Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern.

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues.

A phase I clinical trial of CD1c (BDCA-1)+ dendritic cells pulsed with HLA-A*0201 peptides for immunotherapy of metastatic hormone refractory prostate cancer.

Preclinical studies have suggested that purified populations of CD1c (BDCA-1) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use of CD1c BDC in patients with advanced metastatic hormone refractory prostate cancer. HLA-A*0201-positive patients with advanced metastatic prostate cancer were recruited and consented. The vaccine was manufactured by pulsing autologous CD1c BDC, prepared by magnetic bead immunoselection from apheresed peripheral blood mononuclear cells, with a cocktail of HLA-A*0201-restricted peptides (prostate-specific antigen, prostate acid phosphatase, prostate specific membrane antigen, and control influenza peptide) and keyhole limpet hemocyanin. The vaccine was administered intradermally or intravenously and peripheral blood was taken at predetermined intervals for clinical and immunologic monitoring. The vaccine was manufactured with a median purity of 82% CD1c BDC and administered successfully to 12 patients. Each patient received between 1 and 5 × 10 fresh CD1c BDC on day 0, followed by cryopreserved product in the same dose on days 28 and 56. The vaccine was well tolerated in all patients, with the most frequent adverse events being grade 1-2 fever, pain, or injection-site reactions. Vaccination with CD1c BDC is therefore feasible, safe, and well tolerated in patients with advanced-stage metastatic prostate cancer.

A flow cytometry based assay for the enumeration of regulatory T cells in whole blood.

The analysis of regulatory T cells (T-reg(s)) is becoming an increasingly important consideration in the development of novel immunotherapeutic strategies. Accurate quantification of T-regs during treatment protocols is crucial, particularly where the therapeutic strategy is targeting T-regs. The TruCOUNT™ method has utility for enumerating different immune cells but has not been used to detect T-regs. We have utilized this technology to develop an assay to enumerate human T-regs in whole blood, based on CD127 expression. The mean number of CD4(+)CD25(+)CD127(lo) T-regs per µl of whole blood was 48±16.9 with a range of 18 - 79 (n=22) and the average percentage was 6.1±1.9% (range 2.2-10.4%). The percentages of CD4(+)CD25(+)CD127(lo) T-regs were similar when detected in whole blood or density-gradient separated PBMC, and were comparable to those distinguished using the T-reg marker FoxP3. The assay was robust and reliable for enumeration of the lower frequency T-regs, with CV's for intra-assay repeatability and inter-assay precision of <9% and <35%, respectively. The CV's for the detection of total CD4(+) T lymphocytes using this assay were <2% for intra-assay repeatability and <18% for inter-assay precision, providing further evidence for reproducibility. This assay has a number of advantages over current methods, including small sample volume, the ability to determine absolute cell counts, and no need for hematology cell analyzers. This assay will simplify clinical trial immune monitoring and can be used to provide crucial data on patient T-reg numbers before, during, and after therapeutic interventions.

Manufacture of clinical grade human placenta-derived multipotent mesenchymal stromal cells.

Clinical grade human mesenchymal stem cells (MSC) are manufactured and used in clinical trials for a range of regenerative and inflammatory diseases. Human MSC have now been derived from tissues other than bone marrow, such as placenta, as described in this laboratory protocol. It provides instructions for clinical grade MSC manufacturing according to the Code of Good Manufacturing Practice (cGMP) principles and according to policies and procedures of our internal Quality Management System (QMS), which is based on the International Organization for Standardization (ISO) standard requirements. Relevant organizational structure and QMS elements are presented.

Biological diversity from a structurally diverse library: systematically scanning conformational space using a pyranose scaffold.

Success in discovering bioactive peptide mimetics is often limited by the difficulties in correctly transposing known binding elements of the active peptide onto a small and metabolically more stable scaffold while maintaining bioactivity. Here we describe a scanning approach using a library of pyranose-based peptidomimetics that is structurally diverse in a systematic manner, designed to cover all possible conformations of tripeptide motifs containing two aromatic groups and one positive charge. Structural diversity was achieved by efficient selection of various chemoforms, characterized by a choice of pyranose scaffold of defined chirality and substitution pattern. A systematic scanning library of 490 compounds was thus designed, produced, and screened in vitro for activity at the somatostatin (sst(1-5)) and melanin-concentrating hormone (MCH(1)) receptors. Bioactive compounds were found for each target, with specific chemoform preferences identified in each case, which can be used to guide follow-on drug discovery projects without the need for scaffold hopping.

Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials.

Mesenchymal stem cells (MSC) are being used increasingly in clinical trials for a range of regenerative and inflammatory diseases. Bone marrow is the traditional source but is relatively inaccessible in large volume. MSC have now been derived from tissues other than bone marrow including placenta and adipose tissue. We have used placenta obtained after delivery as a source of MSC and have been unable to detect any marked differences from marrow-derived MSC in terms of cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability. This report described our manufacturing process for isolating and expanding placenta-derived human MSC and their safe infusion into the first patient in a clinical trial program of human placenta-derived MSC.

Comparison of human placenta- and bone marrow-derived multipotent mesenchymal stem cells.

Bone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs), but placenta appears to be an alternative and more readily available source. This study comprehensively compared human placenta-derived MSC (hpMSC) and human bone marrow-derived MSC (hbmMSC) in terms of cell characteristics, optimal growth conditions and in vivo safety specifically to determine if hpMSC could represent a source of human MSC for clinical trial. MSC were isolated from human placenta (hpMSC) and human bone marrow (hbmMSC) and expanded ex vivo using good manufacturing practice-compliant reagents. hpMSC and hbmMSC showed similar proliferation characteristics in different basal culture media types, fetal calf serum (FCS) concentrations, FCS heat-inactivation experiments, flask types and media replacement responsiveness. However, hpMSC and hbmMSC differed with respect to their proliferation capabilities at different seeding densities, with hbmMSC proliferating more slowly than hpMSC in every experiment. hpMSC had greater long-term growth ability than hbmMSC. MSC from both sources exhibited similar light microscopy morphology, size, cell surface phenotype, and mesodermal differentiation ability with the exception that hpMSC consistently appeared less able to differentiate to the adipogenic lineage. A comparison of both hbmMSC and hpMSC from early and medium passage cultures using single-nucleotide polymorphism (SNP) GeneChip analysis confirmed GTG-banding data that no copy number changes had been acquired during sequential passaging. In three of three informative cases (in which the gender of the delivered baby was male), hpMSC were of maternal origin. Neither hpMSC nor hbmMSC caused any acute toxicity in normal mice when injected intravenously at the same, or higher, doses than those currently used in clinical trials of hbmMSC. This study suggests that human placenta is an acceptable alternative source for human MSC and their use is currently being evaluated in clinical trials.

Public access defibrillation: psychological consequences in responders.

BACKGROUND: Adverse psychological reactions are relatively frequent in professional ambulance crews who attend traumatic events, yet appear unusual in lay persons who attempt resuscitation of victims of out of hospital cardiac arrest. AIM: To investigate the psychological profile of first responders to gain insight into possible factors that might protect them against such reactions. METHODS: Qualitative study of first responders in a community scheme in Barry, South Wales. In depth semi-structured interviews with six subjects were analysed using Interpretive Phenomenological Analysis (IPA). RESULTS: The study identified a resilience phenomenon in first responders accounted for by certain enabling core beliefs about their role, their capacity, and about the meaning of negative and positive outcomes for themselves. A realistic appreciation of their own limitations, confidence in their ability to perform as trained and being able to handle positive and negative outcomes were prominent features. The ability to act with emotional detachment appears a further protective mechanism. This mindset, loosely described as 'a philosophy', protects against the development of adverse reactions to stress or from becoming unduly concerned about negative outcomes. The responders had altruistic motives for undertaking the role yet were capable of operating with a high degree of naturally occurring resilience to stress or undermining anxiety. It is the combination of being motivated by altruism coupled with an inherent resilience that appears to be the crucial protective mechanism. CONCLUSIONS: The group demonstrated an apparently innate resilience to the adverse psychological effects of responding with an AED in a PAD scheme. This enables them to operate optimally in stressful situations without experiencing the negative psychological consequences that might otherwise arise. This information may be used to raise awareness about the psychological requirements for the role and to assist screening or selection processes.

Points to Consider in Designing Mesenchymal Stem Cell-Based Clinical Trials.

SUMMARY: Therapeutic applications of cells are likely to increase greatly in the future. Cell and cell-based gene therapy manufacturing facilities need to be purpose-designed and accredited by their national medicinal regulatory body. Production scientists need to work in close tandem with quality assurance and ethics committees to absolutely ensure the safety of new cellular products. In this review, we consider the need for preclinical safety and efficacy data, tissue source for manufacture of clinical grade human mesenchymal stem cells, aseptic tissue processing, indemnification, and the role of the national medicinal regulatory body in appropriate clinical trial design.

Therapeutic applications of mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. They also have remarkable immunosuppressive properties. We describe their application in settings that include the cardiovascular, central nervous, gastrointestinal, renal, orthopaedic and haematopoietic systems. Manufacturing of MSC for clinical trials is also discussed. Since tissue matching between MSC donor and recipient does not appear to be required, MSC may be the first cell type able to be used as an "off-the-shelf" therapeutic product.