Isolation, proliferation and morphological characteristics of bone-marrow derived mesenchymal stem cells (BM-MSC) from different animal species.
Stem cell biology has attracted tremendous interest recently. It is hoped that it will play a major role in the treatment of a number of incurable diseases via transplantation therapy. Several varities of stem cells have been isolated and identified in vivo and in vitro. Very broadly they comprise of two major classes: embryonic/ fetal stem cells and adult stem cells.
Stem cells are defined by the following three criteria. First, stem cells undergo self-renewing cell divisions; that is, they can give rise to at least one daughter cell that is identical to the initial cell. A characteristic required to maintain the stem cell pool. Second, stem cells undergo lineage commitment and differentiation, giving rise to more differentiated progenitors, precursor cells, and ultimately terminally differentiated cells.
Third, stem cells repopulate in a robust fashion, a given tissue where they differentiate in response to specific cause to differentiate into cell types of that tissue that can take over the function of that tissue. The best example of a stem cell is the bone marrow stem cell that is unspecialized and able to specialize into bone or blood cells under different signals /environment with new special function (1).
Mesenchymal stem cells are multipotential cells capable of proliferation and differentiation into osteogenic, chondrogenic and adipogenic lineages both in vitro and in vivo (2, 3). Minimal criteria for defining MSC according to International Society of Cell Therapy (ISCT) are (a) they must be plastic-adherent when maintained in a standard culture conditions, (b) MSC must express some surface antigen such as CD105, CD73, CD90, CD44 and must not express CD34, CD45, CD 14, CD19, and HLA-DR, and (c) MSC must differentiate in vitro into osteoblast, adipocytes and chondrocytes under specific differentiating condition (4).
Although traditionally MSC isolated from bone marrow, more recent reports have detailed the isolation of cells with MSC characteristics from a variety of tissues including umbilical cord blood, chorionic villi of the placenta, Wharton's jelly, peripheral blood, fetal liver and lung, adipose tissue, skeletal muscle, periosteum, deciduous teeth, amniotic fluid, synovium and the circulatory system. Bone marrow and periosteum sources are richest in young animals with their numbers diminishing, but still present in old age (5).
One of the attractive advantage of BM-MSCs as a source of cell transplantation is their low immunogenecity. Recently, several studies have reported that BM-MSCs may be immune-privileged cells that do not elicit immune response due to an absence of their immunologically relevant cell surface markers. BM-MSCs also are known to inhibit proliferation of T lymphocytes, B lymphocytes, dendritic cells and natural killer cells (6). As MSCs comprise a mere 0.01-0.0001% of total bone marrow nucleated cells, in vitro cell culture expansion was essential in order to get sufficient numbers for clinical applications. Isolation technique generally was based on the adherent properties of the MSC (1).
The purpose of this study was to isolate, proliferate and morphological characterization of mesenchymal stem cells (MSC) collected from bone marrow of sheep, goat, dog, pig and rabbit.
Materials and Methods
Collection of bone marrow aspiration
1. From Rabbit: The animals were anaesthetized by intramuscular injection of xylazine @ 5 mg/kg followed, 10 minutes later by, ketamine @ 50 mg/kg in the thigh muscles. The area of iliac crest on either side was prepared in aseptic manner. The bone marrow aspirate was collected with the help of an 18 G bone marrow biopsy/ aspiration needle from the posterior aspect of iliac crest. For the collection of bone marrow (BM), the biopsy needle was inserted through the skin and the musc1e with little force. Once the needle was making contact with the bone, it was advanced by rotating it slowly until the bone cortex was penetrated. The stylet of the biopsy needle was removed and 2.5 ml of bone marrow was aspirated into a hypodermic syringe containing anti-coagulant. The biopsy needle was then removed and the same procedure was followed in the contra-lateral bone to collect another 2.5 ml of bone marrow aspirate in the same syringe. Thus a total quantity of 5ml of bone marrow aspirate was collected from each animal (Fig.1).
2. From Dog: Animals are anaesthetized with xylazine and ketamine at the rate of 1mg/ kg and 5mg/kg body weight IM respectively. Then the dogs were placed on lateral recumbence, so that the procedure would be easy. The skin over the site of collection was shaved and aseptically prepared. A 14-16 G bone marrow aspiration needle with an interlocking stylet was used for collection. For aspiration from the iliac crest, the greater prominence of the wing of the ileum was palpated. The needle was inserted with slow rotating movement in an angel of 45[degrees] parallel to the long axis of the ilium. Reaching the bone marrow may cause a brief reduction in pain reaction that confirms the location of the needle. After removal of the stylet, a 10ml syringe containing anti-coagulant was attached and bone marrow was aspirated (Fig.2).
3. From Sheep & Goat: Animals were sedated with xylazine at the rate of 0.05mg/kg body weight IM. Then, the skin over the site of collection was shaved and aseptically prepared and then the animals were placed in sitting position. A 14-16 G bone marrow aspiration needle with an interlocking stylet was used for collection. For aspiration from the iliac crest, the greater prominence of the wing of the ilium was palpated. The needle was inserted with slow rotating movement in an angel of 45[degrees] parallel to the long axis of the ilium. Reaching the bone marrow may cause a brief reduction in pain reaction that confirms the location of the needle. After removal of the stylet, a 10ml syringe containing anti-coagulant was attached and bone marrow was aspirated (Figs 3-4).
4. From Pig: Animals were sedated with atropineazaperone-ketamine (1:2:4 ratio) combination the skin over the sternum was shaved and aseptically prepared and then the animals were placed in lateral recumbency. A 14-16 G bone marrow aspiration needle with an interlocking stylet was used for collection. The needle was inserted with slow rotating movement directly to the body of the sternum. After reaching into the sternum, stylet was removed and a 10ml syringe containing anticoagulant was attached and bone marrow was aspirated (Fig.5).
Isolation and culture of mesenchymal stem cells (MSCs)
The marrow sample was diluted with equal amount of Dulbecco's phosphate buffered saline (DPBS) and was layered on density gradient medium at 2:1 ratio. The sample was subjected to gradient centrifuge at 3000 rpm for 30 minutes and the buffy coat containing mononuclear cells were collected carefully from the interface (Fig.6). The buffy coat was taken in the test tube along with the media- Dulbecco's Modified Eagle's Medium (DMEM) containing antibiotic and subjected for centrifugation for 10 minutes. Then the cell pallet were taken and diluted with same media containing antibiotic was again subjected for centrifugation for 10 minutes. The cell pallets were re-suspended, counted and plated at 2 x [10.sup.5] cells/[cm.sup.2] in 25 [cm.sup.2] culture flasks. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing fetal bovine serum (FBS) and antibiotics (mixture of penicillin and streptomycin) in an incubator with atmosphere of 5% CO2, 21% 02, 95% humidity at 370C. After 4-5 days of primary culture, the non adherent cells (RBCs) were removed by changing the medium. The media were changed in every 3 days thereafter still cell confluence was observed.
Expansion of BM-MSC
Once the cells (MSCs) attained 80-90% confluence, the cells (BM-MSCs) were passaged several times to increase cell population. For that, old culture media were removed and cells were washed two times with DPBS (without Ca and Mg). Trypsin/EDTA were added into the flask and were kept at C[0.sub.2] incubator for 5-10 min for detachment of cells (confirmed by microscopic examination).Then equal volume of growth media were added to the flask and mixed gently with 5 strokes. Then it was transfer into a 15ml tube and centrifuge at 2000 rpm for 4 minutes at room temperature, supernatant were removed completely but carefully. Then the cell pellet were resuspended in culture media and subjected for washing and cell suspension was counted. Cells were counted using a hemocytometer and reseeded in flasks with prewarmed growth medium at approximately 100,000 cells per 75 [cm.sup.2] flask (1500 cells per [cm.sup.2]). Cells were examined regularly for its viability, morphological features and confluence under high power microscope. The cells were repeatedly passage and expanded in optimal cultivation conditions in define culture medium until a pure culture was produced (Figs 7-11).
Cell staining and morphology
After the attachment of cells, they were fixed with 4% Para formaldehyde for 10 min and stained with crystal violet stain for 10 min. Excess stain was washed with tap water and after drying the plates were examined under a microscope. The polygonal, star shape and spindle shape cells were seen (Fig 12).
Results and Discussion
Bone marrow collection procedure from rabbits, sheep, goats, dogs and pigs was carried out as per procedure mentioned in the materials and methods and the protocol was repeated and standardized. The mesenchymal stem cells (MSCs) were successfully isolated, cultured and propagated from all the aspirated bone marrow samples of different species of animals. In case of rabbits, 5 ml of bone marrow were collected while in case of sheep, goat, dog and pigs, 10 ml of bone marrow sample were aspirated.
After separation of bone marrow mononuclear cells by density gradient medium, they were seeded onto a T-25 culture flask at 2.2 x [10.sup.5]/[cm.sup.2] along with DMEM medium and incubated at 37[degrees]C with 5% C[O.sub.2] for attachment and growth of MSCs. On day '0' all the seeded cells were found floating in the culture medium and by the next 4-6 days, the cells began to attach at the coated surface of plastic culture flask. After 3-4 days of initial culture, the media was replaced to replenish the depleted nutrients in the old culture media. Out of these species, canine and porcine BM-MSCs showed early attachment and better propagation or expansion property, followed by sheep, goat and rabbit. Canine and porcine BM-MSCs showed attachment by 3-5 days and became fully confluent by 79 days. The cells in monolayer assumed polygonal, star, spindle and fibroblast like morphology. On subsequent passages, all the cells acquired spindle shape with uniform morphology. In case of ovine and caprine BM-MSCs, the attachment began by 6-8 days and became completely confluent by 10-13 days. In primary monolayer, the attached cells showed polygonal shapes, but in the 2nd and 3rd passages they showed a uniform fibroblast like morphology. In case of rabbit MSCs, the cells attachment began late in comparison to canine, porcine, ovine and caprine BM-MSCs i.e. between 8-10 days. Initially they showed polymorphic nature and acquired uniform morphology in subsequent passages. The cells reached confluent growth by 12-15 days.
After all the cells become 80-90% confluent, the cells were passages using trypsin with EDTA for lifting/ detachment the stem cells and reseeded at 30% confluent level or 1000 cells/[cm.sup.2] to a fresh culture flask.
In general, primary colonies of MSCs of different animal species were observed on day 4-6 post seeding. The majority of cells were round, oval-shaped growth; after 7-9 days, adherent cells were increased and gradually extended to the growth of the polygon, star or spindle-shaped. At 12-15 days, adherent cells increased significantly, and a colony formation was observed, adjacent colony and blended them into pieces for more than 70% of cells were arranged in swirling. The cells were ill-defined, elongated spindle-shaped; 15-18 days later, colony of a long spindle cells which were uniformly distributed. Cellular morphology of stem cells varied between monolayer of round, elongated spindle-shaped with shorter/longer cytoplasmic extensions and they were grown in single cell or in cluster form. Proliferation capacity of canine and porcine MSC was much higher than other species. MSCs were characterized morphologically by crystal violet staining.
Mesenchymal stem cells are characterized morphologically by a small cell body with a few cell processes that are long and thin. The cell body contains a large, round nucleus with a prominent nucleolus which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance. The remainder of the cell body contains a small
amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosome. The cells, which are long and thin, are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils
More than three decades ago, Friedenstein and his colleagues experimentally proved for the first time that fibroblast-like cells could be isolated from bone marrow via their inherent adherence to plastic in culture (7). They described these cells as multipotential stromal precursor cells, which were spindle shaped and clonogenic in culture conditions, defining them as colony-forming unit fibroblasts (CFU-F). In addition to this, several studies conducted on MSCs reported that these cells are capable to differentiation into cardiomyocytes, neurons, and astrocytes in vitro and in vivo in addition to the mesoderm lineage (8, 9, 10). Several procedures were proposed for simply harvesting autologous or homologous mesenchymal stem cells, expanding them in culture, inducing differentiation and seeding them on suitable scaffolds in accordance with the targeted tissue type and implanting the construct into the patient's body (4, 11). Density gradient media was used most commonly for isolation of MSCs and their plastic adherent property was exploited for culture expansion.
Based on morphology in monolayer cell culture, mesenchymal stem cells from rabbit, canine, pig, sheep and goat bone marrow were found very similar to MSCs from other species and other sources. BM-MSCs were isolated successfully from these species considered by standard methodology developed for other species (9, 12). Individual colonies of rabbit MSCs became apparent by 7-8 days after plating. Morphologically the undifferentiated rabbit MSCs are fibroblast like cells with spindle-shaped or polygonal cell bodies, similar to their rodent or human counterparts (13). Similar morphology of r-MSC has also been reported (14). In the present study the rabbit MSCs were expanded rapidly and the first passage cells produced 80-90% confluence in 12-15 days. These observations were in concurrence with the other findings (15, 16, 17). In case of canine and porcine, the MSCs were isolated and cultured successfully from the bone marrow as described by previous workers (18, 19). In monolayer, they assume polygonal, star, spindle and fibroblast like morphology, similar to that of rabbit MSCs and BM-MSCs isolated from rodents and humans as described previously (20) and they attached to the culture flask by 3-5 days and became full confluent by 7-9 days. Similar observation has also been reported by other workers (20, 21, 22). The proliferation of cMSC and pMSC was faster in comparison to ovine, caprine and rabbit MSC used in the study. The isolation and culture of ovine and caprine MSCs were successfully done from the bone marrow as described by Niemeyer et al. (23) with slight modification. In primary monolayer, the attached cells show polygonal shapes, which are similar in morphology to that of cMSCs and rMSCs. They started to attach by 4-6 days and became complete confluent by 9-11 days. Similar observations were also reported (24). The proliferation of oMSC was faster in comparison to rMSC used in the study. However, it is well-accepted that the population doubling time of MSC depends on the donor (species, age, and sex) and the initial plating density (25, 26).
The present study underlines the techniques of isolation, culture, proliferation and morphological characteristics of bone marrow derived mesenchymal stem cell from different species of animals. The technique is considered easy and useful for further research or application in different regenerative medicine in animal sciences.
The authors are thankful to Department of Biotechnology (DBT), Ministry of Science & Technology, Government of India for providing financial assistance in the form of "Approved project on Stem cell" for carrying out the present study. Thanks are also due to Director, Indian Veterinary Research Institute, Izatnagar (UP) and Head, Surgery Division, IVRI for the facilities provided during the course of study.
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S.K. Maiti, M.U. Shiva Kumar, Lucky Srivastava, A.R. Ninu and Naveen Kumar
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India
Received 4 January 2013; Accepted 14 January 2013; Available online 14 January 2013
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|Title Annotation:||Original Article|
|Author:||Maiti, S.K.; Kumar, M.U. Shiva; Srivastava, Lucky; Ninu, A.R.; Kumar, Naveen|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jan 1, 2013|
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