In vitro expansion of human cardiac progenitor cells: exploring 'omics tools for characterization of cell-based allogeneic products.

Human cardiac stem/progenitor cells (hCPCs) have been shown to be capable to regenerate contractile myocardium. However, because of their relative low abundance in the heart, in vitro expansion of hCPC is mandatory to achieve necessary quantities for allogeneic or autologous cardiac regeneration therapy applications (10(6)-10(9) cells/patient). Up to now, cell number requirements of ongoing phase I/IIa trials have been fulfilled with production in static monolayer cultures. However, this manufacturing process poses critical limitations when moving to the following clinical phases where hundreds of patients will be enrolled. For this, increased process yield is required, while guaranteeing the quality of the cell-based products. In this work, we developed and validated a robust, scalable, and good manufacturing practice (GMP)-compatible bioprocess for the expansion of high-quality hCPC. We applied platforms extensively used by the biopharmaceutical industry, such as microcarrier technology and stirred systems, and assessed culture conditions' impact on hCPC's quality and potency, as required by regulatory agencies. Complementary analytical assays including gene expression microarrays and mass spectrometry-based approaches were explored to compare transcriptome, proteome, surface markers, and secretion profiles of hCPC cultured in static monolayers and in stirred microcarrier-based systems. Our results show that stirred microcarrier-based culture systems enabled achieving more than 3-fold increase in hCPC expansion, when compared with traditional static monolayers, while retaining cell's phenotype and similar "omics" profiles. These findings demonstrate that this change in the production process does not affect cell's identity and quality, with potential to be translated into a transversal production platform for clinical development of stem-cell therapies.

Nitric oxide regulates clonal expansion and activation-induced cell death triggered by staphylococcal enterotoxin B.

Increased interest has recently been focused on nitric oxide (NO) due to its several biological roles. Apart from being a potential antimicrobial defense and a mediator of autoimmune diseases, NO also appears to be a strong mediator of T-cell responses. In this report, we have characterized the effect of NO on T-cell function. For this purpose, we analyzed in vivo T-cell responses to the bacterial superantigen produced by Staphylococcus aureus, staphylococcal enterotoxin B (SEB), in mice treated with an NO donor (isosorbide dinitrate [ISO]). We show that ISO partially prevents SEB-triggered activation-induced cell death of spleen and lymph node CD4Vbeta8+ T cells but not of CD8Vbeta8+ T cells. SEB-promoted thymic deletion is not abolished by ISO; however, a rapid recovery of thymocyte numbers due to increased double-positive (DP) CD4+ CD8+ thymocyte proliferation was clearly observed in ISO-treated, SEB-injected mice but not in controls (untreated SEB-injected mice). It was also found that ISO inhibits the early SEB-induced cell proliferation (i.e., that found 12 h after SEB injection), accelerating the clonal anergy usually observed 3 days after SEB injection. Inhibition of T-cell proliferation by the NO donor does not appear to be due to inhibition of cytokine production. These results show that NO interferes with apoptosis and facilitates thymic proliferation of DP thymocytes, although it inhibits peripheral T-cell proliferation.

T cell receptor-associated alpha-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck.

The activation of a phosphatidylinositol (PI) 3-kinase is one of the earliest intracellular responses to T lymphocyte stimulation. We have used a human T cell line, Jurkat, and an antibody that recognizes the clonotype of its T cell receptor (TcR) to characterize the association of PI 3-kinase with the TcR and its activation in response to TcR cross-linking. We show that the TcR is constitutively associated with the alpha isoform of the PI 3-kinase p85 regulatory subunit. Stimulation of Jurkat cells through the TcR results in the rapid and transient activation of the associated PI 3-kinase. In addition, our results indicate that the tyrosine kinase pp56lck is essential for PI 3-kinase activation via the TcR.

Induced photolysis of rabbit red blood cells by several photosensitizers.

The photolysis of rabbit red blood cells induced by several photosensitizers has been studied. Membrane photohemolysis was found to be dependent on light dose (J/cm2) for hematoporphyrin (HP), methylene blue (MB) and toluidine blue (TB). No significant hemolysis was detected when mesotetra (4N-methylpyridyl) porphine (T4MPyP) plus light was used. Sodium azide inhibited, whereas deuterium oxide potentiated membrane lysis with HP, MB and TB. In the case of T4MPyP no differences were observed when using sodium azide or deuterium oxide.

Photodynamic induction of DNA-protein cross-linking in solution by several sensitizers and visible light.

The combined effect of several sensitizers and light on H2O or D2O solutions of DNA-histone complexes, as well as the significance of singlet oxygen (1O2), in this photosensitizing reaction has been studied. On H2O solutions, the production of 1O2, as well as the formation of DNA-protein cross-links (DPCs), were found to be dependent on light dose for all the sensitizers. Mesotetra (4N-methylpyridyl) porphine (T4MPyP), methylene blue (MB), and toluidine blue (TB) were the best photosensitizers with regard to tryptophan photolysis, followed by hematoporphyrin (HP), thioflavine T (TT), and pyronin G (PG). The formation of DPCs showed high initial rates, reaching a plateau at doses over 90 J/cm2. Under these irradiation conditions, the percentage of DPCs induced by the sensitizers decreases in the order T4MPyP > MB > TB >> HP approximately TT >> PG (approximately 0). These DPCs were totally destroyed with proteinase K (15 micrograms/ml). The irradiation of the DNA-histone-sensitizer solutions in the presence of L-carnosine (5 x 10(-4) M) produced approximately a 50% of DPCs inhibition for T4MPyP, MB, and TB, and a total inhibition for HP, TT, and PG. The substitution of H2O by D2O as solvent significantly increased the photodegradation of tryptophan, as well as the photoinduction of DPCs by the sensitizers. The results obtained indicate that singlet oxygen is the main agent responsible in the DNA-protein cross-linking formation.