliferation for 2-3 passages Induced Differentiation of MSC: adipocytic. After 2-3 days of initial plating replace media. After 7-12 days in culture Observe for apparent isolated MSC colonies in the culture. Trypsinized: + Trypsin (0.
2 % sodium azide in PBS) Stain on ice Dilute in 1:100 FITC-conjugated goat anti-mouse igG Detect positive cells 3.5 g/L glucose) w/ 20 % FBS 2. α-MEM induction medium) Seed cells at 6000-8000/cm^2 After 1 day incubate in DMEM (10 % FBS. 10nM βglycerophosphate. 100 mM dexamethasone.Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow + fresh medium every 3-4 days @ passage 2: replace media Use DMEM (4. Flow cytometric determination of cell-surface antigen profile Feline MSC + trypsin (0. Osteocytic Differentiation c. 0.05 % trypsin-EDTA) Resuspend in staining media (1 % BSA.35 mM Lascorbic acid) Incubate for 3 weeks Change media 2x/week + supplements: 10 % FBS Stain for Ca w/ Alizarin Red S (10 5 . Induced differentiation a. long-term neuronal Differentiation MSC cells Incubate in α-MEM Note: cell expanded 70 % to 80 % confluency in DMEM w/ 10 % FBS After 24 hours + basic fibroblast growth factor (10ng/ml) Cells remain at confluency for 3-7 days Incubate (Alpha Minimal Essential Medium. 0. Adipocytic Differentiation b.
8. Stain cells For expression of neuron specific proteins: 1. Neurofilament M(NF-M) 2.11.14 – eicosatetraynoic acid % .5 % goat serum w/ neurofilament M (1:4-1:200) trkA (1:200) or BIII-tubulin (1:200) overnight at 4 ºC Visualize (+) cells w/ FITC.or TRITC-conjugated 2º antibody Counter stain w/ propidium iodide 6 .Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow 10 % normal rabbit serum 10 nM dexamethasone 5 µg/ml insulin 50 µM 5. 2 % dimethyl sulfoxide (DMSO) + 200µM butylated hydroxynisole Cytocentrifuge after 3 days Stain for lipid droplets w/ Red O (0.2) Or Test for alkaline phosphatase activity Incubate in neuronal induction medium: α-MEM . immunochemistry study of neuronal differentiated MSC Induced neuronal phenotype MSC Fixed w/ 4% paraformaldehyde for 12 mins Rinse w/ PBS Block in 10 % normal goat serum for 1 hr Stain fixed cells w/ 1.3% in isopropanol w/ 0. Nerve growth factor receptor (trkA) BIIItubulin 4. pH 4.4 % dextrin) 25 mM KCl 2 mM valproic acid 10µM hydrocortisone 5µg/ml insulin 2mM L-glutamine w/ serum After 24 hrs.
Martin. and Henry J. Auburn University. College of Veterinary Medicine. USA (Received 10 January 2002. Auburn. round cells w/ irregular boundaries. Glenn P. Cox. Niemeyer. Hathcock.Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow 5. Ala. Baker The Scott-Ritchey Research Center. bipolar or polyglonal in morphology Contaminating monocyte colonies: large.16 7 % in methanol) Analyze under the microscope: morphological evaluation Differentiate MSC colonies from monocyte contamination Note: In theory Phenotype MSC’s: fibroblast-like cells.. Terri L. Enumeration of MSC in feline bone marrow To evaluate % MSC in feline bone marrow: Mononuclear cells isolated by Percoll centrifugation Plate at 2x10^5cells/cm^2 In 6-well plates Incubate for 10 days Stain isolated colonies w/ methylene blue (0. revised 22 March 2002. Nancy R. large nuclei Source: Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow Douglas R. accepted 18 April 2002) 7 .