Isolation and characterization of feline dental pulp stem cells

Abstract

Objectives: The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability.

Methods: Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P₂, P₄ and P₆, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P₂, and cells were immunophenotyped in P₄ by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P₂, P₄ and P₆.

Results: The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P₂ to P₆. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45^-, CD90⁺ and CD44⁺. Structural and numerical cytogenetic aberrations were observed in P₂-P₄.

Conclusions and relevance: In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.

Keywords: Dental pulp; adult stem cells; explant; stromal cells.

Figure 1

Microscopic image of feline dental pulp stem cells (fDPSCs) (× 4 objective). (a) fDPSC explant with cells adhered to the plastic. The dental pulp explant is indicated with a single asterisk and the lines marked on the bottom of the well to facilitate adherence of the explant are indicated with a double asterisk, cells with an epithelioid appearance are indicated with a white arrow and cells with a spindle-like appearance with a black arrow. (b) fDPSCs in passage 2 (× 4 objective), (c,e) fDPSC of sample A in passage 2 (× 20 and × 40 objective) and (d,f) fDPSC of sample B in passage 2 (× 20 and × 40 objective)

Figure 2

Feline dental pulp stem cell (fDPSCs) colony-forming unit fibroblast assay. (a) Macroscopic image of colonies. (b) Microscopic image (× 20 objective) of an fDPSC colony

Figure 3

Trilineage differentiation of feline dental pulp stem cells in passage 2. Microscopic images of (a) osteogenic differentiation stained with Alizarin red showing calcium deposits (× 10 objective), (c) chondrogenic differentiation stained with Alcian blue showing matrix rich in glycosaminoglycans (× 10 objective) and (e) adipogenic differentiation stained with Oil Red O showing lipid vacuoles (× 40 objective). (b,d,f) Undifferentiated controls at (b,d) × 10 objective and (f) at × 40 objective

Figure 4

Immunophenotypic analysis of feline dental pulp stem cells (fDPSCs). (a) Representative histograms of CD90, CD44 and CD45 expression of fDPSCs (dashed line on each histogram represents non-stained cells). (b) Dot plots representing the gating strategy employed: cells were gated based on forward scatter (FSC) and side scatter (SSC) properties, single cells were then selected and CD90, CD44 and CD45 expression analyzed. Representative dot plot of CD44 and CD90 expression of fDPSC is shown. FITC = fluorescein isothiocyanate

Figure 5

Cytogenetic study of feline dental pulp stem cells (fDPSCs). (a) Normal 38, XX metaphase of fDPSCs. (b) Proposed normal karyotype (2n = 38, XX) stained with Giemsa of fDPSCs from a female cat