BEHAVIOR, MORPHOLOGY AND MORPHOMETRY OF EQUINE ADIPOSE – DERIVED MESENCHYMAL STEM CELLS IN CULTURE

Krzysztof Marycz, Jakub Grzesiak
Electron Microscope Laboratory, Departament of Animal Hygiene and Ichthyology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

Last researches showed multipotent abilities of fat – isolated mesenchymal stem cells. These cells have been successfully used in many locomotive disorders therapies, being even more effective in these kinds of diseases than stem cells from bone marrow. Obtained and maintained in vitro, AD-MSCs were observed, photographed and measured to get a picture of their typical behavior, appearance and sizes.

Key words: AD-MSC, behavior, morphology, morphometry.

INTRODUCTION

Multipotent, progenitor cells could be characterized by few abilities, which distinguish them from other cell types. First, they maintain proliferation capacity, being undifferentiated. Moreover, they have a potential to change into diverse kinds of cells. In general, two different kinds of stem cells were described: namely embryonic and adult ones [8]. Both of them have many advantages, but also disadvantages, as far as their clinical utility is concerned. Despite embryonic stem cells (ES) have enormous proliferative and multilineage potential, they could be unpredictable after use. Contrastive, mature progenitor cells have limited possibility of differentiation, but instead, their development is easier to control. This group could be further divided into hematopoetic cells (eg. cord blood), and mesenchymal cells existing in most adult tissues. Recently, rising interest in mesenchymal stem cells (MSC) isolated from fat tissue autogenic transplantations, as a tool of regenerative medicine, is observed. Adipose tissue is easy obtainable source of these cells, on the contrary to invasive collection methods from other sources, such us bone marrow. It has also higher progenitor cells producing efficiency [13]. Most of MSC applications are autogenic in their nature, which eliminates adverse host reactions. In equine locomotor system diseases, such as osteoarthritis or tendon and ligament injuries, many successes have been documented [1,3,4]. In this study, isolation and culturing of MSCs were performed. Cells were observed with light and electron microscope, with association of dyes or unstained. On the ground of these observations, comparison of morphology and morphometry of equine cells coming from primary and secondary cultures was made.

MATERIAL AND METHODS

Isolation. White adipose tissue was obtained from five horses suffering from orthopedic disorders (tendon and/or ligament injuries) with their owners permission. Fat was collected from tail base area. Approximately 5 grams of tissue was dissected, under aseptic conditions, from each animal, under local anesthesia. Samples were transported immediately to laboratory in sterile Hank's Balanced Salt Solution (HBSS, Sigma). Cells isolation included fine mincing by surgical scissors, washing in HBSS, clearing from any visible blood traces and vessels; finally enzyme digestion was performed [13]. Digestion buffer comprised with 0.2% collagenase type I (Sigma) dissolved in HBSS, in which shredded tissue was incubated for 30 minutes in 37°C, 5%CO₂ incubator. After that, mixture was centrifuged in Falcon tube 1200xg/10 minutes (IEC CL31R, Thermo Scientific), which separated solution into three layers, with nucleated cells located in the lowest one. Supernatant was discarded, cell fraction was carefully collected and transferred to 25ccm T-flask with medium (DMEM + 10%FBS + antibiotic solution, Sigma). Cells were maintained in humidified incubator (FDHI, Thermo Scientific) in 37°C, 5%CO₂ seven days. Just before reaching confluency by cells, they were washed twice in HBSS, trypsinised (0.2% trypsin, Sigma) and incubated till the majority of them had detached from surface. Trypsin solution with suspended cells was collected, neutralized by complete medium addition, transferred into Falcon tube and centrifuged at 300g for 5 minutes. Supernatant was removed; cell pellet was resuspended in complete medium and counted for total and alive cell yield with Thoma counting chamber by trypan blue (0.4%, Sigma) staining. Afterwards, cells were split to desired density (5x10⁴ cells/ml^-1) and seeded in new T-flasks with 15% of FBS in medium. Twenty passages were maintained.

Visualisation. Microscopic observations and measurements were made daily and of each passage of culture, by means of inverted contrast-phase microscope (AxioObserver A1, Zeiss; AxioImager 4.7, Zeiss). Additionally, semiconfluent primary and passaged cell cultures were fixed (in glutaraldehyde/osmium tetroxide), observed and measured in scanning electron microscope (Evo LS15, Zeiss). Photographic documentation and morphometry of equine cells including all culture stages was performed to compare shapes and sizes of suspended cells, diameters of their nuclei, diameters of endosomal vesicles and ranges of perinuclear areas [11]. Thickness of cells' layer was also measured (SE1, 1000x, profile width, Evo LS15 Zeiss). Immunocytochemistry applications for CD44 (Novocastra, 1:200) and CD105 (Abcam, 1:200) antigens were also carried out for confirmation of stem cells' markers existence. Also, Oil Red O (Sigma) staining was made on primary and secondary cell cultures to investigate preadipocytes' presence. Janus green B and methylene blue (Sigma) staining was conducted for mitochondria visualization.

RESULTS

In primary cultures, the diversity of cell types has been observed. Stromal vascular fraction cells (SVF), namely MSCs, preadipocytes, fibroblasts, smooth muscle and endothelial cells were noticed. Despite hematopoietic cells fraction was observed, in contrary for aforementioned cells, they had no adhesive properties. MSCs expanded faster than others, so, after seven days, they had dominated culture vessels. During first passage, about 3x10⁶ alive cells were detached and split to new flasks. Trypan blue staining confirmed almost 100% vitality.

CELLS' BEHAVIOR

Primary culture. Equine individual cells, collected by collagenase digestion, settled out in a few spots (7–15) in T-flask (25 ccm) as particular CFU (colony forming unit). Dividing cells were aiming toward other colonies and dispersing within vessels. After colonies' fusion, cells were creating a storiform patterns, forming in wavelike bundles. Sometimes they accumulated in rounded clumps. This structure was growing so rapidly that clump surrounding cells' layer has been broken (Fig. 1).

Secondary culture. Horses' cells cultures were nearly confluent after 7 days. Detached and transferred, cells were suspended in 15% FBS medium, and after approximately one hour of incubation, they began to attach (Fig. 2). They dispersed in regular way, and created a net-like structure one day after (Fig. 3). The observed growth was relatively rapid and population doubling time was less than 24 hours. Cells' distribution directly after passaging was predominately scattered and random. After approximately 24 hours of incubation cells' pattern could be described as storiform, similar to primary cultures, however they were distributed rarely with free spaces between them (Fig. 4). Process of cell division was morphologically different. Division of particular cells underwent in different planes (longitunal, transversal). Some of daughter cells were spindle shaped and adherent to surface immediately, what was observed mainly in almost confluent cultures. On the other hand, in less dense cultures, another-round-shaped form of daughter cells was observed (Fig. 5).

CELLS MORPHOLOGY

Primary culture. Particular stem cell morphology could be described as bi- or three-angled cells, with high nuclear-cytoplasmic ratio. Animals' cultured cells were spindle-shaped, with prominent nucleoli. Incidentally, cells with intracytoplasmatic abundant vacuoles of different diameter, situated in perinuclear region, were noticed. Application of Oil Red O staining allowed confirming lipid nature of vacuoles' content. Even less frequent, about five times bigger than the rest and flattened cells, with irregular borders were observed. They were characterized by foamy cytoplasm and multiple nuclei (Fig. 6). Contrast-phase microscopy with Janus Green B and methylene blue dyes showed that mitochondria were localized pericentrically (Fig. 7). Most of equine cells always exhibited two nucleoli of approximately equal size.

Secondary culture. Just after seeding, cells were rounded, of gold-brown colour when examined in inverted contrast-phase microscope. First day after passage, morphology of cells changed, they were more flattened, and create relatively long cytoplasmic connections with each other. Next day, cells' morphology was uniform and they were spindle shaped again. Oil Red O staining has not displayed a presence of adipogenic precursors or adipocytes. Immunocytochemistry showed the presence of CD44 and CD105 antigens on cells' surfaces. SEM techniques allowed visualizing precisely the ultrastructure of cultured cells. Not only nuclei with prominent nucleoli, but also mitochondria surrounding nucleus, intercellular connections and secretory granules within individual cells were observed. Cells' nuclei were comparable, including the nucleoli numbers. Everyday observations showed movement of endosomal vesicles in cell's cytoplasm. They were mostly found in the perinuclear compartment, and just before cell's division they migrate toward to opposite margins. In non-dividing cells, they were intensively moving within cell. After 19–20 passages, examined cells were less adhesive, more flattened and they were taking up more place. Intracytoplasmic lipid droplets began to appear again, which was confirmed by means of Oil Red O staining. Cell density decreased (down to 1.9x10⁶ per flask). Their vitality also decreased, in means of trypan blue method (56% vitality while eighteenth time passaged).

Morphometry. Briefly, not adherent cells, nuclei, nucleoli and endosomal vehicles were measured, averaged and compared. Average size of nucleus was 16.4 ľm; average diameter of rounded, not adherent cells was 22.35 ľm; average diameter of endosomes was 0.71 µm; average length of intercellular connections (processes) was 20.64 µm (Fig. 8, 9, 10).

DISCUSSION

Intensive sport activity in horses and mechanical injuries often lead to locomotive system diseases such as: tendon and ligament injuries, arthritis, ostearthrosis, hip joint dysplasia etc. New section of medicine, called regeneration medicine, began to expand more intensively last years [7]. Stem cell – based therapies for animals have proved great therapeutic potential of mature progenitor cells. Clinical efficacy of bone marrow derived MSC (BMD-MSC) in animals are well documented. However, in the course of locomotive disorders, adipose derived MSC (AD-MSC) seems to give better results [3]. Last researches described adipose tissue as the most available and easy obtainable source of MSCs. Simplicity of isolation connected with non-invasiveness is main undeniable advantage of AD-MSC. Moreover, particular isolation from adipose tissue allows to obtain  greater yield of proper cells, as this organ has an attribute of dynamic growth so it must have large number of self-renewal, proliferative cells [12]. In this research, heterogeneous cell population from fat tissue was isolated, including MSCs, preadipocytes, fibroblasts, smooth muscle and endothelium cells, with significant MSCs domination [14]. While further investigations, special attention was put toward progenitor cells subpopulation. The main goal of this study was to show different morphological forms of aforementioned cells with diversity of visualization techniques. Contrast-phase microscopy analysis allowed visualizing the general pattern of culture growth and behavior of particular cells. Observed storiform form of cells' layer and their spindle shape, both in primary and secondary cultures, confirm their mesenchymal origin [6,10].  Among majority of MSCs, presence of big, polynucleated cells was also noticed in primary cultures. They probably represent macrophage line, with phagocytic abilities. Additionally, their common properties and antigen phenotype with preadipocytes is well documented [2]. Sparse cell population, seen only in primary and late passages, with intracytoplasmic vacuoles, through the Oil Red O assays, could be defined as preadipocytes. Other type of cells, such as hematopoetic and endothelium ones, short time after culture establishment, probably decayed without their specific growing factors and were absent at further culture stages. Observations of cells' morphophysiology showed endosomes actively migrating within the cell, what was particulary prominent at early phase of the growth. It could be consistent with high metabolic and mitotic status [9]. In overconfluent cultures, decreased proliferation rate was observed, which could be explained, among others, by contact inhibition [5]. Relatively big size of MSCs, their adhesive properties and improved observation tools allowed to examine and precisely measure them alive in vitro. The specific staining procedures (Oil Red O, Janus Green B, methylene blue) applied created the possibility to label presence and location changes of choosen cells' organelles (lipid vacuoles, mitochondria, ribosomes, nucleoli). Aim of this research was to describe typical appearance of MSCs and to compare cells from different individuals and from different culture stages.

CONCLUSION

Isolated stromal cells from different individuals had same morphology and manifested similar behavior in vitro. For confirming this conclusion, more individuals and other species should be undertaken for researches.

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Accepted for print: 8.04.2011

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