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Background and Aims The long-term safety and efficacy of allogeneic bone marrow-derived mesenchymal stromal cell [bmMSC] therapy in perianal Crohn’s disease [CD] fistulas is unknown. We aimed to provide a 4-year clinical evaluation of allogeneic bmMSC treatment of perianal CD fistulas. Methods A double-blind dose-finding study for local bmMSC therapy in 21 patients with refractory perianal fistulising Crohn’s disease was performed at the Leiden University Medical Center in 2012–2014. All patients treated with bmMSCs [1 x 107 bmMSCs cohort 1, n = 5; 3 × 107 bmMSCs cohort 2, n = 5; 9 × 107 bmMSCs cohort 3, n = 5] were invited for a 4-year evaluation. Clinical events were registered, fistula closure was evaluated, and anti-human leukocyte antigen [HLA] antibodies were assessed. Patients were also asked to undergo a pelvic magnetic resonance imaging [MRI] and rectoscopy. Results Thirteen out of 15 patients [87%] treated with bmMSCs were available for long-term follow-up. Two non-MSC related malignancies were observed. No serious adverse events thought to be related to bmMSC therapy were found. In cohort 2 [n = 4], all fistulas were closed 4 years after bmMSC therapy. In cohort 1 [n = 4] 63%, and in cohort 3 [n = 5] 43%, of the fistulas were closed, respectively. In none of the patients anti-HLA antibodies could be detected 24 weeks and 4 years after therapy. Pelvic MRI showed significantly smaller fistula tracts after 4 years. Conclusions Allogeneic bmMSC therapy for CD-associated perianal fistulas is also in the long-term a safe therapy. In bmMSC-treated patients, fistulas with closure at Week 24 were still closed after 4 years.

Mesenchymal stromal cells (MSC) hold promise as a novel immune‐modulatory therapy in organ transplantation. First clinical studies have used autologous MSCs; however, the use of allogeneic "off‐the‐shelf" MSCs is more sustainable for broad clinical implementation, although with the risk of causing sensitization. We investigated safety and feasibility of allogeneic MSCs in renal transplantation, using a matching strategy that prevented repeated mismatches. Ten patients received two doses of 1.5 × 106/kg allogeneic MSCs 6 months after transplantation in a single‐center nonrandomized phase Ib trial, followed by lowering of tacrolimus (trough level 3 ng/mL) in combination with everolimus and prednisone. Primary end point was safety, measured by biopsy proven acute rejection (BPAR) and graft loss 12 months after transplantation. Immune monitoring was performed before and after infusion. No BPAR or graft loss occurred and renal function remained stable. One patient retrospectively had DSAs against MSCs, formed before infusion. No major alterations in T and B cell populations or plasma cytokines were observed upon MSC infusion. Administration of HLA selected allogeneic MSCs combined with low‐dose tacrolimus 6 months after transplantation is safe at least in the first year after renal transplantation. This sets the stage to further explore the efficacy of third‐party MSCs in renal transplantation., The authors report that administration 6 months after kidney transplantation of allogeneic bone marrow–derived mesenchymal stromal cells selected to have no repeated HLA mismatches with the kidney donor was safe 1 year after transplantation with no instances of biopsy‐proven acute rejection, graft loss, or de novo antibodies against either donor.

Background Cell-based therapies have the potential to become treatment options for many diseases, but efficient scale-out of these therapies has proven to be a major hurdle. Bioreactors can be used to overcome this hurdle, but changing the culture method can introduce unwanted changes to the cell product. Therefore, it is important to establish parity between products generated using traditional methods versus those generated using a bioreactor. Methods Mesenchymal stromal cells (MSCs) are cultured in parallel using either traditional culture flasks, spinner vessels or a new bioreactor system. To investigate parity between the cells obtained from different methods, harvested cells are compared in terms of yield, phenotype and functionality. Results Bioreactor-based expansion yielded high cell numbers (222–510 million cells). Highest cell expansion was observed upon culture in flasks [average 5.0 population doublings (PDL)], followed by bioreactor (4.0 PDL) and spinner flasks (3.3 PDL). Flow cytometry confirmed MSC identity (CD73+, CD90+ and CD105+) and lack of contaminating hematopoietic cell populations. Cultured MSCs did not display genetic aberrations and no difference in differentiation and immunomodulatory capacity was observed between culture conditions. The response to IFNγ stimulation was similar for cells obtained from all culture conditions, as was the capacity to inhibit T cell proliferation. Conclusions The new bioreactor technology can be used to culture large amounts of cells with characteristics equivalent to those cultured using traditional, flask based, methods. Electronic supplementary material The online version of this article (10.1186/s12967-019-1989-x) contains supplementary material, which is available to authorized users.

Highlights • Seventeen EBMT centers participated to a questionnaire on MSC manufacturing. • 88% of centers manufacture MSC from bone marrow and only 2 centers from umbilical cord. • Human platelet lysate has replaced bovine serum as culture medium supplement. • Release criteria extensively differ among centers. • The results highlight the need to harmonize MSC manufacturing., The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients’ stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

CD4(+) regulatory T cells (Tregs) are a distinct population of T cells involved in maintaining peripheral tolerance to self-antigens. Several studies have shown increased frequency and number of Tregs in the elderly. Whether such an increase has any clinical relevance has not been addressed. Here, we have analyzed circulating Tregs in 114 donors between the ages of 18 and 89 years and assessed their implications for survival of the very elderly. In line with previously published data, we observed higher proportions of Tregs in the elderly. Expression of chemokine receptor 4 (CCR4) by Tregs has been shown to characterize antigen-primed activated Tregs with immediate suppressive function. Thus we further analyzed Tregs expressing or lacking this chemokine receptor. There were more CCR4(+) and CCR4(-) Tregs in the elderly than the young. Finally, using a subset of 48 elderly donors participating in the Leiden 85-plus study we documented that people with greater median frequencies of CCR4(+) Tregs enjoyed a better 8-year survival rate than those with lower frequencies of these cells. Our data, demonstrating for the first time a positive correlation between increased frequency of Tregs and survival in the elderly, imply an increasing importance of controlling inappropriate immune responses and inflammation as we grew old.

Myocardial infarction triggers reparative inflammatory processes programmed to repair damaged tissue. However, often additional injury to the myocardium occurs through the course of this inflammatory process, which ultimately can lead to heart failure. The potential beneficial effects of cell therapy in treating cardiac ischemic disease, the number one cause of death worldwide, are being studied extensively, both in clinical trials using adult stem cells as well as in fundamental research on cardiac stem cells and regenerative biology. This review summarizes the current knowledge on molecular and cellular processes implicated in post-infarction inflammation and discusses the potential beneficial role cell therapy might play in this process. Due to its immunomodulatory properties, the mesenchymal stromal cell is a candidate to reverse the disease progression of the infarcted heart towards heart failure, and therefore is emphasized in this review.

BACKGROUND & AIMS: Patients with perianal fistulizing Crohn's disease have a poor prognosis because these lesions do not heal well. We evaluated the effects of local administration of bone marrow-derived mesenchymal stromal cells (MSCs) to these patients from healthy donors in a double-blind, placebo-controlled study. METHODS: Twenty-one patients with refractory perianal fistulizing Crohn's disease were randomly assigned to groups given injections of 1 x 10(7) (n = 5, group 1), 3 x 10(7) (n = 5, group 2), or 9 x 10(7) (n = 5, group 3) MSCs, or placebo (solution with no cells, n = 6), into the wall of curettaged fistula, around the trimmed and closed internal opening. The primary outcome, fistula healing, was determined by physical examination 6, 12, and 24 weeks later; healing was defined as absence of discharge and

COPD is characterised by tissue destruction and inflammation. Given the lack of curative treatments and the progressive nature of the disease, new treatments for COPD are highly relevant. In vitro cell culture and animal studies have demonstrated that mesenchymal stromal cells (MSCs) have the capacity to modify immune responses and to enhance tissue repair. These properties of MSCs provided a rationale to investigate their potential for treatment of a variety of diseases, including COPD. Preclinical models support the hypothesis that MSCs may have clinical efficacy in COPD. However, although clinical trials have demonstrated the safety of MSC treatment, thus far they have not provided evidence for MSC efficacy in the treatment of COPD. In this review, we discuss the rationale for MSC-based cell therapy in COPD, the main findings from in vitro and in vivo preclinical COPD model studies, clinical trials in patients with COPD and directions for further research.

The low percentages of naive T cells commonly observed in elderly people are thought to be causally associated with mortality, primarily from infectious disease, and are taken as a hallmark of “immunosenescence”. Whether low levels of naive cells actually do associate with mortality has, however, not been tested in longitudinal studies. Here, we present correlations between peripheral T-cell phenotypes and 8-year survival in individuals from the population-based prospective Leiden 85-plus Study. Counter-intuitively, we found that a lower frequency of naive CD8+ T cells (characterized as CD45RA+CCR7+CD27+CD28+) at baseline (>88 years) correlated with significantly better survival, while there was a tendency for the reciprocal accumulation of late-differentiated effector memory cells (CD45RA−CCR7−CD27−CD28−) also to associate with better survival. These findings suggest that better retention of memory cells specific for previously encountered antigens may provide a survival advantage in this particular population. Given the prevalence of Cytomegalovirus (CMV) and its reported association with immunosenescence, we tested whether memory for this potential pathogen was relevant to survival. We found that individuals mounting an exclusively pro-inflammatory ex vivo response (TNF, IFN-γ, IL-17) to the major CMV target molecules pp65 and IE1 had a significant survival advantage over those also having anti-inflammatory responses (IL-10). These findings suggest that higher levels of naive T cells may not necessarily be associated with a survival advantage and imply that the nature of immunosurveillance against CMV may be crucial for remaining longevity, at least in the very elderly. Electronic supplementary material The online version of this article (doi:10.1007/s11357-012-9425-7) contains supplementary material, which is available to authorized users.

Item does not contain fulltext Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive and reparative properties in vitro and in vivo. Although autologous bone marrow (BM)-derived MSCs are already clinically tested in transplant recipients, it is unclear whether these BM cells are affected by renal disease. We assessed whether renal failure affected the function and therapeutic potential of BM-MSCs.METHODS: MSCs from 10 adults with end-stage renal disease (ESRD) and 10 age-matched healthy controls were expanded from BM aspirates and tested for phenotype and functionality in vitro.RESULTS: MSCs from ESRD patients were >90% positive for CD73, CD90 and CD105 and negative for CD34 and CD45 and showed a similar morphology and differentiation capacity as MSCs from healthy controls. Of importance for their clinical utility, growth characteristics were similar in both groups, and sufficient numbers of MSCs were obtained within 4 weeks. Messenger RNA expression levels of self-renewal genes and factors involved in repair and inflammation were also comparable between both groups. Likewise, microRNA expression profiling showed a broad overlap between ESRD and healthy donor MSCs. ESRD MSCs displayed the same immunosuppressive capacities as healthy control MSCs, demonstrated by a similar dose-dependent inhibition of peripheral blood mononuclear cell proliferation, similar inhibition of proinflammatory cytokines tumor necrosis factor-α and interferon-γ production and a concomitant increase in the production of interleukin-10.CONCLUSIONS: Expanded BM-MSCs procured from ESRD patients and healthy controls are both phenotypically and functionally similar. These findings are important for the potential autologous clinical application of BM-MSCs in transplant recipients.

Background Mesenchymal stromal cells (MSCs) are investigated for their potential to reduce inflammation and to repair damaged tissue. Inflammation and tissue damage are hallmarks of chronic obstructive pulmonary disease (COPD) and MSC infusion is a promising new treatment for COPD. Inflammatory mediators attract MSCs to sites of inflammation and affect their immune-modulatory properties, but little is known about their effect on regenerative properties of MSCs. This study investigates the effect of the pro-inflammatory cytokines TNF-α and IL-1β on the regenerative potential of MSCs, using an in vitro wound healing model of airway epithelial cells. Methods Standardized circular wounds were created by scraping cultures of the airway epithelial cell line NCI-H292 and primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC), and subsequently incubated with MSC conditioned medium (MSC-CM) that was generated in presence or absence of TNF-α/IL-1β. Remaining wound size was measured up to 72 h. Phosphorylation of ERK1/2 by MSC-CM was assessed using Western blot. Inhibitors for EGFR and c-Met signaling were used to investigate the contribution of these receptors to wound closure and to ERK1/2 phosphorylation. Transactivation of EGFR by MSC-CM was investigated using a TACE inhibitor, and RT-PCR was used to quantify mRNA expression of several growth factors in MSCs and NCI-H292. Results Stimulation of MSCs with the pro-inflammatory cytokines TNF-α and IL-1β increased the mRNA expression of various growth factors by MCSs and enhanced the regenerative potential of MSCs in an in vitro model of airway epithelial injury using NCI-H292 airway epithelial cells. Conditioned medium from cytokine stimulated MSCs induced ERK1/2 phosphorylation in NCI-H292, predominantly via EGFR; it induced ADAM-mediated transactivation of EGFR, and it induced airway epithelial expression of several EGFR ligands. The contribution of activation of c-Met via HGF to increased repair could not be confirmed by inhibitor experiments. Conclusion Our data imply that at sites of tissue damage, when inflammatory mediators are present, for example in lungs of COPD patients, MSCs become more potent inducers of repair, in addition to their well-known immune-modulatory properties.

Background : Mesenchymal stromal cells (MSCs) may reduce inflammation and promote tissue repair in pulmonary emphysema. Aim : To study the safety and feasibility of bone marrow-derived autologous (BM-) MSC intravenous administration to patients with severe emphysema. Design : A phase I, prospective open-label study registered at ClinicalTrials.gov as [NCT01306513][1]. Eligible patients had lung volume reduction surgery (LVRS) on two separate occasions. During the first LVRS bone marrow was collected, from which MSCs were isolated and expanded ex vivo . After 8 weeks, patients received two autologous MSC infusions 1 week apart, followed by the second LVRS procedure at 3 weeks after the second BM-MSC infusion. Methods : Up to 3 weeks after the last MSC infusion adverse events were recorded. Using immunohistochemistry and qPCR for analysis of cell and proliferation markers, emphysematous lung tissue obtained during the first surgery was compared with lung tissue obtained after the second surgical session to assess BM-MSC effects. Results : From 10 included patients three were excluded: two did not receive MSCs due to insufficient MSC culture expansion, and one had no second surgery. No adverse events related to MSC infusions occurred and lung tissue showed no fibrotic responses. After LVRS and MSC infusions alveolar septa showed a 3-fold increased expression of the endothelial marker CD31 ( P = 0.016). Conclusions : Autologous MSC treatment in severe emphysema is feasible and safe. The increase in CD31 expression after LVRS and MSC treatment suggests responsiveness of microvascular endothelial cells in the most severely affected parts of the lung. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01306513&atom=%2Fqjmed%2Fearly%2F2016%2F02%2F03%2Fqjmed.hcw001.atom

High-grade osteosarcoma is a tumor with a complex genomic profile, occurring primarily in adolescents with a second peak at middle age. The extensive genomic alterations obscure the identification of genes driving tumorigenesis during osteosarcoma development. To identify such driver genes, we integrated DNA copy number profiles (Affymetrix SNP 6.0) of 32 diagnostic biopsies with 84 expression profiles (Illumina Human-6 v2.0) of high-grade osteosarcoma as compared with its putative progenitor cells, i.e., mesenchymal stem cells (n ¼ 12) or osteoblasts (n ¼ 3). In addition, we performed paired analyses between copy number and expression profiles of a subset of 29 patients for which both DNA and mRNA profiles were available. Integrative analyses were performed in Nexus Copy Number software and statistical language R. Paired analyses were performed on all probes detecting significantly differentially expressed genes in corresponding LIMMA analyses. For both nonpaired and paired analyses, copy number aberration frequency was set to >35%. Nonpaired and paired integrative analyses resulted in 45 and 101 genes, respectively, which were present in both analyses using different control sets. Paired analyses detected >90% of all genes found with the corresponding nonpaired analyses. Remarkably, approximately twice as many genes as found in the corresponding nonpaired analyses were detected. Affected genes were intersected with differentially expressed genes in osteosarcoma cell lines, resulting in 31 new osteosarcoma driver genes. Cell division related genes, such as MCM4 and LATS2, were overrepresented and genomic instability was predictive for metastasis-free survival, suggesting that deregulation of the cell cycle is a driver of osteosarcomagenesis. V C 2012 Wiley Periodicals, Inc.

Background and aims Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I–III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays Methods The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied Results MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment Conclusions This study demonstrates that, in vitro , MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy.

Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells.Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining.Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were approximately 90-fold enriched in the MSCA-1(+)CD56(-) fraction and approximately 180-fold in the MSCA-1(+)CD56(+) fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1(+)CD56(-) mesenchymal stem cells subset and CD166 to MSCA-1(+)CD56(+/-) mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1(+)CD56(+/-) cells whereas adipocytes emerged exclusively from MSCA-1(+)CD56(-) cells. The culture of single sorted MSCA-1(+)CD56(+) cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities.Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1(+)CD56(+) subset is an attractive starting population for autologous chondrocyte transplantation.

Background: Severe graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic transplantation with haemopoietic stem cells. Mesenchymal stem cells modulate immune responses in vitro and in vivo. We aimed to assess whether mesenchymal stem cells could ameliorate GVHD after haemopoietic-stem-cell transplantation. Methods: Patients with steroid-resistant, severe, acute GVHD were treated with mesenchymal stem cells, derived with the European Group for Blood and Marrow Transplantation ex-vivo expansion procedure, in a multicentre, phase II experimental study. We recorded response, transplantation-related deaths, and other adverse events for up to 60 months' follow-up from infusion of the cells. Findings: Between October, 2001, and January, 2007, 55 patients were treated. The median dose of bone-marrow derived mesenchymal stem cells was 1·4×106 (min-max range 0·4-9×106) cells per kg bodyweight. 27 patients received one dose, 22 received two doses, and six three to five doses of cells obtained from HLA-identical sibling donors (n=5), haploidentical donors (n=18), and third-party HLA-mismatched donors (n=69). 30 patients had a complete response and nine showed improvement. No patients had side-effects during or immediately after infusions of mesenchymal stem cells. Response rate was not related to donor HLA-match. Three patients had recurrent malignant disease and one developed de-novo acute myeloid leukaemia of recipient origin. Complete responders had lower transplantation-related mortality 1 year after infusion than did patients with partial or no response (11 [37%] of 30 vs 18 [72%] of 25; p=0·002) and higher overall survival 2 years after haemopoietic-stem-cell transplantation (16 [53%] of 30 vs four [16%] of 25; p=0·018). Interpretation: Infusion of mesenchymal stem cells expanded in vitro, irrespective of the donor, might be an effective therapy for patients with steroid-resistant, acute GVHD. Funding: Swedish Cancer Society, Children's Cancer Foundation, Swedish Research Council, Cancer Society in Stockholm, Cancer and Allergy Foundation, Karolinska Institutet, Istituto Superiore di Sanità, European Union, Regione Lombardia, Fondazione CARIPLO, Associazione Italiana Ricerca contro il Cancro, Compagnia di San Paolo Torino, Progetto CARIGE Cellule Staminali, European Commission, Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Ricerca Finalizzata Regione Liguria 2005 Assistenza Domiciliare, Dutch Programme for Tissue Engineering. © 2008 Elsevier Ltd. All rights reserved.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells and interest in MSC therapy has been raised by the observation that MSCs are able to modulate immune responses in vitro and in vivo. Here, we show that MSCs are not intrinsically immune privileged and are capable of inducing memory T cell responses following injection in vivo in immunocompetent hosts. After cotransplantation in recipients that have received sublethal irradiation, allogeneic MSCs can still induce an alloresponse that may result in graft rejection, suggesting that the immunogenicity of allogeneic MSCs are not fully prevented by a nonmyeloablative conditioning regimen. It is still unclear whether the immunogenicity of allogeneic MSCs is also preserved following a fully myeloablative conditioning regimen.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into several mesodermal lineages. These cells have been isolated from various tissues, such as adult bone marrow, placenta, and fetal tissues. The comparative potential of these cells originating from different tissues to differentiate into the chondrogenic lineage is still not fully defined. The aim of our study was to investigate the chondrogenic potential of MSCs isolated from different sources. MSCs from fetal and adult tissues were phenotypically characterized and examined for their differentiation capacity, based on morphological criteria and expression of extracellular matrix components. Our results show that both fetal and adult MSCs have chondrogenic potential under appropriate conditions. The capacity of bone marrow-derived MSCs to differentiate into chondrocytes was reduced on passaging of cells. MSCs of bone marrow origin, either fetal or adult, exhibit a better chondrogenesis than fetal lung- and placenta-derived MSCs, as demonstrated by the appearance of typical morphological features of cartilage, the intensity of toluidine blue staining, and the expression of collagen type II, IX, and X after culture under chondrogenic conditions. As MSCs represent an attractive tool for cartilage tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for these therapeutic approaches.

Background Mesenchymal stromal cells (MSC) may serve as an attractive therapy in renal transplantation due to their immunosuppressive and reparative properties. While most studies have used autologous MSCs, allogeneic MSCs offer the advantage of immediate availability for clinical use. This is of major importance for indications where instant treatment is needed, for example allograft rejection or calcineurin inhibitor toxicity. Clinical studies using allogeneic MSCs are limited in number. Although these studies showed no adverse reactions, allogeneic MSCs could possibly elicit an anti-donor immune response, which may increase the incidence of rejection and impact the allograft survival in the long term. These safety issues should be addressed before further studies are planned with allogeneic MSCs in the solid organ transplant setting. Methods/design 10 renal allograft recipients, 18–75 years old, will be included in this clinical phase Ib, open label, single center study. Patients will receive two doses of 1.5 × 106 per/kg body weight allogeneic bone marrow derived MSCs intravenously, at 25 and 26 weeks after transplantation, when immune suppression levels are reduced. The primary end point of this study is safety by assessing biopsy proven acute rejection (BPAR)/graft loss after MSC treatment. Secondary end points, all measured before and after MSC infusions, include: comparison of fibrosis in renal biopsy by quantitative Sirius Red scoring; de novo HLA antibody development and extensive immune monitoring; renal function measured by cGFR and iohexol clearance; CMV and BK infection and other opportunistic infections. Discussion This study will provide information on the safety of allogeneic MSC infusion and its effect on the incidence of BPAR/graft loss. Trial registration: NCT02387151

Cotransplantation of CD34(+) hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs) enhances HSPC engraftment. For these applications, MSCs are mostly obtained from bone marrow (BM). However, MSCs can also be isolated from the Wharton's jelly (WJ) of the human umbilical cord. This source, regarded to be a waste product, enables a relatively low-cost MSC acquisition without any burden to the donor. In this study, we evaluated the ability of WJ MSCs to enhance HSPC engraftment. First, we compared cultured human WJ MSCs with human BM-derived MSCs (BM MSCs) for in vitro marker expression, immunomodulatory capacity, and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSCs have a more restricted differentiation capacity, both WJ MSCs and BM MSCs expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Most importantly, cotransplantation of either WJ MSCs or BM MSCs with CB CD34(+) cells into NOD SCID mice showed similar enhanced recovery of human platelets and CD45(+) cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood, and spleen 6 weeks after transplantation when compared to transplantation of CD34(+) cells alone. Upon coincubation, both MSC sources increased the expression of adhesion molecules on CD34(+) cells, although stromal cell-derived factor-1 (SDF-1)-induced migration of CD34(+) cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34(+) cells were cultured on monolayers of WJ MSCs in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSCs replaced WJ MSCs in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34(+) cell engraftment.

// Johnny Suijker 1 , Hans J. Baelde 1 , Helene Roelofs 2 , Anne-Marie Cleton-Jansen 1 , Judith V.M.G. Bovee 1 1 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Immuno-Haematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands Correspondence to: JVMG Bovee, e-mail: j.v.m.g.bovee@lumc.nl Keywords: isocitrate dehydrogenase, enchondroma, mesenchymal stem cells, ollier disease, maffucci syndrome Received: March 24, 2015 Accepted: May 13, 2015 Published: May 25, 2015 ABSTRACT Mutations in isocitrate dehydrogenase 1 ( IDH1 ) and IDH2 are found in a somatic mosaic fashion in patients with multiple enchondromas. Enchondromas are benign cartilaginous tumors arising in the medulla of bone. The mutant IDH1/2 causes elevated levels of D-2-hydroxyglutarate (D-2-HG). Mesenchymal stem cells (MSC) are the precursor of the osteoblastic, chondrogenic and adipocytic lineage and we hypothesized that increased levels of D-2-HG cause multiple enchondromas by affecting differentiation of MSCs. Bone marrow derived MSCs from different donors were differentiated towards osteoblastic, chondrogenic and adipocytic lineage in the presence or absence of 5 mM D-2-HG. Three of four MSCs showed near complete inhibition of calcification after 3 weeks under osteogenic differentiation conditions in the presence of D-2-HG, indicating a block in osteogenic differentiation. Two of four MSCs showed an increase in differentiation towards the chondrogenic lineage. To evaluate the effect of D-2-HG in vivo we monitored bone development in zebrafish, which revealed an impaired development of vertebrate rings in the presence of D-2-HG compared to control conditions ( p -value < 0.0001). Our data indicate that increased levels of D-2-HG promote chondrogenic over osteogenic differentiation. Thus, mutations in IDH1/2 lead to a local block in osteogenic differentiation during skeletogenesis causing the development of benign cartilaginous tumors.

Background: Consistent average length differences between species and chromosome arm differences within species indicate that telomere length is genetically determined. This seems to contradict an observed large variation in lengths of the same human telomere between metaphases of the same individual. We examined the extent to which the variation in the telomeres of the human X and Y chromosomes is heritable, induced, or technical in origin. Methods: Metaphase chromosomes were stained by fluorescence in situ hybridization with a telomere repeatspecific probe, and fluorescence intensities of the X and Y chromosomes were measured. If telomere length variation is predominantly genetically determined and a 50% probability of meiotic recombination between the pseudoautosomal regions of Yp and Xp in the father is taken into account, one expects an equal chance that the Yp telomere of a son is derived from his father’s Xp or Yp telomere. This implies that the Yp/Yq telomere ratios in fathers and sons will be identical in the absence of paternal meiotic recombination and different when recombination occurs. Results: Among five father-son pairs, four showed similar Yp/Yq ratios (P 0.05), whereas one pair exhibited a large difference in the Yp/Yq ratio that was attributable to a significantly longer Xp than Yp telomere in the father and a presumptive meiotic exchange between X and Y during paternal meiosis. Further, the Xq telomere exhibited a generally shorter telomere length than the others. Conclusions: The high variation in telomere length appeared to be intracellular (between sister chromatids) and, hence, technical in nature. We found no measurable induced variation in the cells studied, implying that, if induced variation exists, it is small compared with the technical variation. © 2005 Wiley-Liss, Inc. Key terms: telomeric length; inheritance; fluorescence in situ hybridization; technical error

It is generally believed that homeostatic responses regulate T-cell recovery after peripheral stem cell transplantation (PSCT). We studied in detail immune recovery in relation to T-cell depletion and clinical events in a group of adult patients who underwent PSCT because of hematologic malignancies. Initially, significantly increased proportions of dividing naive, memory, and effector CD4+and CD8+ T cells were found that readily declined, despite still very low numbers of CD4+ and CD8+ T cells. After PSCT, increased T-cell division rates reflected immune activation because they were associated with episodes of infectious disease and graft-versus-host disease (GVHD). T-cell receptor excision circles (TRECs) were measured to monitor thymic output of naive T cells. Mean TREC content normalized rapidly after PSCT, long before naive T-cell numbers had significantly recovered. This is compatible with the continuous thymic production of TREC+ naive T cells and does not reflect homeostatic increases of thymic output. TREC content was decreased in patients with GVHD and infectious complications, which may be explained by the dilution of TRECs resulting from increased proliferation. Combining TREC and Ki67 analysis with repopulation kinetics led to the novel insight that recovery of TREC content and increased T-cell division during immune reconstitution after transplantation are related to clinical events rather than to homeostatic adaptation to T-cell depletion.

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

Testicular germ cell tumours (TGCTs) may arise through a process of multi-step carcinogenesis, and loss of heterozygosity (LOH) at specific loci is likely to be an important early event, although this has not been studied in detail. In order to explore the pathogenetic relationships among TGCTs, we investigated the genetic changes in testicular tumours that exhibit a disease continuum through the precursor carcinoma in situ (CIS) to either seminoma (SE) and/or non-seminomatous germ cell tumour (NSGCT). Universal amplification has been performed on 87 TGCT specimens and 36 samples of CIS cells microdissected from single paraffin-embedded tumour sections from 40 patients, including multiple specimens of CIS and TGCT cells of varied histology microdissected from 24 individual patients. Seventy-seven microsatellite markers were used to assay these samples for LOH at candidate regions selected from the literature, mapping to 3q, 5q, 9p, 11p, 11q, 12q, 17p and 18q. Construction of deletion maps for each of these regions identified common sites of deletion at 3q27–q28, 5q31, 5q34–q35, 9p22–p21 and 12q22, which correlate with allelic losses we have also observed in the precursor CIS cells. Evidence for allelic loss at 3q27–q28 was observed in all of the embryonal carcinoma samples analysed. We conclude that inactivation of gene(s) within these regions are likely to be early events in the development and progression of TGCTs. These results also provide molecular evidence in support of the hypothesis that SE is an intermediate stage of development within a single neoplastic pathway of progression from CIS precursor cells to NSGCT. © 2000 Cancer Research Campaign

Steroid-nonresponsive acute graft-versus-host disease (aGVHD) after hematopoietic stem cell transplantation carries a poor prognosis. Various groups have reported beneficial effects of mesenchymal stromal cell (MSC) infusion as salvage treatment. Response to treatment is typically evaluated using the diagnostic clinical criteria for aGVHD. In this study, we evaluated the usefulness of additional gastrointestinal biopsy specimens paired with serum biomarkers. In a cohort of 22 pediatric patients, persistent or recurrent diarrhea was seen in 18 children treated with MSC infusion for steroid-refractory aGVHD. To exclude other causes of gastrointestinal pathology, patients were biopsied for histological analysis. Eight of 12 patients exhibited residual tissue damage and villous atrophy, but no active aGVHD. Serum biomarkers have been identified as an alternative tool for monitoring the response to aGVHD treatment. The value of biomarkers for inflammation, tissue, and endothelial cell damage was evaluated in our cohort. Although predictive of response to treatment and survival, these markers lack the necessary specificity and sensitivity to predict response to MSC therapy. Objective endpoints for clinical trials on the treatment of steroid-refractory aGVHD remain to be defined. Thus, we recommend including biopsies and biomarkers to discriminate between ongoing aGVHD and postinflammatory malabsorption.

Mesenchymal stromal cell (MSC) infusions have been reported to be effective in patients with steroid-refractory, acute graft-versus-host disease (aGvHD) but comprehensive data on paediatric patients are limited. We retrospectively analysed a cohort of 37 children (aged 3months-17years) treated with MSCs for steroid-refractory grade III-IV aGvHD. All patients but three received multiple MSC infusions. Complete response (CR) was observed in 24 children (65%), while 13 children had either partial (n=8) or no response (n=5). Cumulative incidence of transplantation-related mortality (TRM) in patients who did or did not achieve CR was 17% and 69%, respectively (P=0·001). After a median follow-up of 2·9years, overall survival (OS) was 37%; it was 65% vs. 0% in patients who did or did not achieve CR, respectively (P=0·001). The median time from starting steroids for GvHD treatment to first MSC infusion was 13d (range 5-85). Children treated between 5 and 12d after steroid initiation showed a trend for better OS (56%) and lower TRM (17%) as compared with patients receiving MSCs 13-85d after steroids (25% and 53%, respectively; P=0·22 and 0·06, respectively). Multiple MSC infusions are safe and effective for children with steroid-refractory aGvHD, especially when employed early in the disease course. © 2013 John Wiley & Sons Ltd.

In experimental studies, mesenchymal stem cell (MSC) transplantation in acute myocardial infarction (AMI) models has been associated with enhanced neovascularization and myogenesis. Clinical data however, are scarce. Therefore, the present study evaluates the safety and feasibility of intramyocardial MSC injection in nine patients, shortly after AMI during short-term and 5-year follow-up. Periprocedural safety analysis demonstrated one transient ischemic attack. No other adverse events related to MSC treatment were observed during 5-year follow-up. Clinical events were compared to a nonrandomized control group comprising 45 matched controls. A 5-year event-free survival after MSC-treatment was comparable to controls (89 vs. 91 %, P = 0.87). Echocardiographic imaging for evaluation of left ventricular function demonstrated improvements up to 5 years after MSC treatment. These findings were not significantly different when compared to controls. The present safety and feasibility study suggest that intramyocardial injection of MSC in patients shortly after AMI is feasible and safe up to 5-year follow-up.

We thank Campioni et al. for their response to our recent publication[1][1] in which they focus on the relevance of other molecules besides IL-6 and IL-10 for the immunomodulatory functions of multipotent stromal cells (MSC). In our publication, we had focused on the mechanistic roles for IL-6 and

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4+CD25hiFoxP3+ T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-β1 mediates induction of Tregs. TGF-β1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-β1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-β1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC.

Adipose tissue-derived multipotent stromal cells (AT-MSCs) are studied as an alternative to bone marrow-derived multipotent stromal cells (BM-MSCs) for immunomodulatory treatment. In this study, we systematically compared the immunomodulatory capacities of BM-MSCs and AT-MSCs derived from age-matched donors. We found that BM-MSCs and AT-MSCs share a similar immunophenotype and capacity for in vitro multilineage differentiation. BM-MSCs and AT-MSCs showed comparable immunomodulatory effects as they were both able to suppress proliferation of stimulated peripheral blood mononuclear cells and to inhibit differentiation of monocyte-derived immature dendritic cells. However, at equal cell numbers, the AT-MSCs showed more potent immunomodulatory effects in both assays as compared with BM-MSCs. Moreover, AT-MSCs showed a higher level of secretion of cytokines that have been implicated in the immunomodulatory modes of action of multipotent stromal cells, such as interleukin-6 and transforming growth factor-β1. This is correlated with higher metabolic activity of AT-MSCs compared with BM-MSCs. We conclude that the immunomodulatory capacities of BM-MSCs and AT-MSCs are similar, but that differences in cytokine secretion cause AT-MSCs to have more potent immunomodulatory effects than BM-MSCs. Therefore, lower numbers of AT-MSCs evoke the same level of immunomodulation. These data indicate that AT-MSCs can be considered as a good alternative to BM-MSCs for immunomodulatory therapy.

Multipotent stromal cells have immunomodulatory capacities and have been used in transplantation and autoimmune diseases. One of the effects of multipotent stromal cells involves the inhibition of dendritic cell differentiation. Since interleukin-6 and interleukin-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines may also mediate the inhibitory effect of human multipotent stromal cells in immature dendritic cell differentiation. In order to test this hypothesis monocytes were cultured with interleukin-4 and granulocyte-monocyte colony-stimulating factor in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of interleukin-6 and interleukin-10. Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14(-)CD1a(+)) and resulted in the generation of CD14(+)CD1a(-) cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of interleukin-6 and interleukin-10. Multipotent stromal cells produced interleukin-6 and neutralizing this interleukin-6 reversed the inhibitory effect of the multipotent cells. Interleukin-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cell-produced interleukin-6. In conclusion, through constitutive production of interleukin-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an anti-inflammatory interleukin-10-producing cell type.

Despite excellent short-term results, long-term survival of transplanted kidneys has not improved accordingly. Although alloimmune responses and calcineurin inhibitor-related nephrotoxicity have been identified as main drivers of fibrosis, no effective treatment options have emerged. In this perspective, mesenchymal stromal cells (MSCs) are an interesting candidate because of their immunosuppressive and regenerative properties. Of importance, no other clinical studies have investigated their effects in allograft rejection and fibrosis. We performed a safety and feasibility study in kidney allograft recipients to whom two intravenous infusions (1 million cells per kilogram) of autologous bone marrow (BM) MSCs were given, when a protocol renal biopsy at 4 weeks or 6 months showed signs of rejection and/or an increase in interstitial fibrosis/tubular atrophy (IF/TA). Six patients received MSC infusions. Clinical and immune monitoring was performed up to 24 weeks after MSC infusions. MSCs fulfilled the release criteria, infusions were well-tolerated, and no treatment-related serious adverse events were reported. In two recipients with allograft rejection, we had a clinical indication to perform surveillance biopsies and are able to report on the potential effects of MSCs in rejection. Although maintenance immunosuppression remained unaltered, there was a resolution of tubulitis without IF/TA in both patients. Additionally, three patients developed an opportunistic viral infection, and five of the six patients displayed a donor-specific downregulation of the peripheral blood mononuclear cell proliferation assay, not reported in patients without MSC treatment. Autologous BM MSC treatment in transplant recipients with subclinical rejection and IF/TA is clinically feasible and safe, and the findings are suggestive of systemic immunosuppression.

Background Human mesenchymal stromal cells (MSCs) have been reported to preserve cardiac function in myocardial infarction (MI) models. Previously, we found a beneficial effect of intramyocardial injection of unstimulated human MSCs (uMSCs) on cardiac function after permanent coronary artery ligation. In the present study we aimed to extend this research by investigating the effect of intramyocardial injection of human MSCs pre-stimulated with the pro-inflammatory cytokine interferon-gamma (iMSCs), since pro-inflammatory priming has shown additional salutary effects in multiple experimental disease models. Methods MI was induced in NOD/Scid mice by permanent ligation of the left anterior descending coronary artery. Animals received intramyocardial injection of uMSCs, iMSCs or PBS. Sham-operated animals were used to determine baseline characteristics. Cardiac performance was assessed after 2 and 14 days using 7-Tesla magnetic resonance imaging and pressure-volume loop measurements. Histology and q-PCR were used to confirm MSC engraftment in the heart. Results Both uMSC and iMSC therapy had no significant beneficial effect on cardiac function or remodelling in contrast to our previous studies. Conclusions Animal models for cardiac MSC therapy appear less robust than initially envisioned.

Item does not contain fulltext Detailed understanding of mesenchymal stromal cells (MSC) migration is imperative for future cellular therapies. To identify genes involved in the process of MSC migration, we generated gene expression profiles of migrating and nonmigrating fetal bone marrow MSC (FBMSC). Only 12 genes showed differential expression in migrating versus nonmigrating FBMSC. The nuclear receptors Nur77 and Nurr1 showed the highest expression in migratory MSC. Nur77 and Nurr1 are members of NR4A nuclear orphan receptor family, and we found that their expression is rapidly increased upon exposure of FBMSC to the migratory stimuli stromal-derived factor-1alpha (SDF-1alpha) and platelet-derived growth factor-BB. Lentiviral expression of Nur77 or Nurr1 resulted in enhanced migration of FBMSC toward SDF-1alpha compared with mock-transduced FBMSC. Analysis of the cell cycle, known to be involved in MSC migration, revealed that expression of Nur77 and Nurr1 decreases the proportion of cells in S-phase compared with control cells. Further, gain-of-function experiments showed increased hepatocyte growth factor expression and interleukin (IL)-6 and IL-8 production in MSC. Despite the altered cytokine profile, FBMSC expressing Nur77 or Nurr1 maintained the capacity to inhibit T-cell proliferation in a mixed lymphocyte reaction. Our results demonstrate that Nur77 and Nurr1 promote FBMSC migration. Modulation of Nur77 and Nurr1 activity may therefore offer perspectives to enhance the migratory potential of FBMSC which may specifically regulate the local immune response.

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P = 0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM. Bone Marrow Transplantation (2011) 46, 200-207; doi: 10.1038/bmt.2010.87; published online 19 April 2010

The TNF promoter is silenced in mesenchymal stem cells able to respond to LPS by NFκB translocation and cytokine production yet without TNF. Previously, we demonstrated that several TLRs are expressed on cord blood-derived USSC. Stimulation of USSC with TLR agonists resulted in a marked increase of IL-6 and IL-8 production. Interestingly, TNF was undetectable after TLR stimulation, which appeared to be a result of an inactivated TNF promoter in USSC. Here, we elaborate this study by demonstrating that although USSC do not produce TNF, they are susceptible to TNF stimulation, resulting in NF-κB translocation and cytokine production. Additionally, we compared different stem cell sources for their ability to produce TNF. Interestingly, we found that the TNF promoter in BM-MSC is inactivated as well. Like USSC, they are able to respond to TNF stimulation, but they are not able to produce TNF, even not after LPS stimulation. This limited cytokine response in combination with the well-studied immunosuppressive properties of MSC makes these cells ideal for immune-suppressive treatment modalities such as graft-versus-host disease.

Item does not contain fulltext Stem cells are widely studied to enable their use in tissue repair. However, differences in function and differentiation potential exist between distinct stem cell populations. Whether those differences are due to donor variation, cell culture, or intrinsic properties remains elusive. Therefore, we compared 3 cell lines isolated from 3 different niches using the Affymetrix Exon Array platform: the cord blood-derived neonatal unrestricted somatic stem cell (USSC), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and adult adipose tissue-derived stem cells (AdAS). While donor variation was minimal, large differences between stem cells of different origin were detected. BM-MSC and AdAS, outwardly similar, are more closely related to each other than to USSC. Interestingly, USSC expressed genes involved in the cell cycle and in neurogenesis, consistent with their reported neuronal differentiation capacity. The BM-MSC signature indicates that they are primed toward developmental processes of tissues and organs derived from the mesoderm and endoderm. Remarkably, AdAS appear to be highly enriched in immune-related genes. Together, the data suggest that the different mesenchymal stem cell types have distinct gene expression profiles, reflecting their origin and differentiation potential. Furthermore, these differences indicate a demand for effective differentiation protocols tailored to each stem cell type. 01 april 2010

Haploidentical hematopoietic stem-cell transplantation (HSCT) is associated with an increased risk of graft failure. Adult bone marrow-derived mesenchymal stromal cells (MSCs) have been shown to support in vivo normal hematopoiesis and to display potent immune suppressive effects. We cotransplanted donor MSCs in 14 children undergoing transplantation of HLA-disparate CD34+ cells from a relative. While we observed a graft failure rate of 15% in 47 historic controls, all patients given MSCs showed sustained hematopoietic engraftment without any adverse reaction. In particular, children given MSCs did not experience more infections compared with controls. These data suggest that MSCs, possibly thanks to their potent immunosuppressive effect on alloreactive host T lymphocytes escaping the preparative regimen, reduce the risk of graft failure in haploidentical HSC transplant recipients. © 2007 by The American Society of Hematology.

Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell–depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.

Accelerated telomere shortening has been proposed as a possible long-term risk of allogeneic bone marrow transplantation (allo-BMT). In this study we monitored telomere length in white blood cells (WBC), granulocytes, and naïve and memory CD4+ T lymphocytes in recipients of allo-BMT at long-term follow-up. Peripheral blood was collected from 10 allo-BMT recipients and donors at a median interval of 18 years after allo-BMT. Telomere length was determined using Southern blot analysis. Similar to results previously reported at short-term follow-up, a small difference in telomere length (0.1-0.3 kb) between recipients and donors was detected in WBC, granulocytes and naïve CD4+ T cells. Our data therefore provide no evidence for sustained telomere shortening in leucocytes, and render the possibility of long-term haematopoietic graft failure unlikely. In addition, we observed two phenomena that may be related to involution of the thymus. First, the number of naïve CD4+ T cells in the blood was significantly lower in recipients (0.4 x 10(9)/l) than in donors (0.7 x 10(9)/l) (P0.05). Second, telomeres in memory CD4+ T cells from recipients were on average 0.6 kb shorter than those from donors (P = 0.01). The latter may be related to the reported rapid peripheral expansion of memory T cells immediately after transplantation.

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is approximately 8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.

We previously proposed that a local duplication, not the loss of the subsequently amplified marker from its original site, might be the first step in gene amplification in human cells. It is important to investigate this issue in naturally occurring amplification and when copy numbers are relatively low. We have examined the location of single-copy and amplified 11q13 sequences in cell lines from human breast cancers and squamous cell carcinomas using fluorescence in situ hybridization both with a probe specific for the 11q13 amplifying region and with a chromosome 11-specific library. We show that in most cell lines the 11q13 amplicons are physically linked to chromosome 11 or to a chromosome derived from chromosome 11 by various rearrangements near the 11q13 region. In none of the cell lines were interstitial deletions of 11q13 detected. These results indicate that 11q13 amplification in human tumor cells generally does not involve deletion as the initial step. One cell line with chromosomally located amplified 11q13 sequences contained double minutes that harbored the MYC gene but no 11q13 sequences. This suggests that the genetic outcome and the mechanism of gene amplification are probably dependent on specific DNA sequences rather than on the origin of the cells.

Although gene amplification, a process that is markedly enhanced in tumor cells, has been studied in many different cell systems, there is still controversy about the mechanism(s) involved in this process. It is still unclear what happens to the DNA sequences that become amplified, whether they remain present at their original location (conservative gene amplification) or whether gene amplification necessarily results in a deletion at the original location (non-conservative gene amplification). We have studied gene amplification in a human osteosarcoma cell line, starting from a cell clone which contains only one copy of a plasmid integrate. Independent amplificants, originating from this clone and containing elevated plasmid copy numbers, were isolated and analyzed. Based on previous observations, encompassing the persistence of single-copy DNA sequences besides amplified DNA sequences clustered at a different location in the independent amplificants, we proposed an amplification pathway including a local duplication step and transposition of the duplicated DNA to other chromosomal positions. Now we have extended our study to more independent amplificants. We prove that the single-copy plasmid-containing chromosomes in the different amplificants and the single-copy plasmid-containing chromosome in the original parental cell clone are indeed identical, namely a translocation chromosome composed of at least three parts of which two originate from chromosomes 14 and 17. We show that the unit of amplification and the unit of the proposed transposition event are at least 1.5 Mb. We also demonstrate that the amplified DNA sequences, present at genomic locations other than the original single-copy DNA sequences, are preferentially associated with chromosome 16. We find that the amplified DNA sequences are often located at or near a site of chromosome translocation involving chromosome 16. In one cell clone we detect the amplified DNA sequences in most of the cells to be located within a complete chromosome 16 while in a minority of cells the amplified sequences are located at or near a breakpoint on a translocation chromosome 16. This indicates that this amplification region is highly unstable and frequently gives rise to translocation events.

The immune system of patients with severe combined immunodeficiency (SCID) reconstitutes to a large extent during the first years after hematopoietic stem cell transplantation (HSCT). It was suggested, however, that accelerated loss of thymus output may cause impaired immune function at the long term. To address this issue, we studied patients with SCID who underwent allogeneic HSCT 5 to 32 years earlier and identified early determinants of long-term T-cell reconstitution. A variety of immune parameters were analyzed both early (1-4 years) and late (5-32 years) after HSCT. Late after HSCT, a clear distinction could be made between a group of 8 patients with impaired T-cell reconstitution and 11 patients with good immune reconstitution. Importantly, in patients with decreased long-term T-cell reconstitution, T-cell recovery was already poor early after HSCT, demonstrating that long-term immune failure was not caused by accelerated loss of thymus output or long-term graft failure, but resulted from poor early grafting. The number of T-cell receptor excision circles (TRECs) early after HSCT was most predictive for long-term T-cell reconstitution. Frequent monitoring of T-cell immunity and TREC numbers early after HSCT may thus serve to timely identify patients who will fail to reconstitute properly and who may need additional treatment.