In the present study, the differentiation and phenotypic properties of MSCs isolated from bone marrow (BM) were studied. In the present study, the initial events controlling the adipogenic and osteogenic differentiation of mesenchymal stem cells were analyzed.
Materials
Methods
Co-culture of mesenchymal stem cells with leukemic cells
MSC differentiation into adipocytes and osteocytes
Mesenchymal stem cells
Isolation and source of mesenchymal stem cells
The frequency of MSCs in adult bone marrow has been estimated to be approximately one per 3.4 x 104 BM mononuclear cells (Wexler, Donaldson et al. 2003). MSCs can be expanded in vitro in DMEM containing 10% fetal bovine serum without any loss of proliferative or differentiation potential (Ayatollahi, Salmani et al. 2012).
Differentiation and proliferation potential of mesenchymal stem cells
Surface Antigens Expressed on Cultured Human Mesenchymal Stem Cells Surface Antigens Expression Levels Reference CD13, CD29, CD44,.
Therapeutic and clinical applications of mesenchymal stem cells
No long-term toxicity was observed and the patients showed a disease-free survival (Koc, Gerson et al. 2000). Apart from migrating to sites of tissue injury to facilitate tissue repair, MSC have been reported to migrate to tumor sites enabling the use of these cells as vehicles for drug delivery (Kidd, Spaeth et al. 2009).
MSC for therapeutic applications: the challenges
- Differentiation of transplanted MSC in vivo
- Migration of transplanted MSC
- Transformation during ex vivo expansion
- Modulation of tumor microenvironment in vivo
Even formation of bone tissue in the heart of mesenchymal stem cells after infusion for myocardial infarction was reported (Breitbach, Bostani et al. 2007). The transformation process was found to be a highly coordinated event involving different molecular signatures (Rubio, Garcia et al. 2008).
Controlling differentiation of mesenchymal stem cells
Interactions with the microenvironment, mechanical signals and differentiation
ECMs translate into cytoskeletal modifications which are further translated into biochemical signals that influence cellular processes (Ross, Coon et al. 2013). Cytoskeletal organization and especially actin filaments regulated the differential behavior of stem cells in response to changing environmental cues and play an important role in cell differentiation (Treiser, Yang et al. 2010).
Cytoskeleton of a cell: sensing the mechanical signals
Cell shape, actin cytoskeleton and differentiation of mesenchymal stem cells The importance of cell shape in the process of differentiation of mesenchymal
Actin severing has been reported to alter the elastic modulus of mesenchymal stem cells (Titushkin and Cho 2009). Changes in contractile properties of the actin cytoskeleton were reported to control the fate of mesenchymal stem cells.
Cytoskeleton related signaling pathways during differentiation
Hematopoiesis and the hematopoietic stem cell niche
Two definitive HSC niches have been reported in the bone marrow, the endosteal niche and the vascular niche (Zhang, Niu et al. 2003). The endosteal niche lies in close proximity to the bone cells or osteocytes and helped maintain HSC quiescence, while the vascular niche consisted of highly vascularized areas and signaled HSC proliferation and differentiation (Winkler, Barbier et al.
Hematologic malignancies and the hematopoietic stem cell niche
Leukemia is postulated to originate from a primitive stem cell population called leukemic stem cell (LSC) (Bonnet and Dick 1997; Johnsen, Kjeldsen et al. 2009). The homeostatic BM microenvironment has been reported to provide a survival advantage to the leukemic stem cells (Lane, Scadden et al. 2009).
An altered hematopoietic stem cell niche during malignancies protects the leukemic cells
Thus, change in bone marrow microenvironment during hematological malignancies plays an important role during disease progression and also affects hematopoietic recovery after therapy. Diagrammatic representation of the interaction of leukemic cells with stromal cells in the bone marrow.
Mesenchymal stem cells: an important component of the bone marrow microenvironment
Osteoblasts have been reported to secrete factors that aid in the maintenance of HSC in vitro. Mature osteocytes were reported to create a calcium gradient in the BM that aided migration of HSC into the bone marrow (Adams, Chabner et al. 2006).
Properties of mesenchymal stem cells from bone marrow of patients with hematologic disorders
- Proliferation and hematopoietic support
- Genetic properties
- Phenotypic properties
- Differentiation potential
- Cytokine and gene expression properties
Decreased expression of CD73, CD90 and CD105 was reported in MSC isolated from patients with hematological disorders (Campioni, Moretti et al. 2006). An increase in adipogenic differentiation potential was reported in MSC isolated from ALL patients (Vicente Lopez, Vazquez Garcia et al. 2014).
Aims and objectives of the present investigation
MATERIALS
- General laboratory chemicals
- Composition of reagents and buffers (i). Phosphate buffered saline (1X)
- Cell culture media
- Dyes and stains
Dissolve the components in deionized water with stirring, adjust the pH to 7.2-7.4 with 1N HCl and sterilize by autoclaving at 121°C and 15psi for 20 minutes. A 4% paraformaldehyde solution was prepared by dissolving powdered paraformaldehyde powder in PBS over a hot plate or water bath set at 55 °C. When the solution became clear and completely dissolved, the pH of the solution was adjusted to the range of 7.2-7.4 and aliquots were stored at -20 °C until use.
250 µl sodium nitrate solution was mixed with 250 µl FBB alkaline solution and added to 11.25 ml deionized water. Finally, 250µl Napthol AS-Bl-Alkaline solution was added and the substrate was ready for use.
METHODS
- Isolation of mesenchymal stem cells
- Differentiation of mesenchymal stem cells
- Phenotyping of mesenchymal stem cells
- Karyotyping of mesenchymal stem cells
- Scanning electron microscopy
- Actin staining with phalloidin TRITC
- Staining with phosphoprotein-specific antibodies for flow cytometry
- Propidium iodide staining of MSC for cell cycle analysis
- Inhibition of actin polymerization
- Inhibition and recovery of actin polymerization during differentiation
- RNA isolation
- Reverse transcription
- Real time PCR
- Primers for real time PCR
- Analysis of Real time PCR data
- Coating of tissue culture flask and glass surfaces
- Drug treatment of MSC
- MTT assay
- Co-culture of mesenchymal stem cells with leukemic cells
- Maintenance of cells in culture (i). Mesenchymal stem cells
- Data analysis
The cells were then washed with deionized water and nuclei were counterstained with neutral red. 3-5 drops of pre-chilled Carnoy's fixative were added and the cells were pelleted by centrifugation (1600 rpm x 10 minutes). 10 ml of freshly prepared Carnoy's fixative was added and the cells were pelleted by centrifugation.
Briefly, medium was removed and cells were washed 2-3 times with 0.2 µM filtered phosphate-buffered saline (PBS). The cells were then sputter coated with gold and analyzed by SEM (Leo 1430vp, Germany). Cells were then washed with PBS and stained with propidium iodide (Sigma) (50 µg/ml) and analyzed by flow cytometry.
After that, the normal induction medium was added and the cells were allowed to differentiate for the required time period.
MSC differentiation into adipocytes and osteocytes 1. Cell morphology and size
- Actin modifications during MSC differentiation
- Actin polymerization inhibition decreases osteogenesis
- Inhibition of actin polymerization enhances adipogenesis
- Integrin expression during MSC differentiation into adipocytes and osteocytes
- Actin modification signals through p38MAPK in osteocytes
Minimal actin polymerization was observed after addition of CYD at various time points in the presence or absence of osteogenic induction medium (Figure 4.1.12). At all time points tested, there was a significant decrease in the percentage of cells that differentiated into osteocytes when CYD was added to the induction medium (Figure 4.1.13.A). There was an eight-fold decrease in OSTEOCALCIN expression after 7 days when MSCs were differentiated in the presence of CYD, and a 30-fold decrease after 14 days (Figure 4.1.13.B).
MSCs were differentiated into osteocytes in the presence (CYD) or absence (CONTROL) of CYD for the indicated time periods. Second, when MSCs were treated with CYD for 3 days during the 7-day induction period, there was a 3-fold reduction in osteogenic differentiation potential compared to cells differentiated for 7 days without CYD (Figure 4.1.14.B). Consistent with the decreased number of ALP positive cells during CYD treatment, there was a significant decrease in OSTEOCALCIN levels during the above treatment conditions in the presence of CYD (Figure 4.1.14.C).
A significant increase in phosphorylated levels of both p38MAPK (Figure 4.1.21.A) and ERK1/2 MAPK (Figure 4.1.22) was observed when MSCs were differentiated into osteocytes.
Isolation and characterization of mesenchymal stem cells from patients with bone marrow disorders
- Morphology
- Differentiation of MSC
- Phenotype of MSC
In addition, a significant difference in the cell surface expression of CD90 was found between different patient groups. Nevertheless, there was a significant increase in CD90 expression levels in patients with acute lymphoblastic leukemia (ALL) after chemotherapeutic treatment (30% at diagnosis and ~80% post-chemotherapeutic treatment) (Figure.4.2.8.B). CD90 expression in MSC isolated from patients at diagnosis (PRE-TR) and after treatment (POST-TR) for (A) Acute Myeloid Leukemia (AML) (B) Acute Lymphoblastic Leukemia (ALL).
There was a significant reduction in CD90 expression in MSCs obtained from patients belonging to an older age group of 51-80 years. Flow cytometric analysis of CD90 expression in MSCs isolated from bone marrow of young (5–20) and aged (51–80) donors. The level of cell surface expression of CD90 was determined at different time points during differentiation.
MSCs were cultured for several passages and cell surface expression levels of CD90 were analyzed.
Discussion
Role of cytoskeleton in MSC differentiation
Interestingly, it was observed that inhibition of actin polymerization during the process of differentiation led to decrease in osteogenic differentiation and increase in adipogenic differentiation potential of MSC. Few or no actin filaments were observed in MSC treated with CYD during osteogenic differentiation. Differences in pattern of actin polymerization recovery and related differentiation potential of MSC after removal of CYD during osteogenic differentiation clearly indicated that actin cytoskeleton modifications play an important role during osteogenic differentiation of MSC.
CD49e was found to be upregulated during osteogenic differentiation of MSC as reported by others (Hamidouche, Fromigue et al. 2009). Thus, both actin polymerization and CD49e were affected by CYD during MSC osteogenic differentiation, and CD49e was reported to be an important factor during osteogenesis. However, the addition of the p38MAPK inhibitor (SB208530) during differentiation had no effect on MSC osteogenic differentiation.
No difference in phosphorylated ERK1/2 levels was observed during adipogenic and osteogenic differentiation of MSCs after CYD treatment.
Properties of mesenchymal stem cells isolated from bone marrow of patients with hematologic disorders
Reduced osteogenic and adipogenic differentiation potential was also reported in MSC from children with aplastic anemia (Chao, Peng et al. 2010). Without any description of the functional role, reduction in CD90 expression was already reported in MSC from patients with aplastic anemia (Shevela EY, et al., 2013). Apart from this, low levels of CD90 expression were reported in MSC from MDS patients with no difference in expression of other markers such as CD29 and CD105 (Flores-Figueroa, Arana-Trejo et al. 2005).
The expression of CD90 has been reported to be developmentally and posttranscriptionally regulated (Xue, Calvert et al. 1990; Xue and Morris 1992). Campioni et al., reported reduced CD90 expression in MSC from hematological malignancies, but no difference in adipogenic, osteogenic and chondrogenic differentiation potential was observed (Campioni, Rizzo et al. 2009). Some drugs were reported to be toxic while others were not (Li, Law et al. 2004).
Factors secreted by cancer cells have been reported to induce morphological and genetic changes in MSCs (Al-Toub, Almusa et al. 2013).
Conclusions
These findings may provide insight into an altered bone marrow microenvironment, which is postulated to influence leukemia progression and hematopoietic recovery during hematological malignancies. The results also indicate a possible correlation between CD90 expression and osteogenic differentiation of mesenchymal stem cells from patients with hematological disorders. Silencing CD90 expression during osteogenic differentiation will provide insight into the role of CD90 during osteogenic differentiation of MSC.
In addition, analyzing the osteogenic differentiation potential of CD90 negative and CD90 positive MSC fractions and studying the signaling pathways affected during differentiation will provide further insight into the signaling pathways affected by CD90 during MSC differentiation.
Bibliography
34;Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy." Cell Prolif. 34;Human endometrial mesenchymal stem cells do not undergo spontaneous transformation during long-term culture.". 34;Tailored integrin-extracellular matrix interactions to direct human mesenchymal stem cell differentiation." Stem Cells Dev.
34;Infusion of allogeneic mesenchymal stem cells for the treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH).". J Cell Physiol. 34; Cytoskeletal organization of human mesenchymal stem cells (MSCs) changes during their osteogenic differentiation. " J Cell Biochem.
34;Mesenchymal stem cell-mediated cancer therapy: A dual-target strategy of personalized medicine." World J Stem Cells.
Doctoral dissertation project (July-2009 onwards)
Master’s dissertation project (July-2008 to May-2009)
Short term training on “Introduction to Bioinformatics and its Implications”
