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The placenta has a lifelong impact on the health of both the mother and fetus. Despite its significance, human early placental development is poorly understood due to the limited models. The models that can reflect the key features of early human placental development, especially at early gestation, are still lacking. Here, the authors report the generation of trophoblast-like tissue model from human pluripotent stem cells (hPSCs) in three-dimensional (3D) cultures. hPSCs efficiently self-organize into blastocoel-like cavities under defined conditions, which produce different trophoblast subtypes, including cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs), and invasive extravillous trophoblasts (EVTs). The 3D cultures can exhibit microvilli structure and secrete human placenta-specific hormone. Single-cell RNA sequencing analysis further identifies the presence of major cell types of trophoblast-like tissue as existing in vivo. The results reveal the feasibility to establish 3D trophoblast-like tissue model from hPSCs in vitro, which is not obtained by monolayer culture. This new model system can not only facilitate to dissect the underlying mechanisms of early human placental development, but also imply its potential for study in developmental biology and gestational disorders., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The amnion serves to create a protective environment for a growing fetus, and the study of amniotic development will greatly facilitate our understanding of normal and abnormal pregnancies. However, this remains a poorly studied field due to the lack of ideal human models. Herein, we present an integrative strategy to generate amnion-like cavity tissue from human pluripotent stem cells (hPSCs) in an amnion-on-a-chip device through combining a bioengineering approach and developmental biology principles. hPSCs could self-organize into an amnion epithelial cavity in a perfusable 3D culture microchip, resembling human amniotic development in mid-gestation. These cavities exhibited the critical features of amnion tissue based on morphological characteristics, marker expression, and transcriptome analysis. RNA-seq revealed that a set of amnion-specific genes were highly expressed in the obtained cavities, suggesting that the amnion epithelium was derived from hPSCs. Moreover, the amnion-specific mid-gestation marker KRT24 was highly expressed at the mRNA and protein levels, verifying the high maturation of amnion tissues after long-term 3D culturing and differentiation for up to 20 days. These new findings demonstrate the potential of this new amnion-on-a-chip model for investigating essential biological events in human amnions in normal and diseased states via integrating microengineering technology and stem cell biology.

Organized cardiomyocyte alignment is critical to maintain the mechanical properties of the heart. In this study, we present a new and simple strategy to fabricate a biomimetic microchip designed with an onion epithelium-like structure and investigate the guided behavior of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on the substrate. The hiPSC-CMs were observed to be confined by the three dimensional surficial features morphologically, analogous to the in vivo microenvironment, and exhibited an organized anisotropic alignment on the onion epithelium-like structure with good beating function. The calcium imaging of hiPSC-CMs demonstrated a more mature Ca 2+ spark pattern as well. Furthermore, the expression of sarcomere genes (TNNI3, MYH6 and MYH7), potassium channel genes (KCNE1 and KCNH2), and calcium channel genes (RYR2) was significantly up-regulated on the substrate with an onion epithelium-like structure instead of the surface without the structure, indicating a more matured status of cardiomyocytes induced by this structure. It appears that the biomimetic micropatterned structure, analogous to in vivo cellular organization, is an important factor that might promote the maturation of hiPSC-CMs, providing new biological insights to guide hiPSC-CM maturation by biophysical factors. The established approach may offer an effective in vitro model for investigating cardiomyocyte differentiation, maturation and tissue engineering applications.

Organoids derived from self‐organizing stem cells represent a major technological breakthrough with the potential to revolutionize biomedical research. However, building high‐fidelity organoids in a reproducible and high‐throughput manner remains challenging. Here, a droplet microfluidic system is developed for controllable fabrication of hybrid hydrogel capsules, which allows for massive 3D culture and formation of functional and uniform islet organoids derived from human‐induced pluripotent stem cells (hiPSCs). In this all‐in‐water microfluidic system, an array of droplets is utilized as templates for one‐step fabrication of binary capsules relying on interfacial complexation of oppositely charged Na‐alginate (NaA) and chitosan (CS). The produced hybrid capsules exhibit high uniformity, and are biocompatible, stable, and permeable. The established system enables capsule production, 3D culture, and self‐organizing formation of human islet organoids in a continuous process by encapsulating pancreatic endocrine cells from hiPSCs. The generated islet organoids contain islet‐specific α‐ and β‐like cells with high expression of pancreatic hormone specific genes and proteins. Moreover, they exhibit sensitive glucose‐stimulated insulin secretion function, demonstrating the capability of these binary capsules to engineer human organoids from hiPSCs. The proposed system is scalable, easy‐to‐operate, and stable, which can offer a robust platform for advancing human organoids research and translational applications. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell (hPSC)-derived islet cells provide promising resources for diabetes studies, cell replacement treatment and drug screening. Recently, hPSC-derived organoids have represented a new class of in vitro organ models for disease modeling and regenerative medicine. However, rebuilding biomimetic human islet organoids from hPSCs remains challenging. Here, we present a new strategy to engineer human islet organoids derived from human induced pluripotent stem cells (hiPSCs) using an organ-on-a-chip platform combined with stem cell developmental principles. The microsystem contains a multi-layer microfluidic device that allows controllable aggregation of embryoid bodies (EBs), in situ pancreatic differentiation and generation of heterogeneous islet organoids in parallel under perfused 3D culture in a single device. The generated islet organoids contain heterogeneous islet-specific α and β-like cells that exhibit favorable growth and cell viability. They also show enhanced expression of pancreatic β-cell specific genes and proteins (PDX1 and NKX6.1) and increased β-cell hormone specific INS gene and C-peptide protein expressions under perfused 3D culture conditions compared to static cultures. In addition, the islet organoids exhibit more sensitive glucose-stimulated insulin secretion (GSIS) and higher Ca2+ flux, indicating the role of biomimetic mechanical flow in promoting endocrine cell differentiation and maturation of islet organoids. This islet-on-a-chip system is robust and amenable to real-time imaging and in situ tracking of islet organoid growth, which may provide a promising platform for organoid engineering, disease modeling, drug testing and regenerative medicine. [ABSTRACT FROM AUTHOR]

Liver organoids derived from human pluripotent stem cells (PSCs) represent a new type of in vitro liver model for understanding organ development, disease mechanism and drug testing. However, engineering liver organoids with favorable functions in a controlled cellular microenvironment remains challenging. In this work, we present a new strategy for engineering liver organoids derived from human induced PSCs (hiPSCs) in a 3D perfusable chip system by combining stem cell biology with microengineering technology. This approach enabled formation of hiPSC-based embryoid bodies (EBs), in situ hepatic differentiation, long-term 3D culture and generation of liver organoids in a perfusable micropillar chip. The generated liver organoids exhibited favorable growth and differentiation of hepatocytes and cholangiocytes, recapitulating the key features of human liver formation with cellular heterogeneity. The liver organoids in perfused cultures displayed improved cell viability and higher expression of endodermal genes (SOX17 and FOXA2) and mature hepatic genes (ALB and CYP3A4) under perfused culture conditions. In addition, the liver organoids showed a marked enhancement of hepatic-specific functions, including albumin and urea production and metabolic capabilities, indicating the role of mechanical fluid flow in promoting the functions of the liver organoids. Moreover, the liver organoids exhibited hepatotoxic response after exposure to acetaminophen (APAP) in a dose- and time-dependent manner. The established liver organoid-on-a-chip system may provide a promising platform for engineering stem cell-based organoids with applications in regenerative medicine, disease modeling and drug testing. [ABSTRACT FROM AUTHOR]

Brain organoids derived from human induced pluripotent stem cells can recapitulate the early stages of brain development, representing a powerful in vitro system for modeling brain development and diseases. However, the existing methods for brain organoid formation often require time-consuming procedures, including the initial formation of embryoid bodies (EBs) from hiPSCs, and subsequent neural induction and differentiation companied by multi-steps of cell transfer and encapsulation in a 3D matrix. Herein, we propose a simple strategy to enable in situ formation of massive brain organoids from hiPSCs on a micropillar array without tedious manual procedures. The optimized micropillar configurations allow for controlled EB formation, neural induction and differentiation, and generation of functional human brain organoids in 3D culture on a single device. The generated brain organoids were examined to imitate brain organogenesis in vivo at early stages of gestation with specific features of neuronal differentiation, brain regionalization, and cortical organization. By combining microfabrication techniques with stem cells and developmental biology principles, the proposed method can greatly simplify brain organoid formation protocols as compared to conventional methods, overcoming the potential limitations of cell contamination, lower throughput and variance of organoid morphology. It can also provide a useful platform for the engineering of stem cell organoids with improved functions and extending their applications in developmental biology, drug testing and disease modeling. [ABSTRACT FROM AUTHOR]

There is a growing interest in using paper as a biomaterial scaffold for cell-based applications. In this study, we made the first attempt to fabricate a paper-based array for the culture, proliferation, and direct differentiation of human induced pluripotent stem cells (hiPSCs) into functional beating cardiac tissues and create “a beating heart on paper.” This array was simply constructed by binding a cured multi-well polydimethylsiloxane (PDMS) mold with common, commercially available paper substrates. Three types of paper material (print paper, chromatography paper and nitrocellulose membrane) were tested for adhesion, proliferation and differentiation of human-derived iPSCs. We found that hiPSCs grew well on these paper substrates, presenting a three-dimensional (3D)-like morphology with a pluripotent property. The direct differentiation of human iPSCs into functional cardiac tissues on paper was also achieved using our modified differentiation approach. The cardiac tissue retained its functional activities on the coated print paper and chromatography paper with a beating frequency of 40–70 beats per min for up to three months. Interestingly, human iPSCs could be differentiated into retinal pigment epithelium on nitrocellulose membrane under the conditions of cardiac-specific induction, indicating the potential roles of material properties and mechanical cues that are involved in regulating stem cell differentiation. Taken together, these results suggest that different grades of paper could offer great opportunities as bioactive, low-cost, and 3D in vitro platforms for stem cell-based high-throughput drug testing at the tissue/organ level and for tissue engineering applications. [ABSTRACT FROM AUTHOR]