Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature

Abstract

The International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell (ISCT MSC) committee offers a position statement to clarify the nomenclature of mesenchymal stromal cells (MSCs). The ISCT MSC committee continues to support the use of the acronym “MSCs” but recommends this be (i) supplemented by tissue-source origin of the cells, which would highlight tissue-specific properties; (ii) intended as MSCs unless rigorous evidence for stemness exists that can be supported by both in vitro and in vivo data; and (iii) associated with robust matrix of functional assays to demonstrate MSC properties, which are not generically defined but informed by the intended therapeutic mode of actions.

Introduction

The International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell (ISCT MSC) committee issued a position paper in 2005 [1] clarifying that the term mesenchymal stem cell is not equivalent or interchangeable with mesenchymal stromal cell (MSC). The former refers to a stem cell population with demonstrable progenitor cell functionality of self-renewal and differentiation [2], [3], whereas the latter refers to a bulk population with notable secretory [4], immunomodulatory [5] and homing [6] properties. The ISCT's MSC committee further issued a minimal criteria to define multipotent MSCs as being plastic adherent, expressing CD73, CD90 and CD105, lacking the expression of hematopoietic and endothelial markers CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR and capable of in vitro differentiation into adipocyte, chondrocyte and osteoblast lineages [7].

These markers largely remain as minimal markers for defining in vitro–expanded MSCs. Nevertheless, there are issues that need to be considered. For example, CD34 negativity has been used to define MSCs; however, this is not as definitive. Depending on donors and passages, certain percentages of Adipose derived stem cells are CD34 positive. It should be noted that the Stro-1 antibody, which is also useful for the identification and isolation of MSCs, was actually produced with CD34+ bone marrow cells as immunogen [8]. It is likely that MSCs in vivo are more CD34 positive than in vitro–expanded counterparts, and inclusion of Insulin-like growth factor 1 in the culture media has been shown to increase CD34 expression [9]. The negativity of CD34 in MSCs could be related to plastic-adhering culture conditions [10]. Additionally, although MSCs are typically defined by lack of HLA-DR expression, this can be upregulated by exposure to interferon-γ [11] in both mouse and human MSCs, in a cell culture–dependent manner [12], indicating the plasticity of these cell surface markers. Hence, the identity of MSCs as defined by the original cell surface markers is evolving as our understanding grows.

The original position paper was meant to foster harmonization of terminology that was in keeping with the scientific evidence available then supporting functionality of the cells, “without fueling unrealistic expectations, to the general public.” However, the interchangeable use of MSCs as mesenchymal stem cells or mesenchymal stromal cells has propagated, not diminished; there are in fact 54 858 results for a search string of “mesenchymal stem cells” versus 58 111 results for search string of “mesenchymal stromal cells” queried on Pubmed (February 2019). Additionally, many direct-to-consumer clinics have marketed mesenchymal stem cells to patients, sometimes even resulting in adverse reactions affecting patient safety [13]. This has prompted calls for disuse of the term “stem cells” in MSC nomenclature [14], [15], reiterating the original position of the 2005 and 2006 Cytotherapy publications. In fact, there have been further suggestions to disqualify MSC research under the “stem cell” umbrella by funding bodies, publications and conferences organized by stem cell societies to emphasize the difference between MSCs and mesenchymal stem cells. However, the solution to this nomenclature quandary lies in more rigorous scientific examination, discussion, discourse and exchange of ideas and not exclusion of valuable research on MSC properties, function and definitions from esteemed scientific conferences and funding bodies [16]. With the advent of next-generation sequencing tools at the single-cell level and the mass cytometry technology, it is possible to decipher epigenomic, transcriptomic and proteomic level differences in heterogeneous populations composed of bulk MSCs and rarer progenitor/stem cell populations.

The aim of this position paper is to further consolidate and clarify ISCT's MSC committee position on functional definitions of mesenchymal stem versus stromal cells.

Part of the confusion lies in the use of the acronym, MSCs, which can be expanded to imply both mesenchymal stromal cells, mesenchymal stem cells, multipotent stem cells or medicinal signalling cells. The original ISCT's MSC committee position papers sought to retain the MSC acronym to provide continuity to “scientific discourse, electronic literature databases” because the term has been in use for more than three decades now. Even calls for the disuse of the “stem cell” terminology agree that retaining the MSC acronym is vital to support the continuity of scientific research and discourse [14].

Part of the contention with this terminology rises from the use of the term “mesenchymal” derived from the word mesenchyme, which is used to describe loosely organized tissue during embryonic development broadly associated with connective and bone marrow tissues. In fact, in the absence of prospective and definitive markers for MSCs, the in vivo equivalence of in vitro expanded MSCs has been much debated. There is evidence that MSCs share markers and functionality with perivascular and mural cells (pericytes and vascular smooth cells) [17]. This has led to the hypothesis that MSCs originate from perivascular cell populations and emigrate into capillary walls in surrounding fibrous tissues during times of development and injury, contributing to the repair process [18], [19], [20], [21]. Others have highlighted that although there are similarities in between pericytes and MSCs, important differences are also present [22], [23], [24], and, in fact, MSCs share many similarities with adventitial cells [25]. Nonetheless, the controversy is less about the “mesenchymal” origin of these cells, and more centred on the “stem” versus “stromal” designation [14]. The ISCT's MSC committee does not favor abandoning the “mesenchymal” terminology, and recommends that the “MSC” acronym remain in use, but be additionally annotated with functional definitions to further clarify the designation.

Specifically, as part of this annotation, the tissue of origin of all MSCs should be provided. For example, bone marrow–derived, adipose tissue–derived and umbilical cord tissue–derived MSCs may be abbreviated appropriately as BM-MSCs, AD-MSCs and UC-MSCs, respectively. This context-dependent approach is necessary because MSCs from different tissues exhibit varied phenotypic, functional [26] and secretome behavior [27], emphasizing the need to specify tissue source ontogeny.

Second, the ISCT MSC committee recommends that functional definitions be used to clarify whether the MSC acronym stands for mesenchymal stromal cells or mesenchymal stem cells. This is particularly important because there are currently no cell surface markers to distinguish bulk populations of mesenchymal stromal cells from rarer populations of mesenchymal stem cells. Although CD146+ is a minimal surface marker that is enriched in BM-MSCs with clonogeneic colony forming unit-fibroblasts (CFU-F) potential that demonstrates in vivo ossicle formation and supports bone marrow hematopoiesis [2], this marker overlaps with expression of other generic MSC markers including CD49a, CD90 and CD105 [2], rendering it common to both stromal and stem cell populations. Unless there is rigorous functional evidence in vitro and in vivo to demonstrate the self-renewal and differentiation properties, the term mesenchymal stem cells should not be used.

Previously, progenitor self-renewal and differentiation properties in vitro and in vivo have been demonstrated for mesenchymal stem cells isolated from BM, periosteum, umbilical cord blood, muscle [28], umbilical cord Wharton's jelly [29], dental pulp [30], adipose tissue [31] and growth plate [3]. Importantly, the differentiation potential of these stem/progenitor cells has varied according to the tissue of origin, method of isolation and in vitro propagation of the cells. Rigorous in vitro demonstration of self-renewal and/or multilineage differentiation should involve clonogeneic experiments as shown by Russell et al. [29], [32], [33], [34], [35], [36]. Rigorous in vivo demonstration has not always been possible with clonogeneic cells; it has typically been limited to bone formation by skeletal stem cells under heterotropic transplantation conditions with an osteoconductive carrier [2], muscle regeneration upon local transplantation in injured-muscle tissue [37], [38] or bone and cartilage formation in an intra-femoral transplantation model [39], [40]. CFU-F progenitor assays and in vitro tri-lineage differentiation assays using inductive medium and cell staining, especially under clonal conditions, are indicative of progenitor status but not sufficient to demonstrate self-renewal capacity of mesenchymal stem cells in the absence of in vivo data.

Third, the ISCT MSC suggests that the moniker mesenchymal stromal cells be used to describe bulk unfractionated populations. We acknowledge that this is a heterogeneous population that includes fibroblasts, myofibroblasts and even a small proportion of stem/progenitor cells [30], [33], [41], but excludes hematopoietic and endothelial cells. Given this heterogeneity, we recommend the ongoing need to characterize MSCs using a matrix of assays to demonstrate secretion of trophic factors [42], [43], [44], modulation of immune cells [5], [45], [46], [47] and other relevant properties including promoting angiogenesis [48], [49], [50], [51]. Importantly, we recognize this limitation in functionally defining stromal cells, and advocate that the matrix of functional assays [52] be carefully selected based on the proposed utility of MSCs to demonstrate functionality of these cells both in vitro and in vivo. It is important to recognize that a matrix of functional assays will be needed to best capture the multimodal properties of this heterogeneous population of MSCs for their intended use. The ISCT's MSC committee had put out a consensus paper on functional matrix assays recommending three approaches, including quantitative RNA analyses of selected genes, flow cytometry of cell surface markers and protein analysis of MSC secretome [44]. The paper also speculated that characterization of exosomes and/or microRNA may be included as part of this multi-modal approach with increasing demonstration of functional roles for MSC-secreted exosomes [53], [54], [55] and microRNAs [56], [57].

Use of licensed MSCs by interferon-γ or tumor necrosis factor-α or other cytokines in these functional assays is recommended because it mimics an in vivo diseased inflammatory environment that MSCs are likely to encounter [44]. Licensing of MSCs was previously recommended in an ISCT MSC position paper on interrogating MSC modulation of immune cells and addressing issues of culture-condition–driven immune plasticity [47]. The ISCT MSC committee also recommends using resting MSCs as internal controls or other reference materials as previously discussed [58].

Importantly, it is the paracrine and immunomodulatory properties of MSCs that largely serve as the basis for their clinical utility as exemplified in the Food and Drug Administration–authored review of 66 investigational new drug (IND) applications of MSC-based products submitted to the agency prior to 2013 [59]. Approximately half of the IND applications described MSC potency in terms of secretion of factors or expression of surface proteins on MSCs or on target cells such as lymphocytes [59]. Referring to MSCs as mesenchymal stromal cells, therefore, does not diminish their clinical utility and may more accurately reflect their functional benefit. This is especially true because many of the applications include non-homologous indications, which do not reply on the “stemness” or differentiation capacity of MSCs to connective tissue lineages, but in their interaction with tissue-specific progenitors and immune cells [60]. This is reinforced by the recent approval of Alofisel by Takeda and Tigenix for treatment of complex perianal fistulas in adult patients [61]. The primary mechanisms of action are speculated to be impairment of proliferation of activated lymphocytes and reduction in the release of pro-inflammatory cytokines, which allow the tissue around the fistula track to heal [62] in line with current understanding of the immunomodulatory properties of MSCs. Similarly, the 2017 approval by the Drug Controller General of India of Stempeucel for treatment for patients with critical limb ischemia due to Buerger's disease is predicated on the cells’ anti-inflammatory and immunomodulatory properties, including inducing angiogenesis in ischemic muscle [63], [64], [65], in line with current functional definitions of MSCs.

Ultimately, the questions of whether mesenchymal stem versus stromal cells are functionally distinct subpopulations and whether the stem and stromal cells have overlapping paracrine and immunomodulatory functions are best answered based on single-cell sequencing experiments, statistical clustering and analyses of data. From a clinical translational perspective, the secretory and immunomodulatory functions associated with clinical benefits of MSC-based therapies are thought to reside in the bulk, heterogeneous stromal cell fraction. The ISCT MSC committee strongly recommends that such cells continued to be termed and characterized as mesenchymal stromal cells through a matrix of functional assays with appropriate controls. For research and clinical translation applications, the ISCT MSC committee recommends the continued use of the acronym MSC, but with detailed annotation of tissue source and a robust matrix approach to demonstrate relevant functionality. Importantly, stringent evidence for stem cell functionality should be provided to justify the use of the “stem” moniker. The ISCT MSC committee recommends that functional distinction around definitions of stromal and stem cells be maintained. Further analysis should be focused on their secretomes and related functionalities. This will provide clarity to the field, enable stringency and continue to fuel science-based evidence for furthering research and development on both mesenchymal stromal and stem cells.