This international phase III, randomized, placebo-controlled, double-blind study assessed the efficacy and safety of lenalidomide in RBC transfusion-dependent patients with International Prognostic ...Scoring System lower-risk non-del(5q) myelodysplastic syndromes ineligible for or refractory to erythropoiesis-stimulating agents.
In total, 239 patients were randomly assigned (2:1) to treatment with lenalidomide (n = 160) or placebo (n = 79) once per day (on 28-day cycles). The primary end point was the rate of RBC transfusion independence (TI) ≥ 8 weeks. Secondary end points were RBC-TI ≥ 24 weeks, duration of RBC-TI, erythroid response, health-related quality of life (HRQoL), and safety.
RBC-TI ≥ 8 weeks was achieved in 26.9% and 2.5% of patients in the lenalidomide and placebo groups, respectively (P < .001). Ninety percent of patients achieving RBC-TI responded within 16 weeks of treatment. Median duration of RBC-TI with lenalidomide was 30.9 weeks (95% CI, 20.7 to 59.1). Transfusion reduction of ≥ 4 units packed RBCs, on the basis of a 112-day assessment, was 21.8% in the lenalidomide group and 0% in the placebo group. Higher response rates were observed in patients with lower baseline endogenous erythropoietin ≤ 500 mU/mL (34.0% v 15.5% for > 500 mU/mL). At week 12, mean changes in HRQoL scores from baseline did not differ significantly between treatment groups, which suggests that lenalidomide did not adversely affect HRQoL. Achievement of RBC-TI ≥ 8 weeks was associated with significant improvements in HRQoL (P < .01). The most common treatment-emergent adverse events were neutropenia and thrombocytopenia.
Lenalidomide yields sustained RBC-TI in 26.9% of RBC transfusion-dependent patients with lower-risk non-del(5q) myelodysplastic syndromes ineligible for or refractory to erythropoiesis-stimulating agents. Response to lenalidomide was associated with improved HRQoL. Treatment-emergent adverse event data were consistent with the known safety profile of lenalidomide.
Mesenchymal stromal cells are a promising option to treat knee osteoarthritis. Their safety and usefulness must be confirmed and the optimal dose established. We tested increasing doses of bone ...marrow mesenchymal stromal cells (BM-MSCs) in combination with hyaluronic acid in a randomized clinical trial.
A phase I/II multicenter randomized clinical trial with active control was conducted. Thirty patients diagnosed with knee OA were randomly assigned to intraarticularly administered hyaluronic acid alone (control), or together with 10 × 10(6) or 100 × 10(6) cultured autologous BM-MSCs, and followed up for 12 months. Pain and function were assessed using VAS and WOMAC and by measuring the knee motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage.
No adverse effects were reported after BM-MSC administration or during follow-up. BM-MSC-administered patients improved according to VAS during all follow-up evaluations and median value (IQR) for control, low-dose and high-dose groups change from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 4 (3, 5), 2 (1, 3) and 2 (0,4) respectively at 12 months (low-dose vs control group p = 0.005 and high-dose vs control group p < 0.009). BM-MSC-administered patients were also superior according to WOMAC, although improvement in control and low-dose patients could not be significantly sustained beyond 6 months. On the other hand, the BM-MSC high-dose group exhibited an improvement of 16.5 (12, 19) points at 12 months (p < 0.01). Consistent with WOMAC and VAS values, motion ranges remained unaltered in the control group but improved at 12 months with BM-MSCs. X-ray revealed a reduction of the knee joint space width in the control group that was not seen in BM-MSCs high-dose group. MRI (WORMS protocol) showed that joint damage decreased only in the BM-MSC high-dose group, albeit slightly.
The single intraarticular injection of in vitro expanded autologous BM-MSCs together with HA is a safe and feasible procedure that results in a clinical and functional improvement of knee OA, especially when 100 × 10(6) cells are administered. These results pave the way for a future phase III clinical trial.
gov identifier NCT02123368. Nº EudraCT: 2009-017624-72.
Mesenchymal stromal cells (MSCs) are a promising option to treat knee osteoarthritis (OA). Their safety and usefulness have been reported in several short-term clinical trials but less information is ...available on the long-term effects of MSC in patients with osteoarthritis. We have evaluated patients included in our previous randomized clinical trial (CMM-ART, NCT02123368) to determine their long-term clinical effect.
A phase I/II multicenter randomized clinical trial with active control was conducted between 2012 and 2014. Thirty patients diagnosed with knee OA were randomly assigned to Control group, intraarticularly administered hyaluronic acid alone, or to two treatment groups, hyaluronic acid together with 10 × 10
or 100 × 10
cultured autologous bone marrow-derived MSCs (BM-MSCs), and followed up for 12 months. After a follow up of 4 years adverse effects and clinical evolution, assessed using VAS and WOMAC scorings are reported.
No adverse effects were reported after BM-MSCs administration or during the follow-up. BM-MSCs-administered patients improved according to VAS, median value (IQR) for Control, Low-dose and High-dose groups changed from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 7 (6, 7), 2 (2, 5) and 3 (3, 4), respectively at the end of follow up (Low-dose vs Control group, p = 0.01; High-dose vs Control group, p = 0.004). Patients receiving BM-MSCs also improved clinically according to WOMAC. Control group showed an increase median value of 4 points (- 11;10) while Low-dose and High-dose groups exhibited values of - 18 (- 28;- 9) and - 10 (- 21;- 3) points, respectively (Low-dose vs Control group p = 0.043). No clinical differences between the BM-MSCs receiving groups were found.
Single intraarticular injection of in vitro expanded autologous BM-MSCs is a safe and feasible procedure that results in long-term clinical and functional improvement of knee OA.
Within the myelodysplastic syndrome (MDS) work package of the European LeukemiaNet, an Expert Panel was selected according to the framework elements of the National Institutes of Health Consensus ...Development Program. A systematic review of the literature was performed that included indexed original papers, indexed reviews and educational papers, and abstracts of conference proceedings. Guidelines were developed on the basis of a list of patient- and therapy-oriented questions, and recommendations were formulated and ranked according to the supporting level of evidence. MDSs should be classified according to the 2008 World Health Organization criteria. An accurate risk assessment requires the evaluation of not only disease-related factors but also of those related to extrahematologic comorbidity. The assessment of individual risk enables the identification of fit patients with a poor prognosis who are candidates for up-front intensive treatments, primarily allogeneic stem cell transplantation. A high proportion of MDS patients are not eligible for potentially curative treatment because of advanced age and/or clinically relevant comorbidities and poor performance status. In these patients, the therapeutic intervention is aimed at preventing cytopenia-related morbidity and preserving quality of life. A number of new agents are being developed for which the available evidence is not sufficient to recommend routine use. The inclusion of patients into prospective clinical trials is strongly recommended.
Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has ...been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer.
Human MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed.
EV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 μm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP. Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively.
We described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.
Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify ...CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.
Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC‐EV. ...MSC‐EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC‐EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)‐STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho‐STAT5 were confirmed by WES Simple in CD34+ cells with MSC‐EV. In addition, these cells displayed a higher colony‐forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC‐EV was significantly increased in the injected femurs. In summary, the incorporation of MSC‐EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357–1368
Schematic representation of the experiments carried out in this work. CD34+ cells isolated from leukapheresis (for in vitro experiments) or from cord blood (for in vivo experiments) were cocultured with mesenchymal stromal cell‐extracellular vesicles (MSC‐EV) for 24 hours. After that, gene expression profile, viability, proliferation capacity, and colony‐forming capacity of CD34+ cells were assessed in both CD34+ cocultivated alone or with MSC‐EV. Finally, their engraftment ability was analyzed in nonobese diabetic/severe combined immunodeficient mice.
This trial evaluated the feasibility and efficacy of the infusion of mesenchymal stem cells expanded using human serum for the treatment of refractory acute or chronic graft-versus-host disease. ...Twenty-eight expansions were started. In 22, a minimum of more than 1 x 10⁶ mesenchymal stem cells/kg were obtained after a median of 26 days; this threshold was not obtained in the remaining cases. Ten patients received cells for the treatment of refractory or relapsed acute graft-versus-host disease and 8 for chronic disease. One patient treated for acute graft-versus-host disease obtained a complete response, 6 had a partial response and 3 did not respond. One of the chronic patients achieved complete remision, 3 a partial response, and 4 did not respond. The current study supports the use of this approach in less heavily treated patients for both acute and chronic graft-versus-host disease. The trial has been registered at ClinicalTrials.gov: identifier NCT00447460.
This phase 3, randomized, double-blind study assessed the efficacy and safety of lenalidomide in 205 red blood cell (RBC) transfusion-dependent patients with International Prognostic Scoring System ...Low-/Intermediate-1-risk del5q31 myelodysplastic syndromes. Patients received lenalidomide 10 mg/day on days 1-21 (n = 69) or 5 mg/day on days 1-28 (n = 69) of 28-day cycles; or placebo (n = 67). Crossover to lenalidomide or higher dose was allowed after 16 weeks. More patients in the lenalidomide 10- and 5-mg groups achieved RBC-transfusion independence (TI) for ≥ 26 weeks (primary endpoint) versus placebo (56.1% and 42.6% vs 5.9%; both P < .001). Median duration of RBC-TI was not reached (median follow-up, 1.55 years), with 60% to 67% of responses ongoing in patients without progression to acute myeloid leukemia (AML). Cytogenetic response rates were 50.0% (10 mg) versus 25.0% (5 mg; P = .066). For the lenalidomide groups combined, 3-year overall survival and AML risk were 56.5% and 25.1%, respectively. RBC-TI for ≥ 8 weeks was associated with 47% and 42% reductions in the relative risks of death and AML progression or death, respectively (P = .021 and .048). The safety profile was consistent with previous reports. Lenalidomide is beneficial and has an acceptable safety profile in transfusion-dependent patients with Low-/Intermediate-1-risk del5q myelodysplastic syndrome. This trial was registered at www.clinicaltrials.gov as #NCT00179621.
Introduction
The ability of mesenchymal stromal cells (MSC) to suppress T‐cell function has prompted their therapeutic use for graft‐versus‐host disease (GVHD) control. However, as MSC also modulate ...the activity of NK cells, which play an important role in graft‐versus‐leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity.
Objective
The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM‐CSM) or adipose tissue (AT‐MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL.
Methods
Human NK cells were stimulated with IL‐15 in the presence of BM‐MSC or AT‐MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays.
Results
Although both BM‐MSC and AT‐MSC induced a similar inhibition of NK cell proliferation, only BM‐MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN‐γ secretion.
Conclusion
These results suggest that, in the context of GVHD inhibition, the use of AT‐MSC rather than BM‐MSC could further preserve NK cell activity and, thus, favor GVL.
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