Proteomic profiling of distinct clonal populations of bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing MSC self-renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub-cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow-derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two-Dimensional Liquid Chromatography/Matrix-Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self-renewal and differentiation.

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 with the increasing number of older people with degenerative diseases of the skeletal system such as osteoarthritis (OA). Because 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 (MSCs) from bone marrow samples of patients with OA by characterizing MSCs at the single cell level. Single-cell clonal cultures were established in 96-well plates by limiting dilution of bone marrow stromal cells (BMSCs) from three patients with OA. Fourteen clones were established for subsequent characterization. There was a wide variation in cell doubling times, with the time taken to reach 20 population doublings 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 BMSCs 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 patients with OA. Therefore, despite a supposedly smaller stem cell compartment in these patients, we demonstrate here that they can still yield a potentially therapeutically useful source of syngeneic MSCs.

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 homogeneous 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 population. 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 vivo.

Stem cell-related gene expression in clonal populations of mesenchymal stromal 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 osteogenic 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 two-dimensional and three-dimensional culture systems. When transforming growth factor beta3 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, resulting in detectable expression of osteopontin and osteocalcin. These chondrogenic and osteogenic differentiation markers were significantly enhanced in the three-dimensional cultures compared to the two-dimensional 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.