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In an attempt to further decrease experimental variability, several labs have tried to increase the purity of isolated MSCs through positive and negative selection. The success of positive and negative selection varies depending on the specificity of the marker, the starting cell population, and the isolation strategy.
Although negative and positive selection of MSCs has gained acceptance in the field, there are some concerns over using such an isolation strategy. Second, any type of selection increases the risk of isolating a distinct cell population, which may preferentially differentiate towards a specific lineage.
Despite these limitations, cells isolated by positive selection have been reported to have increased clonogenicity compared to cells isolated based on plastic adhesion. Negative selection of cells is often preferred over positive selection since the isolated cells remain untouched by antibodies, beads, and magnets.
MSCs can be negatively selected by using a cocktail of antibodies to proteins expressed by hematopoietic cells Figure 5. To improve the purity of negatively selected human MSCs, Modder et al.
Sorting of bone marrow cells based on GD2 expression yielded cells with the ability to differentiate into adipocytes, chondrocytes, and osteocytes. MSCs have a finite replicative capacity in culture.
The decrease in differentiation potential of MSCs in culture emphasizes the need to verify MSC identity and multipotency both after isolation and after the cells have been passaged in culture. If the reduction of specific MSC markers can be correlated with reduced differentiation potential or increasing age, then the markers could be used to simultaneously verify the identity and quality of MSCs. While more research is needed to examine the potential correlation between marker expression and MSC multipotency, several non-MSC markers and assays are commonly used to assess cell quality and replicative capacity.
Histochemical detection of senescence-associated lysosomal beta-galactosidase SA-beta-Gal expression by senescent cells is the one of the most common assays used to assess senescence of MSCs and several other cell types. Cells are typically fixed and incubated with a SA-beta-Gal staining solution containing X-gal which generates a blue precipitate when cleaved by beta-galactosidase.
Recently Hildebrand and colleagues identified alpha-L-Fucosidase alpha-Fuc as a novel marker of senescence that is upregulated in response to replicative, DNA damage-, and oncogene-induced senescence. If additional publications support this claim, then alpha-Fuc activity may become the preferred method for assessing senescence.
The colony-forming unit-fibroblast assay can be used to estimate the proliferative and clonogenic potential of MSCs in culture. Bone marrow stromal cells are plated on plastic culture dishes at a low density and cultured under conditions that allow individual CFU-F to adhere and proliferate.
By plating the cells at a low density, single colony clusters arise from the clonal expansion of a single CFU-F. The number of colonies present indicates the number of CFU-F present in the original sample. However, the colonies are only clonal when plated at a low density since at higher densities adherent cells with limited growth potential can be observed. MSCs isolated from animals such as mice, pigs, sheep, goats, horses, and dogs are used in preclinical trials and in the veterinarian clinic for the treatment of conditions such as osteoarthritis in dogs and musculoskeletal conditions in horses.
While all MSCs display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens that has been described for human MSCs. For some animals, the limited availability of species-specific antibodies has made it difficult to establish positive and negative expression of MSC markers. In the absence of species-specific antibodies, anti-human antibodies are often used to examine the marker expression profile of non-human MSCs. However, if the antibodies are not examined for cross-reactivity, a negative finding may indicate that the marker is not expressed or that the antibody does not recognize the antigen.
Results by Catherine De Shawer and colleagues emphasize the importance of verifying cross-reactivity. Table 3 includes results from individual publications that examined surface antigen expression of non-human MSCs. Mouse Mesenchymal Stem Cells were stained with the primary monoclonal antibodies indicated in each panel and supplied in the Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel Catalog SC filled histograms or isotype controls empty histograms.
MSC identification is made by phenotypic assays, which assess marker expression and functional assays, which assess the differentiation potential of the cell population. Table 4 includes the most common methods for verifying MSC identity, as well as the advantages and disadvantages of each method. For analysis of marker expression, flow cytometry and immunocytochemistry are efficient methods that reveal the marker profile of individual cells, whereas protein arrays and immunoblotting demonstrate the average marker expression by a population of cells.
Mesenchymal stem cells are defined by their ability to differentiate into adipocytes, chondrocytes, and osteocytes in culture. MSCs can be efficiently differentiated using a combination of media and supplements to induce adipogenesis , chondrogenesis , and osteogenesis.
Additionally, small molecules can be used to enhance differentiation. Confirmation of tri-lineage differentiation provides excellent evidence for the verification of MSC identity.
Furthermore, functional analysis is a well-accepted method of verification that is not dependent on the tissue or species type. Figure 8 shows an example of functional verification of human MSCs.
Flow cytometry is an efficient method that can be used to examine the phenotype of cells at the single cell level. Multi-color flow cytometry allows users to examine multiple MSC markers simultaneously, which can reveal heterogeneity in a population that might not be obvious when examining multiple samples stained for individual markers. Specifically, multi-color flow cytometry can reveal the percentage of cells that express several MSC markers, whereas single-color flow cytometry can only reveal the percentage of cells that express each MSC marker individually.
Additionally, many flow cytometers are equipped with a cell sorting function, which can be used to enrich for MSCs. Figure 9 demonstrates verification of human MSCs by multi-color flow cytometry. In contrast, minimal expression of antigens recognized by the Negative Marker Cocktail was detected.
Similar to flow cytometry, immunocytochemistry can be used to examine the phenotype of cells at the single cell level. Additionally, multi-color immunocytochemistry can reveal heterogeneity in a population of cells that might not otherwise be detected when staining cells for MSC markers individually.
Unlike standard flow cytometry, immunophenotyped MSCs can be recovered after performing live cell immunocytochemistry. Media formulations used for growing MSCs can vary by application as well as by research stage ie, basic or translational.
For many researchers, deciphering which variant of MSC media best suits their needs can be a daunting prospect. Specific formulations of MSC media now exist to address these various applications, including serum-containing, serum-free, xeno-free, and GMP-grade media.
The cells were harvested and stained for the expression of positive and negative MSC markers. Quadrants were set based on isotype controls. Mesenchymal stem cells have been isolated from a wide range of species and tissues using several techniques. MSCs are isolated as a heterogeneous population of cells that differ in growth kinetics and differentiation potentials and may be contaminated with terminally or partially differentiated cells.
Prior to , the heterogeneity of the cell population combined with the different techniques used to isolate, culture, and define MSCs led to experimental variability, inefficient differentiation, and contradictory data. Since the criteria, several studies suggest that the minimal criteria should be revised to account for newly identified markers as well as for species and tissue-specific variations in MSC phenotype.
While the functional definition of MSCs is unlikely to change, the phenotypic definition appears to vary depending on the species and tissue source. Tissue-specific and species-specific MSC markers will likely emerge as source-specific MSCs are systematically characterized and as a wider variety of species-specific antibodies become available. As MSCs become better characterized for their in vitro properties and their in vivo abilities, it is likely that the definition and nomenclature will continue to evolve.
What markers do you find most useful for MSC isolation and characterization? Skip to main content. To some scientists, the defining features of MSCs, as proposed by the ISCT are little more than characteristics shared by connective tissues and perivascular cells.
Multipotent differentiation potential as demonstrated by staining of in vitro differentiated cells. In Arnold Caplan proposed that the acronym MSC represent 'Medicinal Signaling Cell' to reflect that the therapeutic benefit of MSCs is primarily due to the secretion of bioactive molecules rather than tissue regeneration. CD34 20 , Primitive hematopoietic cells and endothelial cells Oxford University Press is a department of the University of Oxford.
It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Close mobile search navigation Article navigation. Abstract Biologists rely on morphology, function and specific markers to define the differentiation status of cells.
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Markers & Methods to Verify Mesenchymal Stem Cell Identity, Potency, & Quality
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