Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Veterinary Clinical Sciences
Non Technical Summary
Power Analysis: The sample size was determined based on preliminary experiments in ourlaboratory. The mean cell doubling time for adult stem cells from healthy and laminitic hoovesare 27.8 and 45 hours, respectively, with a standard deviation of 12.4 hours. With thisinformation, the required sample size was computed. Based on these calculations, 5 individualsper group should give a power of 0.8035 to detect a difference between treatment cohorts atsignificant level of 0.05. Statistical Analysis: All statistical analyses will be performed using theSAS 9.1.2 software package (SAS Institute Inc., Cary, NC) and the Type I error will bemaintained at 0.05 for all comparisons. The expansion rates, CFU frequencies, percentage ofcells positive for each antibody and gene expression will be compared between normal andlaminitic hooves within passages and among passages within hoof condition. Cell doubling datafor each passage will be compared using PROC GLM and the Scheffe's test for pairwisemultiple comparisons of the means. The same mean comparisons between passages will beperformed for the limit dilution assay results after log transformation of the data. The modelingbehavior of the adult stem cell doubling number from passages 0 to 3 will be determined byregression analysis using the PROC REG procedure.We anticipate that the in vitro expansion rate and plasticity of adult progenitor cells fromlaminitic hooves will be significantly lower than those from normal hooves. Additionally, weexpect a higher percentage of progenitor and lower number of keratinocyte proteins thatcorrespond to to upregulation and down regulation of mRNA messages, respectively, in cellsfrom laminitic hooves. Whether or not the hypotheses are true, cell immunophenotypepopulations composing 2-D, in vitro models of stem cells from normal and laminitic hooves willbe established. The models will support future studies directed toward the pathophysiology ofand therapies for equine laminitis. Additionally, application of adult stem cells for treatment ofhooves damaged by laminitis holds great promise to treat equine companions affected by thecondition.Antibodies directed against non-equine clusters of differentiation or keratins may havevariable cross-reactivity with the homologous proteins in equine tissue. The majority ofantibodies to be used in this study have been validated for the horse. However, shouldunforeseen problems arise, additional antibodies will be tested and validated for the targetproteins. Stem cells isolated from the hoof require unique differentiation medium. Media havebeen designed and tested in the laboratory with the exception of the keratinocyte medium. Thetime and funds necessary for differentiation medium development has been factored into theproposal. Differentiation medium used to induce adult stem cells into keratinoblasts will betested initially. If the medium cannot be developed within the time allocated for the aim, theremaining differentiation methods will be sufficient to test the proposed hypotheses. EquineRTPCR primer design may require use of nucleotide sequences of homologous genes in othermammals. All primers will be validated with amplicon sequencing and comparison to knownmRNA sequence data with the basic local alignment search tool (BLAST).
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
This study is designed to address the unmet medical need of abnormal hoof growth and functionin horses that suffer from laminitis. The overall goal is to develop in vitro models composed ofcharacterized cells for the study of laminitis. The overarching hypothesis to be tested is thatabnormal stem cell expansion and differentiation in the equine hoof contributes to thepathophysiology of laminitis. This hypothesis will be tested using cells harvested from normaland laminitic hooves. Cell in vitro expansion rate and differentiation capabilities will bequantified. Additionally, cells will be characterized by protein and gene expression. Our overarching hypothesis is that changes in expansion potential and plasticity of hoof stemcells compromise tissue regeneration after laminitic damage. Cells will be harvestedimmediately post-mortem from horses euthanized for reasons unrelated to this study.Hypothesis 1: In vitro expansion rates and plasticity are significantly lower in progenitor cellisolates from laminitic versus normal adult equine hooves.Hypothesis 2: A greater percentage of progenitor cells from laminitic hooves are positive forprogenitor cell markers and a lower percentage are positive for intracellular keratins compared tothose from normal hooves after short term culture.Hypothesis 3: Levels of mRNA for progenitor target genes are higher and levels of keratin targetgenes are lower in progenitor cells from laminitic hooves compared to those from normal hoovesafter short term culture.Objectives 1: In vitro expansion rates of cells isolated from laminitic and normal hooves(n=5/condition) will be quantified with doubling time and cell doublings for cell passages 0-3.Limit dilution assays will be used to quantify the frequency of stromal, osteogenic, adipogenic,neurogenic and keratinogenic colony forming units for cell passages 0 and 3 for each cell isolate.Objective 2: Flow cytometry will be used to quantify the percentage of cells positive forprogenitor cell surface markers CD44, CD29 and CD105, and, following cell wall solubilization,keratins K14, K15 and K19 for cell passages 0 and 3.Objective 3: Levels of mRNA for target genes CD44, CD29, CD105, K14, K15 and K19 will bequantified in cell passages 0 and 3 with semi-quantitative RT-PCR.
Project Methods
Hooves will be harvested from five horses with laminitis and five horses without laminitis thatare euthanatized for reasons unrelated to this study. Inclusion criteria will be as follows - Age: 7- 15 years; Gender: any; Weight: 900 - 1100 lb; breed: thoroughbred, quarter horse, arabian, ormixed breeds. Laminitis group inclusion criteria will be - 1) Previous subjective diagnosis ofObel grade 2 laminitis; and 2) 13 mm of vertical P3 displacement (sinking) or 15º of capsularrotation in standard radiographs. Exclusion criteria will include - 1) Draft, warm-blood, or ponybreeds; 2) Evidence of hoof disease or damage in the normal group or evidence of hoof diseaseor damage other than laminitis in the laminitis group. Adult stem cells will be harvested andstem cell tissue cell density determined for each hoof. Expansion rates will be determined withdoubling time and cell doubling numbers. Plasticity will be quantified with limit dilution assaysafter no induction, keratinogenic, neurogenic, adipogenic and osteogenic induction. Progenitorsurface protein and intra-cellular keratin profiles will be determined with both flow cytometryand mRNA levels. Outcomes will be compared between normal and laminitic hooves as well asbetween cell passages within disease cohorts.Adult Stem Cell Harvest: The surface of each hoof will be disinfected with 30 % bleach. Hooveswill then be placed in 1 % chlorhexidine until tissue harvest. A reciprocating saw with sterileblades will be used to expose the laminar tissue. Laminar tissue will be harvested using a sterilescalpel then washed three times with phosphate-buffered saline (PBS) containing penicillin,streptomycin, and amphotericin. The tissue will be weighed, minced and transferred to a tubecontaining collagenase digest (1%g Bovine Serum Albumin (BSA) and 0.1% collagenase type-1in 50ml DMEM-Ham's F12 (Hyclone, Logan, UT)) with 2% antibiotic/antimycotic andincubated at 37°C for 4 hours with shaking. After incubation, the solution with cells andcollagenase will be transferred into a new 15 ml tube and centrifuged at 1200 rpm for 5 minutes.The supernatant will be discarded and the cells will be washed with PBS. Cells will beresuspended in culture medium consisting of DMEM-Ham's F12 supplemented withantibiotic/antimycotic solution and 10 % fetal bovine serum (Hyclone, Logan, UT). Trypan blueexclusion and a hemocytometer will be used to determine viable cell numbers and tissue densitycalculated.Cell Expansion: Primary cells will be seeded at a density of 105 nucleated cells/cm2 in T25culture flasks (Corning Inc., Corning, NY) in stromal culture medium. The cells will beincubated for 2-3 days before the first medium change, allowing cell adherence and facilitatingremoval of the nonadherent cell fraction. Thereafter, the medium will be changed every 3-4 daysuntil the adherent cell population reaches about 80% confluence. At this point, the adherentprimary stem cells will be passaged by incubation with 0.05% trypsin and reseeded at5x103cells/cm2 in 6 well plates (Nunclon, Rochester, NY). The first three passages (P0-P3) willbe evaluated after 2, 4, and 6 days of culture. Cell doubling times (DT) and numbers (CD) willbe calculated using the following two formulae: 1. CD= ln(Nf /Ni)/ln(2) and 2. DT= CT/CD.CT=culture time. Where DT is the cell-doubling time, CT the cell culture time, CD the celldoublings,Nf the final number of cells, Ni the initial number of cells.Colony-Forming Unit (CFU) (limiting dilution) Assays: CFU limiting dilution assays will beperformed according to previous methods to quantify stromal (CFU-F) osteogenic (CFU-Ob),adipogenic (CFU-Ad), neurogenic (CFU-n) and keratinogenic (CFU-K) differentiationcapabilities for cell passages 0 and 3. Medium exchanges will be performed every 2 days. Cellswill be added to a 96-well plate at the following dilutions: 5 x 103, 2.5 x 103, 1.25 x 103, 6.25 x102, 3.12 x 102, and 1.56 x 102, suspended in 100 μL of stromal medium/well. One plate for eachCFU assay per passage per animal will be seeded with cells. The plates will be incubated at 37ºCin a 5% CO2 humidified incubator and the medium will be changed every 2-3 days. Cells will becultured for 2-3 days in stromal medium to allow cell attachment and then cultured in theappropriate induction medium. Digital images will be obtained with an inverted phase contrastmicroscope (Nikon TS100F; Nikon Instruments Inc., Lewisville TX) and CoolSpot CCD Camera(Nikon Instruments Inc.) at each passage and subjectively evaluated for colony morphology anddistribution. The frequency of CFUs will be calculated as F=e-x, where F is the ratio of negativeto positive wells within a row, e is the natural logarithm constant 2.71, and x is CFU number perwell.Flow Cytometry: Cells will be fixed in 10% buffered formalin, and then incubated with FITC orPE labeled antibodies against CD44, 29 and 105. Flow cytometry for intracellular keratins (K)14, 15 and 19 (Abcam Inc. Cambridge, MA) with similarly labeled antibodies will be performedfollowing cell permeabilization with the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA)according to manufacturer's instructions. The flow cytometry assay will be performed using aFACScan flow cytometer (Becton Dickinson, San Jose, CA) and data will be analyzed with aMacintosh workstation (Apple Computer, Cupertino, CA), that contains CellQuest graphicssoftware (Becton Dickinson, San Jose, CA) for data acquisition and analysis.RT-PCR: Total RNA will be isolated from cells (RNAqueous Kit, Ambion, Inc., Austin, TX),DNase-treated, and reverse-transcribed using oligo (dT) primers and Moloney murine leukemiavirus (M-MLV) reverse transcriptase. Target gene mRNA levels will be quantified with RT-PCRusing SYBR Green technology (MJ Research Chromo4 Detector, Bio-Rad Laboratories) underoptimized cycling conditions. The dissociation stage (melting curve) will be performed with onecycle at 95°C for 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds. Serial dilutions ofall cDNA samples will be analyzed for each target gene to confirm primer efficiency. Relativegene expression will be evaluated against the reference gene GAPDH.