A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods.We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine

Characterization of cell lines derived from breast cancers and normal mammary tissues for the study of the intrinsic molecular subtypes

Five molecular subtypes (luminal A, luminal B, HER2-enriched, basal-like, and claudin-low) with clinical implications exist in breast cancer. Here, we evaluated the molecular and phenotypic relationships of (1) a large in vitro panel of human breast cancer cell lines (BCCLs), human mammary fibroblasts (HMFs), and human mammary epithelial cells (HMECs); (2) in vivo breast tumors; (3) normal breast cell subpopulations; (4) human embryonic stem cells (hESCs); and (5) bone marrow-derived mesenchymal stem cells (hMSC). First, by integrating genomic data of 337 breast tumor samples with 93 cell lines we were able to identify all the intrinsic tumor subtypes in the cell lines, except for luminal A. Secondly, we observed that the cell lines recapitulate the differentiation hierarchy detected in the normal mammary gland, with claudin-low BCCLs and HMFs cells showing a stromal phenotype, HMECs showing a mammary stem cell/bipotent progenitor phenotype, basal-like cells showing a luminal progenitor phenotype, and luminal B cell lines showing a mature luminal phenotype. Thirdly, we identified basal-like and highly migratory claudin-low subpopulations of cells within a subset of triple-negative BCCLs (SUM149PT, HCC1143, and HCC38). Interestingly, both subpopulations within SUM149PT were enriched for tumor-initiating cells, but the basal-like subpopulation grew tumors faster than the claudin-low subpopulation. Finally, claudin-low BCCLs resembled the phenotype of hMSCs, whereas hESCs cells showed an epithelial phenotype without basal or luminal differentiation. The results presented here help to improve our understanding of the wide range of breast cancer cell line models through the appropriate pairing of cell lines with relevant in vivo tumor and normal cell counterparts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-013-2743-3) contains supplementary material, which is available to authorized users

Distribution and diet of juvenile Patagonian toothfish on the South Georgia and Shag Rocks shelves (Southern Ocean)

The distribution and diet of juvenile (<750 mm) Patagonian toothfish are described from 4 annual trawl surveys (2003-06) around the island of South Georgia in the Atlantic sector of the Southern Ocean. Recruitment of toothfish varies inter-annually, and a single large cohort dominated during the four years surveyed. Most juveniles were caught on the Shag Rocks shelf to the NW of South Georgia, with fish subsequently dispersing to deeper water around both the South Georgia and Shag Rocks shelves. Mean size of juvenile toothfish increased with depth of capture. Stomach contents analysis was conducted on 795 fish that contained food remains and revealed that juvenile toothfish are essentially piscivorous, with the diet dominated by notothenid fish. The yellow-finned notothen, Patagonotothen guntheri, was the dominant prey at Shag Rocks whilst at South Georgia, where P. guntheri is absent, the dominant prey were Antarctic krill and notothenid fish. The diet changed with size, with an increase in myctophid fish and krill as toothfish grow and disperse. The size of prey also increased with fish size, with a greater range of prey sizes consumed by larger fish

Lanternfish (myctophidae) zoogeography off Eastern Australia: a comparison with physicochemical biogeography

In this first attempt to model the distributions of a mesopelagic fish family at this scale in the eastern Australian region (10°S to 57°S), lanternfish species occurrence data spanning a period from 1928 to 2010 were modelled against environmental covariates. This involved: (1) data collation and taxonomic quality checking, (2) classification of trawls into “horizontal” (presence-absence) and “oblique” (presence-only) types, and classification of vertical migration patterns using existing literature and the species occurrence database, (3) binomial GAMs using presence-absence data for representative temperate, subtropical and tropical species to examine depth interactions with environmental covariates and refine the selection of environmental layers for presence-only MAXENT models, (4) Presence-only MAXENT modelling using data from all trawls and the reduced environmental layers, and (5) Multivariate analysis (area-wise and species-wise) of the resulting matrix of logistic score by geographic pixel. We test the hypothesis that major fronts in the region (Tasman Front, Subtropical Convergence, Subantarctic Front) represent zoogeographic boundaries. A four-region zoogeographic scheme is hypothesised: Coral Sea region, Subtropical Lower Water region, Subtropical Convergence/South Tasman region and Subantarctic region. The Tasman Front, Subtropical Convergence and Subantarctic Front represented zoogeographic boundaries. An additional boundary at ~25°S (coined the ‘Capricorn’ boundary) was adopted to delineate the Coral Sea from Subtropical Lower Water regions. Lanternfish zoogeographic regions are congruent with some aspects of two prevailing physicochemical biogeographic schema in the region, but neither of these schema alone accurately predicts lanternfish distributions. As lanternfishes integrate vertical ocean processes, the hypothesised lanternfish zoogeography may represent a useful model for a generalised pelagic biogeography that should be tested for other oceanic groups

Microbioreactor arrays for full factorial screening of exogenous and paracrine factors in human embryonic stem cell differentiation

Timed exposure of pluripotent stem cell cultures to exogenous molecules is widely used to drive differentiation towards desired cell lineages. However, screening differentiation conditions in conventional static cultures can become impractical in large parameter spaces, and is intrinsically limited by poor spatiotemporal control of the microenvironment that also makes it impossible to determine whether exogenous factors act directly or through paracrine-dependent mechanisms. We detail here the development of a continuous flow microbioreactor array platform that combines full-factorial multiplexing of input factors with progressive accumulation of paracrine factors through serially-connected culture chambers, and further, the use of this system to explore the combinatorial parameter space of both exogenous and paracrine factors involved in human embryonic stem cell (hESC) differentiation to a MIXL1-GFP(+) primitive streak-like population. We show that well known inducers of primitive streak (BMP, Activin and Wnt signals) do not simply act directly on hESC to induce MIXL1 expression, but that this requires accumulation of surplus, endogenous factors; and, that conditioned medium or FGF-2 supplementation is able to offset this. Our approach further reveals the presence of a paracrine, negative feedback loop to the MIXL1-GFP(+) population, which can be overcome with GSK-3 beta inhibitors (BIO or CHIR99021), implicating secreted Wnt inhibitory signals such as DKKs and sFRPs as candidate effectors. Importantly, modulating paracrine effects identified in microbioreactor arrays by supplementing FGF-2 and CHIR in conventional static culture vessels resulted in improved differentiation outcomes. We therefore demonstrate that this microbioreactor array platform uniquely enables the identification and decoding of complex soluble factor signalling hierarchies, and that this not only challenges prevailing strategies for extrinsic control of hESC differentiation, but also is translatable to conventional culture systems