Theranostic barcoded nanoparticles for personalized cancer medicine.

Personalized medicine promises to revolutionize cancer therapy by matching the most effective treatment to the individual patient. Using a nanoparticle-based system, we predict the therapeutic potency of anticancer medicines in a personalized manner. We carry out the diagnostic stage through a multidrug screen performed inside the tumour, extracting drug activity information with single cell sensitivity. By using 100 nm liposomes, loaded with various cancer drugs and corresponding synthetic DNA barcodes, we find a correlation between the cell viability and the drug it was exposed to, according to the matching barcodes. Based on this screen, we devise a treatment protocol for mice bearing triple-negative breast-cancer tumours, and its results confirm the diagnostic prediction. We show that the use of nanotechnology in cancer care is effective for generating personalized treatment protocols.

Variability in progeny production and virulence of cyanophages determined at the single-cell level.

Little information regarding viral progeny production (burst size) and host mortality (viral virulence) is currently available for environmentally relevant phages. This is partially due to the difficulty in accurately measuring these infection properties with existing methods. Here, we set up a simple system for determining viral virulence and burst size at the single-cell level following flow cytometric separation of infected cells. We applied this assay to two distinct cyanomyoviruses, Syn9 and S-TIM5, during infection of two marine Synechococcus strains each. We found that virulence ranged from 44%-82%, differing for the same phage on different hosts. Average burst sizes ranged from 21-43 infective viruses/cell, and differed with host for Syn9, whereas the burst size of S-TIM5 was similar for both hosts. In addition, virulence and burst sizes were different for the two phages when infecting their common host. Furthermore, wide-ranging cell-to-cell variability was found for single-cell burst sizes in each of the four interactions, ranging from 2 to over 100 infective viruses/cell. This variability, discerned at both the population and single-cell levels under controlled laboratory conditions, is likely to be much more complex in natural environments.

The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications.

Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells.

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS-derived cardiomyocytes), in the present study we investigated in iPS-derived cardiomyocytes, the functional properties related to [Ca(2+) ](i) handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca(2+) release to contraction and the b-adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Our major findings showed that iPS-derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force-frequency relations and mild (compared to adult) post-rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR-Ca(2+) handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF-derived iPS, the functional properties related to excitation-contraction coupling, resemble in part those of adult cardiomyocytes.

A pure population of ectodermal cells derived from human embryonic stem cells.

Embryonic stem (ES) cells represent a unique cellular model to recapitulate in vitro early steps of embryonic development and an unlimited cellular source in therapy for many diseases, as well as targets for drug discovery and toxicology screens. Although previous studies have reported epidermal differentiation of mouse and human embryonic stem (huES) cells, the heterogeneity of the resulting cell culture impairs the evaluation of differentiated cells for cell therapy. We report here the reproducible isolation of a homogenous ectodermal cell population, IT1, from human ES cells. Like primary cells, IT1 cells remain homogenous over 15 passages, expand up to 60 population doublings, and then die through senescence. Accordingly, IT1 cells display a normal karyotype and a somatic cell cycle kinetics and do not produce teratoma in nude mice. The production of K14-expressing epithelial cells driven by p63 expression strengthens the ectodermal nature of IT1 cells. Since IT1 can be isolated from different huES cell lines, it may provide a ready source of ectodermal progenitors for the development of a toxicology cell model, new-drug-screening strategies, and cell therapy transplantation.

Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.

Leishmania donovani are the causative agents of kala-azar in humans. They undergo a developmental program following changes in the environment, resulting in the reversible transformation between the extracellular promastigote form in the sand fly vector and the obligatory intracellular amastigote form in phagolysosomes of macrophages. A host-free differentiation system for L. donovani was used to investigate the initial process of promastigote to amastigote differentiation. Within an hour after exposing promastigotes to differentiation signal (concomitant exposure to 37 degrees C and pH 5.5), they expressed the amastigote-specific A2 protein family. At 5 h they started to transform to amastigote-shaped cells, a process that was completed 7 h later. This morphological transformation occurred synchronously, while cells arrested at G1. By sequential exposure to elevated temperature (for 24 h) and then acidic pH, we found that heat was responsible for the growth arrest and acidic pH to its release and subsequent route to differentiation into amastigotes. Lastly, ethanol and Azetidine 2 carboxylic acid (a synthetic proline analog) that induced heat shock response in promastigotes were capable of replacing heat in the differentiation signal.