Healthy human CSF promotes glial differentiation of hESC-derived neural cells while retaining spontaneous activity in existing neuronal networks.

The possibilities of human pluripotent stem cell-derived neural cells from the basic research tool to a treatment option in regenerative medicine have been well recognized. These cells also offer an interesting tool for in vitro models of neuronal networks to be used for drug screening and neurotoxicological studies and for patient/disease specific in vitro models. Here, as aiming to develop a reductionistic in vitro human neuronal network model, we tested whether human embryonic stem cell (hESC)-derived neural cells could be cultured in human cerebrospinal fluid (CSF) in order to better mimic the in vivo conditions. Our results showed that CSF altered the differentiation of hESC-derived neural cells towards glial cells at the expense of neuronal differentiation. The proliferation rate was reduced in CSF cultures. However, even though the use of CSF as the culture medium altered the glial vs. neuronal differentiation rate, the pre-existing spontaneous activity of the neuronal networks persisted throughout the study. These results suggest that it is possible to develop fully human cell and culture-based environments that can further be modified for various in vitro modeling purposes.

Comparative analysis of targeted differentiation of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells reveals variability associated with incomplete transgene silencing in retrovirally derived hiPSC lines.

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

Sildenafil after cardiac arrest and infarction; an experimental rat model.

OBJECTIVES: Resuscitation after cardiac arrest may lead to ischemia-reperfusion injury and infarction. We evaluated whether sildenafil, a phosphodiesterase-5 inhibitor, has an impact on recovery after cardiac arrest in a rat cardiac transplantation model. DESIGN: Sixty-one Fischer344 rats underwent syngeneic heterotopic cardiac transplantation after ischemia and ligation of the left anterior coronary artery of the heart to yield myocardial infarction (IRI + MI). Of these, 22 rats received subcutaneously injected sildenafil (1 mg/kg/day) (IRI +MI + S). Twenty-three additional grafted animals with transplantation only served as controls with ischemia reperfusion injury (IRI). After 2 days, immunohistochemistry for eNOS, and RT-PCR for iNOS and Aquaporin-7 were performed after graft harvesting and histology. RESULTS: Two days after transplantation, remote intramyocardial arteries were more preserved in IRI + MI + S as compared with IRI +MI and IRI (0.74 ± 0.14, 0.56 ± 0.23 and 0.55 ± 0.22, PSU, p < 0.05, respectively). Decreased eNOS staining confirmed the presence of developing infarction in IRI + MI and IRI + MI + S. The expression of iNOS was significantly lower during IRI + MI +S as compared with IRI + MI (0.02 ± 0.01 and 1.02 ± 0.02, FC, p < 0.05). CONCLUSIONS: Administered at the onset of reperfusion and developing infarction, sildenafil has an impact on myocardial recovery after cardiac arrest and ischemia.

Chemical and topographical patterning of hydrogels for neural cell guidance in vitro.

This review focuses on hydrogels and their patterning techniques in relation to central nervous system applications, with emphasis on synthetic and natural materials and chemical and topographical patterning techniques. We describe the properties of hydrogel materials and various techniques used in hydrogel patterning methods. Also, the applicability and utilization of patterned hydrogels with neural cells is discussed. Surface chemistry and topography significantly affect cell behaviour, including cell attachment, migration and maturation. Although several patterning techniques are described in the literature, a review of techniques applicable to hydrogel materials is needed. Use of these patterned cell-hydrogel constructs might provide novel ways to treat central nervous system deficits in the future.

Aquaporin-7 expression during coronary artery bypass grafting with diazoxide.

OBJECTIVES: Aquaporin-7 is a water-channel protein that controls tissue glycerol supply after ischemia. A burden of experimental studies suggests that diazoxide, a mitochondrial K(ATP)-channel opener, may decrease myocardial edema during coronary artery bypass grafting (CABG). We evaluated whether diazoxide has an impact on atrial aquaporin-7 expression during CABG. DESIGN: Sixteen patients with a history of stable coronary artery disease were enrolled in the study. Eight patients were treated during cardiopulmonary bypass with diazoxide, while the rest eight patients remained as controls. Histopathology was evaluated from biopsies procured before and during CABG from the right atrium. From fresh atrial tissue biopsies, Aquaporin-7 was quantified by RT-PCR. RESULTS: Histological differences were apparent between individual patients already before operation at base line reflecting differences in severity of myocardial ischemia. As compared with fold change values before operation, Aquaporin-7 expression after operation was positive in all but one control, whereas aquaporin-7 expression was positive in only two patients receiving diazoxide. The relative aquaporin-7 expression was significantly lower in patients treated with diazoxide as compared with controls (p < 0.05). CONCLUSIONS: Diazoxide may have an impact on myocardial water balance and glycerol energy supply by decreasing relative aquaporin-7 expression during CABG.

Human cell-based micro electrode array platform for studying neurotoxicity.

At present, most of the neurotoxicological analyses are based on in vitro and in vivo models utilizing animal cells or animal models. In addition, the used in vitro models are mostly based on molecular biological end-point analyses. Thus, for neurotoxicological screening, human cell-based analysis platforms in which the functional neuronal networks responses for various neurotoxicants can be also detected real-time are highly needed. Microelectrode array (MEA) is a method which enables the measurement of functional activity of neuronal cell networks in vitro for long periods of time. Here, we utilize MEA to study the neurotoxicity of methyl mercury chloride (MeHgCl, concentrations 0.5-500 nM) to human embryonic stem cell (hESC)-derived neuronal cell networks exhibiting spontaneous electrical activity. The neuronal cell cultures were matured on MEAs into networks expressing spontaneous spike train-like activity before exposing the cells to MeHgCl for 72 h. MEA measurements were performed acutely and 24, 48, and 72 h after the onset of the exposure. Finally, exposed cells were analyzed with traditional molecular biological methods for cell proliferation, cell survival, and gene and protein expression. Our results show that 500 nM MeHgCl decreases the electrical signaling and alters the pharmacologic response of hESC-derived neuronal networks in delayed manner whereas effects can not be detected with qRT-PCR, immunostainings, or proliferation measurements. Thus, we conclude that human cell-based MEA platform is a sensitive online method for neurotoxicological screening.