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After renal transplantation, there is a need for immunosuppressive regimens which effectively prevent allograft rejection, while preserving renal function and minimizing side effects. From this perspective, mesenchymal stromal cell (MSC) therapy is of interest. In this randomized prospective, single‐center, open‐label trial, we compared MSCs infused 6 and 7 weeks after renal transplantation and early tacrolimus withdrawal with a control tacrolimus group. Primary end point was quantitative evaluation of interstitial fibrosis in protocol biopsies at 4 and 24 weeks posttransplant. Secondary end points included acute rejection, graft loss, death, renal function, adverse events, and immunological responses. Seventy patients were randomly assigned of which 57 patients were included in the final analysis (29 MSC; 28 controls). Quantitative progression of fibrosis failed to show benefit in the MSC group and GFR remained stable in both groups. One acute rejection was documented (MSC group), while subclinical rejection in week 24 protocol biopsies occurred in seven patients (four MSC; three controls). In the MSC group, regulatory T cell numbers were significantly higher compared to controls (p = .014, week 24). In conclusion, early tacrolimus withdrawal with MSC therapy was safe and feasible without increased rejection and with preserved renal function. MSC therapy is a potentially useful approach after renal transplantation., TRITON, a randomized, prospective, single‐center, open‐label study, shows that autologous mesenchymal stromal cell therapy and early tacrolimus withdrawal was feasible and safe, without increased rejection and with preserved renal function.

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.

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.

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.

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 aim Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive effects both in vitro and in experimental colitis. Promising results of MSC therapy have been obtained in patients with severe graft versus host disease of the gut. Our objective was to determine the safety and feasibility of autologous bone marrow derived MSC therapy in patients with refractory Crohn9s disease. Patients and intervention 10 adult patients with refractory Crohn9s disease (eight females and two males) underwent bone marrow aspiration under local anaesthesia. Bone marrow MSCs were isolated and expanded ex vivo. MSCs were tested for phenotype and functionality in vitro. 9 patients received two doses of 1–2×10 6 cells/kg body weight, intravenously, 7 days apart. During follow-up, possible side effects and changes in patients9 Crohn9s disease activity index (CDAI) scores were monitored. Colonoscopies were performed at weeks 0 and 6, and mucosal inflammation was assessed by using the Crohn9s disease endoscopic index of severity. Results MSCs isolated from patients with Crohn9s disease showed similar morphology, phenotype and growth potential compared to MSCs from healthy donors. Importantly, immunomodulatory capacity was intact, as Crohn9s disease MSCs significantly reduced peripheral blood mononuclear cell proliferation in vitro. MSC infusion was without side effects, besides a mild allergic reaction probably due to the cryopreservant DMSO in one patient. Baseline median CDAI was 326 (224–378). Three patients showed clinical response (CDAI decrease ≥70 from baseline) 6 weeks post-treatment; conversely three patients required surgery due to disease worsening. Conclusions Administration of autologous bone marrow derived MSCs appears safe and feasible in the treatment of refractory Crohn9s disease. No serious adverse events were detected during bone marrow harvesting and administration.

Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.

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.

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.

Introduction: Autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) are evaluated for clinical use in COPD patients, but it is unclear whether COPD affects BM-MSC function. Aim: To investigate if BM-MSC function and therapeutic potential is affected by underlying COPD. Methods: BM-MSCs from 9 COPD patients and 9 non-COPD controls were investigated for several functional parameters, including differentiation into adipocytes, osteoblasts and chondrocytes, response to pro-inflammatory stimuli and oxidative stress, and their ability to increase wound repair and EGFR ligands in NCI-H292 bronchial epithelial cells. Results: The morphology of BM - MSCs obtained from COPD patients did not differ from that of controls. In MSC differentiation assays, adipocyte differentiation was higher in COPD donors (p = 0.046), whereas osteoblast and chondrocyte differentiation appeared somewhat more pronounced in non-COPD donors (p = 0.2). In response to TNF-α and IL-1β mRNA expression of several growth factors and cytokines was induced, but this did not differ between groups. Also, cigarette smoke condensate and sulforaphane induced expression of oxidative stress genes (HMOX1, SCAL and NQO1) to a similar extent in both groups. No differences between groups were observed in their potential to induce EGFR ligands and wound repair in NCI-H292. Conclusion: BM-MSCs from COPD donors are phenotypically and functionally comparable to those from non-COPD controls and show no signs of impaired function. This finding is relevant for the potential clinical application of autologous MSCs for treatment of COPD.

Background In vitro expanded mesenchymal stromal cells (MSCs) are increasingly used as experimental cellular therapy. However, there have been concerns regarding the safety of their use, particularly with regard to possible oncogenic transformation. MSCs are the hypothesized precursor cells of high-grade osteosarcoma, a tumor with often complex karyotypes occurring mainly in adolescents and young adults. Methods To determine if MSCs from osteosarcoma patients could be predisposed to malignant transformation we cultured MSCs of nine osteosarcoma patients and five healthy donors for an average of 649 days (range 601–679 days). Also, we compared MSCs derived from osteosarcoma patients at diagnosis and from healthy donors using genome wide gene expression profiling. Results Upon increasing passage, increasing frequencies of binucleate cells were detected, but no increase in proliferation suggestive of malignant transformation occurred in MSCs from either patients or donors. Hematopoietic cell specific Lyn substrate 1 (HLCS1) was differentially expressed (fold change 0.25, P value 0.0005) between MSCs of osteosarcoma patients (n = 14) and healthy donors (n = 9). Conclusions This study shows that although HCLS1 expression was downregulated in MSCs of osteosarcoma patients and binucleate cells were present in both patient and donor derived MSCs, there was no evidence of neoplastic changes to occur during long-term culture. Electronic supplementary material The online version of this article (doi:10.1186/s13569-015-0031-1) contains supplementary material, which is available to authorized users.

// 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

Introduction: For clinical applications, Mesenchymal Stromal Cells (MSC) can be isolated from bone marrow and adipose tissue of autologous or allogeneic origin. Allogeneic cell usage has advantages but may harbor the risk of sensitization against foreign HLA. Therefore, we evaluated whether bone marrow and adipose tissue-derived MSC are capable of inducing HLA-specific alloreactivity. Methods: MSC were isolated from healthy human Bone Marrow (BM-MSC) and adipose tissue (ASC) donors. Peripheral Blood Mononuclear Cells (PBMC) was co-cultured with HLA-AB mismatched BM-MSC or ASC precultured with or without IFNy. After isolation via FACS sorting, the educated CD8+ T effector populations were exposed for 4 hours to Europium labeled MSC of the same HLA make up as in the co-cultures or with different HLA. Lysis of MSC was determined by spectrophotometric measurement of Europium release. Results: CD8+ T cells educated with BM-MSC were capable of HLA specific lysis of BM-MSC. The maximum lysis was 24% in an effector:target (E:T) ratio of 40:1. Exposure to IFNγ increased HLA-I expression on BM-MSC and increased lysis to 48%. Co-culturing of PBMC with IFNγ-stimulated BM-MSC further increased lysis to 76%. Surprisingly, lysis induced by ASC was significantly lower. CD8+ T cells educated with ASC induced a maximum lysis of 13% and CD8+ T cells educated with IFNγ-stimulated ASC of only 31%. Conclusion: Allogeneic BM-MSC, and to a lesser extend ASC, are capable of inducing HLA specific reactivity. Our results suggest that clinical therapy with allogeneic MSC should be carefully considered.

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

In humans, alkaline phosphatases are encoded by one tissue-non-specific alkaline phosphatase (TNAP) gene and three tissue-specific alkaline phosphatase genes, intestinal, placental (PLAP), and germ cell-specific alkaline phosphatase (GCAP). Although the presence of alkaline phosphatases in testicular germ cell tumours (TGCTs) of adolescents and adults has been utilized for both detection and patient monitoring, it is not known in detail which isozymes are expressed. Since alkaline phosphatase is detected in carcinoma in situ (CIS), the common precursor of all TGCTs, it might provide a marker for the early diagnosis of TGCTs. Testicular cancers of germ cell and non-germ cell origin along with testicular parenchyma with and without CIS have been analysed for the expression of the different alkaline phosphatase isozymes. Antibodies to TNAP and PLAP/GCAP showed positivity in CIS, seminoma, and embryonal carcinoma. The heterogeneous staining pattern detected in frozen tissue sections was similar to the pattern found in formalin-fixed, paraffin-embedded material, indicating a biological phenomenon and not a handling artefact. Since PLAP and GCAP cannot be distinguished using immunohistochemistry, the expression of these isozymes was studied at the molecular level using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, in combination with a primer extension assay. The results show that CIS and seminoma predominantly express GCAP, while in embryonal carcinoma the expression of GCAP versus PLAP varies. Due to the presence of alkaline phosphatase transcripts in normal testicular parenchyma, an RT-PCR-based analysis of alkaline phosphatase is not informative for the early detection of TGCTs in biopsy samples.

Testicular germ cell tumors (TGCTs) of adolescents and adults have been shown to contain proteins of the human endogenous retrovirus type K family. In a recent study, expression of these retroviral sequences was confirmed using in situ hybridization, which also showed expression in carcinoma in situ, the precursor of all TGCTs. Because of the clinical significance of a test for early diagnosis of TGCTs, we studied whether expression of human endogenous retrovirus type K genes could be an informative parameter. Therefore, we investigated TGCTs of various histologies and testicular parenchyma with and without carcinoma in situ using reverse transcription-polymerase chain reaction for expression of the gag, env, and prt genes. The gag and prt genes were expressed in all samples tested. The env transcripts were not found in TGCTs showing somatic differentiation only but could be detected in most normal testicular parenchyma samples. Therefore, detection of human endogenous retrovirus type K transcripts cannot be used for early diagnosis of TGCTs. Simultaneous expression of multiple gag sequences was found both in normal parenchyma and TGCTs, and we demonstrated that expression of gag sequences with an extra G, necessary to generate a functional protein, was not limited to TGCTs.

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.

Co-transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) enhances HSPC engraftment. For these applications, MSC are mostly obtained from bone marrow. However, MSC can also be sourced from Wharton's jelly (WJ) of the human umbilical cord, which as a 'waste product', is cheaper to acquire and without significant burden to the donor. Here, we evaluated the ability of WJ MSC to enhance HSPC engraftment. First, we compared cultured human WJ MSC with human bone marrow-derived MSC (BM MSC) for in vitro marker expression, immunomodulatory capacity and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSC have a more restricted differentiation capacity, both WJ MSC and BM MSC expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Co-transplantation of either WJ MSC or BM MSC with CB CD34+ cells into NOD-SCID mice showed faster recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood and spleen six weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon co-incubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although 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 MSC in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSC replaced WJ MSC 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. Disclosures No relevant conflicts of interest to declare.

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.

High-grade osteosarcoma is a tumor with a complex genomic profile, occurring primarily in adolescents. The extensive genomic alterations obscure the identification of genes driving tumorigenesis in osteosarcoma progenitor cells. In order 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 osteosarcoma as compared with its putative progenitor cells, i.e. mesenchymal stem cells (n=12) or osteoblasts (n=3). In addition, we performed paired analyses between copy number and expression profiles of a subset of 29 patients for which we had both DNA and mRNA profiles. Integrative analyses were performed in Nexus Copy Number Software and statistical language R. Paired analyses were performed on all probes which were significantly differentially expressed in corresponding LIMMA analyses. For both non-paired and paired analyses, copy number aberration frequency was set to >35%. Non-paired integrative analyses resulted in 45 genes that were present in both analyses using different control sets, i.e. MSCs and osteoblasts. 101 genes overlapped between the two paired integrative analyses. Paired analyses detected >90% of all genes found with the corresponding non-paired analyses. Remarkably, approximately twice as many genes as found in the corresponding non-paired analyses were detected. Affected genes were compared with differential expression in osteosarcoma cell lines. An overrepresentation of altered cell division related genes was found, such as MCM4 and LATS2, suggesting that deregulation of the cell cycle is an initial driver of osteosarcomagenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5128. doi:1538-7445.AM2012-5128

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.

by the Modification of Diet in Renal Disease (MDRD) Study Equation and CockcroftGault (CG) Equation at baseline (the first recorded value) and > 1 yr of follow-up. A control group included pts with IBD not treated with mesalamine with > 1 yr follow-up. Results: There were 155 pts with IBD (mean duration: 12.9 yrs, range: 1-45 yrs) who were treated with mesalamine identified (96 with Crohn's disease and 69 with ulcerative colitis). Among them, 75 were female and the mean age was 40.0 yrs (range 18-81 yrs). The mean follow-up in this group was 4.2 yrs (range: 0.6-9.5 yrs). The mean duration of therapy with mesalamine was 10.7 yrs. The control group included 30 pts with IBD (mean duration: 13.9 yrs, range: 2-37 yrs). Among them, 14 were female and mean age was 38.9 yrs (range: 20-66 yrs). The mean follow-up in this cohort was 3.8 yrs (range: 1.0-8.6 yrs). Results are in table 1 (mean values ± SD). There was a statistically significant difference between pts on low and medium dose mesalamine regarding creatinine clearance monitored by CG equation both at baseline and after follow-up (p