The mitochondrial genome (mtDNA) is an important source of inherited extranuclear variation. Clonal increases in mtDNA mutation heteroplasmy have been implicated in aging and disease, although the impact of this shift on cell function is challenging to assess. Reprogramming to pluripotency affects mtDNA mutation heteroplasmy. We reprogrammed three human fibroblast lines with known heteroplasmy for deleterious mtDNA point or deletion mutations. Quantification of mutation heteroplasmy in the resulting 76 induced pluripotent stem cell (iPSC) clones yielded a bimodal distribution, creating three sets of clones with high levels or absent mutation heteroplasmy with matched nuclear genomes. iPSC clones with elevated deletion mutation heteroplasmy show altered growth dynamics, which persist in iPSC-derived progenitor cells. We identify transcriptomic and metabolic shifts consistent with increased investment in neutral lipid synthesis as well as increased epigenetic age in high mtDNA deletion mutation iPSC, consistent with changes occurring in cellular aging. Together, these data demonstrate that high mtDNA mutation heteroplasmy induces changes occurring in cellular aging.
© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.
© 2023. Crown.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.
Crown Copyright © 2023.

A reproducible imaging protocol should include four main detailed sections. The first should describe the sample preparation and include details about the tissue and/or cell culture preparation, the staining procedure, the optical grade of the coverslip, and the type of mounting media used to mount the sample. The second section should describe the configuration and components of the microscope and include the type of stand, stage, illumination, and detector, as well as the emission (EM) and excitation (EX) filters, objective, and immersion medium specifications. Specialized microscopes may have other important components in the optical path to include. The third section should describe the settings used to acquire an image like the exposure and/or dwell time, final magnification and optical resolution, the pixel and field of view (FOV) sizes, time intervals for any time lapse, total power at the objective (i.e., directed at your sample) and number of planes and step size used to collect a 3-dimensional image, and order of operations used in multi-dimensional image acquisitions. The final section should include details about the image analysis workflow such as the image processing steps, segmentation and measurement methods used to extract information from the image, data size, and necessary computing hardware and networking requirements if data sets are >1 GB, as well as citations and versions for the software and code used to perform any of these steps. Every effort should be made to make an example dataset with accurate metadata available online. Finally, specifics about the type of replicates included in the experiment and details about the statistical analysis conducted are also necessary.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.
© 2022 Wiley Periodicals LLC.