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. In the past few years, sophisticated methodologies have been proposed to allow the design of organoids with improved biological fidelity and physiological relevance. Here, we summarize these emerging technologies and provide insights into how they can be utilized to fulfill the potential of stem cells.

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.

Production of Human Pluripotent Stem Cell-Derived Hepatic Cell Lineages and Liver Organoids: Current Status and Potential Applications.

Liver disease is one of the leading causes of death worldwide, leading to the death of approximately 2 million people per year. Current therapies include orthotopic liver transplantation, however, donor organ shortage remains a great challenge. In addition, the development of novel therapeutics has been limited due to the lack of in vitro models that mimic in vivo liver physiology. Accordingly, hepatic cell lineages derived from human pluripotent stem cells (hPSCs) represent a promising cell source for liver cell therapy, disease modelling, and drug discovery. Moreover, the development of new culture systems bringing together the multiple liver-specific hepatic cell types triggered the development of hPSC-derived liver organoids. Therefore, these human liver-based platforms hold great potential for clinical applications. In this review, the production of the different hepatic cell lineages from hPSCs, including hepatocytes, as well as the emerging strategies to generate hPSC-derived liver organoids will be assessed, while current biomedical applications will be highlighted.

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. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-ß/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.

Design Principles for Pluripotent Stem Cell-Derived Organoid Engineering.

Human morphogenesis is a complex process involving distinct microenvironmental and physical signals that are manipulated in space and time to give rise to complex tissues and organs. Advances in pluripotent stem cell (PSC) technology have promoted the in vitro recreation of processes involved in human morphogenesis. The development of organoids from human PSCs represents one reliable source for modeling a large spectrum of human disorders, as well as a promising approach for drug screening and toxicological tests. Based on the "self-organization" capacity of stem cells, different PSC-derived organoids have been created; however, considerable differences between in vitro-generated PSC-derived organoids and their in vivo counterparts have been reported. Advances in the bioengineering field have allowed the manipulation of different components, including cellular and noncellular factors, to better mimic the in vivo microenvironment. In this review, we focus on different examples of bioengineering approaches used to promote the self-organization of stem cells, including assembly, patterning, and morphogenesis in vitro, contributing to tissue-like structure formation.