Massimo Dominici

Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.

Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement.

The plastic-adherent cells isolated from BM and other sources have come to be widely known as mesenchymal stem cells (MSC). However, the recognized biologic properties of the unfractionated population of cells do not seem to meet generally accepted criteria for stem cell activity, rendering the name scientifically inaccurate and potentially misleading to the lay public. Nonetheless, a bona fide MSC most certainly exists. To address this inconsistency between nomenclature and biologic properties, and to clarify the terminology, we suggest that the fibroblast-like plastic-adherent cells, regardless of the tissue from which they are isolated, be termed multipotent mesenchymal stromal cells, while the term mesenchymal stem cells is used only for cells that meet specified stem cell criteria. The widely recognized acronym, MSC, may be used for both cell populations, as is the current practice; thus, investigators must clearly define the more scientifically correct designation in their reports. The International Society for Cellular Therapy (ISCT) encourages the scientific community to adopt this uniform nomenclature in all written and oral communications.

Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT)

BACKGROUND AIMS Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. METHODS Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. RESULTS In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. CONCLUSIONS The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

BACKGROUND Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. METHODS As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. RESULTS We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). DISCUSSION Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

HUMAN MULTIPOTENT MESENCHYMAL STROMAL CELLS USE GALECTIN-1 TO INHIBIT IMMUNE EFFECTOR CELLS

Human multipotent mesenchymal stromal cells (MSCs) suppress proliferation and alloreactivity of T cells. Several signaling molecules and enzymes contribute to this effect. We focused on carbohydrate-protein interactions and investigated whether lectins are involved in immune modulation by MSC. Gene expression profiling of MSCs revealed that one of the most important lectins in this setting, galectin-1, was highly expressed. Galectin-1 protein was detected intracellularly and on the cell surface of MSCs. In addition, galectin-1 was released into the cell culture supernatant by MSCs. To analyze the functional role of galectin-1, a stable knockdown of galectin-1 in MSCs with use of a retroviral transfection system was established. Galectin-1 knockdown in MSCs resulted in a significant loss of their immunomodulatory properties, compared with MSCs infected with nontargeting control sequences. The galectin-1 knockdown partially restored the proliferation of CD4(+) and CD8(+) T cells. By contrast, the effect of MSCs on nonalloreactive natural killer (NK) cells was unaffected by down-regulation of galectin-1 expression. Furthermore, MSC-derived galectin-1 significantly modulated the release of cytokines involved in graft-versus-host disease (GVHD) and autoimmunity (eg, tumor necrosis factor-α [TNFα], IFNγ, interleukin-2 [IL-2], and IL-10. These results identify galectin-1 as the first lectin mediating the immunomodulatory effect of MSCs on allogeneic T cells.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.

How do mesenchymal stromal cells exert their therapeutic benefit

In recent years mesenchymal stromal cells (MSC) have emerged as a major new form of cell therapy. While the original perception was that MSC were stem/progenitor cells with the potential to contribute to the regeneration of tissue, more recent data suggest that the principal mechanism of MSC activity is through the release of soluble mediators that elicit the observed biologic response. Future studies are needed to identify more completely the spectrum of therapeutic applications and delineate better the associated molecular and cellular mechanisms.

Development and functional characterization of human bone marrow mesenchymal cells immortalized by enforced expression of telomerase

Summary. To create immortal mesenchymal cell lines, we transduced primary human bone marrow mesenchymal cells with telomerase reverse transcriptase (TERT). TERT+ mesenchymal cells continued to grow for > 2 years; parallel TERT– cultures underwent senescence after 15 weeks. TERT+ mesenchymal cells did not form foci in soft agar, had a normal karyotype and could differentiate into osteoblasts and chondrocytes. Their capacity to support leukaemic lymphoblasts and normal CD34+ haematopoietic cells was equal to or greater than that of primary cells; 42 TERT+ mesenchymal cell clones varied in their supporting capacity. Immortalized mesenchymal cells offer a promising tool for identifying molecules that regulate human haematopoiesis.

Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms

Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.