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Markers and Methods to Verify Mesenchymal Stem Cell Identity, Potency, and Quality Scott Schachtele, Ph.D., Christine Clouser, Ph.D., and Joy Aho, Ph.D

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Markers and Methods to Verify Mesenchymal Stem Cell Identity, Potency, and Quality Scott Schachtele, Ph.D., Christine Clouser, Ph.D., and Joy Aho, Ph.D WHITE PAPER Markers and Methods to Verify Mesenchymal Stem Cell Identity, Potency, and Quality Scott Schachtele, Ph.D., Christine Clouser, Ph.D., and Joy Aho, Ph.D. ABSTRACT Mesenchymal stem cells (MSCs) are multipotent cells that are functionally defined by their capacity to self-renew and their ability to differentiate into multiple cell types including adipocytes, chondrocytes, and osteocytes. Translation of MSC-based therapies has been confounded by MSC population heterogeneity as well as non-standardized methods for their definition and characterization. This white paper begins by defining and discussing the evolution of MSC nomenclature. It then provides a detailed description of MSC markers and how they may vary by tissue source and species. Ultimately, this review discusses how to standardize MSC characterization by selecting markers for isolation, characterization, and validation. KEY TOPICS • Defining Mesenchymal Stem Cells • MSC Marker Variation by Species • Established and New MSC Markers • Methods for MSC Identification and Characterization • Methods for MSC Isolation • Media Definitions for MSC Expansion INTRODUCTION Mesenchymal stem cells (MSCs) are multipotent cells that adhere to plastic, have a Box 1. Minimal Experimental Criteria for MSC fibroblast-like morphology, express a specific set of surface antigens, and differentiate into as proposed by the International Society for adipocytes, chondrocytes, and osteocytes.1 Clinically, MSCs are of interest for their ability to Cellular Therapy 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 • Adherence to plastic. cells as well as the lack of standardized methods for their definition and characterization. At • ≥95% of the MSC population must the forefront of these issues is the lack of a MSC-specific marker that can be used to express CD73/5′-Nucleotidase, definitively isolate and characterize MSCs. The absence of a MSC-specific marker limits the CD90/Thy1, and CD105/Endoglin purity of MSCs isolated by methods such as positive and negative selection. As a result, MSCs as measured by flow cytometry. are typically isolated by plastic adherence, which generates a heterogeneous population of • ≤2% of the MSC population must cells that differ in their growth kinetics and differentiation potential. This heterogeneity can express CD34, CD45, CD11b/ increase experimental variability, decrease the efficiency of differentiation, and contribute to Integrin alpha M or CD14, CD79 conflicting data in the literature. In an attempt to increase consistency in the definition and alpha or CD19, and HLA Class II. 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 • Multipotent differentiation potential expression of a panel of surface antigens (Box 1).1 Since proposing the minimal criteria in as demonstrated by staining of 2006, several advances in MSC characterization and marker identification have been made. in vitro differentiated cells. 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. RnDSy-lu-2945 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 Because MSCs isolated from bone marrow have been shown to self-renew and regenerate “Mesenchymal Stem Cells” (MSCs) refers to skeletal tissue in vivo, some scientists suggest that these cells are more accurately multipotent, self-renewing cells that express defined by the term “skeletal stem cell.”3,4,5 Alternatively, the term “medicinal signaling cell” a specific set of surface antigens and can has been proposed as a replacement for “mesenchymal stem cell” as a way to indicate that differentiate into adipocytes, chondrocytes, the primary therapeutic benefit of MSCs may be attributed to secretion of bioactive molecules.6 and osteocytes when cultured in vitro. 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. The definition also suggests that MSCs isolated from different tissues are phenotypically The defining features of MSCs, as and functionally equivalent, which would imply that the cells share the same regenerative proposed by the ISCT. are little more capabilities in vivo. Thus, MSCs isolated from bone marrow and MSCs isolated from placenta than characteristics shared by connective would be expected to have the same ability to regenerate skeletal tissue in vivo. Some tissues and perivascular cells. 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 thein vivo properties of MSCs isolated from different tissues sources. History and Timeline of Mesenchymal Stem Cell Nomenclature CFU-F and Multipotent Mesenchymal Osteogenic Stem Cells Mesenchymal Stem Cells Skeletal Stem Cells Stromal Cells (Arnold Caplan) (Paolo Bianco and Pamela Gehron Robey) (Friedenstein) (ISCT) 1988 1999 2002 2010 1970s 1991 2000 2006 Multipotent Adult Progenitor Cells Stromal Stem Cells Mesenchymal Progenitor Cells and Mesodermal Progenitor Cells Medicinal Signaling Cells (Maureen Owen and colleagues) (James Dennis and colleagues) (Yuehua Jiang and colleagues) (Arnold Caplan) Figure 1. The History and Timeline of Mesenchymal Stem Cell Nomenclature. MSCs were originally identified by Alexander Friedenstein as ©colony R&D Systems, forming Inc. 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
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