Mechanisms facilitating regenerative therapies with multipotent marrow stromal cells
Florian Tögel, Christof Westenfelder
Department of Medicine/Nephrology and VA Medical Center, University of Utah, USA
University of Utah, Department of Medicine/Nephrology and VA Medical Center, Nephrology Research Laboratory (151M), 500 Foothill Blvd, Salt Lake City, UT 84148, USA
E-mail: Florian.Toegel@hsc.utah.edu or Christof.Westenfelder@hsc.utah.edu
Accepted 07 March 2008
Published 22 May 2008
Cell therapy has become a promising new treatment approach for a large number of different diseases, and applications are continually being developed. Bone marrow derived stem cells are currently being tested in clinical trials and have been shown to be promising new therapeutic vehicles. Multipotent marrow stromal cells (MSCs) are a bone marrow derived cell type that can be easily cultured and expanded in vitro and have a broad range of potential and actual therapeutic applications. The mechanism of action of MSCs in the therapeutic situation depends on the disease, and involves differentiation, immunomodulation, paracrine, and anti-apoptotic mechanisms. These mechanisms are discussed in detail in this manuscript.
Overview, differentiation, immunomodulation, clinical trials, cytokines, cell therapy
Cell therapy in medicine
The idea of utilizing cells for therapeutic purposes is by no means new. The Swiss physician Paul Niehans propagated, as early as 1931, different cell types as tools for rejuvenation and cure against diseases, a therapy he called ‘Zellulartherapie’, which has also been called ‘Frischzelltherapie’ . Niehans treated a patient with tetany with injections of the parathyroid glands of an ox and the patient recovered. He also successfully treated Pope Pious XII. However, the use of animal cells was hampered by considerable side effects and this form of therapy subsequently was banned in Germany .
The best-known and most successful example of cell therapy is bone marrow transplantation. Lorenz showed in 1949 that lethally irradiated mice could be rescued by bone marrow cell infusion . A first patient report of intravenous infusion followed by transient engraftment was published in 1957  and in 1968-9 the first matched sibling transplantations were reported . Bone marrow transplantation (BMT) is now established as the first successful cell therapy as a routine procedure for the treatment of formerly incurable leukemias and the pioneer of this therapy, E. Donnall Thomas, received the Nobel Price in Physiology in 1990 .
The concept of cell therapy
The initial intention of BMT was to replace the lethally injured and ablated organ with a new one to rescue the patient. However, it was recognized later that the infused bone marrow also has anti-leukemic properties, a phenomenon called graft-versus leukemia effect . This represents a fundamental advantage of cell therapy over pharmaceutical approaches. Furthermore, cells are able to react in vivo depending on the different circumstances under normal and pathophysiological conditions, systemically through secretion of growth factors, cytokines or chemokines as well as through paracrine and local actions at the site of injury. Additionally, cells are able to integrate into tissues, either as differentiated parenchymal cells or as undifferentiated stromal cells, thereby affecting the organ of engraftment in the long run. These versatile properties make the development of cellular therapies promising and attractive.
Multipotent marrow stromal cells (MSCs)
Friedenstein showed that fibroblast like cells could be generated and propagated in vitro from bone marrow and called these cells ‘marrow stromal cells’ . Due to their differentiation into osteocytes, chondrocytes and adipocytes they have also been called ‘mesenchymal stem cells’. These cells support hematopoietic stem cells (HSCs) by growth factor and cytokine secretion and differentiate into bone, cartilage and fat. MSCs have now been recognized as the second stem cell population in the bone marrow, next to HSCs, but can also be generated from almost any organ . They are now defined by plastic adherence, positivity for the surface markers CD73, CD90 and CD105 and absence of CD45, CD34, HLA-DR .
Extensive in vitro and in vivo studies have shown that MSCs exhibit a potential to differentiate into various cell types (Graph 1). Lineage analyses of cloned MSCs showed that their natural differentiation pathway (‘default pathway’) is the osteogenic lineage and different clones exhibit different lineage potential in vitro . Differentiation of MSCs into different cell types in vitro can be induced through culture conditions and addition of endogenous substances like steroids, growths factors or PPARs or by demethylation agents like 5-azacytidine . MSCs are not a homogeneous population of cells, in vitro cultures are phenotypically different in size and shape, and can be generated from most organs . MSCs are readily generated from bone marrow aspirations, can be expanded in culture on a large scale without the addition of xenogenic additives like fetal calf serum  and are susceptible to transduction with viral vectors which makes them ideal vehicles for cell therapy.
MSCs have been originally derived from the bone marrow by a protocol from Friedenstein, however, with subsequent variation in culture conditions different cell populations with similar but not identical properties like MSCs have been described by various groups. It is currently not entirely clear how these different ‘brands’ of MSCs are related in vivo or if they are derived from a “basic” MSC and how culture conditions, e.g. the addition of growth factors like EGF, a low oxygen environment, low serum conditions and seeding density influence propagation and differentiation potential after several passages in vitro. Verfaillie’s group generated multipotent adult progenitor cells (MAPCs) from the bone marrow under low density and low serum conditions and could show that they have embryonic stem cell like properties when injected into blastocysts . So-called marrow-isolated adult multilineage inducible cells (MIAMI) were generated under low-oxygen tension on fibronectin from bone marrow cells . Lange et al described a population of bone marrow-derived adult stem cells, separated on a Percoll gradient with low density, that showed an extraordinary high proliferative potential and a conserved phenotype characteristic of MSCs . MSCs not only have been derived from bone marrow but from almost any organ .
Unrestricted somatic stem cells (USSCs) have been cultured from human cord blood . The authors state that USSCs have a wider differentiation potential and differ in immunophenotype and in their mRNA expression profile. Not all groups have been successful in generating stem cell like cells from cord blood . In contrast to Mareschi, Lee et al. found a mesenchymal stem cell like population derived from cord blood cells with classical characteristics of MSCs as well as differentiation into neuroglial- and hepatocyte-like cells under appropriate induction conditions. Adipose tissue contains MSCs that are easy to obtain from lipoaspirates . Because they are easy to culture and readily available from different sources, MSCs continue to be a popular research subject with steadily increasing numbers of publications and applications.
Mechanisms of action of MSCs used in cell therapy and regenerative medicine
1. Replacement of injured cells by MSCs through differentiation and integration into organ parenchyma
Cellular differentiation is not an irreversible process. Pathologists know the phenomenon of differentiation of one cell type into another due to prolonged exposure to un-physiological stimuli in epithelia, e.g. gastric reflux causes the squamous epithelium of the esophagus to differentiate into gastric mucosa, and have termed it ‘metaplasia’. In the kidney, tubular cells de-differentiate after ischemic injury, re-express embryonic and developmental markers like Pax-2, and start dividing to repopulate the denuded tubular parts, thereby regenerating a sublethally injured tubule [75, 25].
In the late 1990s researchers described so far unknown and unexpected differentiation of HSCs into a number of cell types, e.g. liver and muscle [17, 55]. These results were surprising because a long held dogma stated that adult stem cells are lineage restricted and can only differentiate into tissue from their lineage and that differentiation is terminal . This so called transdifferentiation was immediately recognized as a new and promising way of regeneration of injured tissue and proposed as a mechanism of action for cell therapy. However, initial enthusiasm led researchers to overlook some problems associated with early studies. These studies utilized crude cell preparations, e.g. whole bone marrow, and therefore it was not clear, which cell type was responsible for the observed phenomenon. Furthermore, transdifferentiation was very rare and only some dispersed single cells could be detected after a meticulous search, calling into question the therapeutic value of this approach. Krause et al in a very carefully conducted study showed, that a prospectively isolated HSCs are indeed the cell type responsible for tissue contribution and differentiation into most organ cells, but the contribution was below 0.1% .
Some time later two groups described fusion of cells in vitro and it was discussed, that this could be a potential explanation for the phenomena described in the early stem cell studies [67, 78]. Indeed, some groups attributed cell fusion as the main mechanism for organ regeneration in certain disease models [47, 72, 74].
Meticulous studies by Wagers and Balsam showed that transdifferentiation is an extremely rare event under steady state and ischemic conditions and HSCs do not contribute much to tissue turnover [73, 5].
The lessons learned from these studies with HSCs are:
• Transdifferentiation or plasticity is a real phenomenon but exceedingly rare in most disease models.
• Replacement of damaged tissue is therefore not a major mechanism for tissue regeneration.
• Under steady state conditions tissue replacement is rare and in disease conditions it is dependent on the model and kinetics used to study transdifferentiation.
Based on initial observations with whole bone marrow and the fact that MSCs can be differentiated into a large number of differentiated cells in vitro and in vivo, e.g. neurons , cardiomyocytes , myocytes , endothelial cells [54, 41], pulmonary cells  and liver cells [34-36], it was hypothesized that differentiation of MSCs into organ parenchymal cells is a major mechanism of tissue protection and regeneration after injury. However, MCSs exhibited tissue repair capacity despite low or transient engraftment in vivo, e.g. in the treatment of osteogenesis imperfecta it was less than 1% , and therefore differentiation into target tissues is most likely only a minor mechanisms of tissue protection and regeneration. The fact that tissue protection is observed without evidence of long-term engraftment also argues against differentiation as a main mechanism of action .
2. Paracrine mechanisms
MSCs produce a number of cytokines, growth factors and adhesion molecules that have been shown to be involved in tissue homeostasis and regeneration . Furthermore, transcriptome analysis by serial analysis of gene expression (SAGE) revealed a large number of transcripts for proteins involved in wound repair, immunological regulation, neural factors as well as angiogenesis [70, 56] implying a role for these factors in MSC mediated tissue regeneration. These factors stimulate cell proliferation (growth factors like IGF ) and are anti-apoptotic . The advantage of administering MSCs rather than growth factors directly lies in the fact that MSCs act on a local level and are able to interact with damaged tissue, which means they probably respond to cytokines like TNF-a secreted by damaged tissue with more or less secretion of a number of growth factors or modulatory cytokines and thereby influence the local environment directly and better than any systemically administered growth factors [41, 62]. In the kidney, MSCs regenerate renal function after acute kidney injury mainly by secreting epidermal growth factor (EGF), insulin-like growth factor (IGF-1), VEGF and by changing the cytokine expression profile of the injured kidney towards a more favorable anti-inflammatory state with higher IL-10 levels [60, 69]. In the heart, MSCs stimulate angiogenesis by secretion of VEGF [1, 29]. Endogenous cell proliferation in the brain is stimulated by MSCs through paracrine mechanisms after injury mediated by different factors [44, 8].
3. Vasculo- and angiogenesis
Blood supply is the most critical factor for tissue survival and most injury mechanisms involve the vasculature in one way or another. Ischemic injury is the most common mechanism of tissue damage for every organ system and fast restoration of regular blood supply is critical for tissue survival. The microvascular bed can be damaged in many ways, but endothelial dysfunction or apoptosis are major factors. MSCs express a number of angiogenic and vasculogenic factors and proteins that have been shown to increase endothelial cell survival and proliferation [2, 23]. In vivo studies have shown that vasculo- and angiogenesis by MSCs is either mediated directly by integration into vascular structures or through paracrine mechanisms stimulating angiogenesis, e.g. secretion of VEGF, angiopoietin or other growth factors [2, 3, 65, 64, 77]. MSCs can be genetically engineered, using different strategies like bcl-2, Akt or erythropoietin expression, to enhance endogenous angiogenic activity [16, 21].
MSCs exhibit low immunogenity due to low or absent MHC-II expression, low MHC-I expression and negativity for costimulatory molecules CD80, CD86 and CD40. Therefore infusion of MSCs do not trigger a direct rejection reaction, although several groups have shown that MSCs are not neutral towards the immune system and antibodies can be measured as well as T cell activation but not proliferation [30, 58]. A large body of data shows immunomodulatory properties of MSCs in vitro on different cell types such as T-cells, B-cells and NK cells . MSCs suppress T-cell proliferation in vitro, interfere with dendritic cell differentiation, inhibit B-cell proliferation and suppress the proliferation and cytokine production of natural killer cells . The in vivo relevance of these in vitro finding has been demonstrated in humans with acute graft versus host (GvHD) and Crohn’s disease [61, 66]. In animal models MSCs have been shown to modify experimental autoimmune encephalitis, a model of multiple sclerosis, and prolonged skin graft survival in baboons [6, 71]. There are conflicting data about effects of MSCs in organ transplantation models. Inoue reported that MSCs were ineffective at prolonging allograft survival and tended to promote rejection . In a different model cardiac allograft survival was prolonged . MSCs also favored tumour survival in animal models .
While the currently known immunomodulatory effects of MSCs show promise for the treatment of a number of diseases, data have to be interpreted carefully dependent on the animal model or in vitro strategy. Culture conditions and numerous other factors not least the model that is studied play important roles and have to be considered carefully to avoid preliminary conclusions.
5. Other applications for MSCs
MSCs are ideally suited as cellular delivery systems in tumor treatment. They can be engineered to express the interferon-beta (IFN-beta) gene and deliver therapeutic doses of IFN-beta directly into the tumor through infiltration thereby suppressing tumor growth and metastasis . In metabolic diseases and enzyme defects, either allogeneic or genetically engineered MSCs are able to function as enzyme replacement therapy  by providing necessary concentrations of an enzyme that is lacking due to a genetic defect. MSCs are rapidly transducable with different viral vectors and are thereby ideal vehicles for therapeutic genes . Other important applications include adjunct infusion to enhance hematopoietic engraftment  and as a source for engineered tissue such as cartilage and bone in tissue engineering .
In the ongoing story of stem cell treatment for patients MSCs have been so far the most promising development and have rapidly advanced from bench to bedside with several products already in late stage trials. Although little is known about MSCs in vivo, they have been characterized extensively in vitro and clinical studies have been finished and new ones are on their way. The mechanisms of action of MSCs are currently investigated in detail and there is still a large number of questions to be addressed until the full therapeutic benefit of these cells can be utilized but the field is rapidly advancing and is giving a shining example for the whole stem cell community.
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Accepted 07 March 2008
Published 22 May 2008