Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Biophysical study of human induced Pluripotent Stem Cell-Derived cardiomyocyte structural maturation during long-term culture.

Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) have an enormous potential for the development of drug screening and modeling cardiac disease platforms. However, early hiPSC-CMs usually exhibit low structural development, precluding the applicability of these cells. Here, we follow during 120 days the progressive structural maturation of hiPSC-CM microtissues obtained using the Wnt signaling modulation protocol. For this purpose, we designed a user friendly custom-written program to quantify cardiac fiber alignment and sarcomere length. Cardiomyocyte shape, cardiac fiber density and multinucleation were also analyzed. Derived cardiomyocytes showed significant progression in cardiomyocyte fiber density and sarcomere length during the long-term culture, with a peak at day 90 of 40% multinucleated cells. In addition, cardiomyocyte microtissues remained functional with progressive maturation leading to a decrease in the percentage of cTnT positive cells from 59% to 22% at day 120, a value similar to the content present in tissues of the adult left ventricle. These data and the framework that we provide to quantify cardiomyocyte structural features can be important to set new metrics to develop applications for drug screening and disease modeling.

Human multilineage pro-epicardium/foregut organoids support the development of an epicardium/myocardium organoid.

The epicardium, the outer epithelial layer that covers the myocardium, derives from a transient organ known as pro-epicardium, crucial during heart organogenesis. The pro-epicardium develops from lateral plate mesoderm progenitors, next to septum transversum mesenchyme, a structure deeply involved in liver embryogenesis. Here we describe a self-organized human multilineage organoid that recreates the co-emergence of pro-epicardium, septum transversum mesenchyme and liver bud. Additionally, we study the impact of WNT, BMP and retinoic acid signaling modulation on multilineage organoid specification. By co-culturing these organoids with cardiomyocyte aggregates, we generated a self-organized heart organoid comprising an epicardium-like layer that fully surrounds  a myocardium-like tissue. These heart organoids recapitulate the impact of epicardial cells on promoting cardiomyocyte proliferation and structural and functional maturation. Therefore, the human heart organoids described herein, open the path to advancing knowledge on how myocardium-epicardium interaction progresses during heart organogenesis in healthy or diseased settings.

3D Microwell Platform for Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.

The generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) represents a valuable tool for a myriad of in vitro applications, including drug screening, disease modeling and regenerative medicine. However, the success of these applications is dependent on the establishment of reliable, efficient, simple, and cost-effective differentiation methods. In this chapter, we describe an efficient and robust 3D platform for the generation of hPSC-CMs based on the use of a microwell culture system, which can be applied in any laboratory environment. Additionally, we will also describe protocols for the structural and functional characterization of the obtained CMs for further quality control upon differentiation.

Xeno-Free Integrated Platform for Robust Production of Cardiomyocyte Sheets from hiPSCs.

Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (CMs), which can be used for cardiac disease modeling, for drug screening, and to regenerate damaged myocardium. Implementation of xeno-free culture systems is essential to fully explore the potential of these cells. However, differentiation using xeno-free adhesion matrices often results in low CM yields and lack of functional CM sheets, capable of enduring additional maturation stages. Here, we established a xeno-free CM differentiation platform using TeSR/Synthemax, including a replating step and integrated with two versatile purification/enrichment metabolic approaches. Results showed that the replating step was essential to reestablish a fully integrated, closely-knit CM sheet. In addition, replating contributed to increase the cTnT expression from 65% to 75% and the output from 2.2 to 3.1 CM per hiPSC, comparable with the efficiency observed when using TeSR/Matrigel. In addition, supplementation with PluriSin1 and Glu-Lac+ medium allowed increasing the CM content over 80% without compromising CM sheet integrity or functionality. Thus, this xeno-free differentiation platform is a reliable and robust method to produce hiPSC-derived CMs, increasing the possibility of using these cells safely for a wide range of applications.

The Azurin-Derived Peptide CT-p19LC Exhibits Membrane-Active Properties and Induces Cancer Cell Death.

Peptides have been thoroughly studied as new therapeutic strategies for cancer treatment. In this work, we explored in vitro the anticancer potential of three novel peptides derived from the C-terminal of azurin, an anticancer bacterial protein produced by Pseudomonas aeruginosa. CT-p26, CT-p19 and CT-p19LC peptides were previously obtained through an in silico peptide design optimization process, CT-p19LC being the most promising as it presented higher hydrophobicity and solubility, positive total charge and, most importantly, greater propensity for anticancer activity. Therefore, in this study, through proliferation and apoptosis assays, CT-p19LC was tested in four cancer cell lines-A549, MCF-7, HeLa and HT-29-and in two non-cancer cell lines-16HBE14o- and MCF10A. Its membrane-targeting activity was further evaluated with zeta potential measurements and membrane order was assessed with the Laurdan probe. The results obtained demonstrated that CT-p19LC decreases cell viability through induction of cell death and binds to the plasma membrane of cancer cells, but not to non-cancer cells, making them less rigid. Overall, this study reveals that CT-p19LC is an auspicious selective anticancer peptide able to react with cancer cell membranes and cause effective action.

p28-functionalized PLGA nanoparticles loaded with gefitinib reduce tumor burden and metastases formation on lung cancer.

Lung cancer is still the main cause of cancer-related deaths worldwide. Its treatment generally includes surgical resection, immunotherapy, radiotherapy, and chemo-targeted therapies such as the application of tyrosine kinase inhibitors. Gefitinib (GEF) is one of them, but its poor solubility in gastric fluids weakens its bioavailability and therapeutic activity. In addition, like all other chemotherapy treatments, GEF administration can cause damage to healthy tissues. Therefore, the development of novel GEF delivery systems to increase its bioavailability and distribution in tumor site is highly demanded. Herein, an innovative strategy for GEF delivery, by functionalizing PLGA nanoparticles with p28 (p28-NPs), a cell-penetrating peptide derived from the bacterial protein azurin, was developed. Our data indicated that p28 potentiates the selective interaction of these nanosystems with A549 lung cancer cells (active targeting). Further p28-NPs delivering GEF (p28-NPs-GEF) were able to selectively reduce the metabolic activity of A549 cells, while no impact was observed in non-tumor cells (16HBE14o-). In vivo studies using A549 subcutaneous xenograft showed that p28-NPs-GEF reduced A549 primary tumor burden and lung metastases formation. Overall, the design of a p28-functionalized delivery nanosystem to effectively penetrate the membranes of cancer cells while deliver GEF could provide a new strategy to improve lung cancer therapy.

Multifactorial Modeling Reveals a Dominant Role of Wnt Signaling in Lineage Commitment of Human Pluripotent Stem Cells.

The human primed pluripotent state is maintained by a complex balance of several signaling pathways governing pluripotency maintenance and commitment. Here, we explore a multiparameter approach using a full factorial design and a simple well-defined culture system to assess individual and synergistic contributions of Wnt, FGF and TGFß signaling to pluripotency and lineage specification of human induced pluripotent stem cells (hiPSC). Hierarchical clustering and quadratic models highlighted a dominant effect of Wnt signaling over FGF and TGFß signaling, drawing hiPSCs towards mesendoderm lineages. In addition, a synergistic effect between Wnt signaling and FGF was observed to have a negative contribution to pluripotency maintenance and a positive contribution to ectoderm and mesoderm commitment. Furthermore, FGF and TGFß signaling only contributed significantly for negative ectoderm scores, suggesting that the effect of both factors for pluripotency maintenance resides in a balance of inhibitory signals instead of proactive stimulation of hiPSC pluripotency. Overall, our dry-signaling multiparameter modeling approach can contribute to elucidate individual and synergistic inputs, providing an additional degree of comprehension of the complex regulatory mechanisms of human pluripotency and commitment.

New insights into the mechanisms of embryonic stem cell self-renewal under hypoxia: a multifactorial analysis approach.

Previous reports have shown that culturing mouse embryonic stem (mES) cells at different oxygen tensions originated different cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support the pluripotency state. Herein we provide new insights into the mechanisms by which oxygen is influencing mES cell self-renewal and pluripotency. A multifactorial approach was developed to rationally evaluate the singular and interactive control of MEK/ERK pathway, GSK-3 inhibition, and LIF/STAT3 signaling at physiological and non-physiological oxygen tensions. Collectively, our methodology revealed a significant role of GSK-3-mediated signaling towards maintenance of mES cell pluripotency at lower O(2) tensions. Given the central role of this signaling pathway, future studies will need to focus on the downstream mechanisms involved in ES cell self-renewal under such conditions, and ultimately how these findings impact human models of pluripotency.