Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow

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: MSCs are exposed to appropriate induction media) 4. Frequency: C. Conclusion 1. Feline MSCs possess several traits similar from other species MSCs. 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

Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow

c. Capable of substantial proliferation and expansion in culture d. Characteristics consistent with those of mouse, rat, dog and human counterpart C. Stem Cell Research, Background 1. Cells usu. derived from rodent and human tissues. 2. Progress: Stem cells from nonrodent like mammals like cats 3. Domestic Cats as Source Advantages: a. Substantial knowledge: hematopoiesis (prenatal, postnatal), immunology (fetal, neonatal, adult) and bone marrow transplantation in domestic cats b. Heterologous for histocompatibility loci (more closely mimic the challenges & limitations encountered in human patients receiving allogeneic cell transplants) c. Studies about nervous system (stem cell transplantation therapy of neurological disorders) d. Cats were used as models for: - 60 inherited human diseases e.g. diabetes & retinal atropy - acquired immunodeficiency diseases e. Gives more info. on cell markers e.g. species related to hematopoietic & immune system - Importance: provide opportunities for experimental assessment of the differentiation fate of transplanted stem cells - Several feline viral diseases adversely affect bone marrow and other organ systems. f. Cats can be easily maintained. - 4 to 6 progeny/breeding - Multiple breeding cycles/year with short gestation period of 631 days g. Cats respond well to intrauterine surgery. h. Cats are large enough to permit freq. sampling of tissue and body fluids. 4. Common Cell Surface Antigen in Feline & Rodent/Human MSCs Cell surface antigen (expression) a. CD9 (myeloid) b. CD44 (brain, erythrocyte, leukocyte, platelet) c. MHC (almost all nucleated cells)

IV. Materials and Methods A. Materials 1. Tissue culture media 2. Supplements 3. Differentiation reagents 4. Percoll 5. Antibodies to CD9 & CD45 6. Antibodies to neuronal proteins: a. mouse anti-pig neurofilament M (160 kDa) 2

Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow

b. rabbit anti-human trkA (nerve growth factor receptor) c. mouse anti-human BIII-tubulin B. General Steps/Overview of the Procedure 1. isolation of mesenchymal stem cells (MSC) from feline marrow 2. flow cytometric determination of cell-surface antigen profile 3. induced differentiation 4. immunochemistry study of neuronal differentiated MSC 5. enumeration of MSC in feline bone marrow C. Procedure 1. 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. Iscoves Modified Dulbeccos Medium (IMDM) w/ 200 units/ ml heparin

Filter @ 100m

Pelletize: centrifuged @ 900 g

Rinse w/ phosphate-buffered salive (PBS) 2X

Load 10^8 cells in 12.5 ml Percoll (1.073 g/ml in 0.15 M NaCl)

Centrifuge @ 1100 g for 30 mins.

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 Dulbeccos Modified Eagles 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, 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 . 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 (0.05 % trypsin-EDTA)

Replate at 8000/cm^2

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 (4.5 g/L glucose) w/ 20 % FBS 2. Flow cytometric determination of cell-surface antigen profile Feline MSC

+ trypsin (0.05 % trypsin-EDTA)

Resuspend in staining media (1 % BSA; 0.2 % sodium azide in PBS)

Stain on ice

Dilute in 1:100 FITC-conjugated goat anti-mouse igG

Detect positive cells

3. Induced differentiation a. Adipocytic Differentiation

b. Osteocytic Differentiation

c. long-term neuronal Differentiation MSC cells Incubate in -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, -MEM induction medium)

Seed cells at 6000-8000/cm^2 After 1 day incubate in DMEM (10 % FBS, 100 mM dexamethasone, 10nM glycerophosphate, 0.35 mM Lascorbic acid) Incubate for 3 weeks Change media 2x/week

+ supplements: 10 % FBS

Stain for Ca w/ Alizarin Red S (10 5

Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow

10 % normal rabbit serum 10 nM dexamethasone 5 g/ml insulin

50 M 5,8,11,14 eicosatetraynoic acid

% , pH 4.2) Or Test for alkaline phosphatase activity

Incubate in neuronal induction medium: -MEM , 2 % dimethyl sulfoxide (DMSO)

+ 200M butylated hydroxynisole

Cytocentrifuge after 3 days

Stain for lipid droplets w/ Red O (0.3% in isopropanol w/ 0.4 % dextrin)

25 mM KCl 2 mM valproic acid 10M hydrocortisone 5g/ml insulin 2mM L-glutamine w/ serum After 24 hrs. Stain cells For expression of neuron specific proteins: 1. Neurofilament M(NF-M) 2. Nerve growth factor receptor (trkA) BIIItubulin

4. 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/ 1.5 % goat serum w/ neurofilament M (1:4-1:200) trkA (1:200) or BIII-tubulin (1:200) overnight at 4 C

Visualize (+) cells w/ FITC- or TRITC-conjugated 2 antibody

Counter stain w/ propidium iodide 6

Report Outline: Isolation and Characterization of Multipotential Mesenchymal Stem Cells from Feline Bone Marrow

5. 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 (0.16 7 % in methanol)

Analyze under the microscope: morphological evaluation

Differentiate MSC colonies from monocyte contamination

Note: In theory Phenotype MSCs: fibroblast-like cells, bipolar or polyglonal in morphology Contaminating monocyte colonies: large, round cells w/ irregular boundaries, large nuclei

Source:

Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow
Douglas R. Martin, Nancy R. Cox, Terri L. Hathcock, Glenn P. Niemeyer, and Henry J. Baker
The Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, Ala., USA (Received 10 January 2002; revised 22 March 2002; accepted 18 April 2002)