Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

Exogenous DNA length and quantity affect the transfection rate, but not sperm viability during sperm-mediated gene transfer

Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mechanisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 × 106 cells/mL) were incubated for 1 h at 38.5 °C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p ˂ 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to influence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to increase SMGT protocols, without altering the sperm viability.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

BACKGROUND: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. METHODS AND RESULTS: Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of ß1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. CONCLUSIONS: Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.

The relationship among HOXA10, estrogen receptor α, progesterone receptor, and progesterone receptor B proteins in rectosigmoid endometriosis: a tissue microarray study.

BACKGROUND: Very few studies have evaluated the expression of homeobox A10 (HOXA10) and steroid (estrogen and progesterone) receptors exclusively in deep endometriosis. Conclusions drawn from studies evaluating peritoneal and ovarian endometriosis are usually generalized to explain the pathogenesis of the disease as a whole. We aimed to evaluate the expression of HOXA10, estrogen receptor α (ER-α), progesterone receptor (PR), and PR-B in rectosigmoid endometriosis (RE), a typical model of deep disease. METHODS: We used RE samples from 18 consecutive patients to construct tissue microarray blocks. Nine patients each were operated during the proliferative and secretory phases of the menstrual cycle. We quantified the expressions of proteins by immunohistochemistry using the modified Allred score. RESULT: The HOXA10 was expressed in the stroma of nodules during the secretory phase in 5 of the 18 patients. Expression of ER-α (in 16 of 18 patients), PR (in 17 of 18 patients), and PR-B (17 of 18 patients) was moderate to strong in the glands and stroma of nodules during both phases. Expression of both PR (P = .023) and PR-B (P = .024) was significantly greater during the secretory phase. CONCLUSION: The HOXA10 is expressed in RE, where it likely imparts the de novo identity of endometriotic lesions. The ER-α, PR, and PR-B are strongly expressed in RE, which differs from previous studies investigating peritoneal and ovarian lesions. This suggests different routes of pathogenesis for each of the 3 types of endometriosis.

Kinetics of changes in plasma membrane related to apoptosis and necrosis in bovine sperm cells at different incubation times

Incubation time induce damages in sperm cells by necrosis and/or apoptosis. The aim of this study was the evaluation of changes in plasma membrane related to apoptosis and necrosis in bovine sperm cells through 2 hours of incubation. Sperm cells were incubated at 5% (v/v) CO subscribed to 2 in air for 0, 30, 60, 90 and 120 minutes. After each period, sperm cells were incubated with fluorescent probes Yo-pro and propidium iodide (PI) to detect change in plasma membrane related to apoptosis and necrosis respectively. Using Yo-pro/PI assay, three different subpopulations of sperm cells were detected by flow cytometry: a) necrotic sperm cells (PI superscript + and Yo-pro superscript to -/+); b) apoptotic sperm cells (Yo-pro superscript to+ and PI superscript to-) and c) living cells (Yo-pro superscript to- and PI superscript to-). The percentage of live cells (plasma membrane integrity) significantly decreases over 2 hour of incubation, on the other hand, the percentage of necrotic and apoptotic cells increase during incubation. Changes in plasma membrane integrity were correlated to incubation time. While live cells were negatively correlated with the increase of incubation time, necrosis and apoptosis were positively correlated. It was also observed that necrosis was the main damage in sperm cells in all incubation times. In conclusion, incubation time induces changes in plasma membrane integrity related to necrosis and apoptosis, whether necrosis is present in higher quantity in all incubation times.

O tempo de incubação causa danos nas células espermáticas relacionados a necrose e/ou apoptose. O objetivo deste estudo foi avaliar as mudanças na membrana plasmática relacionadas a apoptose e necrose em espermatozóides bovinos durante 2 horas de incubação. Os espermatozóides foram incubados a 5% (v/v) CO subscrito por 2 em ar por 0, 30, 60, 90 e 120 minutes. Depois de cada periodo, as células espermáticas foram incubadas com as sondas fluorescentes Yo-pro e iodito de propideo (PI) para detectar mudanças na membrana plasmática relacionadas a apoptose e a necrose, respectivamente. Usando Yo-pro/PI assay, três subpopulações diferentes de células espermáticas são detectadas pelo citômetro de fluxo: a) células espermáticas em necrose (PI elevado a+ and Yo-pro elevado a-/+); b) células espermáticas em apoptose (Yo-pro elevado a+ and PI elevado a-) e c) células espermáticas vivas (Yo-pro- and PI elevado a-). A porcentagem de células vivas (membrana plasmática integra) significativamente diminui durante 2 horas de incubação, por outro lado, a porcentagem de espermatozóides em necrose e apoptose aumentaram durante a incubação. As mudanças na integridade da membrana plasmática foram correlacionadas com o tempo de incubação. Enquanto as células vivas foram correlacionadas negativamente com o aumento do tempo de incubação, necrose e apoptose foram correlacionadas positivamente. Também foi observado que necrose foi o principal dano causado pelo tempo de incubação nas células espermáticas. Conclui-se que o tempo de incubação causa alteração na integridade da membrana plasmática relacionadas a necrose e apoptose nas células espermáticas, sendo que necrose foi observada em maior quantidade em todos os tempos de incubação.