Mesenchymal stromal cells from donors varying widely in age are of equal cellular fitness after in vitro expansion under hypoxic conditions

Background aimsMesenchymal stromal cells (MSC) are gaining in popularity as an experimental therapy for a number of conditions that often require expansion ex vivo prior to use. Data comparing clinical-grade MSC from various ages of donors are scant. We hypothesized that MSC from older donors may display differences in cellular fitness when expanded for clinical use.MethodsWe evaluated the expression of several markers of aging, oxidative stress and growth kinetics, and telomere length, in MSC obtained from a wide age range (8 months to 58 years).ResultsTo evaluate cellular fitness we compared MSC expanded from younger (8 months–6 years) versus older (38–58 years) donors in terms of selected cell-surface markers, lipofuscin, migration ability, telomere length and expression of iNOS, PGE₂, p16INK and SOD. Results did not differ between these groups. Neither SOD activity (0.025 versus 0.028 U/mL) nor death after oxidative challenge was significantly different (1% versus 1.5%, P = 0.14). We did find that, although MSC from older individuals produced slightly fewer cells over a 28-day culture period and had a slightly longer doubling time (54 h versus 42 hr, a satisfactory clinical product could still be obtained regardless of age cohort.ConclusionsCollectively, these data show that MSC can be expanded without significant alterations in expansile properties or obvious changes in parameters associated with senescence. Because cellular fitness was equivalent in these cohorts, MSC from donors up to age 58 years can be used as a source of cells for cellular therapy.     

Original article anti-oxidant pathways are stimulated by mesenchymal stromal cells in renal repair after ischemic injury

Background aimsIschemia-reperfusion (IR) injury is a common cause of acute renal failure. Bone marrow (BM)-derived mesenchymal stromal cells (MSC) delivered after renal IR are renoprotective, but knowledge of the protective mechanism is still in development. This investigation analyzed the protective molecular mechanisms of MSC, in particular relating to modulated oxidative stress.MethodsIn vivo and in vitro models of renal IR were analyzed with and without MSC. In vivo, adult male Sprague–Dawley rats were subjected to 40-min unilateral renal IR. Rat BM-derived MSC were administered at 24 h post-IR (IR + MSC). Other groups had IR but no MSC, or MSC but no ischemia (all groups n = 4). Apoptosis, inflammation, oxidative stress and reparative signal transduction molecules or growth factors were studied 4 days post-IR. In vitro, protection by MSC against oxidative stress (0.4 mm hydrogen peroxide) was investigated using rat renal tubular epithelial cells (NRK52E) with or without MSC in co-culture (tissue culture trans-well inserts), followed by similar analyses to the in vivo investigation.ResultsIn vivo, kidneys of IR + MSC animals had significantly increased cell proliferation/regeneration (cells positive for proliferating cell nuclear antigen, expression of epidermal growth factor), increased heme-oxygenase-1 (improved cell survival, anti-oxidant) and decreased 8-OHdG (decreased oxidative stress). In vitro, MSC delivered with oxidative stress significantly decreased apoptosis and Bax (pro-apoptotic protein), and increased mitosis and phospho-ERK1/2, thereby minimizing the damaging outcome and maximizing the regenerative effect after oxidative stress.ConclusionsThe benefits of MSC, in IR, were primarily pro-regenerative, sometimes anti-apoptotic, and novel anti-oxidant mechanisms were identified.     

Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate

Background aimsMesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC).MethodsSections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3).ResultsWe isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10¹⁰ cells (ranging from 1.0 × 10¹⁰ to 29.0 × 10¹⁰). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo.ConclusionsUC constitute a convenient and very rich source of MSC for the production of third-party ‘clinical doses’ of cells under good manufacturing practice (GMP) conditions.     

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses

Background aimsThe development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horsesMethodsSixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injectionResultsMSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSCConclusionsThe healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.     

In vivo therapy of myocardial infarction with mesenchymal stem cells modified with prostaglandin I synthase gene improves cardiac performance in mice

AimIntra-myocardial injection of adult bone marrow-derived stem cells (MSC) has recently been proposed as a therapy to repair damaged cardiomyocytes after acute myocardial infarction (AMI). PGI₂ has vasodilatation effects; however, the effects of combining both MSC and PGI₂ therapy on AMI have never been evaluated.Main methodsWe genetically enhanced prostaglandin I synthase (PGIS) gene expression in mouse mesenchymal stem cells (MSC) using lentiviral vector transduction (MSC_PGIS). Mice were subjected to an AMI model and injected (intra-myocardially) with either 5 × 10⁴ MSCs or MSC_PGIS before surgery. Fourteen days post AMI, mice were analyzed with echocardiography, immunohistochemistry, and apoptotic, and traditional tissue assays.Key findingsLenti-PGIS transduction did not change any characteristic of the MSCs. PGIS over-expressed MSCs secreted 6-keto-PGF1α in the culture medium and decreased free radical damage during hypoxia/re-oxygenation and H₂O₂ treatment. Furthermore, splenocyte proliferation was significantly suppressed with MSC_PGIS as compared with MSCs alone. Fourteen days post AMI, echocardiography showed more improvement in cardiac function of the MSC_PGIS group than the MSC alone group, sham-operated group, or artery ligation only group. The histology of MSC_PGIS treated hearts revealed MSCs in the infarcted region and decreased myocardial fibrosis/apoptosis with limited cardiac remodeling. Furthermore, the level of the vascular endothelial growth factor was elevated in the MSC_PGIS group as compared to the other three groups.SignificanceIn summary, our results provide both in vitro and in vivo evidence for the beneficial role of MSC_PGIS in limiting the process of detrimental cardiac remodeling in a mouse AMI model during early stages of the disease.     

Mesenchymal stromal cells alone or expressing interferon-β suppress pancreatic tumors in vivo,an effect countered by anti-inflammatory treatment

Background aimsBecause of the inflammatory nature and extensive stromal compartment in pancreatic tumors, we investigated the role of mesenchymal stromal cells (MSC) to engraft selectively in pancreatic carcinomas and serve as anti-tumor drug delivery vehicles to control pancreatic cancer progression.MethodsHuman pancreatic carcinoma cells, PANC-1, expressing renilla luciferase were orthotopically implanted into SCID mice and allowed to develop for 10 days. Firefly luciferase-transduced MSC or MSC expressing interferon (IFN)-β were then injected intraperitoneally weekly for 3 weeks. Mice were monitored by bioluminescent imaging for expression of renilla (PANC-1) and firefly (MSC) luciferase.ResultsMSC selectively homed to sites of primary and metastatic pancreatic tumors and inhibited tumor growth (P = 0.032). The production of IFN-β within the tumor site by MSC–IFN-β further suppressed tumor growth (P = 0.0000083). Prior studies indicated that MSC home to sites of inflammation; therefore, we sought to alter the tumor microenvironment through treatment with a potent anti-inflammatory agent. After treatment, inflammation-associated mediators were effectively down-regulated, including NFκB, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 as well as chemokines involved in MSC migration (CCL3 and CCL25). Treatment with the anti-inflammatory agent CDDO-Me before and after MSC–IFN-β injections resulted in reduction of MSC in the tumors and reversed the positive effect of tumor inhibition by MSC–IFN-β alone (P = 0.041).ConclusionsThese results suggest that MSC exhibit innate anti-tumor effects against PANC-1 cells and can serve as delivery vehicles for IFN-β for the treatment of pancreatic cancer. However, these beneficial effects may be lost in therapies combining MSC with anti-inflammatory agents.     

CXCR4 and matrix metalloproteinase-2 are involved in mesenchymal stromal cell homing and engraftment to tumors

Background aimsBone marrow-derived mesenchymal stromal cells (BMSC) have been shown to migrate to injury, ischemia and tumor microenvironments. The mechanisms by which mesenchymal stromal cells (MSC) migrate across endothelium and home to the target tissues are not yet fully understood.MethodsWe used rat BMSC to investigate the molecular mechanisms involved in their tropism to tumors in vitro and in vivo.ResultsBMSC were shown to migrate toward four different tumor cells in vitro, and home to both subcutaneous and lung metastatic prostate tumor models in vivo. Gene expression profiles of MSC exposed to conditioned medium (CM) of various tumor cells were compared and revealed that matrix metalloproteinase-2 (MMP-2) expression in BMSC was downregulated after 24 h exposure to tumor CM. Chemokine (C–X–C motif) Receptor 4 (CXCR4) upregulation was also found in BMSC after 24 h exposure to tumor CM. Exposure to tumor cell CM enhanced migration of BMSC toward tumor cells. Stromal Cell-Derived Factor (SDF-1) inhibitor AMD3100 and MMP-2 inhibitor partly abolished the BMSC migration toward tumor cells in vitro.ConclusionsThese results suggest that the CXCR4 and MMP-2 are involved in the multistep migration processes of BMSC tropism to tumors.     

Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects

Background aimsCombining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions.MethodsMSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy.ResultsEffective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (≤42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC.ConclusionsMSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.     

Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.Main methodsMice were divided into sham, I/R, Rb1 + I/R (Rb1 postconditioning, 20 mg/kg, i.p. after ischemia), sham + L-NAME, I/R + L-NAME, and Rb1 + I/R + L-NAME groups using 60 min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p < 0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p < 0.05), while it dramatically reduced the concentration of MDA (p < 0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p < 0.05).SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.     

Receptor for hyaluronic acid- mediated motility (RHAMM) regulates HT1080 fibrosarcoma cell proliferation via a β-catenin/c-myc signaling axis

BackgroundHigh levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated cell proliferation.MethodsReal-time PCR, in vitro cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized.ResultsThe reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 fibrosarcoma cell growth (p ≤ 0.01). LMWHA, in a RHAMM-dependent manner increases cell growth of HT1080 cells ((p ≤ 0.01). Both basal and LMWHA dependent growth of HT1080 cells was attenuated by β-catenin deficiency (p ≤ 0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p ≤ 0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intracellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 fibrosarcoma cell proliferation.ConclusionsLMWHA/RHAMM downstream signaling regulates fibrosarcoma cell growth in a β-catenin/c-myc dependent manner.General significanceThe present study suggests that RHAMM is a novel β-catenin intracellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key cellular mediator, which results in c-myc activation and enhanced fibrosarcoma cell growth.     

Improved motor function in dko mice by intravenous transplantation of bone marrow-derived mesenchymal stromal cells

Background aimsWe explored the potential therapeutic value of transplanting bone marrow (BM)-derived mesenchymal stromal cells (MSC) into utrophin/dystrophin-deficient double knock-out (dko) mice, a murine model of Duchenne muscular dystrophyMethodsMSC from male rats were isolated and transplanted into female dko mice via the caudal vein. Behavior and locomotor function were later evaluated, along with the expression of dystrophin and utrophin in the sarcolemma of myofiber tissues. The presence of grafted cells was confirmed via polymerase chain reaction for the sex-determining region of the Y-chromosomeResultsLocomotor activity improved significantly (P < 0.05) from 5 to 15 weeks after cell transplantation, as measured by traction, rotating rod and running wheel tests. We also found that the expression of dystrophin and utrophin increased significantly (P < 0.05) and progressively in the sarcolemma from 5 to 15 weeks after transplantation. The median lifespan of mice in the normal group (74.1 weeks) was significantly (P < 0.001) higher than those in the control (22.0 weeks) and transplantation (35.0 weeks) groups, and the median lifespan of mice in the transplantation group was significantly (P < 0.001) higher than that in the control groupConclusionsResults of this study demonstrate that BM MSC have potential value in xenogeneic transplantation therapy for muscular dystrophy.     

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy

Background aimsTransplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model.MethodsASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10⁵ cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed.ResultsASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group.ConclusionsCompared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.     

Intramyocardial and intracoronary autologous bone marrow-derived mesenchymal stromal cell treatment in chronic severe dilated cardiomyopathy

Background aimsMesenchymal stromal cells (MSC) may improve cardiac function following myocardial infarction. MSC can differentiate into cardiomyocytes and endothelial cells while exerting additional paracrine effects. There is limited information regarding the efficacy of route for MSC treatment of severe dilated cardiomyopathy (DCM). The aim of this study was to demonstrate the clinical safety, feasibility and efficacy of direct intramyocardial and intracoronary administration of autologous bone marrow-derived MSC treatment for no-option patients with chronic severe refractory DCM.MethodsTen symptomatic patients with DCM and refractory cardiac function, despite maximum medical therapy, were selected. Five had ischemic DCM deemed unlikely to benefit from revascularization alone and underwent bypass operations with concurrent intramyocardial MSC injection (group A). Two patients had previous revascularization and three had non-ischemic DCM and received intracoronary MSC injection (group B).ResultsGroup A and B patients received 0.5–1.0 × 10⁶ and 2.0–3.0 × 10⁶ MSC/kg body weight, respectively. All patients remained alive at 1 year. There were significant improvements from baseline to 6 and 12 months in left ventricular ejection fraction and other left ventricular parameters. Scar reduction was noted in six patients by 12 months.ConclusionsAutologous bone marrow MSC treatment is safe and feasible for treating chronic severe refractory DCM effectively, via intracoronary or direct intramyocardial administration at prescribed doses.     

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use

Background aimsThe manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) ‘quality’ with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.MethodsAspirated MSC were enumerated using the CD45^?/low CD271^bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit–fibroblast (CFU-F) assay. The phenotype of CD45^?/low CD271^bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.ResultsCD45^?/low CD271^bright cells had a classic MSC phenotype (CD73⁺ CD105⁺ CD90⁺ ). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34 ⁺ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45^?/low CD271^bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.ConclusionsAbsolute quantification of CD45^?/low CD271^bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.     

Gene expression profiles following intracoronary injection of mesenchymal stromal cells using a porcine model of chronic myocardial infarction

Background aimsWe evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model.Methods and ResultsMyocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10⁶ 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo groupConclusionsInfarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.     

Comparison of two CD40-ligand/interleukin-2 vaccines in patients with chronic lymphocytic leukemia

Background aimsSeveral studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies.MethodsWe compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity.ResultsWe found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine.ConclusionsCLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.     

Co-transplantation of olfactory ensheathing glia and mesenchymal stromal cells does not have synergistic effects after spinal cord injury in the rat

Background aimsOlfactory ensheathing glia (OEG) and mesenchymal stromal cells (MSC) are suitable candidates for transplantation therapy of spinal cord injury (SCI). Both facilitate functional improvement after SCI by producing trophic factors and cytokines. In this study, the co-transplantation of both types of cells was studied to clarify their additive and/ or synergistic effects on SCI.MethodsA balloon-induced compression lesion was used to produce SCI in rats. OEG, MSC or both OEG and MSC (3 × 10⁵ cells of each cell type) were implanted by intraspinal injection 1 week after SCI. The effect of transplantation was assessed using behavioral, electrophysiologic and histologic methods.ResultsHindlimb function was examined with Basso, Beattie and Bresnahan (BBB) and Plantar tests. Improvement was found in all three groups of transplanted rats with different time–courses, but there was no significant difference among the groups at the end of the experiment. Motor-evoked potentials after SCI decreased in amplitude from 7 mV to 10 µV. Linear regression analysis showed a modest recovery in amplitude following transplantation, but no change in the control rats. Histologic findings showed that the white and gray matter were significantly spared by transplantation after SCI.ConclusionsFunctional improvement was achieved with transplantation of OEG and/or MSC, but the co-transplantation of OEG and MSC did not show synergistic effects. The poor migration of OEG and MSC might prevent their concerted action. Pre-treatment with a Rho antagonist and a combination of intraspinal and intravenous injection of the cells might be beneficial for SCI therapy.