Cardiac fibrosis is the most common pathway of many cardiac diseases. To date, there has been no suitable in vitro cardiac fibrosis model that could sufficiently mimic the complex environment of the human heart. Here, a three-dimensional (3D) cardiac sphere platform of contractile cardiac microtissue, composed of human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) and mesenchymal stem cells (MSCs), is presented to better recapitulate the human heart. We hypothesized that MSCs would develop an in vitro fibrotic reaction in response to treatment with transforming growth factor-β1 (TGF-β1), a primary inducer of cardiac fibrosis. The addition of MSCs improved sarcomeric organization, electrophysiological properties, and the expression of cardiac-specific genes, suggesting their physiological relevance in the generation of human cardiac microtissue model in vitro. MSCs could also generate fibroblasts within 3D cardiac microtissues and, subsequently, these fibroblasts were transdifferentiated into myofibroblasts by the exogenous addition of TGF-β1. Cardiac microtissues displayed fibrotic features such as the deposition of collagen, the presence of numerous apoptotic CMs and the dissolution of mitochondrial networks. Furthermore, treatment with pro-fibrotic substances demonstrated that this model could reproduce key molecular and cellular fibrotic events. This highlights the potential of our 3D cardiac microtissues as a valuable tool for manifesting and evaluating the pro-fibrotic effects of various agents, thereby representing an important step forward towards an in vitro system for the prediction of drug-induced cardiac fibrosis and the study of the pathological changes in human cardiac fibrosis.

Directed differentiation of hPSCs and transition from 3D intestinal organoids into functional hIECs.

Although brain organoids are an innovative technique for studying human brain development and disease by replicating the structural and functional properties of the developing human brain, some limitations such as heterogeneity and long-term differentiation (over 2 months) impede their application in disease modeling and drug discovery. In this study, we established simplified brain organoids (simBOs), composed of mature neurons and astroglial cells from expandable hPSC-derived primitive neural stem cells (pNSCs). simBOs can be rapidly generated in 2 weeks and have more homogeneous properties. Transcriptome analysis revealed that three-dimensional (3D) environment of simBOs facilitates the conversion of pNSCs to mature neuronal systems compared to a two-dimensional environment in the context of neurotransmitter release, synaptic vesicle formation, ion channels, calcium signaling, axonal guidance, extracellular matrix organization, and cell cycle. This result was correlated with the translocation of YAP1 into the cytoplasm by sensing matrix stiffness on the 3D models. Furthermore, we demonstrated that simBOs could easily be specified into midbrain-like simBOs by treatment with Shh and FGF8. Midbrain-like simBOs from a Parkinson’s disease patient (LRRK2^G2019S)-derived pNSCs and gene-corrected (LRRK2^WT) control pNSCs represented disease-associated phenotypes in terms of increased LRRK2 activity, decreased dopaminergic neurons, and increased autophagy. Treatment with the LRRK2 inhibitor, PFE-360, relieved the phenotype of Parkinson’s disease in midbrain-like simBOs. Taken together, these approaches could be applied to large-scale disease models and alternative drug-testing platforms.

Within the human lung, interactions between alveolar epithelial cells and resident macrophages shape lung development and function in both health and disease. To study these processes, we develop a co-culture system combining human pluripotent stem cell-derived alveolar epithelial organoids and induced macrophages to create a functional environment, termed induced alveolar assembloids. Using single-cell RNA sequencing and functional analyses, we identify alveolar type 2-like cells producing GM-CSF, which supports macrophage tissue adaptation, and macrophage-like cells that secrete interleukin-1β and interleukin-6, express surfactant metabolism genes, and demonstrate core immune functions. In response to alveolar epithelial injury, macrophage-like cells efficiently eliminate damaged cells and absorb oxidized lipids. Exposure to bacterial components or infection with Mycobacterium tuberculosis reveals that these assembloids replicate key aspects of human respiratory defense. These findings highlight the potential of induced alveolar assembloids as a platform to investigate human lung development, immunity, and disease.

Brain organoids are valuable research models for human development and disease since they mimic the various cell compositions and structures of the human brain; however, they have challenges in presenting aging phenotypes for degenerative diseases. This study analyzed the association between aging and the gut metabolite trimethylamine N-oxide (TMAO), which is highly found in the midbrain of elderly and Parkinson’s disease (PD) patients. TMAO treatment in midbrain organoid induced aging-associated molecular changes, including increased senescence marker expression (P21, P16), p53 accumulation, and epigenetic alterations. In addition, TMAO-treated midbrain organoids have shown parts of neurodegeneration phenotypes, including impaired brain-derived neurotrophic factor (BDNF) signaling, loss of dopaminergic neurons, astrocyte activation, and neuromelanin accumulation. Moreover, we found TMAO treatment-induced pathophysiological phosphorylation of α-synuclein protein at Ser-129 residues and Tau protein at Ser202/Thr205. These results suggest a role of TMAO in the aging and pathogenesis of the midbrain and provide insight into how intestinal dysfunction increases the risk of PD. Furthermore, this system can be utilized as a novel aging model for induced pluripotent stem cell (iPSC)-based modeling of late-onset diseases.