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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. Figure 5. Sorting of bone marrow cells based on GD2 expression yielded cells with the ability to differentiate into adipocytes, chondrocytes, and osteocytes. Sivasubramaniyan et al.

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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.

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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. Figure 6. 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.

Figure 7. 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.

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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.

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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.


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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. Figure The cells were harvested and stained for the expression of positive and negative MSC markers.


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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.

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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? All Rights Reserved. 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 CD45 20 , CD11b and CD14 20 , Monocytes and macrophages 28 , CD79 alpha and CD19 alpha Others show strong reactivity in the proliferative but not secretory stage in biopsy tissue Qi et al.

It is possible that basalis endometrium was also present if samples were obtained in the early proliferative stage. Differences to our results may be technical due to the use of different antibodies or paraffin sections with the associated antigen retrieval versus fresh frozen tissue. Nevertheless, we consistently showed strong apical-lateral epithelial-specific immunoreactivity in the bases of glands with two different N-cadherin monoclonal antibodies using frozen sections and confocal microscopy in a large number of Pre-M and Post-M endometrial samples.

N-cadherin also has roles in the niche of hematopoietic stem cells Zhang et al. In these progenitors, N-cadherin interacts with the Wnt signalling pathway. The differential expression of N- and P-cadherin in the current study, and other Wnt molecules Nguyen et al. However, they were used across the range of techniques, and were always outnumbered by non-adenomyotic samples. Such an assay is technically difficult and likely requires inclusion of progenitor cell niche factors, which are currently unknown for endometrial epithelial progenitors.

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In both studies other cell types endothelial, stromal were present that may have acted as niche cells. We believe that the xenografting of organoids recently generated in 3D culture this study Valentijn et al. The identification of a human endometrial epithelial progenitor marker allows the investigation of these cells in endometrial proliferative disorders which may originate from basalis epithelium. For example, in endometriosis where dislocation of basalis endometrium Leyendecker et al. Studies showing altered immunoreactive N-cadherin in endometriotic Bartley et al. In conclusion, we have identified the first specific surface marker identifying a progenitor population in human endometrial epithelium using a gene profiling approach comparing Pre-M and Post-M epithelium and verified by in vitro stem cell functional assays, clonogenicity self-renewal serial cloning , proliferation and differentiation.

However, the identification of N-cadherin as an endometrial epithelial progenitor marker paves the way for investigations of the role of these cells in gynaecological disorders associated with abnormal endometrial proliferation such as endometriosis, adenomyosis and endometrial cancer.

Supplementary data are available at Human Reproduction online. The authors thank gynaecologists from Monash Health and Waverley Private Hospital who have provided endometrial tissues and research nurses Francis Walker, Yao Han, Germana Ryan and Kellie Woodclarke for consenting patients and collecting tissues.