F.M. Sanchez-Guijo

Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q-syndrome

The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS–MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype: CD45−/CD73++/CD34−/CD271++. They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinical–biological data an unsupervized hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5q− syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5q− syndrome.

Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors.

It is an open question whether in multiple myeloma (MM) bone marrow stromal cells contain genomic alterations, which may contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) analysis to compare the extent of unbalanced genomic alterations in mesenchymal stem cells from 21 myeloma patients (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro culture expansion. Whereas ND-MSCs were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb size) were detected in MM-MSCs. Using real-time reverse transcription PCR, we found correlative deregulated expression for five genes encoded in regions for which genomic imbalances were detected using array-CGH. In addition, only MM-MSCs showed a specific pattern of ‘hot-spot’ regions with discrete (<1 Mb) genomic alterations, some of which were confirmed using fluorescence in situ hybridization (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM patients. We also explored whether cytogenetic abnormalities present in myelomatous plasma cells (PCs) were shared by matching MSCs from the same patients using FISH. All MM-MSCs were cytogenetically normal for the tested genomic alterations. Therefore we cannot support a common progenitor for myeloma PCs and MSCs.

Peripheral endothelial progenitor cells (CD133 +) for therapeutic vasculogenesis in a patient with critical limb ischemia. One year follow-up.

We present a patient with critical limb ischemia who was successfully treated with the injection of autologous peripheral blood (PB) CD133+ purified stem cells (SC) into the gastrocnemius muscle. No serious adverse events related to G-CSF administration, mononuclear cells harvest or CD133+ SC administration was observed. After 17 months of follow-up, our patient has experienced limb salvage, symptomatic relief and functional improvement. Moreover, we have observed the appearance of flow in the right posterior tibial artery that was absent before the procedure. To our knowledge, this is the first case of critical limb ischemia treated with PB CD133+ SC.

Risk factors for thrombotic microangiopathy in allogeneic hematopoietic stem cell recipients receiving GVHD prophylaxis with tacrolimus plus MTX or sirolimus

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a feared complication of allogeneic hematopoietic SCT (HSCT) owing to its high mortality rate. The use of calcineurin inhibitors or sirolimus (SIR) for GVHD prophylaxis has been suggested as a potential risk factor. However, the impact of tacrolimus (TAC) and SIR combinations on the increased risk of TA-TMA is currently not well defined. We retrospectively analyzed the incidence of TA-TMA in 102 allogeneic HSCT recipients who consecutively received TAC plus SIR (TAC/SIR) (n=68) or plus MTX (TAC/MTX)±ATG (n=34) for GVHD prophylaxis. No significant differences were observed in the incidence of TA-TMA between patients receiving TAC/SIR vs TAC/MTX±ATG (7.4% vs 8.8%, P=0.8). Only grade III–IV acute GVHD, previous HSCT and serum levels of TAC >25 ng/mL were associated with a greater risk of TA-TMA. Patients developing TA-TMA have significantly poorer survival (P<0.001); however, TA-TMA ceased to be an independent prognostic factor when it was included in a multivariate model. In conclusion, the combination of TAC/SIR does not appear to pose a higher risk of TA-TMA. By contrast, we identified three different risk groups for developing TA-TMA.

Effects of MSC coadministration and route of delivery on cord blood hematopoietic stem cell engraftment

Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immunofluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche.

CD34?+ cell dose and outcome of patients undergoing reduced-intensity-conditioning allogeneic peripheral blood stem cell transplantation

While in bone marrow allogeneic transplantation the infusion of high doses of progenitor stem cells has a favourable impact on outcome, due to a faster hematopoietic and immune recovery, in the peripheral blood allo-setting the infusion of a high number of CD34 cells increases the risk of extensive chronic graft vs. host disease (cGVHD). This higher incidence of extensive cGVHD has an adverse impact on outcome due to a higher transplant related mortality, specially among patients receiving T-cell depleted allogeneic transplantation with myeloablative conditioning. By contrast, patients undergoing reduced intensity conditioning regimen may benefit from increasing higher CD34 + cell doses, especially those categorized as high risk according to disease status at transplant. Thus, the source of progenitors cells, type of conditioning and GVHD prophylaxis, among other factors, may influence the effect of the progenitor cell dose on outcome after allogeneic transplant.

Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

Purpose: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use.

Influence of donor age in allogeneic stem cell transplant outcome in acute myeloid leukemia and myelodisplastic syndrome

The impact of donor age in patients with acute myeloid leukemia and myelodysplastic syndrome who underwent allogeneic hematopoietic stem cell transplant (HSCT) remains unclear. In the current study, we evaluate 179 consecutive patients who received an HSCT, from January 2000 to January 2013, in our Institution. Most of the HSCT (91%) were HLA-matched. Patient and donor median age were 51 years (18-69) and 47 years (12-75) respectively, and 81 donors (45%) were older than 50 years. The median follow-up was 38 months (range 1-138), Kaplan-Meier estimated 3-year overall survival (OS) was 63% and disease free survival (DFS) was 56%. Interestingly, patients who received an HSCT from a donor older age (>50 y) showed a poorer OS (51% vs 73%; p=0.01), as well as a higher TRM (20% vs 8%; p=0.038) and higher relapse rate (28% vs 39%; p=0.03). In a stratified subanalysis, 3-year estimated OS was significantly lower among patients undergoing an HSCT from >50 years sibling donors compared to those receiving an HSCT from <50 years unrelated donor (54% vs 72%; p<0.001). In summary, we can conclude that receiving an HSCT from a donor over 50 years old is associated with poorer outcome in patients diagnosed with MDS and AML, and this information may be incorporated into the complex process of donor selection.

Usefulness of eltrombopag for treating thrombocytopenia after allogeneic stem cell transplantation

Prolonged isolated thrombocytopenia (PT) and secondary failure of platelet recovery (SFPR) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are wellknown complications that can lead to potentially lethal bleeding. PT was defined as the engraftment of all peripheral blood cell lines but with a platelet counts < 20 × 10/L or dependence on platelet transfusions for >60 days after allo-HSCT [1]. In contrast, SFPR was defined as a decline in platelet count to < 20 × 10/L for 7 consecutive days, or a requirement for transfusion support after achieving a sustained platelet count ≥ 50 × 10/L without transfusions for 7 consecutive days post-HSCT [2]. Previous studies have reported an incidence of SFPR between 12 and 20% and PT is around 20–40% [1, 2]. Currently, there are no specific guidelines for its treatment and platelet transfusion is the standard of care in this setting. However, transfusion support is associated with several adverse events, such as infusion reactions, platelet refractoriness, acute lung injury, cardiac failure due to volume overload, and viral transmission, all of which impose a heavy financial burden [3]. Eltrombopag is effective and safe for treating primary immune thrombocytopenia (ITP) [4], and other conditions such as aplastic anemia [5]. In this letter, we described the characteristics our series of patients who were given eltrombopag for PT and SFPR after allo-HSCT. We retrospectively studied of 467 patients who underwent allo-HSCT between November 2010 and March 2017 in our center. Eltrombopag was given to 14 patients with severe thrombocytopenia after allo-HSCT as compassionate use. Before starting eltrombopag, a bone marrow (BM) aspirate was collected in order to evaluate the number of megakaryocytes. All patients were in complete remission (CR), with full BM chimerism. In addition, none of them had thrombotic microangiopathy. Eltrombopag was started at a dose of 50 mg once daily with dose escalation at a rate of 50 mg weekly to a maximum of 150 mg/day [6]. This study was conducted in accordance with the Declaration of Helsinki and approved by the Local Hospital Ethics Committee. Response to eltrombopag was evaluated by achieving platelet counts ≥50 × 10/L for 7 consecutive days without transfusion [7]. No response was defined as the inability to achieve at least platelet counts ≥20 × 10/L for at least 4 weeks at a dose of 150 mg/day [6]. Safety assessments included evaluation of adverse events graded in accordance with the National Cancer Institute Common Toxicity Criteria (version 4.0). Patients characteristics and outcomes are detailed in Table 1. The median age was 56 years (25–69); the median dose of CD34 cells was 6 × 10/kg (4.5–8.4). Stem cells were collected from peripheral blood in all cases. Graftversus host disease (GVHD) prophylaxis consisted on regimens previously reported [8]. Two patients presented with active acute GVHD while receiving eltrombopag. Five patients developed prior chronic GVHD, which was in CR in all patients during treatment. Concerning thrombocytopenia after allo-HSCT, 3 patients developed PT, whereas 11 patients had SFPR. Prior to starting eltrombopag, the median platelet count was 11.5 × 10/L (4–33), the median hemoglobin level was 9.1 g/dL (8–14), and the median neutrophil count was 1.21 × 10/L (0.08–4.3). The median time from onset of thrombocytopenia to the initiation of eltrombopag was 21 days (1–155). Additionally, a median of two prior treatment lines were administered, including: mesenchymal stem cells (MSCs, n= 3), immunoglobulins These authors contributed equally: D. Rivera, J.M. Bastida.