Differentiation Protocol for Hyaline-cartilage-producing Mesenchymal Stem Cells
Abstract
Abstract
Recently, the ability of stem cells to differentiate into daughter tissues has become the focus of numerous experiments. After successfully using stem cells in experiments conducted in cardiology and neurology, there has been an increase in experimental studies examining the possible ways of exploiting stem cells in orthopaedics and traumatology. Stem cells capable of differentiating into hyaline cartilage can be derived not only from an embryonic stem cell; it is also possible to use mesenchymal stem cells (MSCs), which are found in the bone marrow. Following the isolation of MSCs and the stabilisation of a stem-cell colony, it is necessary to ‘direct’ the differentiation process towards chondrocytes. The procedure includes properly performed bone-marrow collection and isolation and the identification of a cell line of MSCs by means of CD marker expression. Among the subsequent steps are cell-line cultivation and, particularly, a correct differentiation protocol. In our experimental study, we preferred using transforming growth factor (TGF)-β1 (100ng/ml) to affect MSCs in medium for 30 minutes. For the transfer of MSCs into the tissue of the experimental animal tissue, adhesive and type I collagen were utilised. The whole process, comprising the differentiation of MSCs into mature chondrocytes in vivo and the formation of new hyaline cartilage, requires validation by histological, histochemical and immunohistochemical examination. There are numerous ways of exploiting MSCs directed towards differentiation into chondrocytes. An autologous cell transplant can be prepared from the bone marrow and used in the treatment of surface defects of hyaline cartilage and growth-plate defects due to injury or osteosynthesis. As the differentiation cascade of factors is not complete, much research will be necessary to identify the individual factors responsible for successful differentiation and to discover their mutual interaction and sequencing.
Keywords
Mesenchymal stem cells, chondrogenesis, cartilage, differentiation, growth factor
Disclosure: This work was supported by the Ministry of Health of the Czech Republic (Research Projects of IGA MZ CR NR9296-2/2007) and the Ministry of Education, Youth and Sport of the Czech Republic (NPV II 2B06130). The authors have no conflicts of interest to declare.
Received: 18 July 2008 Accepted: 13 October 2008
Correspondence: Ladislav Planka, Faculty Hospital Brno, Cernopolni 9, 613 00 Brno, Czech Republic. E: lplanka@fnbrno.cz
Recently, the ability of stem cells to differentiate into daughter tissues has become the core of numerous experiments. After successfully using stem cells in experiments conducted in cardiology and neurology, there has been an increase in experimental studies examining the possible ways of exploiting stem cells in orthopaedics and traumatology. Their aim is to obtain well-differentiated hyaline cartilage, which is invaluable in the area of articular surface and the growth plate in an immature skeleton. In case of injuries sustained at this site, healing proceeds by means of scar tissue, i.e. fibrocartilage or a scar, and the patient suffers permanent consequences associated with a disorder of function.
Stem cells capable of differentiating into hyaline cartilage can be derived not only from an embryonic stem cell; it is also possible to use adult stem cells, i.e. mesenchymal stem cells (MSCs), which are found throughout the body and can be easily obtained from the bone marrow. Following their isolation and the stabilisation of a stem-cell colony, it is necessary to ‘direct’ the differentiation process towards chondrocytes. In our experiments, we found the differentiation protocol particularly useful and achieved good experimental results using it.1 However, the differentiation itself (chondrogenic, osteogenic, etc.) has been the subject of complex studies and it is obviously a combination of several factors (the surrounding tissue, the humoral immune response, the mechanical loading of tissue, etc.) that matters in natural conditions (in vivo).
Methods
Isolation and Separation of Mononuclear Cells from Rabbit Bone Marrow
In order to obtain valid results, we chose a tried and tested methodology of collecting, culturing and differentiating autogenous MSCs. Our MSCs were separated from bone-marrow blood taken from the ilium, which is the most common site of MSC collection. 2–4 Other published methods of MSC collection include isolation of cells from periosteal blocks 5,6 or from abdominal wall subcutaneous fat. 7 In our study, a collection of bone marrow from the iliac wing was selected in the knowledge that this method is established and commonly used in the Institute of Animal Physiology and Genetics in Libechov.
The bone-marrow blood was also aspirated from os illium (tuber coxae ala osis illi) into a 5ml syringe with 2ml Dulbecco’s phosphate buffered saline (PBS) with 2% foetal bovine serum (FBS) (StemCell Technologies, Vancouver) and 5IU heparin/ml connected with a hypodermic needle (20G/40mm). Under sterile conditions, the bone marrow blood (about 4ml) was deposited over 3ml of Ficoll- Paque PLUS (StemCell Technologies). After centrifugation at 400g for 30 minutes at room temperature, the dense gradient separated erythrocytes and granulocytes as a pellet in the bottom part of the tube; mononuclear cells were situated in an opalescent layer between Ficoll and blood plasma. This layer was taken out, washed in a culture medium (see below) and used for propagation under in vitro conditions. The average amount of mononuclear cells from each isolation was 20x106 cells. Cell number and viability were analysed on Vi-CELL (Series Cell Viability Analyzers) and about 90% of viable cells were detected. It is a well-known fact that MSCs are multipotent cells capable of differentiation not only into chondrocytes, but also into osteoblasts, adipocytes, tenocytes and myoblasts. 8,9,10
After sampling of bone-marrow blood by aspiration, MSCs can be isolated and, in terms of their proliferative capacity, can be cultivated and expanded to large numbers. 11–13 MSCs have a characteristic immunological phenotype and specific cell-surface markers, for example SH-2, 3 and 4 in rabbits. However, their surface lacks haematopoietic markers such as CD34 and CD45. 10,14 The cultured cells were tested on a pig model by means of the established positive markers for MSCs (CD29, CD44, CD90 and CD105; CD147 has recently been added in several studies). The panel of antibodies used was anti-CD29 (clone MEM-101A), anti- CD105 (clone MEM 229), anti-CD147 (clone MEM-M6/2, Exbio Praha a.s., Prague, Czech Republic), anti-CD44 (clone IM7), anti-CD90 (clone 5E10, BD Biosciences, San Jose, California) and anti-CD45 (clone K252-1E4, AbD Serotec, Kidlington, UK). The expression of CD29, CD44 and CD90 resulted in cell populations where more than 95% were marked, and they can be considered homogeneous. The expression of CD105 and CD147 was low, and we noticed a negative cell population in several cases. It is highly probable that these markers tend to reflect with increased sensitivity the contemporary state of the cultured cells. This is particularly the case with incipient spontaneous differentiation, which can be induced by a number of external factors, such as a higher cell density and cell interaction.
