Methods to extract and analyze fluid from human pluripotent stem cell-derived choroid plexus organoids.

The choroid plexus (ChP) is a highly vascularized tissue lining the ventricular space of the brain. The ChP generates cerebrospinal fluid (CSF) and forms a protective barrier in the central nervous system (CNS). Recently, a three-dimensional human pluripotent stem cell (hPSC)-derived ChP organoid model has been developed. This model generates cystic structures that are filled with a fluid resembling CSF and are surrounded by an epithelial layer expressing ependymal choroid plexus-specific markers. Here we describe a method to generate these choroid plexus organoids using a commercial kit and methods to extract the CSF-like fluid for use in downstream analysis.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

Immunomodulation of Pluripotent Stem Cell-Derived Mesenchymal Stem Cells in Rotator Cuff Tears Model.

BACKGROUND: Rotator cuff tears (RCTs) induce chronic muscle weakness and shoulder pain. Treatment of RCT using surgery or drugs causes lipid infiltration and fibrosis, which hampers tissue regeneration and complete recovery. The pluripotent stem cell-derived multipotent mesenchymal stem cells (M-MSCs) represent potential candidate next-generation therapies for RCT. METHODS: The difference between M-MSCs and adult-MSCs was compared and analyzed using next-generation sequencing (NGS). In addition, using a rat model of RCT, the muscle recovery ability of M-MSCs and adult-MSCs was evaluated by conducting a histological analysis and monitoring the cytokine expression level. RESULTS: Using NGS, it was confirmed that M-MSC was suitable for transplantation because of its excellent ability to regulate inflammation that promotes tissue repair and reduced apoptosis and rejection during transplantation. In addition, while M-MSCs persisted for up to 8 weeks in vivo, they significantly reduced inflammation and adipogenesis-related cytokine levels in rat muscle. Significant differences were also confirmed in histopathological remission. CONCLUSIONS: M-MSCs remain in the body longer to modulate immune responses in RCTs and have a greater potential to improve muscle recovery by alleviating acute inflammatory responses. This indicates that M-MSCs could be used in potential next-generation RCT therapies.

Transposable Elements in Pluripotent Stem Cells and Human Disease.

Transposable elements (TEs) are mobile genetic elements that can randomly integrate into other genomic sites. They have successfully replicated and now occupy around 40% of the total DNA sequence in humans. TEs in the genome have a complex relationship with the host cell, being both potentially deleterious and advantageous at the same time. Only a tiny minority of TEs are still capable of transposition, yet their fossilized sequence fragments are thought to be involved in various molecular processes, such as gene transcriptional activity, RNA stability and subcellular localization, and chromosomal architecture. TEs have also been implicated in biological processes, although it is often hard to reveal cause from correlation due to formidable technical issues in analyzing TEs. In this review, we compare and contrast two views of TE activity: one in the pluripotent state, where TEs are broadly beneficial, or at least mechanistically useful, and a second state in human disease, where TEs are uniformly considered harmful.

Heart in a Dish: From Traditional 2D Differentiation Protocols to Cardiac Organoids.

Human pluripotent stem cells (hPSCs) constitute a valuable model to study the complexity of early human cardiac development and investigate the molecular mechanisms involved in heart diseases. The differentiation of hPSCs into cardiac lineages in vitro can be achieved by traditional two-dimensional (2D) monolayer approaches or by adopting innovative three-dimensional (3D) cardiac organoid protocols. Human cardiac organoids (hCOs) are complex multicellular aggregates that faithfully recapitulate the cardiac tissue's transcriptional, functional, and morphological features. In recent years, significant advances in the field have dramatically improved the robustness and efficiency of hCOs derivation and have promoted the application of hCOs for drug screening and heart disease modeling. This review surveys the current differentiation protocols, focusing on the most advanced 3D methods for deriving hCOs from hPSCs. Furthermore, we describe the potential applications of hCOs in the pharmaceutical and tissue bioengineering fields, including their usage to investigate the consequences of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV2) infection in the heart.

Mislocalization of Nucleocytoplasmic Transport Proteins in Human Huntington's Disease PSC-Derived Striatal Neurons.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene (HTT). Disease progression is characterized by the loss of vulnerable neuronal populations within the striatum. A consistent phenotype across HD models is disruption of nucleocytoplasmic transport and nuclear pore complex (NPC) function. Here we demonstrate that high content imaging is a suitable method for detecting mislocalization of lamin-B1, RAN and RANGAP1 in striatal neuronal cultures thus allowing a robust, unbiased, highly powered approach to assay nuclear pore deficits. Furthermore, nuclear pore deficits extended to the selectively vulnerable DARPP32 + subpopulation neurons, but not to astrocytes. Striatal neuron cultures are further affected by changes in gene and protein expression of RAN, RANGAP1 and lamin-B1. Lowering total HTT using HTT-targeted anti-sense oligonucleotides partially restored gene expression, as well as subtly reducing mislocalization of proteins involved in nucleocytoplasmic transport. This suggests that mislocalization of RAN, RANGAP1 and lamin-B1 cannot be normalized by simply reducing expression of CAG-expanded HTT in the absence of healthy HTT protein.

Hypothesis and Theory: Characterizing Abnormalities of Energy Metabolism Using a Cellular Platform as a Personalized Medicine Approach for Alzheimer's Disease.

Sporadic or late-onset Alzheimer's disease (LOAD) is characterized by slowly progressive deterioration and death of CNS neurons. There are currently no substantially disease-modifying therapies. LOAD pathology is closely related to changes with age and include, among others, accumulation of toxic molecules and altered metabolic, microvascular, biochemical and inflammatory processes. In addition, there is growing evidence that cellular energy deficits play a critical role in aging and LOAD pathophysiology. However, the exact mechanisms and causal relationships are largely unknown. In our studies we tested the hypothesis that altered bioenergetic and metabolic cell functions are key elements in LOAD, using a cellular platform consisting of skin fibroblasts derived from LOAD patients and AD-unaffected control individuals and therefrom generated induced pluripotent stem cells that are differentiated to brain-like cells to study LOAD pathogenic processes in context of age, disease, genetic background, cell development, and cell type. This model has revealed that LOAD cells exhibit a multitude of bioenergetic and metabolic alterations, providing evidence for an innate inefficient cellular energy management in LOAD as a prerequisite for the development of neurodegenerative disease with age. We propose that this cellular platform could ultimately be used as a conceptual basis for a personalized medicine tool to predict altered aging and risk for development of dementia, and to test or implement customized therapeutic or disease-preventive intervention strategies.

Genome Editing Human Pluripotent Stem Cells to Model ß-Cell Disease and Unmask Novel Genetic Modifiers.

A mechanistic understanding of the genetic basis of complex diseases such as diabetes mellitus remain elusive due in large part to the activity of genetic disease modifiers that impact the penetrance and/or presentation of disease phenotypes. In the face of such complexity, rare forms of diabetes that result from single-gene mutations (monogenic diabetes) can be used to model the contribution of individual genetic factors to pancreatic ß-cell dysfunction and the breakdown of glucose homeostasis. Here we review the contribution of protein coding and non-protein coding genetic disease modifiers to the pathogenesis of diabetes subtypes, as well as how recent technological advances in the generation, differentiation, and genome editing of human pluripotent stem cells (hPSC) enable the development of cell-based disease models. Finally, we describe a disease modifier discovery platform that utilizes these technologies to identify novel genetic modifiers using induced pluripotent stem cells (iPSC) derived from patients with monogenic diabetes caused by heterozygous mutations.

Organelle Cooperation in Stem Cell Fate: Lysosomes as Emerging Regulators of Cell Identity.

Regulation of stem cell fate is best understood at the level of gene and protein regulatory networks, though it is now clear that multiple cellular organelles also have critical impacts. A growing appreciation for the functional interconnectedness of organelles suggests that an orchestration of integrated biological networks functions to drive stem cell fate decisions and regulate metabolism. Metabolic signaling itself has emerged as an integral regulator of cell fate including the determination of identity, activation state, survival, and differentiation potential of many developmental, adult, disease, and cancer-associated stem cell populations and their progeny. As the primary adenosine triphosphate-generating organelles, mitochondria are well-known regulators of stem cell fate decisions, yet it is now becoming apparent that additional organelles such as the lysosome are important players in mediating these dynamic decisions. In this review, we will focus on the emerging role of organelles, in particular lysosomes, in the reprogramming of both metabolic networks and stem cell fate decisions, especially those that impact the determination of cell identity. We will discuss the inter-organelle interactions, cell signaling pathways, and transcriptional regulatory mechanisms with which lysosomes engage and how these activities impact metabolic signaling. We will further review recent data that position lysosomes as critical regulators of cell identity determination programs and discuss the known or putative biological mechanisms. Finally, we will briefly highlight the potential impact of elucidating mechanisms by which lysosomes regulate stem cell identity on our understanding of disease pathogenesis, as well as the development of refined regenerative medicine, biomarker, and therapeutic strategies.

Cellular metabolism and homeostasis in pluripotency regulation.

Pluripotent stem cells (PSCs) can immortally self-renew in culture with a high proliferation rate, and they possess unique metabolic characteristics that facilitate pluripotency regulation. Here, we review recent progress in understanding the mechanisms that link cellular metabolism and homeostasis to pluripotency regulation, with particular emphasis on pathways involving amino acid metabolism, lipid metabolism, the ubiquitin-proteasome system and autophagy. Metabolism of amino acids and lipids is tightly coupled to epigenetic modification, organelle remodeling and cell signaling pathways for pluripotency regulation. PSCs harness enhanced proteasome and autophagy activity to meet the material and energy requirements for cellular homeostasis. These regulatory events reflect a fine balance between the intrinsic cellular requirements and the extrinsic environment. A more complete understanding of this balance will pave new ways to manipulate PSC fate.

Cellular metabolism and homeostasis in pluripotency regulation

Pluripotent stem cells (PSCs) can immortally self-renew in culture with a high proliferation rate, and they possess unique metabolic characteristics that facilitate pluripotency regulation. Here, we review recent progress in understanding the mechanisms that link cellular metabolism and homeostasis to pluripotency regulation, with particular emphasis on pathways involving amino acid metabolism, lipid metabolism, the ubiquitin-proteasome system and autophagy. Metabolism of amino acids and lipids is tightly coupled to epigenetic modification, organelle remodeling and cell signaling pathways for pluripotency regulation. PSCs harness enhanced proteasome and autophagy activity to meet the material and energy requirements for cellular homeostasis. These regulatory events reflect a fine balance between the intrinsic cellular requirements and the extrinsic environment. A more complete understanding of this balance will pave new ways to manipulate PSC fate.

Cellular metabolism and homeostasis in pluripotency regulation

Pluripotent stem cells (PSCs) can immortally self-renew in culture with a high proliferation rate, and they possess unique metabolic characteristics that facilitate pluripotency regulation. Here, we review recent progress in understanding the mechanisms that link cellular metabolism and homeostasis to pluripotency regulation, with particular emphasis on pathways involving amino acid metabolism, lipid metabolism, the ubiquitin-proteasome system and autophagy. Metabolism of amino acids and lipids is tightly coupled to epigenetic modification, organelle remodeling and cell signaling pathways for pluripotency regulation. PSCs harness enhanced proteasome and autophagy activity to meet the material and energy requirements for cellular homeostasis. These regulatory events reflect a fine balance between the intrinsic cellular requirements and the extrinsic environment. A more complete understanding of this balance will pave new ways to manipulate PSC fate.

Cellular metabolism and homeostasis in pluripotency regulation

Pluripotent stem cells (PSCs) can immortally self-renew in culture with a high proliferation rate, and they possess unique metabolic characteristics that facilitate pluripotency regulation. Here, we review recent progress in understanding the mechanisms that link cellular metabolism and homeostasis to pluripotency regulation, with particular emphasis on pathways involving amino acid metabolism, lipid metabolism, the ubiquitin-proteasome system and autophagy. Metabolism of amino acids and lipids is tightly coupled to epigenetic modification, organelle remodeling and cell signaling pathways for pluripotency regulation. PSCs harness enhanced proteasome and autophagy activity to meet the material and energy requirements for cellular homeostasis. These regulatory events reflect a fine balance between the intrinsic cellular requirements and the extrinsic environment. A more complete understanding of this balance will pave new ways to manipulate PSC fate.

Chemically controlled aggregation of pluripotent stem cells.

Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical-based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension-based manufacturing of PSC cultures that are critical for the translation of regenerative medicine strategies to clinical products.