Involvement of extracellular vesicle microRNA clusters in developing healthy and Rett syndrome brain organoids.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control.

Unsupervised analysis of whole transcriptome data from human pluripotent stem cells cardiac differentiation.

The main objective of the present work was to highlight differences and similarities in gene expression patterns between different pluripotent stem cell cardiac differentiation protocols, using a workflow based on unsupervised machine learning algorithms to analyse the transcriptome of cells cultured as a 2D monolayer or as 3D aggregates. This unsupervised approach effectively allowed to portray the transcriptomic changes that occurred throughout the differentiation processes, with a visual representation of the entire transcriptome. The results allowed to corroborate previously reported data and also to unveil new gene expression patterns.

Developmental Toxicity Studies: The Path towards Humanized 3D Stem Cell-Based Models.

Today, it is recognized that medicines will eventually be needed during pregnancy to help prevent to, ameliorate or treat an illness, either due to gestation-related medical conditions or pre-existing diseases. Adding to that, the rate of drug prescription to pregnant women has increased over the past few years, in accordance with the increasing trend to postpone childbirth to a later age. However, in spite of these trends, information regarding teratogenic risk in humans is often missing for most of the purchased drugs.

Advancing organoid design through co-emergence, assembly, and bioengineering.

Human adult stem cells and patient-derived induced pluripotent stem cells represent promising tools to understand human biology, development, and disease. Under a permissive environment, stem cell derivatives can self-organize and reconstruct their native milieu, resulting in the creation of organ-like entities known as organoids. Although organoids represent a breakthrough in the stem cell field, there are still considerable shortcomings preventing their widespread use, namely their variability, limited function, and reductionist size.

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.

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.

Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies.

Modeling Rett Syndrome With Human Patient-Specific Forebrain Organoids.

Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity.

Functionalization of Electrospun Nanofibers and Fiber Alignment Enhance Neural Stem Cell Proliferation and Neuronal Differentiation.

Neural stem cells (NSCs) have the potential to generate the cells of the nervous system and, when cultured on nanofiber scaffolds, constitute a promising approach for neural tissue engineering. In this work, the impact of combining nanofiber alignment with functionalization of the electrospun poly-ε-caprolactone (PCL) nanofibers with biological adhesion motifs on the culture of an NSC line (CGR8-NS) is evaluated. A five-rank scale for fiber density was introduced, and a 4.

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.

From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges.

The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment.

Stem Cell Bioprocessing and Manufacturing.

The next healthcare revolution will apply regenerative medicines using human cells and tissues [...

Maturation of Human Pluripotent Stem Cell-Derived Cerebellar Neurons in the Absence of Co-culture.

The cerebellum plays a critical role in all vertebrates, and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models, cerebellar neurons differentiated from pluripotent stem cells have been used.

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 TGFβ 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 TGFβ signaling, drawing hiPSCs towards mesendoderm lineages.

Transcriptomic analysis of 3D Cardiac Differentiation of Human Induced Pluripotent Stem Cells Reveals Faster Cardiomyocyte Maturation Compared to 2D Culture.

Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture.

Dissolvable Microcarriers Allow Scalable Expansion And Harvesting Of Human Induced Pluripotent Stem Cells Under Xeno-Free Conditions.

The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno-free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated.

Towards Multi-Organoid Systems for Drug Screening Applications.

A low percentage of novel drug candidates succeed and reach the end of the drug discovery pipeline, mainly due to poor initial screening and assessment of the effects of the drug and its metabolites over various tissues in the human body. For that, emerging technologies involving the production of organoids from human pluripotent stem cells (hPSCs) and the use of organ-on-a-chip devices are showing great promise for developing a more reliable, rapid and cost-effective drug discovery process when compared with the current use of animal models. In particular, the possibility of virtually obtaining any type of cell within the human body, in combination with the ability to create patient-specific tissues using human induced pluripotent stem cells (hiPSCs), broadens the horizons in the fields of drug discovery and personalized medicine.

Three-Dimensional Cell-Based Microarrays: Printing Pluripotent Stem Cells into 3D Microenvironments.

Cell-based microarrays are valuable platforms for the study of cytotoxicity and cellular microenvironment because they enable high-throughput screening of large sets of conditions at reduced reagent consumption. However, most of the described microarray technologies have been applied to two-dimensional cultures, which do not accurately emulate the in vivo three-dimensional (3D) cell-cell and cell-extracellular matrix interactions.Herein, we describe the methodology for production of alginate- and Matrigel-based 3-D cell microarrays for the study of mouse and human pluripotent stem cells on two different chip-based platforms.

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.

Defined and Scalable Differentiation of Human Oligodendrocyte Precursors from Pluripotent Stem Cells in a 3D Culture System.

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system.

Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.

Human pluripotent stem cells (hPSC) have attracted a great attention as an unlimited source of cells for cell therapies and other in vitro biomedical applications such as drug screening, toxicology assays and disease modeling. The implementation of scalable culture platforms for the large-scale production of hPSC and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of cells for these applications. Microcarrier technology has been emerging as an effective approach for the large scale ex vivo hPSC expansion and differentiation.

Correction: Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.

[This corrects the article DOI: 10.1371/journal.pone.

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions.

A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.

Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield.