Search

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.

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.

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.

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.

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

Mesenchymal stem cells (MSC's) can be used in the experimental transplant setting for treatment of life threatening GvHD or to improve engraftment. Presently, the European MSC Consortium has three ethically approved multi center clinical protocols in use. MSC's expanded ex vivo from donor bone marrow are mostly of parental origin. We reviewed donation data from two participating centers, to establish donor commitment, donation and procedural related complications. Parents of children undergoing haploidentical PBSCT or a cord blood transplant were asked to donate 50 cc of bone marrow 4–5 weeks prior to HSCT. Parents of children with non-steroid responsive aGvHD (grade III-IV) were asked at the time of treatment failure. Bone marrow was aspirated either under general or local anesthesia. Haploidentical PBSC donors received in addition 5 days of G-CSF immediately before stem cell harvest. Donors were asked to consider consent for additional MSC's to be donated for research or use as 3rd party donations for other patients. Donors were cleared for donation by an independent physician. Thirty three donors (14 mothers, 18 fathers, 1 brother) of 34 relatives asked, agreed to donate bone marrow for MSC expansion and all additional uses. One parent whose child died before receiving MSC's requested they be used for 3rd party donations. Fourty five patients (included 6 adults) were treated with MSC's (see table 1 for details). Thirty one patients (68%) received MSC's from a parent, the remaining treatments being 3rd party for acute GvHD. All donors were considered suitable for PBSC or BM donation, although excess weight, mild hypertension and depression were documented in a minority of donors. No adverse events were documented after bone marrow donation, but one parent suffered severe bone pain, requiring opiate analgesia, during G-CSF administration. At 6 weeks follow up no adverse effects were reported. MSC donation either alone or in combination with stem cell mobilization can be achieved without major problems. Parental donor commitment is definitely related to the needs of the child. However, donor altruism to include other unknown patients including adult recipients was uniformly evident in our studies. Donation of stem cells for parents is a stressful situation and requests for multiple donations of various types of cell products can raise questions about medical and ethical considerations. Our protocols show that well informed parents have a propensity to commit to others needs as well as their offspring's. It is therefore essential when introducing experimental treatments to establish a balance between donors commitment and patient needs. Table 1 . Patient and donor characteristics PATIENTS n=45 OUTCOME DONORS n=33 Male 29 21 alive 19 Female 16 12 alive 14 Age (range years) 7.0 (0.6–16.4) 38 (26–53.3) Hematological malignancy 35 Immune deficiency 3 Others 7 Haplo+ MSC 21 15 alive Own child donations Cord + MSC 9 1 rejection 1 death Own child donation aGvHD 15 CR in 11 patients 14/15 3rd party donations Complications 2 a GvHD (grade I) Severe pain 1 donor Infections 14 patients Platelet tx in 4 haplo-donors

Haploidentical CD34+ purified peripheral blood stem cell transplantation (PBSCT) is associated with an increased risk of graft failure/rejection. Animal models suggest that MSCs may promote engraftment after hematopoietic stem cell transplanation (HSCT) but there role in overcoming graft failure in human HSCT is still unclear. We recently reported our initial findings in 13 children receiving MSCs in conjunction with haplo-PBSCT. Here we report our extended series of 20 patients, now including those with MDS known to have an increased risk of rejection. MSCs were isolated from marrow of PBSCT donors and expanded under GMP conditions using same batch FCS containing medium and a standardized protocol, approximately 4–5 weeks before transplantation. MSCs (fresh or cryopreserved) were administered i.v. 4 hours before the allograft. Conditioning depended on underlying disease. No pharmacological GvHD prophylaxis was given after PBSCT. Characteristics of the patients and results are summarized in table 1. In comparison to historical controls (20% graft failure), all patients given MSCs showed successful and stable engraftment (p=0.03). Hematological recovery was accelerated (see table 2). Viral reactivations did not differ between patients and controls. MSC use was feasible and safe without infusion toxicities. In contrast to our initial findings, our extended analysis shows a statistically significant reduction in graft failure rates for patients cotransplanted with MSCs. Moreover, MSC cotransplantation may also prevent graft rejection in patients with MDS. In view of these findings, cotransplantation of MSCs could become standard practice for optimizing the outcome of children given this type of allograft. Table 1: Patient characteristics and summary of results PATIENT (n=20) CONTROL (n=52) P VALUE Male/female 14/6 (70/30%) 31/21 (60/40%) 0.6 Age (range) 8 years (2–16) 8 years (1–17) NS All hematological malignancies 16 (80%) 40 (70%) 1.0 MDS 2 (10%) 5 (10%) 1.0 Immune deficiencies 2 (10%) 2 (4%) NS Other 2 (10%) 10 (9%) NS Donor male/female 10/10 29/23 NS Conditioning TBI/Chemo based 12/8 30/22 1.0 N of CD34+cells infused (median, range) 21.3 (11.2–38.6) 23 (12.1–47.5) NS N. of CD3+ cells infused (mean, SD) 0.3 (0.30) 0.5 (0.7) NS aGvHD 0.2 I-II 2 12 III-IV 0 2 Relapse 4/16 (25%) 8/40 (20%) NS Infectious deaths 2 (10%) 7 (13%) NS GRAFT FAILURE 0 11 0.03 Tabel 2. Patient hematopoietic recovery compared to controls PATIENTS CONTROLS P value (student t-test Median (days, range) SD (days) Median (days, range) SD (days) P value equal or less than 0.05 is considered significant Leucocyte 〉1.0 × 109/L 11.5 (9–16) 2.0 14 (9–26) 4.2 0.01 Neutrophils 〉0.5 × 109/L 12 (10–17) 1.9 13 (9–27) 4.6 0.06 Platelets 〉20 × 109/L 11 (9–24) 3.8 13 (7–100) 22.3 0.11 Reticulocytes 〉20 × 109/L 12 (10–31) 5.0 23 (9–41) 10.2 0.01

Haploidentical peripheral blood stem cell transplantation from a parent is an accepted form of therapy in children lacking a suitable donor who require stem cell transplantation. The risk of graft failure/rejection increases with the intense T cell depletion resulting from positive CD34 selection. Mesenchymal stem cells (MSCs) isolated from bone marrow can be expanded to sufficient numbers for clinical use. MSCs can improve hematological recovery but their role in overcoming graft dysfunction post transplant is unknown. We present results of co-transplantation of MSCs in children undergoing haplo-identical PBSCT compared to 48 historical controls. MSCs were isolated from donor marrow and expanded under GMP conditions approximately 4–5 weeks before transplantation. MSCs (fresh or cryopreserved) were administered i.v. 4 hours before donor G-CSF mobilized PBSCs. To date, 8 children have been treated in two centers.Conditioning depended on underlying disease. No additional GvHD prophylaxis was given. Engraftment and immune recovery was compared to the control group. The local ethical committees approved the study. Characteristics and results are summarized in Table 1. There was no difference for age, gender, transplant indication, donor type or CD34+ or CD3+ cells infused. In comparison to historical controls (23% graft dysfunction) all patients had 100% documented and sustained engraftment. Hematological recovery of leucocytes was faster (p=0,008) with lymphocyte rather than neutrophil recovery accounting for this. Near significance in reticulocyte and platelet recovery (p=0.09; 0.08 resp) was evident. Viral reactivations were common in both patients and controls. Acute GvHD was 12.5% in the patient group compared to 39.5% of eligible controls. Two patients died, one relapse and one infection compared to 17 controls (9 relapse, 5 infection, 3 GvHD). Longer follow-up and immune recovery data are continuing. Our results suggest MSC co-transplantation overcomes graft dysfunction and stabilizes engraftment. MSC use is feasible and safe without infusional toxicities. Based on our findings, where graft dysfunction, delayed hematological and immune recovery compromise the success of stem cell transplant outcome, clinical randomized studies of MSCs are warranted. Table 1. PATIENT/CONTROL CHARACTERISTICS PATIENT CONTROL All patients received MSC between 1–2 x 106/kg MALE 4 (50%) 29 (60%) FEMALE 4 (50%) 19 (40%) AGE (range) 8y 2mo (2–15y 6m) 8y (6mo-17y 7mo) MALIGNANT DISEASES 5 (62%) 34 (70%) IMMUNE DEFICENCIES 2 (25%) 6 (13%) BENIGN CONDITIONS 1 (13%) 8 917%) DONOR MALE 5 24 DONOR FEMALE 3 24 CD34 INFUSED 31.8 (SD 3.1) 20 (SD 6.8) NS CD3 INFUSED 0.3 (SD 0.30) 0.68 (SD 0.7) NS aGvHD 1 13 OVERALL SURVIVAL 6 (75%: FU 3–48mo) 31(64%: FU 2–6Y) Table 2. HEMATOLOGICAL RECOVERY PATIENTS CONTROLS Average (days) SD (days) Average (days) SD (days) P value (T-test) SD: Standard deviation : NS: not significant LEUCOCYTES >1.0 X 109/L 12 1.9 16 4.15 0.008 NEUTROPHILS >0.5 X 109/L 13 1.5 15 3.6 0.14 (NS) PLATELETS >20 X 109/L 13 2.7 21 14.7 0.08 (near significance) RETICULOCYTES >20 16 4.9 27 13.7 0.09

Mesenchymal stem cells (MSC) from adult bone marrow have the capacity to differentiate into several mesenchymal tissues and inhibit T-cell alloreactivity in vitro. Within the EBMT MSC expansion consortium we have used MSC to treat grades III–IV acute graft-versus-host disease (GvHD) in 40 patients. The MSC dose was median 1.0 (range 0.4–9) 10^6 cells/kg body weight of the recipient. No side-effects were seen after MSC infusions. Nineteen patients received one dose, 19 patients received two doses, two patients received three and five doses respectively. MSC donors were in five cases HLA-identical sibling donors, 19 haploidentical donors and 41 third-party HLA-mismatched donors. Among the 40 patients treated for severe acute GvHD, 19 had complete responses, nine showed improvement, seven patients did not respond, four had stable disease and one patient was not evaluated due to short follow-up. Twenty-one patients are alive between six weeks up to 3.5 years after transplantation. Nine of these patients have extensive chronic GvHD. One patient with ALL has recurrent leukaemia and one patient has denovo AML of recipient origin. We conclude that MSC have immunomodulatory and tissue repairing effects and should be further explored as treatment of severe acute GvHD in prospective randomized trials.

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.

Graft failure or rejection is an identified problem in haplo-identical or second attempt transplantation. In the bone marrow, mesenchymal stem cells (MSCs) have been identified and shown in animal models to enhance hematopoietic stem cell (HSC) engraftment. Advances in techniques and higher quality culture components have allowed the development of an MSC expansion procedure resulting in sufficient MSCs for clinical application. Adult studies have suggested that co-transplantation of MSCs and HSCs in the HLA-identical setting, is feasible and safe. Here we present the first combined clinical experience within the EBMT MSC expansion consortium with respect to co-transplantation of haplo-identical MSCs in the pediatric allogeneic transplantation setting. Six weeks before the SCT, 50 cc of bone marrow are sampled under sterile conditions. Density gradient-separated MNCs are plated into tissue culture flasks in low-glucose DMEM supplemented with 10% fetal calf serum and incubated at 370C with 5% CO2. At 70% confluence, the cells are trypsinized and re-plated at 4x103 cells/ cm2 until the target dose of 1–2 x106/kg recipient body weight is obtained. Enrichment and expansion of MSCs is performed under GMP conditions in nationally accredited laboratories. MSCs (either fresh or cryopreserved) are administered i.v. 4 hours before infusion of donor HSCs. To date 4 children have undergone co-transplantation. Patient characteristics are summarized in the table. All toxicities associated with the procedure were documented, as were the engraftment kinetics and immune recovery. The study was carried out with approval of the respective local ethical committees. The data indicate that expansion and co-transplantation of MSCs is feasible and well tolerated. While the study is ongoing initial engraftment and immune recovery data (compared to historical data) is encouraging and to date there have been no episodes of graft rejection or severe adverse events related to this treatment. Patient characteristics Patient demographics HSC characteristics MSC characteristics UPN Gender Age Diagnosis Donor CD34 dose (106/kg) Donor Cell dose (106/kg) 1 M 15 ANLL mother-haplo 16 mother 1.5 2 M 2 XLPD father-haplo 20 father 1.9 3 M 2 XLPD father-haplo 18.7 father 1.56 4 M 8 SAA URD (2 loci mismatched) 2.65 mother 1.0