Characterization of bone marrow stromal clonal populations derived from osteoarthritis patients

This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface marker

Clonal characterization of bone marrow derived stem cells and their application for bone regeneration

Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However, all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single- cell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homo- geneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous popu- lation. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in viv

Clonal Isolation and Characterization of Bone Marrow Stromal Cells from Patients with Osteoarthritis

The demand for treatment strategies for damaged musculoskeletal tissue is continuously growing, especially considering the increasing number of older people with degenerative diseases of the skeletal system such as osteoarthritis (OA). Since depletion of multipotent cells has been implicated in degenerative joint diseases, cell based therapies have been proposed for tissue regeneration, especially for cartilage repair. The aim of the present study is to focus on the possibility of deriving and expanding multipotential mesenchymal stem cells (MSC) from bone marrow samples of OA patients, by characterization of MSC at the single cell level. Single cell clonal cultures were established in 96 well plates by limiting dilution of bone marrow stromal cells (BMSC) from three OA patients. A total of 14 clones were established for subsequent characterization. There was a wide variation in cell doubling times, with the time taken to reach 20 population doublings (PD) ranging from 37 days to more than 100 days. The clones were grouped into fast growing and slow growing clones. All except one of the fast growing stem cell clones were tripotential. However, the slow growing clones showed limited differentiation potential and morphological changes associated with cellular senescence with extended duration in culture. Flow cytometric analysis indicated a strong need to investigate for novel cell surface characteristic markers of BMSC because there was no obvious difference in the expression of the selected characteristic BMSC cell surface markers, CD29, CD44, CD90, CD105, and CD166 between fast growing and slow growing clones. This study has demonstrated the existence of a fast growing multipotential MSC population from bone marrow samples of OA patients. Therefore, despite a supposedly reduced stem cell compartment in these patients, we demonstrate here that they can still yield a potentially therapeutically useful source of syngeneic MSC

Stem cell related gene expression in clonal populations of mesenchymal stronal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs

A minimal common osteochondrocytic differentiation medium for the oesteogenic and chondrogenic differentiation of bone marrow stromal cells in the construction of osteochondral graft

To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approach, controllable differentiation of bone marrow stromal cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. This study proposes to test a minimum common osteochondrocytic differentiation medium (MCDM) formulated by including common soluble supplements (dexamethasone and ascorbic acid) used to induce chondrogenic and osteogenic differentiation. The MCDM coupled with supplemented growth factors was tested for its ability to differentiate BMSCs into osteogenic and chondrogenic lineages in both 2-dimensional (2D) and 3-dimensional (3D) culture systems. When transforming growth factor β3 (TGF-β3) was added to MCDM, BMSCs differentiated to chondrocyte-like cells evidenced by the expression of glycosaminoglycans and type II collagen, whereas osteogenic differentiation was induced by supplementing osteogenic protein-1 (OP-1), resulting in detectable expression of osteopontin and osteocalcin. These chondrogenic and osteogenic differentiation markers were significantly enhanced in the 3D cultures compared to the 2D monolayer cultures. The results achieved in this study lay a foundation for future development of osteochondral graft, which could be engineered from bilayered scaffold with spatially loaded growth factors to control BMSC differentiation