Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue

Background aimsHuman mesenchymal stromal cells (MSC) are multipotent cells possessing self-renewal capacity, long-term viability and multilineage potential. We analyzed the effect of four different medium supplements on the expansion and differentiation of adipose tissue-derived MSC (ADSC) in order to avoid the use of xenogeneic serum.MethodsWe compared fetal bovine serum (FBS) with 10% human platelet-rich plasma (hPRP), 3% human platelet-poor plasma (hPPP) and with a cytokine cocktail composed of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-bb (PDGFbb) added to 3% hPPP. This mixture was developed testing EGF, bFGF, granulocyte–colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF-I), PDGFbb and transforming growth factor (TGF)-β1 added alone or in combination with hPPP.ResultsOur data demonstrate that the addition of EGF, bFGF and PDGFbb, in a medium supplemented with hPPP, obtainable from 150–200 mL whole autologous blood, supports ADSC expansion better than FBS, as confirmed by cumulative population doublings (cPD; 15.0 ± 0.5 versus 9.4 ± 2.8). The addition of human platelet-rich plasma (hPRP) further improved ADSC proliferation (cPD 20.0 ± 1.2), but the achievement of hPRP presented a major drawback, requiring 1000–1200 mL autologous or donor whole blood. The medium supplements did not influence ADSC phenotype: they expressed CD105, CD90 and CD44 lacking hematopoietic antigens. The exposure to the proposed cocktail or to hPRP increased adipogenic and osteogenic differentiation.ConclusionsThe addition of EGF, bFGF and PDGFbb to hPPP could ensure a sufficient number of ADSC for clinical applications, avoiding the use of animal serum and representing a novel approach in regenerative medicine.     

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.     

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.     

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Background aimsAlloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use.MethodsWe describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells.ResultsNK cells (6.0 ± 1.2 × 10⁸) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR⁺ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML.ConclusionsThe approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.     

Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction

BackgroundAdipose tissue is an abundant source of mesenchymal stem cells (MSC), which can be used for tissue-engineering purposes. The aim of our study was to determine the more suitable procedure, surgical resection or liposuction, for harvesting human adipose tissue-derived stem cells (hASC) with regard to viability, cell count and differentiation potential.MethodsAfter harvesting hASC, trypan blue staining and cell counting were carried out. Subsequently, hASC were cultured, analyzed by fluorescence-activated cell sorting (FACS) and differentiated under adipogenic, osteogenic and chondrogenic conditions. Histologic and functional analyzes were performed at the end of the differentiation period.ResultsNo significant difference was found with regard to the cell counts of hASC from liposuction and surgically resected material (P = 0.086). The percentage of viable cells was significantly higher for liposuction aspirates than for resection material (P = 0.002). No significant difference was found in the adipogenic differentiation potential (P = 0.179). A significantly lower number of cultures obtained from liposuction material than from resection material could be differentiated into osteocytes (P = 0.049) and chondrocytes (P = 0.012).DiscussionEven though some lineages from lipoaspirated hASC can not be differentiated as frequently as those from surgically resected material, liposuction may be superior for some tissue-engineering purposes, particularly because of the less invasive harvesting procedure, the higher percentage of viable cells and the fact that there is no significant difference between lipoaspirated and resected hASC with regard to adipogenic differentiation potential.     

The influence of pre-operative risk on the number of circulating endothelial progenitor cells during cardiopulmonary bypass

Background aimsThe number of circulating endothelial progenitor cells (EPC) depends on cytokine release and is also associated with cardiovascular risk factors. During cardiopulmonary bypass (CPB) the endothelium is the first organ to be affected by mechanical and immunologic stimuli. We hypothesized that the magnitude of EPC mobilization by CPB correlates with the pre-operative cardiovascular morbidity profile.MethodsEPC were quantified in blood samples from 30 patients who underwent cardiac surgery by magnetic bead isolation and fluorescence-activated cell sorting (FACS) analysis, based on concomitant expression of CD34, CD133 and CD309. Patients were divided into two groups based on the European System for Cardiac Operative Risk Evaluation (EuroSCORE): low risk (LR) and high risk (HR). Ten healthy volunteers served as controls. Samples were obtained before the start of CPB and at 1 and 24 h post-operatively. Plasma samples were collected for determination of release levels of cytokines and growth factors.ResultsAll CPB patients showed a significantly reduced basal number of EPC compared with healthy individuals (LR 5.60 ± 0.39/mL, HR 3.89 ± 0.34/ mL, versus control 0.807 ± 0.82/mL, P = 0.012 versus LR, P < 0.001 versus HR). CPB induced EPC release that peaked 1 h after surgery (pre-operative 4.79 ± 0.32/mL, 1 h 57.49 ± 5.31/mL, 24 h 6.67 ± 1.05/mL, P < 0.001 pre-operative versus 1 h, P < 0.001 pre-operative versus 24 h) and was associated with the duration of CPB. However, EPC release was significantly attenuated in HR patients (33.09 ± 3.58/mL versus 81.89 ± 4.36/mL at 1 h after CPB, P < 0.0001) and inversely correlated with the pre-operative EuroSCORE. Serum granulocyte–colony-stimulating factor (G-CSF), stem cell factor (SCF) and vascular endothelial growth factor (VEGF) levels increased throughout the observation period and were also correlated with the EPC count.ConclusionsCardiovascular risk factors influence the mobilization of EPC from the bone marrow after stimulation by CPB. This could be secondary to impaired mobilization or the result of increased EPC turnover, and may have implications for future cell therapy strategies in cardiac surgical patients.     

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells

Background aimsHuman multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited.MethodsWe systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC).ResultsNine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10²² and 9.7 × 10²³, with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan.ConclusionsF-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.     

Mesenchymal stem cells as a gene therapy carrier for treatment of fibrosarcoma

Background aimsCell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment.MethodsIn this study, we injected intravenously (i.v.) 1 × 10⁶ MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals.ResultsWe observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group.ConclusionsiNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors.     

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.     

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.     

The clonogenic potential of hematopoietic stem cells and mesenchymal stromal cells in various hematologic diseases: a pilot study

Background aimsMesenchymal stromal cells (MSC) are the most popular cells used in regenerative medicine and biotechnology. The clonogenic potential of these cells is defined by colony-forming unit-fibroblasts (CFU-F). It is well known that there is an interaction between hematopoietic cells and stromal cells in disease formation pathogenesis. Therefore we hypothesized that there should be a quantitative and qualitative relationship between MSC colonies (CFU-F) and hematopoietic stem cell colonies (colony-forming unit-granulocyte-macrophages; CFU-GM) among patients with and without hematologic diseases.MethodsForty-two patients were included in this study. Patients were divided into three groups: group A, patients with hematologic malignancies (n = 20); group B, patients with bone marrow (BM) failure (n = 11); group C, patients without hematologic diseases (n = 11). BM aspirates were plated in different densities for CFU-F culture. The plating density was the same for CFU-GM culture.ResultsCFU-GM colonies grew in 90% of group A cells and all of group B and C cells (P = 0.0001). CFU-F colonies became visible on the ninth day of plating in group A and on the eight day in groups B and C. There was no statistically significant difference between the groups for the duration of CFU-F colony formation (P = 0.12). There were differences in the morphology of the colonies among the groups.ConclusionsThis is the first study that has compared the clonogenic potential of stromal cells and hematopoietic stem cells in the same subjects with and without hematologic diseases. No correlation was shown between the clonogenic potential of stromal cells and hematopoietic cells.     

Mesenchymal stromal cells prolong the lifespan in a rat model of amyotrophic lateral sclerosis

Background aimsAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals’ lifespan.MethodsAt the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fluorescent protein (GFP⁺ MSC) on the same day, intraspinally (10⁵ cells) and intravenously (2 × 10⁶ cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis.ResultsThe combined grafting of MSC into symptomatic rats had a significant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 ± 3.33 days compared with 179 ± 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFP⁺ MSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site.ConclusionsIntraspinal and intravenous transplantation of MSC has a beneficial and possibly synergistic effect on the lifespan of ALS animals.     

Cell proliferation and survival in the mating circuit of adult male hamsters: Effects of testosterone and sexual behavior

The transient actions of gonadal steroids on the adult brain facilitate social behaviors, including reproduction. In male rodents, testosterone acts in the posterior medial amygdala (MeP) and medial preoptic area (MPOA) to promote mating. Adult neurogenesis occurs in both regions. The current study determined if testosterone and/or sexual behavior promote cell proliferation and survival in MeP and MPOA. Two experiments were conducted using the thymidine analog BrdU. First, gonad-intact and castrated male hamsters (n = 6/group) were compared 24 h or 7 weeks after BrdU. In MeP, testosterone-stimulated cell proliferation 24 h after BrdU (intact: 22.8 ± 3.9 cells/mm², castrate: 13.2 ± 1.4 cells/mm²). Testosterone did not promote cell proliferation in MPOA. Seven weeks after BrdU, cell survival was sparse in both regions (MeP: 2.5 ± 0.6 and MPOA: 1.7 ± 0.2 cells/mm²), and was not enhanced by testosterone. In Experiment 2, gonad-intact sexually-experienced animals were mated weekly to determine if regular neural activation enhances cell survival 7 weeks after BrdU in MeP and MPOA. Weekly mating failed to increase cell survival in MeP (8.1 ± 1.6 vs. 9.9 ± 3.2 cells/mm²) or MPOA (3.9 ± 0.7 vs. 3.4 ± 0.3 cells/mm²). Furthermore, mating at the time of BrdU injection did not stimulate cell proliferation in MeP (8.9 ± 1.7 vs. 8.1 ± 1.6 cells/mm²) or MPOA (3.6 ± 0.5 vs. 3.9 ± 0.7 cells/mm²). Taken together, our results demonstrate a limited capacity for neurogenesis in the mating circuitry. Specifically, cell proliferation in MeP and MPOA are differentially influenced by testosterone, and the birth and survival of new cells in either region are not enhanced by reproductive activity.     

Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells

AimsThe phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs).Main methodsAFSCs and DPSCs were cultured in a differentiation medium for 14 or 21 days with or without the addition of Ferutinin at a concentration ranging from 10^? 11 to 10^? 4 M. 17β-Estradiol was used as a positive drug at 10^? 8 M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed.Key findingsMTT assay revealed that Ferutinin, at concentrations of 10^? 8 and 10^? 9 M, enhanced proliferation of both AFSCs and DPSCs after 72 h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14 days of culture in both populations.SignificanceThe enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.     

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.     

GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Background aimsBone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque? PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.MethodsBM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.ResultsNo differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45⁺ fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.ConclusionsBoth GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.     

Antiproliferative,antiandrogenic and cytotoxic effects of novel caffeic acid derivatives in LNCaP human androgen-dependent prostate cancer cells

Caffeic acid and its naturally occurring derivative caffeic acid phenethyl ester (CAPE) have antiproliferative and cytotoxic properties in a variety of cancer cell lines without displaying significant toxicity toward healthy cells, and are considered to be potential anticancer agents. However, little is known about their effects on prostate cancer cells. We synthesized and evaluated the effects of caffeic acid, CAPE (2) and 18 synthetic derivatives on cell viability and androgen-dependent cell proliferation, subcellular localisation and expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) in LNCaP human hormone-dependent prostate cancer cells. Several synthetic derivatives of CAPE were strong, concentration-dependent cytotoxic agents in LNCaP cells with IC₅₀ values in the 6.8–26.6 μM range, potencies that were up to five-fold greater than that of CAPE (33.7 ± 4.0 μM). A number of caffeic acid derivatives were inhibitors of androgen-stimulated LNCaP cell proliferation with concomitant inhibition of DHT-stimulated PSA secretion. Compound 24 was the most cytotoxic and antiproliferative caffeic acid derivative (IC₅₀ values of 6.8 ± 0.3 and 2.4 ± 0.8 μM, respectively) inhibiting DHT-stimulated cell proliferation and PSA secretion statistically significantly at concentrations as low as 0.3 μM. Exposure to DHT increased cytoplasmic and nuclear AR levels and co-treatment with increasing concentrations of compound 24 or CAPE (2), notably, further increased these levels. In conclusion, a number of synthetic derivatives of caffeic acid are potent inhibitors of androgen-dependent prostate cancer cell proliferation and viability, acting, at least in part, via an antiandrogenic mechanism that involves increased nuclear accumulation of (presumably inactive) AR.