Pharmacological and molecular characterization of functional P2 receptors in rat embryonic cardiomyocytes.

Purinergic receptors activated by extracellular nucleotides (adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP)) are well known to exert physiological effects on the cardiovascular system, whether nucleotides participate functionally in embryonic heart development is not clear. The responsiveness of embryonic cardiomyocytes (E) 12 to P2 receptor agonists by measuring Ca(2+) influx did not present response to ATP, but responses to P2 agonists were detected in cardiomyocytes taken from E14 and E18 rats. Photometry revealed that the responses to ATP were concentration-dependent with an EC50 of 1.32 µM and 0.18 µM for E14 and E18 cardiomyocytes, respectively. In addition, other P2 agonists were also able to induce Ca(2+) mobilization. RT-PCR showed the presence of P2X2 and P2X4 receptor transcripts on E14 cardiomyocytes with a lower expression of P2X3 and P2X7 receptors. P2X1 and a low level of P2X5 receptor messenger RNA (mRNA) were also expressed at E18. Immunofluorescence data indicated that only P2X2 and P2X4 receptor proteins were expressed in E14 cardiomyocytes while protein for all the P2X receptor subtypes was expressed in E18, except for P2X3 and P2X6. Responses mediated by agonists specific for P2Y receptors subtypes showed that P2Y receptors (P2Y1, P2Y2, P2Y4 and P2Y6) were also present in both E14 and E18 cardiomyocytes. Dye transfer experiments showed that ATP induces coupling of cells at E12, but this response is decreased at E14 and lost at E18. Conversely, UTP induced coupling with five or more cells in most cells from E12 to E18. Our results show that specific P2 receptor subtypes are present in embryonic rat cardiomyocytes, including P2X7 and P2Y4 receptors that have not been identified in adult rat cardiomyocytes. The responsiveness to ATP stimulation even before birth, suggests that ATP may be an important messenger in embryonic as well as in adult hearts.

Pilot safety study of intrabronchial instillation of bone marrow-derived mononuclear cells in patients with silicosis.

BACKGROUND: Silicosis is an occupational disease for which no effective treatment is currently known. Systemic administration of bone marrow-derived mononuclear cells (BMDMCs) has shown to be safe in lung diseases. However, so far, no studies have analyzed whether bronchoscopic instillation of autologous BMDMCs is a safe route of administration in patients with silicosis. METHODS: We conducted a prospective, non-randomized, single-center longitudinal study in five patients. Inclusion criteria were age 18-50 years, chronic and accelerated silicosis, forced expiratory volume in 1 s <60 % and >40 %, forced vital capacity ≥60 % and arterial oxygen saturation >90 %. The exclusion criteria were smoking, active tuberculosis, neoplasms, autoimmune disorders, heart, liver or renal diseases, or inability to undergo bronchoscopy. BMDMCs were administered through bronchoscopy (2 × 10(7) cells) into both lungs. Physical examination, laboratory evaluations, quality of life questionnaires, computed tomography of the chest, lung function tests, and perfusion scans were performed before the start of treatment and up to 360 days after BMDMC therapy. Additionally, whole-body and planar scans were evaluated 2 and 24 h after instillation. RESULTS: No adverse events were observed during and after BMDMC administration. Lung function, quality of life and radiologic features remained stable throughout follow-up. Furthermore, an early increase of perfusion in the base of both lungs was observed and sustained after BMDMC administration. CONCLUSION: Administration of BMDMCs through bronchoscopy appears to be feasible and safe in accelerated and chronic silicosis. This pilot study provides a basis for prospective randomized trials to assess the efficacy of this treatment approach. CLINICAL TRIALS. GOV IDENTIFIER: NCT01239862 Date of Registration: November 10, 2010.

Functional properties of a Brazilian derived mouse embryonic stem cell line.

Pluripotent mouse embryonic stem cells (mESC) are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos. Since ion channel modulation has been reported to interfere with both growth and differentiation process in mouse and human ESC it is important to characterize the electrophysiological properties of newly generated mESC and compare them to other lines. In this work, we studied the intercellular communication by way of gap junctions in a Brazilian derived mESC (USP-1, generated by Dr. Lygia Pereira's group) and characterized its electrophysiological properties. We used immunofluorescence and RT-PCR to reveal the presence of connexin 43 (Cx43), pluripotency markers and ion channels. Using a co-culture of neonatal mouse cardiomyocytes with mESC, where the heart cells expressed the enhanced Green Fluorescent Protein, we performed dye injections to assess functional coupling between the two cell types observing dye diffusion. The patch-clamp study showed outward currents identified as two types of potassium currents, transient outward potassium current (Ito) and delayed rectifier outward potassium current (Iks), by use of specific drug blockage. Calcium or sodium currents in undifferentiated mESC were not identified. We conclude that USP-1 mESC has functional Cx43 channels establishing intercellular communication among themselves and with cardiomyocytes and has a similar electrophysiological profile compared to other mESC cell lines.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation.

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal ß-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed ß-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells.

The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific 'undifferentiation' markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-ß1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.

Human Menstrual Blood-Derived Mesenchymal Cells as New Human Feeder Layer System for Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) in general require coculture with feeder layers in order to remain undifferentiated. However, the use of animal-derived feeder layers is incompatible with the clinical setting. The objective of this work was to investigate whether human menstrual blood-derived mesenchymal cells (MBMCs) can substitute mouse embryonic fibroblasts (MEFs) as a feeder layer for H9-hESCs. Both feeder cell types were isolated and cultured in DMEM F-12 and high glucose DMEM, respectively. After three passages, they were inactivated with mitomycin C. To test MBMC feeder layer capacity, hESCs were grown over MBMCs and MEFs under standard conditions. hESC growth, proliferation, survival, and maintenance of the undifferentiated state were evaluated. hESCs grown over MBMCs preserved their undifferentiated state presenting standard morphology, expressing alkaline phosphatase, transcription factors OCT3/4, SOX2, and NANOG by RT-PCR and SSEA-4 and OCT3/4 by immunofluorescence assays. It is noteworthy that none of the feeder cells expressed these proteins. The average colony size of the hESCs on MBMCs was higher when compared to MEFs (p < 0.05; mean ± SD, n = 3). Growth factor analysis revealed amplification of the transcripts for FGF-2, BMP4, TGF-ß, VEGF, and PEDF by RT-PCR in MBMCs and MEFs before and after inactivation. Furthermore, similar embryoid body formation, size, and morphology were observed in both feeder layers. In addition, EBs expressed marker genes for the three germ layers cultured on both feeder cells. In conclusion, MBMCs are able to maintain hESCs in an undifferentiated state with comparable efficiency to MEFs. Therefore, MBMCs are a suitable alternative to animal-derived feeder layers for growing hESCs.

Lycopene and beta-carotene induce growth inhibition and proapoptotic effects on ACTH-secreting pituitary adenoma cells.

Pituitary adenomas comprise approximately 10-15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27(kip1) in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27(kip1) in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27(kip1); and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing's disease.

Biodistribution of bone marrow mononuclear cells after intra-arterial or intravenous transplantation in subacute stroke patients.

AIMS: To assess the biodistribution of bone marrow mononuclear cells (BMMNC) delivered by different routes in patients with subacute middle cerebral artery ischemic stroke. PATIENTS & METHODS: This was a nonrandomized, open-label Phase I clinical trial. After bone marrow harvesting, BMMNCs were labeled with technetium-99m and intra-arterially or intravenously delivered together with the unlabeled cells. Scintigraphies were carried out at 2 and 24 h after cell transplantation. Clinical follow-up was continued for 6 months. RESULTS: Twelve patients were included, between 19 and 89 days after stroke, and received 1-5 × 10(8) BMMNCs. The intra-arterial group had greater radioactive counts in the liver and spleen and lower counts in the lungs at 2 and 24 h, while in the brain they were low and similar for both routes. CONCLUSION: BMMNC labeling with technetium-99m allowed imaging for up to 24 h after intra-arterial or intravenous injection in stroke patients.

Human menstrual blood-derived mesenchymal cells as a cell source of rapid and efficient nuclear reprogramming.

Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes-OCT4, KLF4, SOX2, and c-MYC-in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2, and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days of posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2-5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.

Transcriptomic alterations in Trypanosoma cruzi-infected cardiac myocytes.

Trypanosoma cruzi infection is a major cause of cardiomyopathy. Previous gene profiling studies of infected mouse hearts have revealed prominent changes in gene expression within many functional pathways. This variety of transcriptomic changes in infected mice raises the question of whether gene expression alterations in whole hearts are due to changes in infected cardiac myocytes or other cells or even to systemic effects of the infection on the heart. We employed microarrays to examine infected cardiac myocyte cultures 48 h post-infection. Statistical comparison of gene expression levels of 7624 well annotated unigenes in four independent cultures of infected and uninfected myocytes detected substantial (>or=1.5 absolute fold changes) in 420 (5.5%) of the sampled genes. Major categories of affected genes included those involved in immune response, extracellular matrix and cell adhesion. These findings on infected cardiac myocytes in culture reveal that alterations in cardiac gene expression described in Chagas disease are the consequence of both direct infection of the myocytes themselves as well as resulting from the presence of other cell types in the myocardium and systemic effects of infection.