MORPHOLOGY OF IN VITRO EXPANDED HUMAN MUSCLE – DERIVED STEM CELLS

a Institute of Medical Biology and Genetics, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 811 08 Bratislava, Slovak Republic b Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 811 08 Bratislava, Slovak Republic c Institute of Histology and Embryology, Faculty of Medical Specialty Studies, Slovak Medical University, Limbova 12, 833 03 Bratislava, Slovak Republic e-mail: lubos.danisovic@fmed.uniba.sk


INTRODUCTION
Stem cells are characterised as undiff erentiated cells which have been derived from embryonic, foetal and adult organisms [1][2][3] .These cells are unique in their potential to generate various types of tissues under proper conditions in vitro and in vivo 4 .Embryonic and foetal stem cells are considered pluripotent but their utilization is restricted by ethical considerations 5 .For this reason, multipotent adult stem cells are promising for tissue engineering and regenerative medicine.
Over the past few years, adult stem cells have been derived from various types of tissues including bone marrow, umbilical cord blood, adipose tissue, skin, periosteum, dental pulp, etc. [6][7][8][9][10][11] .Adult stem cells are adherent and have a fi broblast-like morphology when cultured in vitro.These cells are heterogeneous and express a variety of surface markers including CD29, CD44, CD90, CD105, STRO-1 and Sca-1.Moreover, they are negative for haematopoietic markers CD34, CD45 and for HLA Class II (ref. 12,13 . Skeletal muscle contains populations of myogenic cells (satellite cells) which are capable of diff erentiation into myoblasts and a population of multipotent stem cells also referred as multipotent muscle-derived stem cells 14,15 .Satellite cells express myogenic markers MyoD, Myf5, desmin and PAX-1, and haematopoietic marker CD34 (ref. 16,17 , while muscle-derived stem cells are predominantly positive for Sca-1, CD13, CD34 and CD56 (ref. 18,19  and negative for CD45 (ref. 20).Muscle-derived stem cells are usually isolated by the serial plating technique which is based on the diff erent propensity of cells to adhere to cultivation substrate and lead into the purifi cation of myogenic cells 21 .
The aim of this study was to isolate and culture in vitro human muscle-derived stem cells and do a morphological and phenotypical analysis of them.

Isolation and cell culture
Human muscle cells were obtained under sterile conditions from a biopsy specimen (sized 2 × 5 mm) of femoral muscle (male individual, 3-month-old).The sampling was indicated for a genetic examination and was performed in accordance with The Helsinki Declaration.The muscle was carefully rinsed with sterile phosphate buff ered saline (PBS, Oxoid, UK) supplemented with gentamycin in fi nal concentration of 200 μg.ml - (Lek, Slovenia).Then cut into small pieces and digested with 0.1% collagenase type I (Pan Biotech, Germany) for 60 min.at 37 °C.The obtained suspension was centrifuged at 1000 rpm for 10 min.The resultant supernatant was aspirated and sediment was resuspended in 10 ml of 0.25 % trypsin-EDTA solution (PAA, Austria) for 30 min at 37 °C.The fi nal suspension was fi ltered through 70 μm pore-size cell strainer (BD Falcon, US) and centrifuged at 1000 rpm for 5 min.The supernatant was carefully removed and sediment was resuspended in Dulbecco's modifi ed Eagle's minimal essential medium (DMEM, Pan Biotech, Germany) containing 10% foetal calf serum (FCS, PAA, Austria) and gentamycin in a fi nal concentration of 80 μg.ml -1 .The suspension was plated on an uncoated Petri dish (Ø 40 mm, Swiss) assigned as PP1 and paced in CO 2 incubator (37 °C, 5 % of CO 2 in air) for 120 min.After 2 h, unattached cells fl oating in medium were collected and plated on other Petri dish assigned as PP2.The procedure was repeated after 24 h incubation and the last Petri dish was designated PP3.Cells from PP3 were cultured for 21 days to obtain a suffi cient number of cells, and the culture medium was refreshed every 48 h.When the cells reached confl uence, they were trypsinized and subpassaged up to the 3 rd passage.Cells from last passage were prepared for histological and histochemical analysis according to standard protocols.The phenotypic characterization was done by fl ow cytometry.

Phenotypical analysis
Bone marrow and adipose tissue derived MSCs from the third passage were analyzed by direct and indirect immunofl uorescence, according to protocols specifi c for each antibody.In each case, 10 000 events were acquired and analyzed by a Coulter Epics ALTRA fl ow cytometer.The following anti-bodies were used for cell staining: anti-CD34-FITC; anti-CD56-FITC; anti-human fi broblast surface protein (Sigma Aldrich, USA); anti-CD-13-PECy5; anti-CD45-PE-Cy5 (Dako Glostrup, Denmark); with a secondary FITC-conjugated donkey anti-mouse IgG antibody (Chemicon, USA).

Morphological analysis
The morphology of the in vitro expanded cells was repeatedly examined under inverted microscope Zeiss Axiovert 100 during cultivation.
Cells intended for immunohistochemical analysis were fi xed with cold methanol for 1 min.After that they were prepared for immunofl urescence staining against α-actin and desmin (Dako Glostrup, Denmark) according to standard protocol.
Cells set aside for transmission electron microscopy observation were fi xed with 2.5% glutaraldehyde (Sigma-Aldrich, Germany) for 4 h.After fi xation, samples were Morphology of in vitro expanded human muscle -derived stem cells rinsed in PBS and postfi xed in 2% osmium tetraoxide (Serva, Germany) for 2 h, then rinsed in distilled water and dehydrated in a graduated series of ethanol.Subsequently, the samples were embedded in EPON and cut into semi-thin sections.Ultra-thin sections were mounted on 200 mesh copper grids, then double stained using uranyl acetate and lead citrate (Serva, Germany) and examined using a Philips Morgagni transmission electron microscope.

RESULTS AND DISCUSSON
In the present study we have performed morphological and phenotypical characterization of human musclederived stem cells.These cells were considered to be multipotent and so they are interesting for cell therapy 16,19,20 .We used the generally accepted serial plating technique to obtain a population of stem cells 21 .Primary isolated muscle-derived stem cells had a fi broblast-like morphology (Fig. 1).During subsequent passages they maintained this morphology.Phenotypical analysis showed that almost all of the analyzed cells were CD13, CD34 and CD56 positive and, CD45 negative.This is in accord with other studies 17,22,23 .They did not express anti-human fi broblast surface protein.The immunohistochemical analysis showed expression of α-actin and desmin (Fig. 2), which indicates their mesenchymal origin as well as their myogenic capacity 24 .TEM analysis showed typical ultrastructural morphology of mesenchymal stem cells (Fig. 3).They had large pale nuclei with a large amount of euchromatine.Nuclei were irregular with noticeable nucleoli.Dilated cisterns of rough endoplasmic reticulum were present in the cytoplasm.In certain parts of the cytoplasm there were aggregates of granules of glycogen.The products of cells were actively secreted into the extracellular matrix.In summary, muscle-derived stem cells exhibit characteristics typical for mesenchymal stem cells.After other analysis they could be used in tissue engineering and regenerative medicine.