Equine adipose-derived mesenchymal stem cells: phenotype and growth characteristics, gene expression profile and differentiation potentials

Abstract

Objective: Because of the therapeutic application of stem cells (SCs), isolation and characterization of different types of SCs, especially mesenchymal stem cells (MSCs), have gained considerable attention in recent studies. Adipose tissue is an abundant and accessible source of MSCs which can be used for tissue engineering and in particular for treatment of musculoskeletal disorders. This study was aimed to isolate and culture equine adipose-derived MSCs (AT-MSCs) from little amounts of fat tissue samples and determine some of their biological characteristics.

Materials and methods: In this descriptive study, only 3-5 grams of fat tissue were collected from three crossbred mares. Immediately, cells were isolated by mechanical means and enzymatic digestion and were cultured in optimized conditions until passage 3 (P3). The cells at P3 were evaluated for proliferative capacities, expression of specific markers, and osteogenic, chondrogenic and adipogenic differentiation potentials.

Results: Results showed that the isolated cells were plastic adherent with a fibroblast-like phenotype. AT-MSCs exhibited expression of mesenchymal cluster of differentiation (CD) markers (CD29, CD44 and CD90) and not major histocompatibility complex II (MHC-II) and CD34 (hematopoietic marker). Cellular differentiation assays demonstrated the chondrogenic, adipogenic and osteogenic potential of the isolated cells.

Conclusion: Taken together, our findings reveal that equine MSCs can be obtained easily from little amounts of fat tissue which can be used in the future for regenerative purposes in veterinary medicine.

Keywords: Adipose; Characterization; Differentiation; Equine; Mesenchymal Stem Cells.

Fig 1

Morphological characteristics of putative mesenchymal stem cells in different days: A. Primary single adherent cells on the 4^th day after seeding (×10), B. Spindle and satellite cell morphology (×40), C. Cell colonies on the 7th day after seeding (×4), D. 80% cofluency at the end of primary culture on day 19 (×4), E. Uniform population of fibroblast-like mesenchymal stem cells (MSCs) at the end of second passage (×4) and F. Formation of some nodular cell aggregations in the primary culture (×20).

Fig 2

The growth curve of the P3 AT-MSCs belonging to the 3 horses. Cells rapidly enter the log phase after a brief lag phase and not reaching the plateau until day 8. P3; Passage 3 and AT-MSCs; Adipose tissue-derived MSCs.

Fig 3

P3 cells of horse 1 are cultured (500 cells per well) in 3 replicates. Colony-forming units are visible (blue) using crystal violet staining after 12 days of culture. P3; Passage 3.

Fig 4

Representative tri-lineage differentiation of equine AT-MSCs. Alizarin Red staining of control group (A) (×10) and osteogenic treatment group (B) (×4). Oil Red O staining of control group (C) (×10) and adipogenic treatment group (D) (×20) where lipid droplets inside the cytoplasm are stained with Oil Red O dye. Alician blue staining of a pellet section of control group (E) (×100) and chondrogenic differentiation group (F) (×100) where proteoglycans are stained in the extracellular matrix. ATMSCs; Adipose tissue-derived mesenchymal stem cells.

Fig 5

Expression of GAPDH, CD29, CD34, MHC-II, CD44 and CD90 mRNA of AT-MSCs was analyzed by RT-PCR. Representative ethidium bromide-stained gel electrophoresis revealed expression of GAPDH, CD29, CD44 and CD90. No expression of CD34 and MHC-II was observed. RT has mRNA instead of cDNA template in PCR to exclude DNA contamination and NTC is negative control. The sizes of the generated products were estimated by comparison with the mobility of the 100 bp DNA step ladder. GAPDH; Glyceraldehyde 3-phosphate dehydrogenase, CD; Cluster of differentiation, MHC; Major histocompatibility complex, AT-MSCs; Adipose tissue-derived MSCs; RT-PCR; Reverse transcriptase- polymerase chain reaction and NTC; Non-template control.