Markers & Methods to Verify Mesenchymal Stem Cell Identity, Potency, & Quality



Introduction to Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent cells that adhere to plastic, have a fibroblast-like morphology, express a specific set of surface antigens, and differentiate into adipocytes, chondrocytes, and osteocytes.1 Clinically, MSCs are of interest for their ability to modulate the immune system as well as their potential to regenerate tissues. However, the translation of MSC-based therapies has been hindered by the heterogeneity of the isolated cells as well as the lack of standardized methods for their definition and characterization. At the forefront of these issues is the lack of a MSC-specific marker that can be used to definitively isolate and characterize MSCs. The absence of a MSC-specific marker limits the purity of MSCs isolated by methods such as positive and negative selection. As a result, MSCs are typically isolated by plastic adherence, which generates a heterogeneous population of cells that differ in their growth kinetics and differentiation potential. This heterogeneity can increase experimental variability, decrease the efficiency of differentiation, and contribute to conflicting data in the literature. In an attempt to increase consistency in the definition and verification of MSCs, the International Society for Cellular Therapy (ISCT) proposed minimal criteria for the definition of human MSCs based on plastic adherence, multipotency, and the expression of a panel of surface antigens (Box 1).1   Since proposing the minimal criteria in 2006, several advances in MSC characterization and marker identification have been made. These advances have led to improved methods for isolating, verifying, and assessing the quality of MSCs. This review summarizes the most recent MSC markers, definitions, and the latest methods used to characterize MSCs.


Defining Mesenchymal Stem Cells

The in vivo identity and characteristics as well as the therapeutic potential of MSCs have been debated since their discovery by Alexander Friedenstein in 1968. As a result, several names have been proposed to reflect the changing understanding of MSC properties (Figure 1). However, newly proposed nomenclature for MSCs is not universally adopted by the field, resulting in the use of several different terms to describe the same cell type (Figure 1). While “mesenchymal stem cell” is commonly used to describe multipotent cells that express specific surface antigens and adhere to plastic, some scientists argue that the definition is vague, encompasses cells isolated from virtually every tissue, and does not necessarily predict the stem cell properties of the cells in an in vivo setting.2

For the purpose of this review, the term “Mesenchymal Stem Cells” (MSCs) refers to multipotent, self-renewing cells that express a specific set of surface antigens and can differentiate into adipocytes, chondrocytes, and osteocytes when cultured in vitro.

Because MSCs isolated from bone marrow have been shown to self-renew and regenerate skeletal tissue in vivo, some scientists suggest that these cells are more accurately defined by the term “skeletal stem cell.”3,4,5 Alternatively, the term “medicinal signaling cell” has recently been proposed as a replacement for “mesenchymal stem cell” as a way to indicate that the primary therapeutic benefit of MSCs may be attributed to secretion of bioactive molecules.6

In this review, the term “mesenchymal stromal cells” is used to describe a heterogeneous population of cells that are adherent to plastic, have a fibroblast-like morphology, and express a specific set of marker proteins. Within this heterogeneous population are a subset of cells referred to as “mesenchymal stem cells,” which are multipotent, self-renewing cells that have the ability to differentiate into adipocytes, chondrocytes, and osteoblasts when cultured in vitro. In addition to the conceptual definitions of MSCs, the ISCT offers an experimental definition of MSCs (Box 1) to facilitate identification of human MSCs.

The controversy over the definition and nomenclature of MSCs centers in part on the minimal criteria proposed by the ISCT. According to the minimal criteria, MSCs are not restricted to bone marrow but can be isolated from a wide range of tissues.

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.

The definition also suggests that MSCs isolated from different tissues are phenotypically and functionally equivalent, which would imply that the cells share the same regenerative capabilities in vivo. Thus, MSCs isolated from bone marrow and MSCs isolated from placenta would be expected to have the same ability to regenerate skeletal tissue in vivo. Some scientists argue that this assumption is unfounded and that the defining features of MSCs, as proposed by the ISCT, are little more than characteristics shared by connective tissues and perivascular cells.2 Therefore, MSCs isolated from adipose tissues may not demonstrate the same regenerative properties in vivo as MSCs isolated from bone marrow. In fact, it has been proposed that MSCs are perivascular cells that are lineage-restricted, tissue-specific progenitors and that the local environment and perivascular position could account for the expression of common antigens.2 If this hypothesis is correct, MSCs isolated from bone marrow would have the ability to regenerate skeletal tissue in vivo, while adipose-derived MSCs would be restricted to the regeneration of adipose tissue in vivo. These issues emphasize the controversies surrounding the nomenclature of MSCs and such controversies will persist in the field until experiments are performed to assess the in vivo properties of MSCs isolated from different tissues sources.


Box 1: Minimal Experimental Criteria for MSC as proposed by the International Society for Cellular Therapy

Figure 1: The History and Timeline of Mesenchymal Stem Cell Nomenclature. MSCs were originally identified by Alexander Friedenstein as colony forming unit fibroblasts (CFU-F) and osteogenic stem cells.7,8 However, since this time, a number of terms have been used to describe mesenchymal stem cells (MSCs) including “stromal stem cells,” which was proposed in 1988 by Maureen Owen, Ph.D. and colleagues to indicate that the cells reside in the stromal rather than the hematopoietic compartment. Rather than highlight the cells’ compartmental origin, Arnold Caplan, Ph.D. proposed the term “mesenchymal stem cells” in 1991 to emphasize the self-renewal property and differentiation potential of the cells.9 However, this nomenclature was challenged by James Dennis, Ph.D. and colleagues who suggested that the cells may be progenitors rather than stem cells. As a result, the term “mesenchymal progenitor cells” was proposed.”10 In 2000, Paolo Bianco, M.D. and Pamela Gehron Robey, Ph.D. coined “skeletal stem cell” to specify that the cells give rise to components of the skeletal system, while  just two years later, the term “multipotent adult progenitor cells” was proposed by Yuehua Jiang, M.D. and colleagues to describe the multipotent nature and potential progenitor status of the cells.3,11 Since no direct evidence demonstrated the ability of MSCs to self-renew and differentiate in vivo, in 2006, the International Society for Cellular Therapy proposed the term “multipotent mesenchymal stromal cells.” 1 More recently, in 2010, Arnold Caplan, Ph.D. suggested that the acronym MSC stand for “medicinal signaling cells” to reflect that the primary therapeutic benefit of MSCs may be attributed to the secretion of bioactive molecules rather than tissue regeneration. 6


Markers for Mesenchymal Stem Cell Isolation & Verification

In 1970, Friedenstein and colleagues described a method for isolating fibroblast-like colonies from rodent bone marrow.7 These clonogenic cells were isolated based on their relatively high adhesion to plastic compared to other cells in the bone marrow compartment. The method used by Friedenstein is still widely used today for isolation of MSCs from unpurified bone marrow or Ficoll™-purified bone marrow mononuclear cells.

In 2010 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.

While isolation by plastic adherence is efficient, it does not yield a homogeneous population of cells. The isolated cells demonstrate different growth kinetics and differentiation capacities and include a small population of contaminating hematopoietic cells. Cell homogeneity can be increased over several weeks by culturing under conditions that preferentially expand MSCs and select against expansion of hematopoietic cells.12 However, the remaining MSCs can still demonstrate a range of proliferation rates and differentiation potentials. This heterogeneity may contribute to experimental variability, contradictory data, and poor pre-clinical outcomes.


Established & New Markers for MSC Identification and Verification

Before the ISCT published the minimal criteria for defining MSCs in 2006 (Box 1), variability in the methods used to isolate, expand, and characterize MSCs made it difficult to compare data from different labs. The ISCT criteria offered a quality control step for researchers to verify MSC identity, which decreased experimental variability and increased the transparency of published data.

The ISCT proposed positive and negative markers that enabled researchers to distinguish MSCs from other cells in the bone marrow compartment. The negative markers were selected to include surface antigens that are expressed by hematopoietic cells, while the positive markers were selected to include surface antigens that are absent from most hematopoietic cells (Table 1). However, ISCT acknowledges that the criteria must be met with some flexibility, particularly as they relate to expression of the negative marker, HLA Class II. Specifically, HLA Class II can be expressed by MSCs under certain conditions, such as cytokine stimulation.1 Therefore, cells that meet all other criteria, but are also positive for HLA Class II, can be designated as MSCs if an adjective is used to indicate that the cells were stimulated.

According to the ISCT, CD34 is a negative marker of MSCs. However, some reports suggest that the CD34 negative status is an artifact of cell culture conditions.13,14,15 In fact, several groups have shown that MSCs isolated from adipose tissue express CD34 at the time of isolation but lose expression while in culture.16,17,18 Expression of CD34 by MSCs is also supported by the fact that the STRO-1 antibody (Clone STRO-1), which is commonly used to identify MSCs, was developed using CD34+ bone marrow as the immunogen.19 The debate over the use of CD34 as a negative marker raises the possibility that markers may vary depending on the MSC tissue source.

Table 1: Negative and Positive MSC Markers as proposed by the International Society for Cell Therapy

Negative HSC Marker Used to Exclude Peer-reviewed References
using R&D Systems Antibodies
CD3420,21 Primitive hematopoietic cells and endothelial cells22 23, 24
CD4520,21 Leukocytes25 26, 27
CD11b and CD1420,21 Monocytes and macrophages28,29 27, 30
CD79 alpha and CD19 alpha31 B cells32 33, 34
HLA Class II31 Antigen presenting cells and lymphocytes1,35* 36, 37
Positive MSC Markers Biological Role Peer-reviewed References
using R&D Systems Antibodies
CD73/5’-Nucleotidase21 Catalyzes production of extracellular adenosine from AMP38 39, 40
CD90/Thy120 Wound repair, cell-cell and cell-matrix interactions41 27, 42
CD105/Endoglin21 Vascular homeostasis; modulates TGF-beta functions via interaction with TGF-beta RI and TGF-beta RII43 24, 36, 44

*HLA Class II may be expressed on stimulated MSCs.


The Latest Markers Used to Verify MSC Identity

Since the ISCT-proposed markers were published in 2006, several additional markers have become widely accepted as additional MSC markers. While no markers have been identified that are exclusively expressed by MSCs, the growing panel of MSC-associated markers enables researchers to simultaneously verify expression of several MSC-associated surface antigens, which increases the confidence in the identification and verification of isolated MSCs.


The STRO-1 monoclonal antibody was the first antibody that was successfully used to enrich CFU-Fs from human bone marrow.19,45 While STRO-1 is a widely used marker of MSCs, little is known about the identity of its cognate antigen. In addition to MSCs, STRO-1 recognizes nucleated erythroid cells.46


CD271, also known as low affinity nerve growth factor receptor (NGF R) or p75NTR, is highly expressed by a fraction of clonogenic human bone marrow MSCs and is currently one of the most common and selective markers for enriching MSCs from bone marrow.47,48 Since CD271/NGF R is expressed at low levels by hematopoietic cells, separation of CD271/NGF R+/bright versus CD271/NGF dim cells is typically used to distinguish MSCs from hematopoietic cells.


CD200 was discovered as part of a screen to identify surface antigens expressed by MSCs but not hematopoietic cells.49 CD200+ cells are clonogenic and differentiate into adipocytes, chondrocytes, and osteocytes. However, a subsequent study showed that the expression of CD200 varied widely among twenty different human MSC lines, suggesting that the use of CD200 as a marker of MSCs may depend on the cell line or tissue source.50

Ganglioside GD2

Ganglioside GD2 is a glycosphingolipid that has been reported as a marker that is expressed by MSCs but not hematopoietic cells. GD2 was expressed by 95% of CD45-CD105+CD73+cells isolated from bone marrow. However, further studies are warranted to examine if GD2 expression is donor-dependent as Sivasubramaniyan et al. were unable to detect GD2 in primary bone marrow cells.45


Frizzled-9, also known as CD348, was identified as part of a screen to detect MSC-specific markers. In bone marrow-derived MSCs, Frizzled-9 is expressed in CD271bright cells,51 whereas in placenta-derived MSCs, Frizzled-9 is expressed by highly clonogenic CD271/NGF dim cells.52 The difference in cell expression is likely due to the fact that MSC expression of CD271/NGF R appears to be dependent on the tissue source used for MSC isolation.

Tissue Non-specific Alkaline Phosphatase (TNAP)

TNAP, also referred to as Alkaline Phosphatase/ALPL, is identical to the Mesenchymal Stem Cell Antigen-1 (MSCA-1) and has been used as a marker for the prospective isolation of endometrium-derived MSCs. However, TNAP+ cells show limited multipotency and expression levels do not correlate with colony forming activity.53,54 Therefore, TNAP may be of limited use for MSC verification.


Use of Markers for Mesenchymal Stem Cell Isolation

The use of markers to verify MSC identity serves as an important quality control step that can save significant time and reduce experimental variability. In an attempt to further decrease experimental variability, several labs have tried to increase the purity of isolated MSCs through positive and negative selection. Antibodies against CD45 or other hematopoietic markers can be used to negatively select MSCs, while antibodies against markers such as CD271/NGF R can be used to positively select MSCs. 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.12 First, positive selection can lead to cross-linking of surface antigens, which may activate signaling pathways and alter cell phenotype. 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.16,19,48,55

Negative Selection of Mesenchymal Stem Cells

Negative selection of cells is often preferred over positive selection since the isolated cells remain untouched by antibodies, beads, and magnets.  In contrast, cells isolated by positive selection are labeled with antibodies and/or beads which may interfere with downstream applications and introduce experimental variability, either by preventing antibody binding or by stimulating signaling pathways. MSCs can be negatively selected by using a cocktail of antibodies to proteins expressed by hematopoietic cells. For human bone marrow stromal cells, common targets of negative selection include CD2, CD3, CD11b/Integrin alpha M, CD14, CD15/Lewis X, CD16/Fc gamma RIII, CD19, CD38, CD56/NCAM-1, CD66b/CEACAM-8, CD123/IL-3 R alpha, and CD235a/Glycophorin while CD45 and TER-119 are common targets for negative selection of murine MSCs. To improve the purity of negatively selected human MSCs, Modder et al. followed negative selection of MSCs (using antibodies to CD2, CD3, CD11b/Integrin alpha M, CD14, CD15/Lewis X, CD16/ Fc gamma RIII, CD19, CD56/NCAM-1, CD123/IL-3 R alpha, and CD235a/Glycophorin A) with fluorescence activated cell sorting (FACS) to isolate lin-/CD34-/CD31/PECAM-1- cells. 56 This second selection step removed endothelial cells and precursors, yielding a cell population that was 97% negative for the hematopoietic marker CD45.56

Positive Selection of MSCs Using Established Markers

MSCs express high levels of CD271/NGF R while hematopoietic progenitor cells of the erythroid lineage express low levels of CD271/NGF R (CD271/NGF dim). Isolation of MSCs by CD271/NGF R positive selection is currently the gold standard to which new putative markers are compared. MSCs isolated by CD271/NGF R positive selection show lower hematopoietic cell contamination and higher clonogenicity compared to MSCs isolated by plastic adherence.16,48 Because CD271/NGF R is expressed by other cells in the bone marrow, selection by CD271/NGF R alone does not yield 100% pure MSCs.

MSCs isolated by CD105/Endoglin positive selection demonstrate tri-lineage differentiation and higher colony-forming units-fibroblasts (CFU-F) compared to unsorted, plastic adherent cells.55 CD105/Endoglin is expressed by MSCs as well as other cells within the bone marrow. Therefore, selection based on CD105/Endoglin does not yield 100% pure MSCs. For example, flow cytometric analysis of freshly isolated human bone marrow cells enriched by CD105/Endoglin positive selection demonstrated a purity of 79%.55

The STRO-1 antibody recognizes MSCs as well as nucleated erythroid cells. Therefore, the isolated cells do not approach 100% purity. To improve purity, FACS can be used to sort cells labeled with STRO-1 and Glycophorin A, a marker of erythroid cells. Isolation of STRO-1+/Glycophorin A- cells has been shown to yield a population with 100-fold enrichment of CFU-F compared to unfractionated bone marrow cells.19

Positive Selection of MSCs Using Newly Identified Markers

Ganglioside GD2
Ganglisoside GD2 is negatively expressed by hematopoietic cells and positively expressed by MSCs, making it a promising marker for MSC isolation. Sorting of bone marrow cells based on GD2 expression yielded cells with the ability to differentiate into adipocytes, chondrocytes, and osteocytes.57 However, Sivasubramaniyan et al. were only able to detect GD2 in cultured MSCs and did not detect expression in primary MSCs.45

Sushi domain containing 2 (SUSD2) is a type 1 transmembrane protein that was recently identified as a marker that can be used for prospective isolation of MSCs.45 Antibodies directed against SUSD2 recognize CD271/NGF Rbright, but not CD271/NGF dim cells, suggesting that SUDS2 can differentiate between MSCs and hematopoietic cells. Sivasubramaniyan et al. showed that SUSD2+ but not SUSD2- cells, gave rise to CFU-F and were able to differentiate into adipocytes, chondrocytes, and osteoblasts.45 If these data are corroborated by other laboratories, GD2 may be revealed as one of the most specific MSC markers.

Positive Selection Using a Combination of Mesenchymal Stem Cell Markers

The purity of MSCs can be increased by using more than one MSC marker for positive selection. As an example, a recent publication demonstrated that selection of mesenchymal stem cells using CD271/NGF R, CD90/Thy1, and CD106/VCAM-1 resulted in a highly clonogenic population of cells.58 Specifically, the addition of CD106/VCAM-1 as a marker for positive selection led to isolation of cells with five times greater clonogenic potential compared to the cells isolated with CD271/NGF R and CD90/Thy1 alone.


Markers & Assays to Evaluate MSC Aging, Senescence, & Potency

MSCs have a finite replicative capacity in culture. From early passage numbers, MSCs begin to demonstrate senescence-associated changes such as reduced proliferative capacity, changes in morphology, shortening of telomeres, and an accumulation of the cyclin-dependent kinase inhibitor p16INK4a/CDKN2A.59 Within approximately 5-12 passages, MSCs demonstrate decreased clonogenicity and differentiation potential.59,60,61,62 In parallel, several publications indicate that the expression of specific MSC markers, including CD106/VCAM-1,63,64,65CD146/MCAM,63 and STRO-1,19 decreases with increasing passage number.

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. The parallel reduction in differentiation potential and MSC marker expression has led to the suggestion that CD106/VCAM-1, CD146/MCAM, and STRO-1 may be used as indicators of MSC age or potency.66 Additional research is needed to determine whether the decrease in MSC marker expression correlates with a reduction in differentiation potential and whether such a correlation is dependent on species or tissue source. 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.

Table 2: The Effect of Time in Culture on MSC Marker Expression.

Markers Reported to be Independent of Passage Number/Time in Culture Markers that are Reported to Decrease with Increasing Passage Number/Time in Culture Marker Reported to be Associated with Differentiation Potential
CD73/5’-Nucleotidase, CD90/Thy1, CD105/Endoglin21 CD106/VCAM-1, STRO-122,63,64,65 CD106/VCAM-167


Use of Non-MSC Markers & Assays to Evaluate Cell Quality

MSC aging or senescence can be assessed by examining the senescence-associated beta-galactosidase (SA-beta-Gal) activity, p16INK4a/CDKN2A levels, and cell clonogenicity by CFU-F.68,69 While telomere shortening is a hallmark of cell senescence, the length of telomeres varies from donor to donor, making telomere length a potentially unreliable measurement of MSC senescence.70


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.59,71 Although the assay was first published in 1995, it continues to be a standard assay for evaluating senescence. 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.72 Furthermore, as a marker of senescence, alpha-Fuc was equivalent or more reliable than SA-beta-Gal at the transcriptional and enzymatic level. If additional publications support this claim, then alpha-Fuc activity may become the preferred method for assessing senescence.


p16INK4a/CDKN2A is a cylcin-dependent kinase inhibitor that is highly expressed in senescent cells.73 Under physiological, non-senescent conditions, p16INK4a/CDKN2A  is repressed by BMI1, a polycomb protein that maintains MSC self-renewal.74 PINK4a expression in senescent MSCs positively correlates with activity of senescence associated beta-Galactosidase (SA-beta-Gal) and negatively correlates with expression of the proliferation marker MKI67/Ki-67.

Colony-Forming Unit-Fibroblast (CFU-F) Assay

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.75

MSC Marker Variation by Species

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.76,77 Despite the widespread use of MSCs from non-humans, there are no established minimal criteria for the identification of MSCs in non-humans. 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. Most non-human MSCs express CD29/Integrin alpha 9 beta 1 and CD44. However, the expression of CD73/5’-Nucleotidase, CD90/Thy1, and CD105/Endoglin varies depending on species and strain.

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.78 Their studies demonstrated that many of the anti-human and anti-mouse antibodies used to assess the phenotype and identity of equine MSCs did not-cross react with the equine antigens. For example, none of the anti-human CD34 antibodies recognized equine CD34, yet equine MSCs are reported to be negative for CD34 expression.78 These studies emphasize the need to include proper controls when using anti-human antibodies to evaluate the expression of non-human MSC markers. Table 3 includes results from individual publications that examined surface antigen expression of non-human MSCs.

Mouse Mesenchymal Stem Cell Markers
While there are no minimal criteria established for the identification of mouse MSCs, the surface expression of mouse MSCs has been well characterized. Mouse MSCs are generally characterized by the positive expression of CD29/Integrin alpha 9 beta 1, CD44, CD73/5’-Nucleotidase, CD105/Endoglin, CD106/VCAM-1, and Sca-1/Ly6 and the negative expression of hematopoietic and endothelial markers Ter-119, CD45, CD11b/Integrin alpha M, and CD31/PECAM-1.79,80,81 However, expression of CD90/Thy1 and Sca-1/Ly6 appear to be strain-dependent.79,82

Table 3: Marker Expression in Non-human MSCs

Species Tissue Source* Positive Markers Negative Markers Reference
Canine BM, AT, Dermal cells CD90, CD105 CD45 83
Canine BM, AT, WJ, UCB CD44, CD73, CD90, CD105 CD14, CD34, CD45 84
Canine BM, AT, Muscle, Periosteum CD44, CD90 CD34, CD45 85
Canine BM CD44, STRO-1 CD34, CD45 86
Canine Dental pulp STRO-1, CD146, Nanog CD73, CD105, CD45 87
Canine Amnion, WJ Oct-4, CD44, CD29, CD184, CD34, CD45, 88
Canine AT CD29, CD44, CD90 CD13, CD14, CD34, CD45, CD73, CD105, CD117 89
Canine WJ CD44, CD90, CD105, CD184 CD29, CD33, CD34, CD45 90
Canine UCB CD29, CD33, CD44, CD105, CD184, Oct-4 CD4, CD8 alpha, CD10, CD14a, CD45, CD49b, CD41, CD61, CD62p, CD73, CD90, CD133, HLA-DR 91
Equine BM, AT, Muscle, Periosteal tissue CD44, CD90 CD45, CD34 92
Equine BM CD44, CD90, Vimentin, PCNA CD13, CD34 93
Equine Amniotic CD44, CD90, CD105 CD19, CD20, CD28, CD38, CD62L, CD200, CD31, CD62P, CD34, CD41a 94
Equine UCB CD29, CD44, CD90 CD45, CD73, CD79 alpha, CD105, MHC Class II 95
Equine AT, BM CD29, CD90   96
Equine PB CD29, CD44, CD90, CD105 CD45, CD79 alpha, MHC Class II 97
Equine PB CD51, CD90, CD105   98
Equine Amniotic, UCB, WJ CD90, CD44, CD105 CD34, CD14, CD45 99
Equine UCB CD29, CD44, CD90 CD79 alpha, MHC Class II 100
Mouse BM CD29, CD44, CD105, Sca-1 CD11b, CD31, CD34, CD45, CD86, MHC Class II 101
Mouse Synovium CD29, CD44, Sca-1 (30% positive) CD34, CD45, CD106, CD140a 102
Mouse Adult AT Sca-1, CD44, CD90 CD34, CD45, CD31 103
Mouse Epiphysis derived MSCs CD29, CD44, CD73, CD105, CD106, Sca-1, SSEA-4 CD11b, CD31, CD35, CD45, MHC I, MHC Class II 104
Mouse BM CD29, CD44, CD105, Sca-1 CD34, CD45 105
Mouse AT, BM, lung CD29, CD44, CD49e CD11b, CD34, CD45, CD117 106
Mouse BM CD29, CD44, CD105, Sca-1 CD11b, CD34, CD45, CD31, CD86 101
Mouse BM CD73/, CD80, Sca-1 CD11b, CD45, CD31, CD34, CD86, CD90, CD105, MHC Class II 107

*Key: AT=adipose tissue; BM=bone marrow; PB=peripheral blood; UCB=umbilical cord blood; WJ= Wharton’s jelly


Methods for MSC Identification, Verification, & Characterization

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.

Functional Verification of Mesenchymal Stem 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. Successful differentiation can be verified by staining the differentiated cells for expression of lineage markers and/or by assessing the functionality of the cells. 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 2 shows an example of functional verification of human MSCs.

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Figure 2: Verification of Multipotency using the Human Mesenchymal Stem Cell Functional Identification Kit. Human mesenchymal stem cells were cultured in StemXVivo™ Mesenchymal Stem Cell Expansion Media (Catalog # CCM004) and differentiation was induced as indicated using the media supplements included in the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006). The kit also contains a Goat Anti-Mouse FABP-4 Antigen Affinity-purified Polyclonal Antibody (adipocytes), a Goat Anti-Human Aggrecan Antigen Affinity-purified Polyclonal Antibody (chondrocytes), and a Mouse Anti-Human Osteocalcin Monoclonal Antibody (osteocytes) for the confirmation of differentiation status. The cells were stained using the NorthernLights™ 557-conjugated Donkey Anti-Goat (Catalog # NL001; red) or Anti-Mouse (Catalog # NL007; red) IgG Secondary Antibodies, The nuclei were counterstained with DAPI (blue).

Phenotypic Verification of MSCs by Multi-Color Flow Cytometry

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 3 demonstrates verification of human MSCs by multi-color flow cytometry.

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Figure 3: Human Bone-Marrow Derived Cells Fulfill the ISCT's Definition of Human MSCs Based on Marker Expression. Human bone marrow-derived mesenchymal stem cells were stained using the antibodies and reagents provided in the Human Mesenchymal Stem Cell Marker Verification Multi-Color Flow Cytometry Kit (Catalog # FMC020). The data shows positive expression of MSC-associated surface antigens CD73, CD90, and CD105. In contrast, minimal expression of antigens recognized by the Negative Marker Cocktail was detected.

Phenotypic Verification of MSCs by Immunocytochemistry

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.

Table 4: Methods for MSC Verification and Characterization

Method Type of Verification Advantages Disadvantages
Flow Cytometry Phenotypic
  • Reveals the phenotype of individual cells
  • Examines several markers simultaneously
  • Can be used to isolate specific populations (when equipped with a cell sorter)
  • High sensitivity
  • High speed analysis
  • Can use gating to remove dead cells from analysis
  • Requires expensive  equipment
  • Requires skilled operator
  • Potential cross-reactivity when using several antibodies
  • Potential autofluorescence
  • Cell sorting by FACS can be time consuming and expensive
Immunofluorescence/ Immunocytochemistry Phenotypic
  • Reveals localization of marker proteins
  • Can assess multiple markers simultaneously
  • More efficient than Western blot analysis
  • Can use live or fixed cells
  • Requires specialized equipment
  • Potential for cross-reactivity when using multiple antibodies
  • Potential autofluorescence
  • Photobleaching
  • More time consuming than flow cytometry
Western Blot to detect
MSC and Differentiation Markers
  • High sensitivity
  • High specificity
  • Does not require specialized equipment
  • Does not reveal heterogeneity of cell population
  • Low throughput
Protein Array Phenotypic
  • High sensitivity
  • High specificity
  • Analyze several markers simultaneously
  • More efficient than individual Western blots
  • Cost effective compared to individual Western blots
  • Does not require specialized equipment
  • Ideal for screening cell populations
  • Does not reveal heterogeneity of cell population
  • Low throughput
Real-time RT-PCR Phenotypic
  • Detects early changes in marker expression induced by differentiation
  • Analyze several markers simultaneously
  • High sensitivity and specificity
  • Yields the average marker expression of a population
  • Does not reveal heterogeneity
Induction of Differentiation Functional
  • Does not rely on MSC markers
  • Not dependent on tissue or species type
  • Time consuming
  • Cannot recover MSCs



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 2006, 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. The publication of the Minimal Criteria to Define Human MSCs in 2006 offered standards by which MSCs could be functionally and phenotypically defined.

Since the 2006 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?



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