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Excitation-contraction (EC) coupling is the coordinated process by which an action potential triggers cardiac myocyte contraction. EC coupling is initiated in dyads where the junctional sarcoplasmic reticulum (jSR) is in tight proximity to the sarcolemma of cardiac myocytes. Existing models of EC coupling critically depend on dyad stability to ensure the fidelity and strength of EC coupling, where even small variations in ryanodine receptor channel and voltage-gated calcium channel-α 1.2 subunit separation dramatically alter EC coupling. However, dyadic motility has never been studied. Here, we developed a novel strategy to track specific jSR units in dissociated adult ventricular myocytes using photoactivatable fluorescent proteins. We found that the jSR is not static. Instead, we observed dynamic formation and dissolution of multiple dyadic junctions regulated by the microtubule-associated molecular motors kinesin-1 and dynein. Our data support a model where reproducibility of EC coupling results from the activation of a temporally averaged number of SR Ca2+ release units forming and dissolving SR-sarcolemmal junctions. These findings challenge the long-held view that the jSR is an immobile structure and provide insights into the mechanisms underlying its motility.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are at the center of new cell-based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC-CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse-tubules (T-tubules) with adult-like Ca2+ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC-CMs promotes T-tubules formation, facilitates CaV 1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca2+ ]i imaging, and super resolution microscopy, we found that BIN1 expression induced T-tubule development in hESC-CMs, while increasing differentiation toward a more ventricular-like phenotype. Voltage-gated CaV 1.2 channels clustered along the surface sarcolemma and T-tubules of hESC-CM. The length and width of the T-tubules as well as the expression and size of CaV 1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased CaV 1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1-expressing cells had more CaV 1.2-ryanodine receptor junctions than control cells. This was associated with larger [Ca2+ ]i transients during excitation-contraction coupling. Our data support the view that BIN1 is a key regulator of T-tubule formation and CaV 1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC-CMs, we show that BIN1 is also important for CaV 1.2 channel clustering, junctional SR organization, and the establishment of excitation-contraction coupling. Stem Cells 2019;37:54-64.

The article focuses on the restoration project of four paintings by Bernardo Bitti SJ (1548-1610) at the Museum of the Cathedral of Sucre in Bolivia. Topics include the analysis of Bitti's artistic technique and materials, historical significance of his works in Jesuit evangelization, and the international collaboration of experts leading the project. The initiative, led by UNIR, also aims to raise awareness of Bitti's contributions to Jesuit and Andean art, both locally and globally.

Purpose: Transplantation ofin vitrocultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. Materials and methods: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. Results: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except forABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. Conclusion: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure. [ABSTRACT FROM PUBLISHER]

Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are at the center of new cell-based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC-CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse-tubules (T-tubules) with adult-like Ca 2+ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC-CMs promotes T-tubules formation, facilitates Ca V 1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca 2+ ] i imaging, and super resolution microscopy, we found that BIN1 expression induced T-tubule development in hESC-CMs, while increasing differentiation toward a more ventricular-like phenotype. Voltage-gated Ca V 1.2 channels clustered along the surface sarcolemma and T-tubules of hESC-CM. The length and width of the T-tubules as well as the expression and size of Ca V 1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased Ca V 1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1-expressing cells had more Ca V 1.2-ryanodine receptor junctions than control cells. This was associated with larger [Ca 2+ ] i transients during excitation-contraction coupling. Our data support the view that BIN1 is a key regulator of T-tubule formation and Ca V 1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC-CMs, we show that BIN1 is also important for Ca V 1.2 channel clustering, junctional SR organization, and the establishment of excitation-contraction coupling. Stem Cells 2019;37:54-64., (© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Purpose: Corneal epithelium is maintained by a population of stem cells (SCs) that have not been identified by specific molecular markers. The objective of this study was to find new putative markers for these SCs and to identify associated molecular pathways., Methods: Real time PCR (rt-PCR) was performed in 24 human limbal and central corneal epithelial samples to evaluate the gene expression profile of known corneal epithelial SC-associated markers. A pool of those samples was further analyzed by a rt-PCR array (RT²-PCR-A) for 84 genes related to the identification, growth, maintenance, and differentiation of SCs., Results: Cells from the corneal epithelium SC niche showed significant expression of ATP-binding cassette sub-family G member 2 (ABCG2) and cytokeratin (KRT)15, KRT14, and KRT5 genes. RT²-PCR-A results indicated an increased or decreased expression in 21 and 24 genes, respectively, in cells from the corneal SC niche compared to cells from the central corneal epithelium. Functional analysis by proprietary software found 4 different associated pathways and a novel network with the highest upregulated genes in the corneal SC niche. This led to the identification of specific molecules, chemokine (C-X-C motif) ligand 12 (CXCL12), islet-1 transcription factor LIM/homeodomain (ISL1), collagen-type II alpha 1 (COL2A), neural cell adhesion molecule 1 (NCAM1), aggrecan (ACAN), forkhead box A2 (FOXA2), Gap junction protein beta 1/connexin 32 (GJB1/Cnx32), and Msh homeobox 1 (MSX1), that could be used to recognize putative corneal epithelial SCs grown in culture and intended for transplantation. Other molecules, NCAM1 and GJB1/Cnx32, potentially could be used to positively purify them, and Par-6 partitioning defective 6 homolog alpha (PARD6A) to negatively purify them., Conclusions: Knowledge of these gene and molecular pathways has provided a better understanding of the signaling molecular pathways associated with progenitor-rich limbal epithelium. This knowledge potentially could give support to the design and development of innovative therapies with the potential to reverse corneal blindness arising from ocular surface failure.