Mesenchymal Stromal Cell Therapy in Hematology: From Laboratory to Clinic and Back Again.

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Stem Cells and Development Mesenchymal stromal cell therapy in haematology: from laboratory to clinic and back again (doi: 10.1089/scd.2014.0564) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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Mesenchymal stromal cell therapy in hematology: from laboratory to clinic and back again

Ann De Becker, and Ivan Van Riet

Department Clinical Hematology – Stem Cell Laboratory, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Belgium

Correspondence: Prof. Dr. Ivan Van Riet. Department Clinical Hematology – Stem Cell Laboratory, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium Email: [email protected] Fax: 003224776728 Tel: 003224776711 1

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Abstract. There is currently major interest to use mesenchymal stromal cells (MSC) for a very diverse of therapeutic applications. This stems mainly from the immunosuppressive qualities and differentiation capacity of these cells. In this review we focus on cell therapy applications for MSC in Haematology. In this domain MSC are used for the treatment or prevention of graft versus host disease, support of haematopoiesis or repair of tissue toxicities after haematopoietic cell transplantation. We critically review the accumulating clinical data and elaborate on complications that might arise from treatment with MSC. In addition we assume that the real clinical benefit of using MSC for these purposes, can only be estimated by a better understanding of the influence of in-vitro expansion on the biological properties of these cells as well as by more harmonization of the currently used expansion protocols.

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Introduction. Mesenchymal stromal cells (MSC) are non-haematopoietic cells first described by Friedenstein in the 1970s. They were characterized by plastic adherence, spindle shape morphology and a differentiation capacity towards osteocytes, adipocytes and chondrocytes in addition to forming the stromal microenvironment were the hematopoietic stem cells reside [1]. They were originally derived from bone marrow, however in recent years groups have described culture of MSC from a wide range of tissues such as umbilical cord blood, Wharton’s jelly, adipose tissue, mobilized peripheral blood, dental pulp, and certain foetal tissues [2-7]. MSC were further characterized in the 1980s and 1990s by Caplan and Pittenger and a specific phenotype was determined: MSCs express CD73, CD90 and CD105 and are negative for haematopoietic markers such as CD14 , CD11b, CD34, CD45, CD79 or CD19. This combination of cell surface markers was retained by the International Society for Cellular Therapy (ISCT) in their standards for MSC [8-10]. The cultured cells do not meet all the criteria for stem cell activity, as there is, for example, senescence of the cultured cells. To remove the inconsistency between nomenclature and biological properties, the ISCT proposed in 2005 to use the name mesenchymal stromal cells and to reserve the term mesenchymal stem cell for the true mesenchymal stem cells. Both however, are abbreviated as MSC. Since that position statement, most scientists have adapted this terminology [11].

Rationale for using MSC in Clinical Haematological Practice. There is a lot of interest to use MSC for a wide variety of clinical applications. When introducing the search term ‘mesenchymal stem cells’ in clinicaltrials.gov, more than 400 results are selected. MSC have been introduced in the clinic fairly quickly after their characterization in the late 1990s [12].

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4 The interest to use MSC in Haematology arises from their three characteristics: MSC were found to have immunosuppressive capacities, they form the stromal microenvironment in which the HSC reside and they can give rise to different cell types. Their multipotent differentiation capacity is probably not limited to osteocytes, chondrocytes, adipocytes and stromal cells since a variety of groups have described differentiation into other cell types such as pancreatic cells, muscle cells, etc. [13-19].

Clinical applications of MSC Treatment/prevention of Graft versus Host Disease The most commonly known and studied application of MSC is their use in the treatment and/or prevention of graft versus host disease. Graft versus host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT) [20]. In 2004 a landmark paper was published by LeBlanc et al describing a complete response of steroid refractory acute GVHD in a 9 year old boy [21]. Four years later, data of a first phase II trial within the EBMT (European Group for Blood and Bone Marrow Transplantation) for the treatment of steroid refractory were published by Le Blanc and colleagues. These data showed a complete response in 30 out of a total of 55 patients and improvement of symptoms in 9 of them. This study also showed that complete responders had lower “1 year non relapse mortality” and higher “2 year overall survival” [22]. Subsequent studies confirm that responders to MSC treatment for steroid refractory acute GVHD fare better than non-responders [23-28]. A recurrent policy among these studies is also the scheduling of repeated MSC infusions [22, 23, 25-27, 29-41]. However, not all data reported on MSC for the treatment of steroid refractory acute GVHD are uniformly positive. Osiris therapeutics developed and patented an MSC formulation for IV administration as Prochymal® in the US and is investigating its use in a variety of domains. They started with trials in GVHD treatment but they have widened their scope and have opened trials in Crohn’s disease, diabetes, chronic obstructive pulmonary disease and acute myocardial infarction. They reported that in a large randomized trial on 4

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5 GVHD treatment with Prochymal®, they could not observe an improved overall response rate. A subgroup analyses was performed and this showed a lack of improvement in patients with skin GVHD, the most common presentation of acute GVHD, however there was significant improvement in patients with gut and liver GVHD. These data were disclosed in a press conference and an abstract was presented at the 2010 BMT tandem meeting [33]. In perception at the time, this was considered a negative result and fuelled scepticism on utility of MSC in GVHD treatment. Given the overall encouraging results of those early trials, studies were also set up to test the role of MSC in the prevention of GVHD and the treatment of chronic GVHD [25, 30, 34-36, 42]. Recently the treatment of steroid-refractory GVHD in paediatric patients with MSC (Prochymal®) was reimbursed in Canada and New Zealand. Table 1a gives an overview of the clinical data of trials published on use of MSC for the treatment or prevention of GVHD. This clinical application relies on the immunosuppressive capacities of MSC. Firstly MSC do not express major histocompatibility class II and can therefore escape immune recognition by the host. This makes it possible to use third party MSC, from unrelated donors irrespective of HLA compatibility [43]. This is advantageous since it takes a few weeks to generate a sufficient amount of cells from MSC cultures starting from fresh donor tissue. In addition to this escape mechanism, immunosuppressive properties have been attributed to MSC. However, the mechanisms through which MSC exert their immunosuppressive effects are not well understood as yet. Several studies dissecting MSC immunosuppressive qualities and capacities suggest that a variety of mechanisms are implicated. MSC can suppress Th1 and Th17 cells and can induce regulatory T-cells [44, 45]. Moreover, they have also been attributed immunomodulating effects on B- and natural killer (NK) cells. MSC exert immunosuppressive effects through cell-cell contact but also through soluble factors such as PGE2, prostaglandin B2 and IL10 [43, 46-49]. One soluble factor that has come to attention in recent years is indoleamine 2,3 dioxygenase (IDO). MSC can be activated by exposure to inflammatory cytokines, most notably interferon γ (IFN γ) and tumor necrosis factor α (TNF α). Several groups have shown that IFN γ and TNF α activated MSC exert their immunosuppressive effect 5

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6 through upregulation of IDO. This increase in IDO is also implicated in the differentiation of monocytes in M2 immunosuppressive macrophages thus enhancing the MSC immunosuppressive effect. The same group also described an IDO independent pathway of immunosuppression by IFN γ activated MSC through the ligands of PD1 [50, 51, 52]. Galectins are β galactoside binding proteins that have also been attributed a role in MSC immunosuppression. Galectin-1 and -3 are constitutively expressed on and secreted by bone marrow MSC. When the gene expression of these molecules is blocked simultaneously an almost complete abrogation of the suppressive effect of MSC on T-cells is observed. Ungerer et al. showed that galectin-9 expression by MSC is upregulated by exposure to IFN γ resulting in suppression of both B- and T-cells. Moreover galectin-9 expression levels appeared to correlate with the immunosuppressive qualities of MSC [53, 54, 55]. A better understanding of MSC working mechanisms spurs on to efforts to improve MSC therapies. For example, murine MSC engineered to express CCR7 show improved homing to secondary lymphoid organs and they improved survival in a GVHD model while maintaining the graft versus leukemia effect [56].

Ex vivo expansion of haematopoietic stem/progenitor cells MSC might be a useful tool to optimize ex vivo expansion of HSC, for instance they secrete a number of haematopoietic cytokines [16]. MSC are also part of the hematopoietic stem cell niche. The concept of the hematopoietic stem cell niche was first introduced in 1978: specialized niche cells are in close contact with HSC and provide specific signals that help maintain their function. The niche is formed by a network of stromal cells including MSC, CXCL12 abundant reticular cells, adipocytes, osteoclasts, osteoblasts, osteocytes and neuronal cells. The location of HSC in the niche is still controversial, however most data suggest that they are mainly located in perivascular regions and in the highly vascularized endosteal region (57, 58, 59). MSC in the bone marrow are identified in vivo as nestin+ cells and are spatially associated with HSCs. Depletion of nestin+ cells in a murine model leads to a significant reduction of BM homing of hematopoietic progenitors. These nestin+ cells also have increased expression levels of HSC maintenance genes (57). Efforts to recreate the BM niche in 6

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7 vitro with MSC have shown that MSC seeded on 3D scaffolds and cocultured with HPC resulted in increased migration of HPC into the scaffolds and HSC were localized in clusters in contact with the MSC. Higher levels of molecules supporting hematopoiesis were found in such 3D coculture settings showing also maintenance of a pool of quiescent HSC (60, 61, 62]. It has been assumed recently that MSC–mediated haematopoiesis support could be improved by engineering MSCs to optimize their bone marrow homing. This could be achieved by modifying the cell surface or by genetic engineering and using the cells as a cargo, to deliver haematopoiesis- supporting cytokines in the bone marrow stroma [63]. Ex vivo expansion of haematopoietic progenitor cells (HPC) is mainly relevant in cord blood transplantation. Cord blood is a well-studied alternative donor source for patients without a matched sibling or unrelated donor. HPC in cord blood are more primitive and a lower cell number is required for SCT. However the cell number of a single cord blood unit is usually not sufficient for transplantation of an adult patient. One way to obtain enough cells is to perform a double cord blood transplant. Another strategy might be ex vivo expansion of HPC. Therefore several strategies to expand cord blood HPC have been tested and one of these strategies is the use of a MSC feeder layer [64]. Almost all data on this application for MSC in Haematology are pre-clinical, i.e. either in vitro or in animal models. A concern about in vitro expansion of HPC is the loss of long-term repopulating cells. Several studies have shown that co-culture of MSC with HPC results in higher cell numbers and more long-term culture-initiating cells [65]. When these cells are transplanted in mouse models, enhanced engraftment is observed. One study showed a decrease in GVHD in a double cord blood transplant setting where a group of mice was transplanted with a combination of an expanded and unexpanded cord blood unit and a control group receiving two unexpanded cord blood units. A significant survival benefit was observed in the mice receiving expanded cord blood what was attributed to a decrease in GVHD incidence. The expanded units contained significantly more regulatory T-cells as compared to unexpanded cords and the authors claim this is a result of expansion on an MSC feeder layer [66]. 7

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8 Robinson et al. reported in 2011 the first clinical data on co-transplantation of an unexpanded cord blood unit and another unit expanded on a layer of bone marrow-derived MSC from a family member [67]. Some data suggest that manipulation of HPC source through the removal of certain lymphocyte subsets improves ex vivo expansion on MSC feeder layers. It was found that removing CD3 and/or CD14 positive cells from the cord bloods before starting the expansion yielded higher numbers of total nucleated cells and CD34 positive cells [68].

Management of complications of (allogeneic) stem cell transplantation Poor graft function As a result of the conditioning regimen and previous chemotherapy, the bone marrow microenvironment can be damaged and this might result in a decreased support of haematopoiesis. Since MSC can form the bone marrow stroma and secrete haematopoietic cytokines, there is interest to use MSC to enhance engraftment in SCT. Graft failure can occur in any type of HCT and is associated with important morbidity and mortality [69, 70]. The earliest report of a clinical application of MSC dates back to 2000 when Koç and colleagues reported co-transplantation of MSC and autologous SCT in breast cancer patients. In this study a rapid neutrophil and platelet engraftment was observed [12]. In the setting of autologous SCT graft, engraftment failure occurs less frequent than in the allogeneic setting. Nonetheless will it lead to increased morbidity and mortality. Recently a study was published describing treatment of 22 patients with MSC vs MSC+cord blood for graft failure after autologous SCT. Neutrophil engraftment was more rapid in the cord blood+MSC group (8 days vs 18 days in MSC group). However, this cannot be attributed to cord blood engraftment since chimerism at day +30 was 100% recipient for all subjects. The authors state that this might be due to stromal cells present in the cord blood units enhancing the supportive effect on haematopoiesis of the bone marrow microenvironment. Unfortunately the study did not have a cord blood only arm allowing to examine whether MSC promote the effect of cord blood [71]. 8

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9 In allogeneic SCT grafting, engraftment failure is of concern in particular in cord blood transplantation and haplo-identical SCT. In cord blood transplantation cell doses are much lower than in peripheral blood SCT and greater HLA disparity is allowed; in haplo-identical HCT mega cell doses are used to overcome the greater HLA disparity. Several co-transplantation studies in these settings have been published so far. They show an improvement in engraftment and some also report a decrease of GVHD incidence [12, 32, 42, 72-83]. Table 2a provides an overview of the clinical data of studies using MSC in the treatment/prevention of poor graft function.

Tissue damage after HCT The main area of interest to use MSC in Haematology remains HCT and its complications. In addition to GVHD and poor graft function MSC might also be of use in the treatment of tissue damage after SCT given their multipotent differentiation capacity. Studies have shown the benefit of MSC in repair of cartilage or bone defects or damaged myocardium [84, 85]. The tissue damage after SCT can be due to the toxicity of the conditioning regimen, GVHD, infections, etc. Interest in this application for MSC arose when some patients included in studies for GVHD treatment with MSC showed remarkable repair of damaged tissues combined with several studies showing that MSC preferably home to sites of tissue injury [8, 21, 86-88] . In 2007 Ringden et al reported the results of a pilot study of 10 patients treated with MSC for haemorrhagic cystitis, pneumomediastinum or colon perforation as complications of allogeneic SCT. These data were promising and moreover, MSC DNA could be detected in the bladder of 1 of the patients [89], indeed suggesting homing of MSC to the injured tissues. However, additional and larger trials are needed to corroborate these findings.

Caveats in using MSC for clinical purposes Suppression of the Graft versus Tumour effect?

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10 Allogeneic SCT relies on two pillars for its therapeutic effect: disease eradication by pre SCT treatment and the conditioning regimen (on one hand) and allo-immune activity called the graft versus tumour effect (on the other hand). Since potent immunosuppressive capacities have been ascribed to MSC, there is some concern in the use of these cells for the treatment of complications of allogeneic SCT. In 2008 Ning et al. reported results of an open label, randomized clinical trial of HPC or HPC+MSC transplantation in patients with haematological malignancies. The authors saw a significantly lower incidence of GVHD but this came at a cost of significantly higher relapse rates in the co-transplantation group (60% vs 20%), resulting in a significantly lower disease free survival in this group (60% vs 66.7%) [90]. Many – if not all – studies on the use of MSC for treatment of late GVHD have also included relapse incidence as an endpoint and so far in these studies no significant increases in relapse were reported. However, to be conclusive, a large randomized controlled trial would be required.

Increased risk of infections. Since MSC suppress immune responses, one might expect that treatment with MSC increases infectious complications [31, 91]. In most recent studies on therapeutic applications of MSC, infectious complications are reported. However in the setting of GVHD treatment or poor graft function, there is already an increased incidence of infectious complications due to prolonged neutropenia, impaired immune recovery and prolonged immunosuppressive treatment. This makes it difficult to determine the impact of MSC treatment on the incidence of infections. There has been concern that MSC treatment leads to increased CMV infections. Lucchini et al. addressed this problem and retrospectively analysed frequency and severity in 24 patients treated with MSC in their centre. They could not find an increase in viral infections, nor in mortality compared with a historical control group [92]. Earlier this year Calkoen et al. reported on viral complications in a paediatric patient population treated with either corticosteroids, MSC or another second line immunosuppressive therapy. They could not find an increased incidence of CMV, EBV or 10

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11 adenovirus infections in the MSC treated population, however MSC treatment correlated with worse survival in adenovirus infection [93].

Tumour formation / malignant transformation of MSC. When pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, are injected in mice they will form teratomas [94]. Of course formation of ectopic tissue after injection of MSC has also been a concern. MSC might not only enhance tumour growth or disease progression in a subject but the MSC itself might transform into a malignant cell as well. This has been shown in murine MSC both in vitro and in vivo [95-98]. There have been reports on malignant transformation of human MSC in vitro; however they were retracted several years later and attributed to contamination with tumour cell lines. DNA fingerprinting of the cells believed to be transformed MSC showed that they did not have the profile of the original MSC but their profiles were comparable to a fibrosarcoma and osteosarcoma cell line in one lab and human glioma cell lines in the other lab, proving cross-contamination of these MSC cultures with tumour cell lines and disproving spontaneous transformation of MSC in culture. [99-102]. Because of these reports, several groups have addressed this issue. Since MSC require ex vivo expansion to obtain sufficient cell numbers for therapeutic applications, one could suppose that under the proliferative strain of the culture conditions, MSC might acquire cytogenetic abnormalities predisposing to malignant transformation of the cells. Therefore most groups concentrated at first on cytogenetic stability of MSC in culture. Indeed, several groups found, already at quite early stages of the cultures (passage 3/4 cells) as well as in later passage MSC, that there were cytogenetic abnormalities. However, in in vivo models, these cells did not form tumours, nor could malignant transformation of these cells in vitro be observed. As expected, MSC cultures showed evidence of senescence, regardless of cytogenetics while telomere length shortened and no evidence of increased telomerase activity could be found in these MSC [103-105].

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12 The most recent studies using MSC for therapeutic applications often include evidence for malignant transformation as an endpoint. So far, in the results of clinical trials in Haematology reported, there is no incidence of malignant transformation of MSC in patients treated with MSC. In 2011 Centeno and colleagues published an update on safety and complications of MSC implantation for orthopaedic interventions. They followed 339 patients with MRI and could not demonstrate evidence of malignant transformation [106]. Von Bahr et al. examined tissues from patients treated with MSC at autopsy. They could not find any ectopic tissue formation. They attribute this to the fact that there does not appear to be a sustained engraftment of MSC, since levels of MSC donor DNA were negatively correlated with time since MSC infusion [107]. As discussed above there is as yet no evidence of malignant transformation of human MSC. Malignant transformation appears to be restricted to murine MSC and to occur after prolonged expansion. In our study with murine MSC, we observed evidence for transformation only after multiple (8-9) passages [95]. However, caution with human MSC is warranted and malignant transformation should remain a point of attention in all clinical trials with MSC.

Foetal bovine serum In order to expand MSC in culture, a growth supplement has to be added to a basal medium. The first groups describing MSC expansion used foetal bovine serum (FBS) in varying concentrations for this purpose [1, 8]. However, this poses some challenges: there is of course the danger of transmission of pathogens (bacterial, viral, mycoplasma, prion), additionally the components are not defined and will vary with different batches, resulting in variable efficacy in cultures. FBS proteins can also give rise to xenogeneic reactions, resulting in allergic infusion reactions in case of repeated infusions and the presence of antibodies to FBS can also threaten viability of MSC after administration [108-110]. Efforts are ongoing to find alternatives: the most promising candidate is the replacement of FBS with platelet lysate (PL), thus humanizing the culture medium [110-112]. In recent years an increasing number of clinical studies with MSC expanded in a culture medium supplemented with PL are 12

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13 performed [28, 35, 41, 113]. However, PL still does not resolve all the issues with FBS: there is still a risk of pathogen transmission and PL batches also have a variable quality. A satisfactory serum free medium however has not been developed yet.

Caveats in the interpretation of MSC clinical trials. The use of MSC in a wide variety of clinical domains is investigated by a great number of research groups. In this review we focused on the application of MSC in the field of Haematology. Since they were first derived from bone marrow, a logical first step was to investigate their potential use in this particular domain. They have certain characteristics that make them promising tools for regenerative medicine (restoration of the bone marrow microenvironment) and immunotherapy. In vitro data confirm these expectations, however clinical trials are not uniformly positive [26, 33]. It should be noted that MSC have been moved quite rapidly from the bench to the bedside. They were already described in the 70s by Friedenstein and colleagues but it was just in the early 90s that Caplan and Pittenger published further characterization of these cells and described the mesengenic process. Already in the late 90s patients were treated with MSC [12]. Working mechanisms of MSC were and are not fully understood though we are gaining more and more insight. The evidence is accumulating in in vitro and in vivo studies that MSC can be a valid treatment option in a number of haematological conditions [44, 114-117]. The number of clinical trials with MSC increases further. In Haematology the majority of clinical trials with MSC focus on prevention or treatment of GVHD and poor graft function. However, it is difficult, if not impossible, to compare the data from these different trials since many parameters are highly variable. Tables 1a and 2a give an overview of the clinical variables and outcome of trials with MSC in GVHD and support of engraftment. Tables 1b and 2b list the variable parameters of the cellular products used in these trials. These vary not only between different trials but often also within one trial. 13

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14 In order to obtain a sufficient amount of MSC for clinical applications MSC have to expand ex vivo. The source of MSC differs, as most groups start MSC cultures from bone marrow or cord blood while a few groups use adipose tissue-derived MSC. In some studies even though MSC are all derived from bone marrow, the source of bone marrow is different between patients. In the pivotal trial of LeBlanc et al. for example, MSC are derived from bone marrow from either the HLA-identical sibling, a haploidentical donor or a third party (unrelated) donor [22]. Although MSC can escape immune recognition there could be a difference in clinical efficacy and MSC survival after systemic administration when MSC and HPC donor are the same. The group from Karolinska Instutet in Sweden has addressed this in a follow-up report of patients treated in their centre with MSC for GVHD or haemorrhagic cystitis and could not correlate MSC source (related or unrelated) with response [118]. For the ex vivo expansion of MSC variations of the protocol originally described are used. The most obvious difference is the replacement of FBS most commonly by PL. One group expanded MSC in serum of the donor [119]. The number of cells seeded to start a culture and after passaging is highly variable. Again in most of the trials patients included in the same trial receive MSC cultured for a different length of time and thus after different passage numbers. Often duration of expansion is a function of the cell number needed and obtained. Although all these culture conditions render cells with the MSC characteristics: spindle shape morphology, the combination of phenotypic markers and at least trilineage (adipocytic, osteocytic, chondrocytic) differentiation potential, it is becoming increasingly clear that biological characteristics are influenced by the culture conditions [120-123]. In 2012 the group of LeBlanc analysed the role of passaging on the clinical outcome in patients treated with MSC in their institution between 2002 and 2007 and found that patients treated with early passage MSC (1-2) had a significantly better response rate and overall survival than those treated with MSC cultured for 3 or more passages [118]. What appear to be small details therefore might have a significant impact on the effector functions of the cells. 14

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15 In addition to the differences in expansion protocols of MSC, published studies also differ in other aspects. The infused number is highly variable, with cell numbers varying between studies from 0,3 to 10 million MSC/kg recipient weight MSC [42, 77]. But even in the same trial the infused cell number can differ up to 10 fold among the different patients [23]. In most studies patients receive 12 million MSC/kg recipient weight, however this appears to be a rather arbitrary number, and again in a number of studies the infused cell number depends on the number of cells obtained after expansion. There are almost no data in literature on dose finding for MSC in the treatment of poor graft function or tissue repair after allogeneic stem cell transplantation or to obtain a maximal effect on GVHD. One study with Prochymal® compared 2 dose levels: 2 and 8 million/kg. Since there was no difference observed the remaining patients were treated with 2 million/kg. The total dose of MSC used also depends on the number of infusions planned. In some trials repeated infusions, e.g. biweekly, are administered irrespective of response, in other trials repeated infusions are planned as a function of response [22, 25-41, 47, 113]. The timing of infusion is quite constant in trials for the support of engraftment. MSC are usually infused 4-6 hours prior to HCT. However, in GVHD trials the infusion time points of the first MSC infusion but also between repeated infusions are highly variable, again between different studies but also in the same trial (table 1a). The variability between repeated infusion stems from the fact that they are often added to improve response or in case of relapse of GVHD. Logically patients might benefit from early treatment with MSC, however given the rather small patient numbers in trials reported as yet and the variety in number of treatment lines [1-8] between patients included in these studies, this cannot be assessed. The majority of studies in Haematology with MSC investigate their role in the treatment in GVHD, based on their immunosuppressive qualities. There are commonly used release criteria for MSC: phenotype, differentiation capacity and microbial screens. However, there is no standard or quality control for the immunosuppressive capacities of culture-expanded MSC. Different batches of MSC

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16 are used in different studies, but also in the same trial, MSC cultured with different batches of FBS or PL might have different immunosuppressive qualities. In recent years researchers are trying to develop and define such a quality control system. Nazarov and colleagues for example devised an in vitro assay to evaluate and quantify immunosuppression of expanded MSC by using murine clonal Tcells determining proliferation rate, cell surface marker expression, expression of mRNA of transcription factors and cytokine secretion expression [124]. Issues have been raised concerning cryopreservation of MSC. This procedure is widely used because in vitro expanded MSC can be stored in cell banks, allowing to offer cryopreserved MSC as an off the shelf product. Moreover, MSC have to be expanded in vitro over several weeks to obtain a sufficient cell number whereas treatment is often required as soon as possible in these clinical settings. Some authors have suggested that cryopreservation hampers the immunosuppressive qualities of MSC [125], while others claim that cryopreservation does not interfere with MSC function [126-128]. Finally there are some general remarks on the studies published. Often the number of patients included in the different studies are small, this renders the interpretation of the results even more difficult given the variability of parameters between patients included. There is also a lack of randomized trials. One large placebo-controlled trial has been performed by Osiris with Prochymal®. Results have only been published as an abstract since there was no difference in overall outcome [33]. Most groups use a historic control as reference. Since MSC in Haematology are mainly studied in the niche that is hematopoietic cell transplantation, it follows naturally that relatively small patient’s populations will be eligible for trials on the use of MSC. As shown in tables 1a, 1b, 2a and 2b and discussed above there is a great variability between different trials rendering interpretation of data difficult. Therefore a more rigorous study design for future studies is necessary to be able to examine the therapeutic potential in well-defined settings. In the development of new drugs these compounds have to prove themselves in trials against a standard or placebo, MSC should also be treated as such. Already in in vitro settings MSC efficacy can be compared to placebo or negative

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17 controls. It is only logical to then go on and use these cellular products that have proven effect in vitro in large, multicentre randomized trials. Such a trial will be easier to set up in co-transplantation studies in CB transplantation or haplo-identical HCT for support of engraftment. In GVHD where there is no golden standard treatment for steroid refractory disease such a trial might be more difficult to design, but efforts should also be made in these trials. The scientific community working with MSC is well aware of these issues and efforts are being made to obtain more uniform expansion protocols, as is proven by recent papers describing standardized, GMP approved, expansion protocols and the development of appropriate quality controls [124, 129135].

Conclusion In conclusion we can state that considerable progress has been made over the last 20 years, since MSC were first introduced in clinical applications. Evidence from both in vitro and in vivo data is accumulating regarding MSC as a valid treatment option in a number of haematological conditions. MSC can escape immune response and they can be banked and are thus easily accessible as an off the shelf product. Several questions regarding the working mechanisms of MSC remain unanswered, but we are gaining more and more insight. With this growing knowledge about the biology of MSC, efforts can be made to optimize the cells for therapeutic purposes or their use as targeted therapeutics. MSC have been rushed from the bench to the bedside but it would be wise to take a step back to the bench again. Clinical trials with MSC can only benefit from a better understanding of MSC working mechanisms and definition of optimal and standardized culture conditions. Moving forwards from the bench to the bedside, clinical trials should be designed rationally and rigorously both in MSC specific parameters as in treatment schedules. With rigorous study design, patient groups most

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18 likely to benefit from MSC therapy, can be identified and an optimal treatment schedule can be defined, allowing MSC to find their way to daily clinical practice in Haematology.

Ackowledgments The authors wants to thank Miss Nicole Arras for assistance in preparing this manuscript. Ann De Becker received a PhD Fellowship form the Free University Brussels (Horizontale onderzoeksaktie).

Author Disclosure Statement No competing financial interests exist

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19 References 1. Friedenstein AJ, RK Chailakhyan, NV Latsinik, AF Panasyuk and IV Keiliss-Borok. (1974). Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 17:331-340.

2. Erices A, P Conget and JJ Minguell. (2000). Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109:235-242.

3. Nekanti U, VB Rao, AG Bahirvani, M Jan, S Totey and M Ta. (2010). Long-term expansion and pluripotent marker array analysis of Wharton's jelly-derived mesenchymal stem cells. Stem Cells Dev 19:117-130.

4. Lee RH, B Kim, I Choi, H Kim, HS Choi, K Suh, YC Bae and JS Jung. (2004). Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 14:311-324.

5. Fernández M, V Simon, G Herrera, C Cao, H Del Favero and JJ Minguell. (1997) Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant 20:265-271.

6. Jo YY, HJ Lee, SY Kook, HW Choung, JY Park, JH Chung, YH Choung, ES Kim, HC Yang and PH Choung. (2007). Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng 13:767-773.

7. Götherström C, O Ringdén, M Westgren, C Tammik and K Le Blanc. (2003). Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells. Bone Marrow Transplant 32:265-272. 19

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8. Caplan AI. (1991). Mesenchymal stem cells. J Orthop Res 9(5):641-650.

9. Pittenger MF, AM Mackay, SC Beck, RK Jaiswal, R Douglas, JD Mosca, MA Moorman, DW Simonetti, S Craig and DR Marshak. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284:143-147.

10. Dominici M, K Le Blanc, I Mueller, I Slaper-Cortenbach, F Marini, D Krause, R Deans, A Keating, DJ Prockop and E Horwitz (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315-317.

11. Horwitz EM, K Le Blanc, M Dominici, I Mueller, I Slaper-Cortenbach, FC Marini, RJ Deans, DS Krause and A Keating; International Society for Cellular Therapy. (2005). Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7(5):393-395.

12. Koç ON, SL Gerson, BW Cooper, SM Dyhouse, SE Haynesworth, AI Caplan and HM Lazarus. (2000). Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307-316.

13. Aggarwal S and MF Pittenger. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses.. Blood 105:1815-1822.

14. Stagg J and J Galipeau. (2013). Mechanisms of immune modulation by mesenchymal stromal cells and clinical translation. Curr Mol Med 13:856-867. 20

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21

15. De Miguel MP, S Fuentes-Julián, A Blázquez-Martínez, CY Pascual, MA Aller, J Arias and F ArnalichMontiel. (2012). Immunosuppressive properties of mesenchymal stem cells: advances and applications. Curr Mol Med 12:574-591.

16. Majumdar MK, MA Thiede, SE Haynesworth, SP Bruder and SL Gerson. (2000). Human marrowderived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother Stem Cell Res 9(6):841-848.

17. Li J, L Zhu, X Qu, J Li, R Lin, L Liao, J Wang, S Wang, Q Xu and RC Zhao. (2013). Stepwise differentiation of human adipose-derived mesenchymal stem cells toward definitive endoderm and pancreatic progenitor cells by mimicking pancreatic development in vivo. Stem Cells Dev 22(10):1576-1587.

18. Ting CH, PJ Ho and BL Yen. (2014). Age-related decreases of serum-response factor levels in human mesenchymal stem cells are involved in skeletal muscle differentiation and engraftment capacity. Stem Cells Dev 23:1206-1216.

19. Datta I, S Mishra, L Mohanty, S Pulikkot, PG Joshi. (2011). Neuronal plasticity of human Wharton's jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells. Cytotherapy 13:918-932.

20. Garnett C, JF Apperley and J Pavlů. (2013). Treatment and management of graft-versus-host disease: improving response and survival. Ther Adv Hematol 4:366-378.

21

Page 22 of 49


22 21. Le Blanc K, I Rasmusson, B Sundberg, C Götherström, M Hassan, M Uzunel and O Ringdén. (2004). Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363:1439-1441.

22. Le Blanc K, F Frassoni, L Ball, F Locatelli, H Roelofs, I Lewis, E Lanino, B Sundberg, ME Bernardo, M Remberger, G Dini, RM Egeler, A Bacigalupo, W Fibbe and O Ringdén; Developmental Committee of the European Group for Blood and Marrow Transplantation. (2008). Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371:1579-1586.

23. Ringdén O, M Uzunel, I Rasmusson, M Remberger, B Sundberg , H Lönnies, HU Marschall, A Dlugosz, A Szakos, Z Hassan, B Omazic, J Aschan, L Barkholt and K Le Blanc. (2006). Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 81:13901397. 24. Baron F, C Lechanteur, E Willems, F Bruck, E Baudoux, L Seidel, JF Vanbellinghen, K Hafraoui, M Lejeune, A Gothot, G Fillet and Y Beguin. (2010). Cotransplantation of mesenchymal stem cells might prevent death from graft-versus-host disease (GVHD) without abrogating graft-versus-tumor effects after HLA-mismatched allogeneic transplantation following nonmyeloablative conditioning. Biol Blood Marrow Transplant 16:838-847. 25. Herrmann R, M Sturm, K Shaw, D Purtill, J Cooney, M Wright, M Phillips and P Cannell. (2012). Mesenchymal stromal cell therapy for steroid-refractory acute and chronic graft versus host disease: a phase 1 study. Int J Hematol 95:182-188. 26. Remberger M and O Ringdén. (2012). Treatment of severe acute graft-versus-host disease with mesenchymal stromal cells: a comparison with non-MSC treated patients. Int J Hematol 96(6):822824. 22

Page 23 of 49


23 27. Ball LM, ME Bernardo, H Roelofs, MJ van Tol, B Contoli, JJ Zwaginga, MA Avanzini, A Conforti, A Bertaina, G Giorgiani, CM Jol-van der Zijde, M Zecca, K Le Blanc, F Frassoni, RM Egeler, WE Fibbe, AC Lankester and F Locatelli. (2013). Multiple infusions of mesenchymal stromal cells induce sustained remission in children with steroid-refractory, grade III-IV acute graft-versus-host disease. Br J Haematol 163:501-509. 28. Sánchez-Guijo F, T Caballero-Velázquez, O López-Villar, A Redondo, R Parody, C Martínez, E Olavarría, E Andreu, F Prósper, M Díez-Campelo, C Regidor, E Villaron, L López-Corral, D Caballero, MC Cañizo and JA Pérez-Simon. (2014). Sequential Third-Party Mesenchymal Stromal Cell Therapy for Refractory Acute Graft-versus-Host Disease. Biol Blood Marrow Transplant 20:1580-1585. 29. Fang B, Y Song, L Liao, Y Zhang and RC Zhao. (2007). Favorable response to human adipose tissuederived mesenchymal stem cells in steroid-refractory acute graft-versus-host disease. Transplant Proc 39:3358-3362. 30. Müller I, S Kordowich, C Holzwarth, G Isensee, P Lang, F Neunhoeffer, M Dominici, J Greil and R Handgretinger. (2008). Application of multipotent mesenchymal stromal cells in pediatric patients following allogeneic stem cell transplantation. Blood Cells Mol Dis 40:25-32. 31. Kebriaei P, L Isola, E Bahceci, K Holland, S Rowley, J McGuirk, M Devetten, J Jansen, R Herzig, M Schuster, R Monroy and J Uberti. (2009). Adult human mesenchymal stem cells added to corticosteroid therapy for the treatment of acute graft-versus-host disease. Biol Blood Marrow Transplant 15:804-811. 32. Gonzalo-Daganzo R, C Regidor, T Martín-Donaire, MA Rico, G Bautista, I Krsnik, R Forés, E Ojeda, I Sanjuán, JA García-Marco, B Navarro, S Gil, R Sánchez, N Panadero, Y Gutiérrez, M García-Berciano, N Pérez, I Millán, R Cabrera and MN Fernández. (2009). Results of a pilot study on the use of third-party donor mesenchymal stromal cells in cord blood transplantation in adults. Cytotherapy 11:278-288.

23

Page 24 of 49


24 33. Martin PJ, JP Uberti, RJ Soiffer, H Klingemann, EK Waller, AS Daly, RP Herrmann and P Kebriaei. (2010). Prochymal improves response rates in patients with steroid-refractory acute graft versus host disease (SR-GVHD) involving the liver and gut: results of a randomized, placebo-controlled, multicentre phase III trial in GVHD. Bone Marrow Transpl 45:S170-S171 34. Weng JY, X Du, SX Geng, YW Peng, Z Wang, ZS Lu, SJ Wu, CW Luo, R Guo, W Ling, CX Deng, PJ Liao and AP Xiang. (2010). Mesenchymal stem cell as salvage treatment for refractory chronic GVHD. Bone Marrow Transplant 45:1732-1740. 35. Lucchini G, M Introna, E Dander, A Rovelli, A Balduzzi, S Bonanomi, A Salvadè, C Capelli, D Belotti, G Gaipa, P Perseghin, P Vinci, E Lanino, P Chiusolo, MG Orofino, S Marktel, J Golay, A Rambaldi, A Biondi, G D'Amico and E Biagi. (2010). Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population. Biol Blood Marrow Transplant 16:1293-1301. 36. Zhou H, M Guo, C Bian, Z Sun, Z Yang, Y Zeng, H Ai and RC Zhao. (2010). Efficacy of bone marrowderived mesenchymal stem cells in the treatment of sclerodermatous chronic graft-versus-host disease: clinical report. Biol Blood Marrow Transplant. 2010 Mar;16(3):403-412. 37. Prasad VK, KG Lucas, GI Kleiner, JA Talano, D Jacobsohn, G Broadwater, R Monroy and J Kurtzberg. (2011). Efficacy and safety of ex vivo cultured adult human mesenchymal stem cells (Prochymal™) in pediatric patients with severe refractory acute graft-versus-host disease in a compassionate use study. Biol Blood Marrow Transplant 17(4):534-541. 38. Resnick IB, C Barkats, MY Shapira, P Stepensky, AI Bloom, A Shimoni, D Mankuta, N Varda-Bloom, L Rheingold, M Yeshurun, B Bielorai, A Toren, T Zuckerman, A Nagler and R Or. (2013). Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC). Am J Blood Res 3:225238.

24

Page 25 of 49


25 39. Muroi K, K Miyamura, K Ohashi, M Murata, T Eto, N Kobayashi, S Taniguchi, M Imamura, K Ando, S Kato, T Mori, T Teshima, M Mori and K Ozawa. (2013). Unrelated allogeneic bone marrow-derived mesenchymal stem cells for steroid-refractory acute graft-versus-host disease: a phase I/II study. Int J Hematol 98206-213. 40. Kurtzberg J, S Prockop, P Teira, H Bittencourt, V Lewis, KW Chan, B Horn, L Yu, JA Talano, E Nemecek, CR Mills and S Chaudhury. (2014). Allogeneic human mesenchymal stem cell therapy (remestemcel-L, Prochymal) as a rescue agent for severe refractory acute graft-versus-host disease in pediatric patients. Biol Blood Marrow Transplant 20:229-235. 41. Introna M, G Lucchini, E Dander, S Galimberti, A Rovelli, A Balduzzi, D Longoni, F Pavan, F Masciocchi, A Algarotti, C Micò, A Grassi, S Deola, I Cavattoni, G Gaipa, D Belotti, P Perseghin, M Parma, E Pogliani, J Golay, O Pedrini, C Capelli, S Cortelazzo, G D'Amico, A Biondi, A Rambaldi and E Biagi. (2014). Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients. Biol Blood Marrow Transplant 20:375-381. 42. Li X-H, C-J Gao, W-M Da, Y-B Cao, Z-H Wang, L-X Xu, Y-M Wu, B Liu, Z-Y Liu, B Yan, S-W Li, X-L Yang, X-X Wu and Z-C Han. (2014). Reduced intensity conditioning, combined transplantation of haploidentical hematopoietic stem cells and mesenchymal stem cells in patients with severe aplastic anemia. PLoS ONE 9: e89666. 43. Hao L, H Sun, J Wang, T Wang, M Wang ad Z Zou. (2012). Mesenchymal stromal cells for cell therapy: besides supporting hematopoiesis. Int J Hematol 95: 34-46. 44. Engela AU, MJ Hoogduijn, K Boer, NHR Litjens, MGH Betjes, W Weimar and CC Baan. (2013). Human adipose-tissue derived mesenchymal stem cells induce functional de-novo regulatory T-cells with methylated FOXP3 gene DNA. Clin Exp Immunol 173: 343-354. 45. Duffy MM, T Ritter, R Ceredig and MD Griffin. (2011). Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell Res Ther 2: 34-42. 25

Page 26 of 49


26 46. Ribeiro A, P Laranjeira, S Mendes, I Velada, C Leite, P Andrade, F Santos, A Henriques, M Graos, C Cardoso, A Martinho, M Pais, C Lobato da Silva, J Cabral, H Trindade and A Paiva. (2013). Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells. Stem Cell Res Ther 4: 125 47. Peng Y, X Chen X, Q Liu, X Zhang, K Huang, L Liu, H Li, M Zhou, F Huang, Z Fan, J Sun, Q Liu, M Ke, X Li, Q Zhang and AP Xiang. (2014). Mesenchymal stromal cells infusions improve refractory chronic graft versus host disease through an increase of CD5+ regulatory B cells producing interleukin 10. Leukemia. (Epub ahead of print) doi: 10.1038/leu.2014.225. 48. Jitschin R, D Mougiakakos, L Von Bahr, S Völkl, G Moll, O Ringden, R Kiessling, S Linder, K Le Blanc. (2013). Alterations in the cellular immune compartment of patients treated with third-party mesenchymal stromal cells following allogeneic hematopoietic stem cell transplantation. Stem Cells 31:1715-1725. 49. van den Berk LC, BJ Jansen, S Snowden, KG Siebers-Vermeulen, C Gilissen, G Kögler, CG Figdor, CE Wheelock and R Torensma.

(2014). Cord blood mesenchymal stem cells suppress DC-T Cell

proliferation via prostaglandin B2. Stem Cells Dev 23:1582-1593. 50. François M, R Romieu-Mourez, M Li and J Gallipeau. (2012). Human MSC suppression correlates with cytokine induction of indeolamine 2,3-dioxygenase and bystander M2 macrophage differentiation. Mol Ther 20: 187-195. 51. Shi Y, G Hu, J Su, W Li, Q Chen, P Shou, C Xu, X Chen, Y Huang, Z Zhu, X Huang, X Han, N Xie and G Ren. (2010). Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Res. 20:510-8. 52. Chinnadurai R, IB Copland, SR Patel and J Galipeau. (2014). IDO-independent suppression of T cell effector function by IFN-γ-licensed human mesenchymal stromal cells. J Immunol 192:1491-501.

26

Page 27 of 49


27 53. Madrigal M, KS Rao and NH Riordan. (2014). A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med 12: 260. [Epub ahead of print] 54. Sioud M. (2011). New insights into MSC mediated T-cell suppression through galectins. Scand J Immunol 73: 79-84 55. Ungerer C, P Quade-Lyssy, HH Radeke, R Henschler, C Königs, U Köhl, E Seifried and J Schüttrumpf. (2014). Galectin-9 is a suppressor of T and B cells and predicts the immune modulatory potential of mesenchymal stromal cell preparations. Stem Cells Dev 23:755-66. 56. Li H, Y Jiang, X Jiang, X Guo, H Ning, Y Li, L Liao, H Yao, X Wang, Y Liu, Y Zhan, H Chen and N Mao. (2014). CCR7 guides migration of mesenchymal stem cell to secondary lymphoid organs: a novel approach to separate GvHD from GvL effect. Stem Cells. 32:1890-903. 57. Méndez-Ferrer S, TV Michurina, F Ferraro, AR Mazloom, BD Macarthur, SA Lira, DT Scadden, A Ma'ayan, GN Enikolopov and PS Frenette. (2010). Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466(7308):829-34. 58. Anthony BA and DC Link. (2014). Regulation of hematopoietic stem cells by bone marrow stromal cells. Trends Immunol 35:32-7. 59. Mendelson A and PS Frenette. (2014). Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat Med.20:833-46. 60. Leisten I, R Kramann, MS Ventura Ferreira, M Bovi, S Neuss, P Ziegler, W Wagner, R Knüchel and RK Schneider. (2012). 3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche. Biomaterials. 33:1736-47. 61. Cook MM, K Futrega, M Osiecki, M Kabiri, B Kul, A Rice, K Atkinson, G Brooke and M Doran. (2012). Micromarrows--three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells. Tissue Eng Part C Methods. 18:319-28. 27

Page 28 of 49


28 62. Sharma MB, LS Limaye and VP Kale. (2012). Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells. Haematologica. 97:651-60. 63. Park JS, S Suryaprakash, YH Lao and KW Leong. (2015). Engineering Mesenchymal Stem Cells for Regenerative Medicine and Drug Delivery. Methods. 2015 [Epub ahead of print] 64. Ballen KK, E Gluckman and HE Broxmeyer. (2013). Umbilical cord blood transplantation: the first 25 years and beyond. Blood 122:491-498. 65. Huang GP, ZJ Pan, BB Jia, Q Zheng, CG Xie, JH Gu, IK McNiece and JF Wang. (2007). Ex vivo expansion and transplantation of hematopoietic stem/progenitor cells supported by mesenchymal stem cells from human umbilical cord blood. Cell Transplant 16: 579-585. 66. Fan X, FP Gay, SY Ong, JM Ang, PP Chu, S Bari, TK Lim and WY Hwang. (2013). Mesenchymal stromal cells supported umbilical cord blood ex vivo expansion enhances regulatory T cells and reduces graft versus host disease. Cytotherapy 15: 610-619. 67. Robinson SN, PJ Simmons, H Yang, AM Alousi, MJ de Lima and EJ Shpall. (2011). Mesenchymal stem cells in ex vivo cord blood expansion. Best Pract Res Clin Haematol 24: 83-92. 68. Yang H, SN Robinson, J Lu, WK Decker, D Xing, D Steiner, S Parmar, N Shah, RE Champlin, M Munsell, A Leen, C Bollard, PJ Simmons and EJ Shpall. (2010). CD3(+) and/or CD14(+) depletion from cord blood mononuclear cells before ex vivo expansion culture improves total nucleated cell and CD34(+) cell yields. Bone Marrow Transplant 45:1000-1007. 69. Quinones RR. (1993). Hematopoietic engraftment and graft failure after bone marrow transplantation. Am J Pediatr Hematol Oncol 15:3-17. 70. Locatelli F, B Lucarelli and P Merli. (2014). Current and future approaches to treat graft failure after allogeneic hematopoietic stem cell transplantation. Expert Opin Pharmacother 15:23-36. 28

Page 29 of 49


29 71. Xiong YY, Q Fan, F Huang, Y Zhang, Y Wang, XY Chen, ZP Fan, HS Zhou, Y Xiao, XJ Xu, M Dai, N Xu, J Sun, P Xiang, XJ Huang and QF Liu. (2014). Mesenchymal stem cells versus mesenchymal stem cells combined with cord blood for engraftment failure after autologous hematopoietic stem cell transplantation: a pilot prospective, open-label, randomized trial. Biol Blood Marrow Transplant. 20:236-42. 72. Lazarus HM, OM Koc, SM Devine, P Curtin, RT Maziarz, HK Holland, EJ Shpall, P McCarthy, K Atkinson, BW Cooper, SL Gerson, MJ Laughlin, FR Loberiza Jr, AB Moseley and A Bacigalupo. (2005). Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11:389398. 73. Ball LM, ME Bernardo, H Roelofs, A Lankester, A Cometa, RM Egeler, F Locatelli and WE Fibbe. (2007). Cotransplantation of ex vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem-cell transplantation. Blood 110:2764-2767. 74. Le Blanc K, H Samuelsson, B Gustafsson, M Remberger, B Sundberg, J Arvidson, P Ljungman, H Lönnies, S Nava and O Ringdén. (2007). Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21:1733-1738. 75. Macmillan ML, BR Blazar, TE DeFor and JE Wagner. (2009). Transplantation of ex-vivo cultureexpanded parental haploidentical mesenchymal stem cells to promote engraftment in pediatric recipients of unrelated donor umbilical cord blood: results of a phase I-II clinical trial. Bone Marrow Transplant 43:447-454. 76. Fang B, N Li, Y Song, J Li, RC Zhao and Y Ma. (2009). Cotransplantation of haploidentical mesenchymal stem cells to enhance engraftment of hematopoietic stem cells and to reduce the risk of graft failure in two children with severe aplastic anemia. Pediatr Transplant 13:499-502.

29

Page 30 of 49


30 77. Liu K, Y Chen, Y Zeng, L Xu, D Liu, H Chen, X Zhang, W Han, Y Wang, T Zhao, J Wang , J Wang, Q Han, C Zhao and X Huang. (2011). Coinfusion of mesenchymal stromal cells facilitates platelet recovery without increasing leukemia recurrence in haploidentical hematopoietic stem cell transplantation: a randomized, controlled clinical study. Stem Cells Dev 20:1679-1685. 78. Wu Y, Z Wang, Y Cao, L Xu, X Li, P Liu, P Yan, Z Liu, D Zhao, J Wang, X Wu, C Gao, W Da and Z Han. (2013). Cotransplantation of haploidentical hematopoietic and umbilical cord mesenchymal stem cells with a myeloablative regimen for refractory/relapsed hematologic malignancy. Ann Hematol 92:1675-1684. 79. Wang H, Z Wang, X Zheng, L Ding, L Zhu, H Yan ad Z Guo. (2013). Hematopoietic stem cell transplantation with umbilical cord multipotent stromal cell infusion for the treatment of aplastic anemia--a single-center experience. Cytotherapy 15:1118-1125. 80. Lee SH, MW Lee, KH Yoo, DS Kim, MH Son, KW Sung, H Cheuh, SJ Choi, W Oh, YS Yang and HH Koo. (2013). Co-transplantation of third-party umbilical cord blood-derived MSCs promotes engraftment in children undergoing unrelated umbilical cord blood transplantation. Bone Marrow Transplant 48:1040-1045. 81. Wu Y, Y Cao, X Li, L Xu, Z Wang, P Liu, P Yan, Z Liu, J Wang, S Jiang, X Wu, C Gao, W Da and Z Han. (2014). Cotransplantation of haploidentical hematopoietic and umbilical cord mesenchymal stem cells for severe aplastic anemia: successful engraftment and mild GVHD. Stem Cell Res 12:132-138. 82. Liu X, M Wu, Y Peng, X Chen, J Sun, F Huang, Z Fan, H Zhou, X Wu, G Yu, X Zhang, Y Li, Y Xiao, C Song, AP Xiang and Q Liu. (2013). Improvement in poor graft function after allogeneic hematopoietic stem cell transplantation upon administration of mesenchymal stem cells from third-party donors: A pilot prospective study. Cell Transplant. (Epub ahead of print) doi: 10.3727/096368912X661319 83. Xiong YY, Q Fan, F Huang, Y Zhang, Y Wang, XY Chen, ZP Fan, HS Zhou, Y Xiao, XJ Xu, M Dai M, N Xu, J Sun, P Xiang, XJ Huang and QF Liu. (2014). Mesenchymal stem cells versus mesenchymal stem 30

Page 31 of 49


31 cells combined with cord blood for engraftment failure after autologous hematopoietic stem cell transplantation: a pilot prospective, open-label, randomized trial. Biol Blood Marrow Transplant 20:236-242. 84. Jorgensen C and D Noël. (2011). Mesenchymal stem cells in osteoarticular diseases. Regen Med 6:44-51. 85. Richardson JD, AJ Nelson, AC Zannettino, S Gronthos, SG Worthley and PJ Psaltis. (2013). Optimization of the cardiovascular therapeutic properties of mesenchymal stromal/stem cells-taking the next step. Stem Cell Rev 9:281-302. 86. Chapel A, JM Bertho, M Bensidhoum, L Fouillard, RG Young, J Frick, C Demarquay, F Cuvelier, E Mathieu, F Trompier, N Dudoignon, C Germain, C Mazurier, J Aigueperse, J Borneman, NC Gorin, P Gourmelon and D Thierry. (2003). Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome. J Gene Med 5:1028-1038. 87. Gao J, JE Dennis, RF Muzic, M Lundberg and AI Caplan. (2001). The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 169:12-20. 88. Rustad KC and GC Gurtner. (2012). Mesenchymal stem cells home to sites of injury and inflammation. Adv Wound Care (New Rochelle) 1:147-152. 89. Ringdén O, M Uzunel, B Sundberg, L Lönnies, S Nava, J Gustafsson, L Henningsohn and K Le Blanc. (2007). Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia 21:2271-2276. 90. Ning H, F Yang, M Jiang, L Hu, K Feng, J Zhang, Z Yu, B Li, C Xu, Y Li, J Wang, J Hu, X Lou and H Chen. (2008). The correlation between cotransplantation of mesenchymal stem cells and higher

31

Page 32 of 49


32 recurrence rate in hematologic malignancy patients: outcome of a pilot clinical study. Leukemia 22:593-599. 91. Forslow U, O Blennow, K LeBlanc, O Ringden, B Gustafsson, J Mattson and M Remberger. (2012). Treatment with mesenchymal stromal cells is a risk factor for pneumonia-related death after allogeneic hematopoietic stem cell transplantation. Eur J Haematol 89: 220-227. 92. Lucchini G, E Dander, F Pavan, I Di Ceglie, A Balduzzi, P Perseghin, G Gaipa, A Algarotti, M Introna, A Rambaldi, A Rovelli, A Biondi, E Biagi and G D'Amico. (2012). Mesenchymal stromal cells do not increase the risk of viral reactivation nor the severity of viral events in recipients of allogeneic stem cell transplantation. Stem Cells Int 2012:690236. 93. Calkoen FG, C Vervat C, AG van Halteren, MJ Welters, LA Veltrop-Duits, AC Lankester, RM Egeler, LM Ball ad MJ van Tol. (2014). Mesenchymal stromal cell therapy is associated with increased adenovirus-associated but not cytomegalovirus-associated mortality in children with severe acute graft-versus-host disease. Stem Cells Transl Med 3:899-910. 94. Fong CY, K Gauthaman and A Bongso. (2010). Teratomas from pluripotent stem cells: A clinical hurdle. J Cell Biochem 111:769-781. 95. Xu S, A De Becker, H De Raeve, B Van Camp, K Vanderkerken, I Van Riet. (2012). In vitro expanded bone marrow-derived murine (C57Bl/KaLwRij) mesenchymal stem cells can acquire CD34 expression and induce sarcoma formation in vivo. Biochem Biophys Res Commun 424: 391-397. 96. Tolar J, AJ Nauta, MJ Osborn, A Panoskaltsis Mortari, RT McElmurry, S Bell, L Xia, N Zhou, M Riddle, TM Schroeder, JJ Westendorf, RS McIvor, PC Hogendoorn, K Szuhai, L Oseth, B Hirsch, SR Yant, MA Kay, A Peister, DJ Prockop, WE Fibbe and BR Blazar. (2007). Sarcoma derived from cultured mesenchymal stem cells. Stem Cells 25: 371-379.

32

Page 33 of 49


33 97. Jeong J, JW Han, J Kim, H Cho, C Park, N Lee, D Kim and Y Yoon. (2011). Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy. Circ Res 108:1340-1347. 98. Xiao W, AB Mohensy, CW Hogendoorn and A Cleton-Jansen. (2013). Mesenchymal stem cell transformation and sarcoma genesis. Clinical Sarcoma Research 3: 310-318. 99. Rubio D, J Carcia-Castro, MC Martin, R de la Fuente, JC Cigudosa, AC Lloyd and A Bernad. (2005). Spontaneous human adult stem cell transformation. Cancer Res 65:3035-3039. 100. de la Fuente R, A Bernad, J Carcia-Castro, MC Martin and JC Cigudosa. (2010). Retraction: Spontaneous human adult stem cell transformation. Cancer Res 70: 6682. 101. Røsland GV, A Svendsen, A Torsvik, E Sobala, E McCormack, H Immervoll, J Mysliwietz, J-C Tonn, R Goldbrunner, PE Lønning, R Bjerkvig and C Schichor. (2009). Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergoe spontaneous malignant transformation. Cancer Res 2009; 69:5331-5339. 102. Torsvik A, GV. Røsland, A Svendsen, A Molven, H Immervoll, E McCormack, PE Lønning, M Primon, E Sobala, J-C Tonn, R Goldbrunner, C Schichor, J Mysliwietz, TT Lah, H Motaln, S Knappskog and R Bjerkvig. (2010). Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: putting the research field back on track. Cancer Res 70: 6393-6396.

103. Bernardo M, N Zaffaroni, F Novara, AM Cometa, MA Avanzini, A Moretta, D Montagna, R Maccario, R Villa, MG Daidone, O Zuffardi and F Locatelli. (2007). Human bone marrow-derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 67: 9142-9149. 104. Wang Y, Z Zhang, Y Chi, Q Zhang, F Xu, Z Yang, L Meng, S Yang, S Yan, A Mao, J Zhang, Y Yang, S Wang, J Cui, L Liang, Z-B Han, X Fang and ZC Han. (2013). Long-term cultured mesenchymal stem cells 33

Page 34 of 49


34 frequently develop genomic mutations but do not undergo malignant transformation. Cell Death Dis 4:e950. 105. Prockop DJ and A Keating. (2012). Relearning the lessons of genomic stability of human cells during expansion in culture: implications for clinical research. Stem Cells 30:1051-1052. 106. Centeno CJ, JR Schultz, M Cheever, M Freeman, S Faulkner, B Robinson and R Hanson. (2011). Safety and complications reporting update on the re-implantation of culture-expanded mesenchymal stem cells using autologous platelet lysate technique. Curr Stem Cell Res Ther 6: 368-378. 107. von Bahr L, Batsis I, Moll G, Hägg M, Szakos A, Sundberg B, Uzunel M, Ringden O, Le Blanc K. (2012). Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation. Stem Cells. 2012 Jul;30(7):1575-8. 108. Mackensen A, R Dräger, M Schlesier, R Mertelsmann and A Lindemann. (2000). Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells. Cancer Immunol Immunother 49; 152-156. 109. Sundin M, O Ringden, B Sundberg, S Nava, C Götherström and K LeBlanc. (2007). No alloantibodies against mesenchymal stromal cells but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica 92: 1208-1215. 110. Gottipamula S, MS Muttigi, U Kolkundkar and RN Seetharam. (2013). Serum-free media for the production of human mesenchymal stromal cells: a review. Cell Prolif 46: 608-627. 111. Iudicone P, D Fioravanti, G Bonanno, M Miceli, C Lavorino, P Totta, L Frati, M Nuti and L Pierelli. (2014). Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells. J Transl Med 12: 28-41. 112. Bieback K. (2013). Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures. Transfus Med Hematother 40: 326-335. 34

Page 35 of 49


35 113. von Bonin M, F Stölzel, A Goedecke, K Richter, N Wuschek, K Hölig, U Platzbecker, T Illmer, M Schaich, J Schetelig, A Kiani, R Ordemann, G Ehninger, M Schmitz and M Bornhäuser. (2009). Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium. Bone Marrow Transplant 43:245-251. 114. Aggarwal S and MF Pittenger. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105:1815-1822. 115. Maitra B, E Szekely, K Gjini, MJ Laughlin, J Dennis, SE Haynesworth and ON Koç. (2004). Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 33:597-604. 116. Tobin LM, ME Healy, K English and BP Mahon. (2013). Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graftversus-host disease. Clin Exp Immunol 172:333-348. 117. Li ZY, CQ Wang, G Lu, XY Pan and KL Xu. (2014). Effects of bone marrow mesenchymal stem cells on hematopoietic recovery and acute graft-versus-host disease in murine allogeneic umbilical cord blood transplantation model. Cell Biochem Biophys 70:115-122. 118. von Bahr L, B Sundberg , L Lönnies , B Sander, H Karbach, H Hägglund, P Ljungman, B Gustafsson, H Karlsson, K Le Blanc and O Ringdén. (2012). Long-term complications, immunologic effects, and role of passage for outcome in mesenchymal stromal cell therapy. Biol Blood Marrow Transplant 18:557-564. 119. Arima N, F Nakamura, A Fukunaga, H Hirata, H Machida, S Kouno and H Ohgushi. (2010). Single intra-arterial injection of mesenchymal stromal cells for treatment of steroid-refractory acute graftversus-host disease: a pilot study. Cytotherapy 12:265-268.

35

Page 36 of 49


36 120. De Becker A, P Van Hummelen, M Bakkus, I Vande Broek, J De Wever, M De Waele and I Van Riet. (2007). Migration of culture-expanded human mesenchymal stem cells through bone marrow endothelium is regulated by matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3. Haematologica 92:440-449. 121. Strioga M, S Viswanathan, A Darinskas, O Slaby and J Michalek. (2012). Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. Stem Cells Dev 21:2724-2752. 122. Kern S, H Eichler, J Stoeve, H Klüter ad K Bieback. (2006). Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24:1294-1301. 123. Peng L, Z Jia, X Yin, X Zhang, Y Liu, P Chen, K Ma and C Zhou. (2008). Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem Cells Dev 17:761773. 124. Nazarov C, J Lo Surdo, SR Bauer and CH Wei. (2013). Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro. Stem Cell Res Ther 4:128. 125. François M, IB Copland, S Yuan, R Romieu-Mourez, EK Waller and J Galipeau. (2012). Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing. Cytotherapy 14:147-152. 126. Mamidi MK, KG Nathan, G Singh, ST Thrichelvam, NA Mohd Yusof, NA Fakharuzi, Z Zakaria, R Bhonde, AK Das and AS Majumdar. (2012). Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation. J Cell Biochem 113:3153-3164.

36

Page 37 of 49


37 127. Ginis I, B Grinblat and MH Shirvan. (2012). Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium. Tissue Eng Part C Methods. 18:453-463. 128. Holubova M, D Lysak, T Vlas, L Vannucci and P Jindra. (2014). Expanded cryopreserved mesenchymal stromal cells as an optimal source for graft-versus-host disease treatment. Biologicals 42:139-144. 129. Nold P, C Brendel, A Neubauer, G Bein and H Hackstein. (2013). Good manufacturing practicecompliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor. Biochem Biophys Res Commun 430:325-330. 130. Fekete N, MT Rojewski, D Fürst, L Kreja, A Ignatius, J Dausend and H Schrezenmeier. (2012). GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC. PLoS One 7:e43255. 131. Rojewski MT, N Fekete, S Baila, K Nguyen, D Fürst, D Antwiler, J Dausend, L Kreja, A Ignatius, L Sensebé and H Schrezenmeier. (2013). GMP-compliant isolation and expansion of bone marrowderived MSCs in the closed, automated device quantum cell expansion system. Cell Transplant 22:1981-2000. 132. Wuchter P, K Bieback, H Schrezenmeier, M Bornhäuser, LP Müller, H Bönig, W Wagner, R Meisel, P Pavel, T Tonn, P Lang, I Müller, M Renner, G Malcherek, R Saffrich, EC Buss, P Horn, M Rojewski, A Schmitt, AD Ho, R Sanzenbacher and M Schmitt. (2014). Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells

for

immunotherapeutic

applications.

Cytotherapy

(e-pub

ahead

of

print).

doi:

10.1016/j.jcyt.2014.04.002. 133. Martins JP, JM Santos, JM Almeida, MA Filipe, MV de Almeida, SC Almeida, A Agua-Doce, A Varela, M Gilljam, B Stellan, S Pohl, K Dittmar, W Lindenmaier, E Alici, L Graça, PE Cruz, HJ Cruz and 37

Page 38 of 49


38 RN Bárcia. (2014). Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data. Stem Cell Res Ther 5:9. 134. Jang YK, M Kim, YH Lee, W Oh, YS Yang and SJ Choi. (2014). Optimization of the therapeutic efficacy of human umbilical cord blood-mesenchymal stromal cells in an NSG mouse xenograft model of graft-versus-host disease. Cytotherapy 16:298-308. 135. Menard C, L Pacelli, G Bassi, J Dulong, F Bifari, I Bezier, J Zanoncello, M Ricciardi, M Latour, P Bourin, H Schrezenmeier, L Sensebé, K Tarte and M Krampera. (2013). Clinical-grade mesenchymal stromal cells produced under various good manufacturing practice processes differ in their immunomodulatory properties: standardization of immune quality controls. Stem Cells Dev 22:17891801. 136. Meuleman N, T Tondreau, I Ahmad, J Kwan, F Crokaert, A Delforge, C Dorval, P Martiat, P Lewalle, L Lagneaux and D Bron. (2009).

Infusion of mesenchymal stromal cells can aid

hematopoietic recovery following allogeneic hematopoietic stem cell myeloablative transplant: a pilot study. Stem Cells Dev 18:1247-1252. 137. Yin F, M Battiwalla, S Ito, X Feng, F Chinian, JJ Melenhorst, E Koklanaris, M Sabatino, D Stroncek, L Samsel, J Klotz, NF Hensel, PG Robey and AJ Barrett. (2014). Bone marrow mesenchymal stromal cells to treat tissue damage in allogeneic stem cell transplant recipients: correlation of biological markers with clinical responses. Stem Cells 32:1278-1288.

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Stem Cells and Development Mesenchymal stromal cell therapy in haematology: from laboratory to clinic and back again (doi: 10.1089/scd.2014.0564) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f Page 39 of 49

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Stem Cells and Development Mesenchymal stromal cell therapy in haematology: from laboratory to clinic and back again (doi: 10.1089/scd.2014.0564) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f

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40

Table 1b: Overview of characteristics of the cellular products used in MSC studies in GVHD 1st author, Year Journal [reference] Ringden O, 2006 Transplant. [23] Fang B, 2007 Transplant Proc. [29] Muller I, 2008 Blood Cells Mol Dis. [30] LeBlanc K, 2008 Lancet [22] Kebriaei P, 2009 BBMT [31] Von Bonin M, 2009 BMT [113] Gonzalo-Daganzo R, 2009 Cytotherapy [32] Baron F, 2010 BBMT [24] Martin P, 2010 BBMT suppl [33] Weng JY, 2010 BMT [34] Lucchini G, 2010 BBMT [35] Arima N, 2010 Cytotherapy [119] Zhou H, 2010 BBMT [36] Prasad V, 2011 BBMT [37] Hermann R, 2012 Int J Hematol [25] Remberger M, 2012 Int J Hematol [26] Resnick IB, 2013 Am J Blood Res [38]

MSC origin (donor)

P

FBS/PL

Cell dose (x106/kg)

BM (4 3rd party, 2 HLA id sib, 6 haplo) AT (haplo, 3rd party)

1 (4), 2 (4), 3 (3), 4(1)

FBS

0.7 - 9

NR

FBS

1-2

BM (HSC donor, 2 haplo parent) BM (HLA id sib 5, haplo 18, 3rd party 69) BM (3rd party)

NR

FBS

0.4 - 3

1-4

FBS

0.4 – 9

5

FBS

2 or 8

BM (3rd party)

1-2

PL

0.6 - 1.1

BM (3rd party)

Max 3

FBS

1 – 3.3

BM (3rd party)

2

FBS

NR

BM (3rd party)

NR

FBS

2

BM (3rd party)

2-3

FBS

0.23-1.42

BM (3rd party)

2

PL

0.7-3.7

BM (HPC donor)

2

Donor serum

5

BM (3rd party)

3-6

FBS

10-20 (total dose)

BM (3rd party)

NR

FBS

2 or 8

BM (MMRD, MRD, 3rd party, haplo) BM (NR)

2-3

FBS

1.7-2.3

NR

FBS

0.65-2

BM (HSC donor, 3rd party)

FBS

0.3-3.1

Ball LM, 2013 Br J Haematol [27] Muroi K, 2013

BM (3rd party, haplo)

1-3 (n=48) 6 (n=1) 7 (n=1) 2-3

FBS

0.9-3

BM (3rd party)

NR

FBS

2

40


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41 Int J Hematol [39] Kurtzberg J, 2014 BBMT [40] Sanchez-Guijo F, 2014 BBMT [28]

BM (3rd party)

5

FBS

2

BM (3rd party)

1(n=12) 2 (n=8) 3 (n=5) 0

PL

0.7 – 1.31

Li X, 2014 CB (3rd party) NA 2.87 – 10 PLoS One [42] Introna M, 2014 BM (3rd party) NR PL 1±0.5 BBMT [41] Peng Y, 2014 BM (3rd party) 4-5 FBS 1 Leukemia [47] Yin F, 2014 BM (3rd party) 4 FBS 2 Stem Cells [137] P= passage number, BM= bone marrow, CB= cord blood, AT= adipose tissue, HLA id sib= HLA identical sibling, FBS= foetal bovine serum, PL= platelet lysate, MRD= matched related donor, MMRD= mismatched related donor, NR = not reported.

Table 1a: Overview of clinical data of studies with MSC in GVHD. 1st author, Year Journal [reference]

N

Repeated infusions?

aGVHD

cGVHD

Infusion time point

Ringden O, 2006 Transplant. [23]

9

1 (n=6) 2 (n=3)

Steroid refractory grade III-IV

NA

d+46 – d+694 post HCT

Fang B, 2007 Transplant Proc. [29]

6

Yes in 2pt

NA

d+65-d+243 post HCT

1-8

Muller I, 2008 Blood Cells Mol Dis.[30]

7

1 (n=4) 2 (n=2) 3 (n=1)

Steroid refractory grIIIIV 2 pt

3 pt

d+46 - d+768 post HCT

3-6

LeBlanc K, 2008 Lancet [22]

55

1 (n=27) 2 (n=22) 3-5 (n= 6)

Steroid refractory gr IIIV

NA

d+27 - d+533 post HCT

1-5

Kebriaei P, 2009

32

2

Grade II-IV

NA

24-48h

NA

N previous treatment lines 2-8

results

Remarks

-Complete resolution of aGVHD in 6/8 pts -Improved survival in MSC recipients -1 pt treated for steroid refractory extensive cGVHD did not respond to MSC treatment -Significantly better survival rate in responders to MSC treatment compared to untreated pts -5/6 CR -1 pt did not respond

1 pt had MSC donor DNA in colon and a lymph node

-1 case of aGVHD did not progress tot cGVHD -Other case of aGVHD died of relapse -1 case of cGVHD did not respond and died from PTLD 18m after MSC infusion -1 case of cGVHD did not repond to MSC – death of cGVHD 12m after MSC treatment -Slight improvement in 1 pt with cGVHD -CR 30/55 -PR 9/55 -Better 1y TRM for CR (37%) vs PR/NR (72%) -Higher 2y OS for CR (53%) vs PR/NR (16%) -Response not related to HLA match -77% CR, 16% PR

-Repeated infusions due to poor proliferation of MSC in vitro -1pt treated for poor graft function, good response -1pt treated for hemophagocytosis improvement in all cell lines after MSC treatment

8% donor colon epithelial cells on biopsy

Concomittant corticosteroids

41


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42 BBMT [31] Von Bonin M, 2009 BMT [113]

13

1

Gonzalo-Daganzo R, 2009 Cytotherapy [32]

9

Yes

Baron F, 2010 BBMT [24]

20

No

steroid refractory gr IIIIV Steroid refractory grII (2pt) Prevention

4-31d after GVHD diagnosis

2-3

-ORR 54% -1CR, 1PR, 5 MR, 1PD

Additional medical therapy if less than PR

NA

NR

NA

CR in both patients

Cotransplantation study in CBT Additional MSC infusions in 2pt with steroid refractory GVHD

Prevention

30-120min before HCT

NA

Steroid refractory

NA

NR

NR

1-5

NA

Refractory

1-5

11

1-4

Refractory (8)

Refractory (3)

3

No

Steroid refractory gut/liver aGVHD

NA

6.4-246.1 w after cGVHD diagnosis 30-300d after HCT 12-37.3m after

-45% aGVHD grII-IV compared to 56% historic control -Probability of dying from GVHD or while on treatment for GVHD10% vs 31% historic control -Overall response Prochymal® 82% vs 73% placebo -Response in gut & liver GVHD better than placebo (76 vs 47% and 82 vs 68%) -Overall response 73.7% -4/19 CR -10/19 PR -Overall response 71.4% -CR 23.8% -PR in 1 patient, recurrent aGVHD after 3m -Idiopathic pneumonia syndrome in 2/3 patients

Martin P, 2010 BMT suppl [33]

24 4

8 infusions biweekly

Weng JY, 2010 BMT [34]

19

Lucchini G, 2010 BBMT [35] Arima N, 2010 Cytotherapy [119] Zhou H, 2010 BBMT [36] Prasad V, 2011 BBMT [37] Hermann R, 2012 Int J Hematol [25]

4

Yes

2-3

Improvement of symptoms in all patients

12

8 biweekly infusions 2-19

Grade III-IV

2-5

Steroid refractory grIIIV

Steroid refractory

-58% CR -17% PR -cGVHD: 2/7 CR, 2/7 PR, 3/7 NR -aGVHD: 7/12 CR, 4/12 PR, 1 NR -CR important in aGVHD for survival, not in cGVHD

Remberger M, 2012 Int J Hematol [26]

28

Yes: 29 doses total

Steroid refractory grIIIIV

NA

12-37.3m after GVHD diagnosis 18-157d after GVHD diagnosis 1-10m after alloHCT in aGVHD 14-89m after alloHCT in cGVHD 0-37d after diagnosis of aGVHD

Resnick IB, 2013 Am J Blood Res [38]

50

1-4

Steroid refractory

NA

≥2

Ball LM, 2013 Br J Haematol [27]

37

1-13

Steroid refractory grIII-

NA

1-136d after aGVHD diagnosis 5-85d after aGVHD

19

Sclerodermatou s

1-5 2-3

1

NR

NR

Placebo controlled trial

13-944d between 2 MSC infusions

Intra-arterial injection

Intra-BM injection of 1-2 x 107 total cells

Concomitant etanercept

-CR 6/15, PR 1/15, SD 1/15, PD 7/15 -marked increase in fungal infections in MSC treated group -trend for better survival and lower TRM in MSC group -no difference in survival between early and late disease -Overall response rate 66% with 34% CR

Pt between 2002-2006 treated with (n=15) or without (n=13) MSC, no randomisation

-65% CR -Higher TRM non CR vs CR; 69% vs 17%

-Chimaerism analysis of BM 1y after HCT showed no donor MSC

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43 IV

diagnosis

Muroi K, 2013 Int J Hematol [39]

14

8 biweekly

Steroid refractory grIIIII

NA

Kurtzberg J, 2014 BBMT [40] Sanchez-Guijo F, 2014 BBMT [28]

75

8 biweekly infusions Yes: min 2 doses/pt, 21 3 doses, 18 planned 4 doses

Grade B-D

NA

Steroid refractory grade II-IV

Li X, 2014 PLoS One [42] Introna M, 2014 BBMT [41]

17

No

40

Yes min 2/pt with 57d interval

25

-OS in CR 65% after 2.9y FU vs 0% in non CR -Trend for better OS and lower TRM when treated early (5-12d) -8/14 CR, 5/14 PR -estimated time to reach 50% CR after the first MSC infusion is 3 weeks -no difference in time to reach CR between grII and III aGVHD -61% OR

-3-43 d between MSC infusions

Within 48h of diagnosis of steroid refractory aGVHD 2-1639d

1

NA

1-7d after refractory GVHD diagnosis

1

-11/25 CR, 6/25 PR -Median time to response 28d after first MSC infusion -Responses were similar for all GVHD grades -Better survival of complete responders

-1pt with angor episode after 1st and 2nd MSC infusion – MSC treatment was discontinued -4 doses planned: day 1, 4, 11, 18

Prevention

Prevention

NA

-Lower incidence of GVHD

-MSC collected and prepared on d0 as fresh preparation

Steroid refractory

Steroid refractory

6h before haploHCT 4-1535d after GVHD diagnosis

1 (17pt) ≥2 (23pt)

-Overall response rate at 28d after infusion 67.5% with 27.5% CR -More CR in grade II aGVHD than higher grades (61.5% vs 11%) 20/23 CR+PR

2- ≥4

-No other second line treatment -additional 4 weekly MSC infusions if PR/NR

Peng Y, 2014 23 3 with 4w NA Steroid NR NR Leukemia [47] interval refractory Yin F, 2014 7 3 with 1w Steroid d+24-d+350 NR 5/7 CR Stem Cells [137] interval refractory post HCT Significantly prolonged survival if CR N= number, pt= patient, aGVHD: acute graft versus host disease, cGVHD= chronic graft versus host disease, d= day, HCT= haematopoietic cell transplantation, CR= complete response, PR= partial response, MR= minore response, NR= no response, SD= stable disease, PD= progressive disease, FU= follow-up, PTLD= post-transplant lymphoproliferative disease, TRM = transplant related mortality, OS= overall survival, CBT = cord blood transplantation, m= month, w= week, NA = not applicable, NR = not reported.

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Stem Cells and Development Mesenchymal stromal cell therapy in haematology: from laboratory to clinic and back again (doi: 10.1089/scd.2014.0564) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f Page 45 of 49

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Table 2b: Overview of cellular product characteristics of MSC used in clinical trials for engraftment First author, Year MSC origin (donor) P FBS/PL Cell dose (x106/kg) Journal [reference] Koç ON, 2000 BM (autologous) NR FBS 1-2.2 JCO [12] Lazarus HM, 2005 BM (HSC donor) 4 FBS 1-5 BBMT [72] Ball LM, 2007 BM (HSC donor) ≤3 FBS 1-5 Blood [73] LeBlanc K, 2007 BM (4 haplo, 3 HLA id sib) 2-3 FBS 1 Leukemia [74] Macmillan ML, 2009 BM (Haplo parent) 1-4 FBS 0.06-10 BMT [75] Gonzalo-Daganzo R, 2009 BM (Haplo parent, haplo relative, ≤3 FBS 1 – 3.3 Cytotherapy [32] 3rd party) Fang B, 2009 AT (Haplo (mothers)) NR FBS 1 Pediatr Transplant [76] Meuleman N, 2009 BM (HSC donor) 2-3 Serum free 1 Stem Cells Dev [136] Liu K, 2011 BM (HSC donor (4pt),3rd party 4-5 Serum free 0.3-0.5 Stem Cells Dev [77] (23pt)) Wu Y, 2013 CB (3rd party) NR NR 0.5 Ann Hematol [78] Wang H, 2013 CB (3rd party) 3 NA 0.27-2.5 Cytotherapy [79] Lee SH, 2013 CB (3rd party) NR FBS 1 or 5 BMT [80] Liu X, 2013 BM (3rd party) 4-5 FBS 1 (additional infusion if Cell Transplant [82] no CR after 28d) Wu Y, 2014 CB (3rd party) NR NR 0.5 Stem Cell Res [81] Li X, 2014 CB (3rd party) 0 NA 2.87 – 10 PLoS One [42] Xiong YY, 2014 BM (3rd party) 4-5 FBS 2.12-5.47 BBMT [83] Yin F, 2014 BM (3rd party) 4 FBS 2 (3 weekly infusions) Stem Cells [137] P= passage number, BM= bone marrow, CB= cord blood, AT= adipose tissue, FBS= foetal bovine serum, PL= platelet lysate, d= day, HSC= haematopoietic stem cell, haplo= haplo-identical, HLA id= HLA identical, sib= sibling, NR= not reported

46


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47

Table 2a: Overview of clinical data of trials using MSC to support engraftment. First author, Year Journal [reference] Koç ON, 2000 JCO [12]

N

HCT

Infusion time point

28

Autologous

1-24h after autoHCT

Lazarus HM, 2005 BBMT [72] Ball LM, 2007 Blood [73]

46

MRD

14

Haplo

4h before HCT

LeBlanc K, 2007 Leukemia [74] Macmillan ML, 2009 BMT [75]

7

MRD, MUD, MMUD

Within 4h before HCT

8

CB

At CBT (in 3pt additional infusion d+21)

Gonzalo-Daganzo R, 2009 Cytotherapy [32]

9

CB+haplo

Fang B, 2009 Pediatr Transplant [76] Meuleman N, 2009 Stem Cells Dev [136] Liu K, 2011 Stem Cells Dev [77] Wu Y, 2013 Ann Hematol [78]

2

Within 4h prior to HCT

55

1 haplo sib, 1 HLA id sib HLA id sib (n=3) Haplo (n=3) Haplo

50

Haplo

4h before HCT

Wang H, 2013 Cytotherapy [79]

22

Haplo BM/PBSC

Lee SH, 2013 BMT [80] Liu X, 2013 Cell Transplant [82]

7

CB

Immediately prior to HCT

20

Primary PGF: 31-35d after HCT 2ary PGF: 31-41d after occurrence of PGF

Wu Y, 2014 Stem Cell Res [81]

21

MRD, MUD 4 matched, 16 mismatched Haplo

6

94-295d after HCT Coinfusion

4h before HCT

MSC marrow chimaerism

18 subjects analysed: 2 mixed chimaerism Recipient

Engraftment -PMN recovery 8d -plt>20000 8.5d -rapid haematologic recovery after autoHCT+MSC -No influence of MSC dose on PMN or platelet recovery -No graft rejection -PMN and plt recovery similar to historic controls -faster leukocyte/lymphocyte recovery -100% hematopoietic engraftment -PMN median 12d, plt >30000/µL 12d -100% haematopoietic engraftment -PMN recovery 19d -Plt > 50000/µl 53d -Engraftment kinetics similar to historic control -PMN recovery 12d -Plt >20000/µl 44d -Engraftment kinetics similar to historic controls -1pt second graft failure – 1pt first graft failure before MSC+HSCT -100% T-cel donor chimaera after cotransplantation -durable response 2/6 -no response 4/6 -No difference in PMN and plt engraftment -but plt >50000/µL 22d vs 28d (MSC vs control) p20000/µL 14d Xiong YY, 2014 22 CB At CBT -Randomisation between MSC vs MSC+CB BBMT [83] -After 1 treatment cycle response 7/11 MSC, 9/11 MSC+CB -More rapid PMN recovery MSC+CB vs MSC Yin F, 2014 2 MRD, MUD NR Delayed marrow failure Stem Cells [137] No improvement N= number, pt= patient, aGVHD: acute graft versus host disease, cGVHD= chronic graft versus host disease, BM= bone marrow, CB= cord blood, AT= adipose tissue, PGF= poor graft function, d= day, h= hour, HCT= haematopoietic cell transplantation, MRD= matched related donor, (M)MUD= (mis)matched unrelated donor, haplo= haplo-identical, HLA id= HLA identical, sib= sibling, PBSC: peripheral blood stem cells, CBT = cord blood transplantation, PMN = neutrophil, plt= platelet, CR= complete response, PR= partial response, OR= overall response

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Stem Cells and Development Mesenchymal stromal cell therapy in haematology: from laboratory to clinic and back again (doi: 10.1089/scd.2014.0564) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 49 of 49

49

49

Stem cell therapy in the horse: from laboratory to clinic.

Pompe disease: from pathophysiology to therapy and back again.

Retinoids and breast cancer: from basic studies to the clinic and back again.

From medicine to butterflies and back again.

From disease to illness and back again.

From NHS to army--and back again.

From people-centered to person-centered services, and back again.

Plant epigenetics: from genotype to phenotype and back again.

From AAA+ to BB- and on the way back again.

From trochophore to pilidium and back again - a larva's journey.

Ways of thinking: from crows to children and back again.

From the motor cortex to the movement and back again.

reperfusion.

Reperfusion Injury.

Plants antioxidants: From laboratory to clinic.

Mesenchymal stromal cell cryopreservation.

Neuroscience. Back together again.

PHYSICAL THERAPY MANAGEMENT OF ICE HOCKEY ATHLETES: FROM THE RINK TO THE CLINIC AND BACK.

MECP2 disorders: from the clinic to mice and back.

Good medical ethics, from the inside out--and back again.

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Dissipation and heating in solar wind turbulence: from the macro to the micro and back again.

Hematology clinic: chronic myelogenous leukemia.

Monkey neurophysiology to clinical neuroscience and back again.