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    RNA-sequencing of single cholangiocyte-derived organoids reveals high organoid-to organoid variability. Life science alliance Over the last decades, organoids have been established from most of the tissue-resident stem and iPS cells. They hold great promise for our understanding of mammalian organ development, but also for the study of disease or even personalised medicine. In recent years, several reports hinted at intraculture organoid variability, but a systematic analysis of such heterogeneity has not been performed before. Here, we used RNA-seq of individual intrahepatic cholangiocyte organoids to address this question. We find that batch-to-batch variation is very low, whereas passage number has a profound impact on gene expression profiles. On the other hand, there is organoid-to-organoid variability within a culture. Using differential gene expression, we did not identify specific pathways that drive this variability, pointing towards possible effects of the microenvironment within the culture condition. Taken together, our study provides a framework for organoid researchers to properly consider experimental design. 10.26508/lsa.202101340
    Human organoids: New strategies and methods for analyzing human development and disease. Cell For decades, insight into fundamental principles of human biology and disease has been obtained primarily by experiments in animal models. While this has allowed researchers to understand many human biological processes in great detail, some developmental and disease mechanisms have proven difficult to study due to inherent species differences. The advent of organoid technology more than 10 years ago has established laboratory-grown organ tissues as an additional model system to recapitulate human-specific aspects of biology. The use of human 3D organoids, as well as other advances in single-cell technologies, has revealed unprecedented insights into human biology and disease mechanisms, especially those that distinguish humans from other species. This review highlights novel advances in organoid biology with a focus on how organoid technology has generated a better understanding of human-specific processes in development and disease. 10.1016/j.cell.2022.06.051
    In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid. Kupfer Molly E,Lin Wei-Han,Ravikumar Vasanth,Qiu Kaiyan,Wang Lu,Gao Ling,Bhuiyan Didarul B,Lenz Megan,Ai Jeffrey,Mahutga Ryan R,Townsend DeWayne,Zhang Jianyi,McAlpine Michael C,Tolkacheva Elena G,Ogle Brenda M Circulation research RATIONALE:One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. OBJECTIVE:To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. METHODS AND RESULTS:We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. CONCLUSIONS:This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting. 10.1161/CIRCRESAHA.119.316155
    Generation of blood vessel organoids from human pluripotent stem cells. Wimmer Reiner A,Leopoldi Alexandra,Aichinger Martin,Kerjaschki Dontscho,Penninger Josef M Nature protocols Blood vessels are fundamental to animal life and have critical roles in many diseases, such as stroke, myocardial infarction and diabetes. The vasculature is formed by endothelial cells that line the vessel and are covered with mural cells, specifically pericytes in smaller vessels and vascular smooth muscle cells (vSMCs) in larger-diameter vessels. Both endothelial cells and mural cells are essential for proper blood vessel function and can be derived from human pluripotent stem cells (hPSCs). Here, we describe a protocol to generate self-organizing 3D human blood vessel organoids from hPSCs that exhibit morphological, functional and molecular features of human microvasculature. These organoids are differentiated via mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I-Matrigel matrix. Blood vessels form within 2-3 weeks and can be further grown in scalable suspension culture. Importantly, in vitro-differentiated human blood vessel organoids transplanted into immunocompromised mice gain access to the mouse circulation and specify into functional arteries, arterioles and veins. 10.1038/s41596-019-0213-z
    Vascularized Organoids: A More Complete Model. Yu Jin International journal of stem cells As an emerging research model , organoids have achieved major progress in recapitulating morphological aspects of organs and personalized precision therapy. Various organoids have been currently constructed (e.g., brain, heart, liver, and gastrointestinal). Though there are prominent advantages on microstructures and partial functions, most of them have been encountering a frustrating challenge that stromal components (e.g., blood vessels) are in short supplement, which has imposed the main dilemma on the application of such model ex vivo. As advanced technologies, co-culturing pluripotent stem cells, mesenchymal stem cells, with endothelial cells on 3D substrate matrix, are leaping forward, a novel model of an organoid with vascularization is formed. The mentioned contribute to the construction of the functional organoids derived from corresponding tissues, making them more reliable in stem cell research and clinical medicine. The present study overall summarizes progress of the evolution, applications and prospects of vascularized organoids. 10.15283/ijsc20143
    Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals. Zhang Qiangzhe,Jiang Junjie,Han Pengcheng,Yuan Qi,Zhang Jing,Zhang Xiaoqian,Xu Yanyan,Cao Henghua,Meng Qingzhang,Chen Li,Tian Tian,Wang Xin,Li Pu,Hescheler Jurgen,Ji Guangju,Ma Yue Cell research Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca(2+) sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals. 10.1038/cr.2010.163
    From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish. Scalise Mariangela,Marino Fabiola,Salerno Luca,Cianflone Eleonora,Molinaro Claudia,Salerno Nadia,De Angelis Antonella,Viglietto Giuseppe,Urbanek Konrad,Torella Daniele International journal of molecular sciences Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research. 10.3390/ijms222413180