Zubair, Abba C.

Abstract Abstract 1184 Lenalidomide, a second generation immunomodulatory derivative of thalidomide, has demonstrated significant clinical benefit in myelodysplastic syndrome, multiple myeloma and B-cell malignancies. Its side effects include myelosuppression, particularly neutropenia, and impaired filgrastim induced stem cell mobilization in patients with myeloma. The precise mechanism of action of lenalidomide on hematopoietic and bone marrow microenvironment remains largely unknown. In this study, we investigated the effect of lenalidomide on survival, lineage commitment and differentiation of normal purified CD34+ hematopoietic stem cells. Mobilized peripheral blood progenitor cells (PBPCs) were obtained from a normal allogeneic stem cell donor with an informed consent, and were subjected to positive selection with the EasySep magnetic cell selection system to purify CD34+ cells (purity of 95%). Unsorted and CD34- accessory cells were used as controls. To assess the direct cytotoxic and proliferative effect of lenalidomide on purified CD34+ cells, lenalidomide (Celgene, NJ) was dissolved in DMSO and was diluted in culture medium to final concentration of 0.1, 1, 10, or 100 μM. Purified CD34+, CD34- and unsorted cells were plated in triplicates at a density of 2×104/mL in serum-free StemSpan® medium supplemented with growth factors and cytokines. 24 hours after plating the cells, lenalidomide or 0.1% DMSO as control was added. 72 hours following treatment, cells were counted with Beckman coulter counter and phenotypically analyzed using Accuri C6 flow-cytometer. The percentage of viable cells was determined using 7AAD staining. While no difference in viability was noted in the treated vs. untreated purified CD34+ cells, the viability of CD34- cells and unsorted cells was reduced to 30% and 40% respectively by lenalidomide. Contrary to our assumption, lenalidomide, in a dose dependant manner, induced proliferation of purified CD34+ cells. Maximal increase in proliferation was observed between 1mM and 10mM concentrations. The rate of growth plateaued beyond the 10mM concentration and 100mM concentration appeared to have no additional effect. At the peak of growth (10mM), the cell count nearly tripled. (Fig. 1) Lenalidomide appeared to be cytotoxic to CD34- and unsorted cells in a dose dependant manner. At 100mM concentration, the CD34- cell proliferation dropped by 4 fold and the unsorted cells by 7 fold. To examine the effect of lenalidomide on the clonogenic potential of purified CD34+ cells, 2,000 cells were seeded onto MethoCult® media (Stem cell technologies) supplemented with growth factors and cytokines. Cells were cultured in the presence of lenalidomide (0.1, 1, 10 and 100 μM) or 0.1 % DMSO as control. BFU-E, CFU-GM and CFU-mix were scored on day 14 with the use of an inverted microscope and standard morphological criteria. Significance of difference between groups was determined by student's t test for paired samples. Lenalidomide induced a significant increase in clonogenic activity of CD34+ cells (148 ± 27 colonies per 103 CD34+ cells, p = 0.01), most evident at the dose of 10 μM with enhanced formation of BFU-E (102 ± 23/ per 103 CD34+ cells, p = 0.01) but demonstrated no significant impact on CFU-GM (36 ± 14 per 103 CD34+ cells, p = 0.83) compared to control. (Fig. 2) This study demonstrates that lenalidomide has pronounced effects on both CD34+ and CD34- components of mobilized PBPCs. It enhances the proliferative and clonogenic potentials of CD34+ cells with lineage commitment towards erythroid differentiation. It also has cytotoxic activity towards CD34- cells. It is likely that neutropenia and impaired mobilization of stem cells in patients treated with lenalidomide is mediated via its impact on CD34- accessory cells in the bone marrow microenvironment. Further studies exploring the impact of lenalidomide on normal bone marrow stromal cells, T and NK cell mediated signaling are needed. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSCs) are becoming an increasingly popular therapeutic option among patients with a broad range of ailments to modulate immunity and induce regeneration. The majority of patients receiving these MSC therapies are on concurrent medication or have ongoing infection. In the present study, we examined the effect of immunosuppressive drugs and lipopolysaccharides (LPS)/endotoxins on the secretory profile, migration towards site of injury, and suppression of lymphocyte proliferation of bone marrow-derived MSCs (BMSCs). Generally, LPS coculture augmented the secretory capacity of BMSCs while exposure to immunosuppressive drugs resulted primarily in no change or attenuated secretion, with some cases of increased secretion, dependent on the cytokine assayed. Among the immunosuppressants evaluated, Hydrocortisone had the most widespread inhibitory effect, while LPS from E. coli O111:B4 had the most potent stimulatory effect. In addition, we also showed that Hydrocortisone or LPS from E. coli O111:B4 affected the migratory and immunosuppressive capacity of BMSCs. Following simulation with Hydrocortisone, BMSC migration was attenuated, and immunosuppressive capacity against T cell proliferation was enhanced, however, the opposite effects were seen with LPS from E. coli O111:B4. Our data suggests that the clinical outcomes of MSC-based therapy are affected by the use of immunosuppressive medication or the presence of endotoxemia in patients.

AbstractBackgroundMobilization regimen choice is a significant contributing factor for successful hematopoietic progenitor cell (HPC) collection by leukocytapheresis and reaching the target CD34+ cell dose. How mobilization regimen affects collection efficiency and the quality of products collected using the Spectra Optia apheresis instrument is not fully known.MethodsWe evaluated the impact of granulocyte‐colony stimulating factor (GCSF) and GCSF/plerixafor mobilization regimens on CE and product composition. We studied 373 leukocytapheresis HPC collections for 147 autologous transplants from January 1, 2010 to December 31, 2014. Patients were categorized in two groups; good mobilizers, mobilized with GCSF only (GM) and poor mobilizers, mobilized with GCSF and Plerixafor (PM).ResultsOverall, compared with PM group, total nucleated cell (TNC) yield was significantly lower in GM group (P = <.001). In contrast, median percent mononuclear cell (MNC) collected from GM (86.5%) was significantly higher than products collected from PM group (79.5%; P < .001). Compared with GM group, CD34+ cell CE was about 10% lower in PM group (P < .008). In addition, daily CD34+ cell/Kg yield was significantly higher in GM (2.08 × 10/Kg) compared with PM group (1.64 x 10/Kg, P = .019). Overall, the median number of collections per patient was two for GM and three for PM (P = .004).ConclusionProducts collected from PM group contained higher TNC content relative to GM group but had lower MNC enrichment, CD34+ cell CE and daily CD34+ cell yield per Kg.

BACKGROUND Expanding quantities of mesenchymal stem cells (MSCs) sufficient to treat large numbers of patients in cellular therapy and regenerative medicine clinical trials is an ongoing challenge for cell manufacturing facilities. STUDY DESIGN AND METHODS We evaluated options for scaling up large quantities of bone marrow–derived MSCs (BM‐MSCs) using methods that can be performed in compliance with Good Manufacturing Practices (GMP). We expanded BM‐MSCs from fresh marrow aspirate in αMEM supplemented with 5% human platelet lysate using both an automated cell expansion system (Quantum, Terumo BCT) and a manual flask‐based method using multilayer flasks. We compared MSCs expanded using both methods and assessed their differentiation to adipogenic and osteogenic tissue, capacity to suppress T‐cell proliferation, cytokines, and growth factor secretion profile and cost‐effectiveness of manufacturing enough BM‐MSCs to administer a single dose of 100 × 10 6 cells per subject in a clinical trial of 100 subjects. RESULTS We have established that large quantities of clinical‐grade BM‐MSCs manufactured with an automated hollow‐fiber bioreactor were phenotypically (CD73, CD90, CD105) and functionally (adipogenic and osteogenic differentiation and cytokine and growth factor secretion) similar to manually expanded BM‐MSCs. In addition, MSC manufacturing costs significantly less and required less time and effort when using the Quantum automated cell expansion system over the manual multilayer flasks method. CONCLUSION MSCs manufactured by an automated bioreactor are physically and functionally equivalent to the MSCs manufactured by the manual flask method and have met the standards required for clinical application.

Objective Bone marrow aspiration and concentration (BMAC) is becoming a more common regenerative therapy for musculoskeletal pathology. In our current pilot study, we studied patients with mild-to-moderate bilateral knee osteoarthritis, compared pain at 12-month follow-up between BMAC-injected and saline-injected knees, and examined cartilage appearance measured by magnetic resonance imaging (MRI) T2 quantitative mapping. Design Twenty-five patients with mild-to-moderate bilateral osteoarthritic knee pain were randomized to receive BMAC into one knee and saline placebo into the other. Bone marrow was aspirated from the iliac crests, concentrated in an automated centrifuge, combined with platelet-poor plasma for knee injection, and compared with saline injection into the contralateral knee. Primary outcome measures were T2 MRI cartilage mapping at 6-month and Visual Analog Scale and Osteoarthritis Research Society International Intermittent and Constant Osteoarthritis Pain scores and radiographs at 12-month follow-up. Results Constant, intermittent, and overall knee pain remained significantly decreased from baseline at 12-month follow-up (all P ⩽ 0.01), with no apparent difference between BMAC- and saline-treated knees (all P ⩾ 0.54). A similar significant increase from baseline to 12-month follow-up regarding quality of life was observed for both BMAC- and saline-treated knees (all P ⩽ 0.04). T2 quantitative MRI mapping showed no significant changes as a result of treatment. Conclusions BMAC is safe to perform and relieves pain from knee arthritis but showed no superiority to saline injection at 12-month follow-up. MRI cartilage sequences failed to show regenerative benefit with single BMAC injection. The mechanisms of action that led to pain relief remain unclear and warrant further studies.

BACKGROUNDClinical trials involving mesenchymal stem cell (MSC) therapy have variable outcomes. We hypothesize this is largely attributed to donor‐to‐donor variability and tissue of origin.STUDY DESIGN AND METHODSWe examined proliferation rates, cytokine secretion profiles, and differentiation capability of seven bone marrow–derived MSCs (BM‐MSCs) and 16 adipose tissue–derived MSCs (AD‐MSCs) from 23 donors.RESULTSAD‐MSCs had the capacity to undergo more than 40 population doublings, while the BM‐MSC proliferation rate was found to be considerably slower. We observed more donor‐to‐donor variability in proliferation rates of BM‐MSCs than with AD‐MSCs. Cytokine analysis revealed that secretion of eight cytokines was significantly increased by AD‐MSCs at Passage (P)3 compared with P1, while for BM‐MSCs at P3 relative to P1, only interleukin‐8 and RANTES secretion was significantly increased. By P5, secretion of all cytokines by AD‐MSCs was either decreased or unchanged relative to P1. In contrast, cytokine secretion by BM‐MSCs at P5 was mostly unchanged, although secretion of six cytokines was significantly increased relative to P1. When we compared cytokine secretion between AD‐MSCs and BM‐MSCs at P3, AD‐MSCs significantly secreted higher concentrations of cytokines than BM‐MSCs while the opposite was observed at P5. This suggests that BM‐MSCs are relatively more potent at P5 while AD‐MSCs are relatively more potent at P3. AD‐MSCs and BM‐MSCs exhibited the capacity for chondrogenic differentiation. AD‐MSCs and BM‐MSCs appeared to display a more enhanced inclination toward adipogenic and osteogenic differentiation, respectively.CONCLUSIONMSC physiology is significantly influenced by donor variability and tissue of origin and this should be considered when designing clinical trials.

BACKGROUND: There has been a surge in clinical trials studying the safety and efficacy of mesenchymal stem cells for the treatment of perianal Crohn’s disease. OBJECTIVE: The purpose of this work was to systematically review the literature to determine safety and efficacy of mesenchymal stem cells for the treatment of refractory perianal Crohn’s disease. DATA SOURCES: Sources included PubMed, Cochrane Library Central Register of Controlled Trials, and Embase. STUDY SELECTION: Studies that reported safety and/or efficacy of mesenchymal stem cells for the treatment of perianal Crohn’s disease were included. Two independent assessors reviewed eligible articles. INTERVENTION: The study intervention was delivery of mesenchymal stem cells to treat perianal Crohn’s disease. MAIN OUTCOMES MEASURES: Safety and efficacy of mesenchymal stem cells used to treat perianal Crohn’s disease were measured. RESULTS: Eleven studies met the inclusion criteria and were included in the systematic review. Three trials with a comparison arm were included in the meta-analysis. There were no significant increases in adverse events (OR = 1.07 (95% CI, 0.61–1.89); p = 0.81) or serious adverse events (OR = 0.53 (95% CI, 0.28–0.98); p = 0.04) in patients treated with mesenchymal stem cells. Mesenchymal stem cells were associated with improved healing as compared with control subjects at primary end points of 6 to 24 weeks (OR = 3.06 (95% CI, 1.05–8.90); p = 0.04) and 24 to 52 weeks (OR = 2.37 (95% CI, 0.90–6.25); p = 0.08). LIMITATIONS: The study was limited by its multiple centers and heterogeneity in the study inclusion criteria, mesenchymal stem cell origin, dose and frequency of delivery, use of scaffolding, and definition and time point of fistula healing. CONCLUSIONS: Although there have been only 3 trials conducted with control arms, existing data demonstrate improved efficacy and no increase in adverse or serious adverse events with mesenchymal stem cells as compared with control subjects for the treatment of perianal Crohn’s disease.

Abstract Feasibility, tolerance, and safety of intravenous infusions of allogeneic mesenchymal stem cell (MSC) therapy in lung transplant recipients with bronchiolitis obliterans syndrome (BOS) are not well established. MSCs were manufactured, cryopreserved, transported to our facility, thawed, and infused into nine recipients with moderate BOS (average drop in forced expiratory volume in 1 second was 56.8% ± 3.2% from post-transplant peak) who were refractory to standard therapy and not candidates for retransplant. Cells were viable and sterile prior to infusion. Patients received a single infusion of either 1 (n = 3), 2 (n = 3), or 4 (n = 3) million MSCs per kg. Patients were medically evaluated before; during; and at 24 hours, 1 week, and 1 month after infusion for evidence of infusion-related adverse events and tolerance of therapy. Vital signs, pulmonary function test results, Borg Dyspnea Index, and routine laboratory data were recorded. Vital signs and O2 saturation did not significantly change during or up to 2 hours after MSC infusion. There were no significant changes in gas exchange variables, pulmonary function test results, or laboratory values at 1, 7, and 30 days postinfusion compared with preinfusion values. Infusion of MSCs in patients with BOS was feasible, safe, and well tolerated and did not produce any significant adverse changes in clinical, functional, or laboratory variables during or up to 30 days after infusion. Manufacturing, transport, and administration of intravenous, allogeneic bone marrow-derived MSCs in doses from 1 to 4 million MSCs per kg is safe in lung transplant recipients with BOS.