Сельскохозяйственная биология, 2012, № 2, с. 32-38.

doi: 10.15389/agrobiology.2012.2.32eng

УДК 636.2:576.3/.7.086.83:591.04

CHARACTERISTIC OF MESENCHYMAL STEM CELLS, ISOLATED FROM BONE MARROW AND FATTY TISSUE OF CATTLE

I.M. Volkova, E.V. Viktorova, I.P. Savchenkova, M.I. Gulyukin

From bone marrow (BM) and fatty tissue (FT) of cattle the authors isolated the cell populations with phenotype, similar to multipotent mesenchimal stem cells (MMSC). The comparative analysis was made for the properties and the features of isolated cell populations. It was established, that cattle MMSC, isolated from BM and FT, capable to form the cells of bone and fatty tissues at the cultivation in induction media in vitro. The obtained results suggested that cattle BM and FT are important source of MMSC, which can be used in agricultural and food biotechnology, veterinary, virology.

Keywords: multipotent mesenchymal stem cells, bone marrow, adipose tissue, cultivation, induction, differentiation, cattle.

Today, multipotent mesenchymal stem cells (MMSC) derived from the bone marrow (BM) and adipose tissue (AT) are in focus of many studies. The most intense researches were conducted on humans (1-4) and rodents (5-6). Farm animals are just recently been involved in these works, as is suggested by scientific reports on MMSC obtained from bovine (cattle) (7-9), pigs (10-11), horses (12) and sheep (13). Along with it, MMSC-like cells were detected in the umbilical cord blood of horses (14) and cattle (15),  as well as in swine AT (16).
MMSC of farm animals is of interest owing to basic functions and features of these cells. Firstly, it was reliably established their capability to self-reproduction in vitro without aneuploidy and malignancy; MMSC culture remains genetically stable during a long-time proliferation and starts a diploid cell line. Secondly, when MMSC is specifically induced in vitro, they differentiate into cells of various tissues thereby providing a unique material for creation of new cellular systems in agricultural biotechnology, veterinary and virology. Biological material (AT and BM) containing the cells with MMSC features and properties is quite available, which facilitates this relevant direction of research. Another advantage of MMSC is their ability to grow on three-dimensional media. They can fill scaffolds and differentiate in a given direction into three-dimensional structures providing in vitro simulation of particular tissues. This allows using MMSC as a promising object for studying in vitro the complex mechanisms of infection occurring at cellular and tissular levels (17-18).
The facts of directed differentiation of MMSC derived from human (19) and rodents (20-21) into muscle cells show a new promising way of research important in many areas - medicine, veterinary, biology and biotechnology.
Food biotechnology is an intensely developed area of agricultural biotechnology. The experiments on obtaining ex vivo the non-human muscle tissue for consumption - so-called “cultured meat” – are performed in many countries (22). In Russia, such work is carried out by I.A. Rogov et al. (23, 24). Cultures of diploid cells derived from mammalian tissues and organs are not suitable for this purpose because aging limits a period of proliferation in vitro (not more than 50 cytogenerations). The continuous process of a large-scale growth of cellular biomass needs a source of cells capable for a long-time proliferation in vitro, such as MMSC of mammals.
The purpose of this study was isolation from the cattle bone marrow and adipose tissue of cells phenotypically related to multipotent mesenchymal stem cells and comparative characteristics of the latter.
Technique. BM (30 ml) was obtained from cows (age - 3 years) at a meat-processing factory; the biological material was derived from the femur under sterile conditions in no later than 30 minutes after slaughter. BM sample was diluted 4 times with saline phosphate-buffered free from Ca²⁺ and Mg²⁺ ions (PBS-2), with Li-heparin (“Sarstedt”, Germany); a mononuclear fraction of cells was isolated by centrifugation for 30 min at 1000 g in Ficoll gradient (Ficoll-Paque, “Pharmacia”, Sweden). These operations were carried out according to the own-developed methodological guidelines (25). The derived cells were counted in Goryaev chamber. The cells (1,1 х 10⁶) were inoculated in a culture vessel (25 cm² area). The primary culture medium was DMEM (Dul-becco's modified Eagle's medium) with a low content of glucose (1 g/l) supplemented with 10% fetal bovine serum (FBS) (HyClone, “Perbio”, Belgium) and antibiotics (final concentration of streptomycin - 100 ug/ml, penicillin - 100 units/ml). After 24 h the medium was discarded with a fresh one and adherent cells were left for further growing.
AT was obtained from the same animals immediately after slaughter. The cells were isolated as described previously (25). To do this, AT was thoroughly washed with PBS-2, cut to fragments with small sharp scissors and a scalpel and treated with 0,01% solution the mixture of collagenase type I and type II based on DMEM at 37 oC for 30 min. Collagenase was neutralized with an equal volume of DMEM medium supplemented with 10% FBS and centrifuged at 1000 g for 10 min. The cells were twice reprecipitated by centrifugation at 800 g for 10 min and resuspended in culture medium. After the last centrifugation, the precipitant was resuspended in DMEM and passed sequentially through filters with a pore size of 80 microns (for the cells of stromal-vascular fraction), followed by 10-7 um (for stem cells). After the account in Goryaev cell-count chamber (1,2 x 10⁶), the derived cells were placed in a culture vessel (area 25 cm²). The base medium for culturing AT-derived MMSC was the same as for ones isolated from BM. After 24 h the medium was discarded with a fresh one, adherent cells were left for growing; later, the medium was changed every 4 days.
The efficiency of clone formation was evaluated at inoculation of the cells (1 ½ 103) in culture vessels (area 25 cm²) as the ratio of the total number of introduced cells to the number of clone cells on the 7-10th day. Viability was determined by standard methods with trypan blue staining (0,1% solution). Morphological characteristics were observed using an inverted phase-contrast-microscope (“Carl Zeiss AG”, Germany) with special software (AxioVision Rel. 4.8). The main criteria were cell size and shape, nuclear-cytoplasmic ratio, cytoplasm homogeneity and the presence of nucleoli in the nucleus. The evaluation was performed in native samples and in samples stained by Romanovsky-Giemsa. The mitotic index in each cell population was calculated at the phase of logarithmic growth as the ratio of mitoses to a total number of accounted cells (at least 1 x 10⁶) multiplied by 1000 (‰).
The ability of MMSC to directed differentiation into osteocytes and adipocytes was studied according to the method of I.P. Savchenkova et al. (25). Osteogenic differentiation was induced by dexamethasone (“KRKA”, Slovenia) (10-7 M), β-glycerophosphate (“Sigma”, USA) (10 mM) and ascorbic acid (“Sigma”, USA) (0,2 mM); adipogenic – by dexamethasone (“KRKA”, Slovenia) (10-7 M) and insulin (“Sigma”, USA) (10-9 M) (26). Osteogenic and adipogenic potential of cattle BM- and AT-derived MMSC was estimated from the efficiency of formation of bone and adipose tissues on the 21st-28th days of culturing  in inducing media in vitro.
Results. MMSC derived from BM and AT of cattle are known to show strong adhesion to a culture plastic and highly efficient development of colonies. On the 2nd day of cultivation, the single cells adherent to a plastic were detected (Fig. 1, b).
Populations of BM-derived cells were morphologically heterogeneous and included cells of three types. The first type was represented by small spindle-shaped cells of 10-15 mm diameter with a homogeneous cytoplasm and low nuclear-cytoplasmic ratio. The second type of cells were larger (15-20 um), more rounded, with a homogeneous cytoplasm and shifted to one edge of the cell nucleus. The third type had a fibroblast-like morphology: flat and large (60 um), flattened cells with numerous outgrowths of the cytoplasm. The number of cells of each type changed in the process of cultivation and depended on the initial rate of inoculation. The period of cytogeneration was 24 hours. The culture of isolated cells manifested high mitotic index (45 ‰). The cells proliferated and a monolayer was formed on the 10th day they formed (Fig. 1c).
AT-derived cells morphologically differed from the BT-derived MMSC. AT-derived cells showed the diversity within a population. A dominant group included large (about 60 um) cells of a fibroblast-like morphology and many cytoplasmic vacuoles and granules. A smaller group was represented by small (near 20 um) round dark cells. A smallest group were the cells with morphology similar to BM-derived MMSC - small (10-15 um diameter) narrow spindle-shaped cells with homogeneous cytoplasm. The time of cytogeneration  was 36 hours. Mitotic index of AT-derived cells equaled to 34 ‰, which was lower than that of BM-derived MMSC. These cells proliferated and formed a monolayer on the 10th day (Fig. 1, d).

Fig. 1-3.

Fig. 1. Multipotent mesenchymal stem cells (MMSC) of cattle derived from the bone marrow (BM) and adipose tissue (AT) at different phases of culturing in vitro: a – BM-derived MMSC, the 2nd day; b – AT-derived MMSC, the 2nd day; c – a monolayer of BM-derived MMSC, the 10th day; d – a monolayer of AT-derived MMSC, the 10th day. A native sample, magnification  x20 (lens), x10 (eyepiece).
Denotation: абвг - abcd

Fig. 2. Clone-forming potentials of multipotent mesenchymal stem cells (MMSC) of cattle cultured in vitro: a – a colony of BM-derived MMSC, the 7th day ; b – a colony of AT-derived MMSC, the 9th day. Native preparation, magnification x20 (lens), x10 (eyepiece).

Fig. 3. Directed differentiation of multipotent mesenchymal stem cells (MMSC) of cattle into the cells of bone tissue and adipose tissue: a – BM-derived MMSC, the 21st day in osteogenic medium (von Kossa staining); b – AT-derived MMSC on the 21st day in adipogenic medium (lipid vesicles are visible). Magnification  x20 (lens), x10 (eyepiece).
Denotations: аб - ab

At low rates of inoculation, the cells derived from both BM and AT developed the colonies of fibroblast-like cells (Fig. 2, a, b). Efficiency of clone formation of MMSC isolated from BM and AT was, respectively, 93 and 88%.

At the 3rd passage, the potential of MMSC for a directed cytodifferentiation was examined. During the culturing in the osteocyte-inducing media, morphological changes in BM-derived cells were recorded on the 14th day, whereas in AT-derived ones – only on the 28th day. Visual assessment was supported by von Kossa staining of experimental samples (Fig. 3a). The obtained data are consistent with results of other authors (2, 7, 8, 10, 13).

MMSC cultured in the adipogenic medium manifested morphological changes peculiar to adipogenic differentiation on the 21st-24th days of cultivation. This differentiation was accompanied by the appearance of clusters of cells with lipid vesicles (Fig. 3b).
Thus, the cells isolated by us from two types of cattle tissues (bone marrow and adipose tissue) are proved as multipotent mesenchymal stem cells (ITCs). The resulting cell populations manifest the properties and features specific to MMSC. This study have demonstrated the potential of these cells for a directed differentiation into the cells of bone and adipose tissues under a specific induction in vitro, which makes them a promising material for agricultural biotechnology.

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