Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.

Rostrocaudal patterning and neural crest differentiation of human pre-neural spinal cord progenitors in vitro.

The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by WNT/fibroblast growth factor (FGF) signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which compromises production of the full range of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity for 7-10 passages, dividing to self-renew and to make neural crest progenitors, while gradually adopting a more posterior identity by activating colinear HOX gene expression. The HOX clock can be halted through GDF11-mediated signal inhibition to produce a PNP and NC population with a thoracic identity that can be maintained for up to 30 passages.

Reverse iontophoresis of amino acids: identification and separation of stratum corneum and subdermal sources in vitro.

PURPOSE: To differentiate the stratum corneum (SC) and subdermal sources of amino acids (AAs) extracted by reverse iontophoresis. METHODS: 13 zwitterionic AAs were quantified in this in vitro study. Repetitive tape-stripping permitted the distribution of the analytes to be determined in the SC. Iontophoresis experiments were performed in which the subdermal chamber contained either phosphate-buffered saline (PBS) only, or a mixture of the 13 AAs in PBS. RESULTS: AAs were homogeneously distributed across the SC and broadly divided into three groups (high, medium, low) in terms of total amount present. As expected, extraction to the cathode for the essentially neutral analytes involved was more efficient. Initial samples obtained during the first hour of iontophoresis primarily extracted AAs from the SC. The fluxes observed in the latter half of the 6-h experiment, on the other hand, correlated well with the corresponding subdermal concentrations. CONCLUSION: A relatively short extraction period (approximately 1 h) by reverse iontophoresis can be used to evaluate the content of AAs in the SC. Once this 'reservoir' has been depleted, reverse iontophoresis can then monitor the subdermal concentrations of the AAs. The latter appears most useful for compounds which are present at lower levels in the SC.

Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state.

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro Amongst these are the pluripotency-linked genes Klf4 and Lin28b The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.

Controlled release of the fibronectin central cell binding domain from polymeric microspheres.

Non-ionic surfactants have been employed as alternatives to PVA for the emulsification-encapsulation of a conformationally labile protein (FIII9'-10) into PLGA microspheres. FIII9'-10 was encapsulated using a w/o/w double emulsification-evaporation technique and the microspheres fabricated were characterized by SEM and CLSM. The peptide backbone integrity of FIII9'-10 was assayed by SDS-PAGE and the degree of unfolding of FIII9'-10 following emulsification-encapsulation was assessed using a fibroblast cell-attachment assay. The encapsulation efficiency for FIII9'-10 was 25% when using PVA, compared to 50-60% when using Igepal CA-630 or Triton-X100, with values below for the other surfactants. FIII9'-10 released from microspheres promoted cell attachment in a concentration-dependent manner, only Igepal CA-630 and Triton X-100 maintaining near-maximal cell attachment, indicating that the conformation of the relatively unstable FIII9' domain was preserved. All non-ionic surfactants reduced microsphere surface porosity, compared to PVA, and an increasing surface rugosity (leading to minor 'ridges') could be correlated with decreasing surfactant HLB. Low surface porosities did not effect the diffusion of FIII9'-10 from the microspheres' internal pores in a 'burst release', as may have been imagined. In summary, non-ionic surfactants should be considered over PVA for the maintenance of biological activity of conformationally labile proteins during encapsulation.

Increased stiffness and cell-matrix interactions of abdominal aorta in two experimental nonhypertensive models: long-term chemically sympathectomized and sinoaortic denervated rats.

RATIONALE: Sinoaortic denervated (SAD) and chemically sympathectomized (SNX) rats are characterized by a decrease in arterial distensibility without hypertension and would, thus, be relevant for analyzing arterial wall stiffening independently of blood pressure level. The fibronectin network, which plays a pivotal role in cell-matrix interactions, is a major determinant of arterial stiffness. We hypothesized that in SAD and SNX rats, arterial stiffness is increased, due to alterations of cell-matrix anchoring leading to spatial reorganization of the extracellular matrix. METHODS: The intrinsic elastic properties of the arterial wall were evaluated in vivo by the relationship between incremental elastic modulus determined by echotracking and circumferential wall stress. The changes of cell-extracellular matrix links in the abdominal aorta were evaluated by studying fibronectin, vascular integrin receptors, and ultrastructural features of the aorta by immunochemistry. RESULTS: In both experimental conditions, wall stiffness increased, associated with different modifications of cell-extracellular matrix adhesion. In SAD rats, increased media cross-sectional area was coupled with an increase of muscle cell attachments to its extracellular matrix via fibronectin and its α5-ß1 integrin. In SNX rats, reduced media cross-sectional area was associated with upregulation of αv-ß3 integrin and more extensive connections between dense bands and elastic fibers despite the disruption of the elastic lamellae. CONCLUSION: In aorta of SNX and SAD rats, a similar arterial stiffness is associated to different structural alterations. An increase in αvß3 or α5ß1 integrins together with the already reported increase in the proportion of less distensible (collagen) to more distensible (elastin) components in both models contributes to remodeling and stiffening of the abdominal aorta.