Human Pluripotent Stem Cell Technology Research Articles

Cardiovascular Toxicity in Cancer Therapy: Protecting the Heart while Combating Cancer.

This review explores the cardiovascular toxicity associated with cancer therapies, emphasizing the significance of the growing field of cardio-oncology. It aims to elucidate the mechanisms of cardiotoxicity due to radiotherapy, chemotherapy, and targeted therapies, and to discuss the advancements in human induced pluripotent stem cell technology (hiPSC) for predictive disease modeling. Recent studies have identified several chemotherapeutic agents, including anthracyclines and kinase inhibitors, that significantly increase cardiovascular risks. Advances in hiPSC technology have enabled the differentiation of these cells into cardiovascular lineages, facilitating more accurate modeling of drug-induced cardiotoxicity. Moreover, integrating hiPSCs into clinical trials holds promise for personalized cardiotoxicity assessments, potentially enhancing patient-specific therapeutic strategies. Cardio-oncology bridges oncology and cardiology to mitigate the cardiovascular side-effects of cancer treatments. Despite advancements in predictive models using hiPSCs, challenges persist in accurately replicating adult heart tissue and ensuring reproducibility. Ongoing research is essential for developing personalized therapies that balance effective cancer treatment with minimal cardiovascular harm.

Recent advances and applications of human brain models.

Recent advances in human pluripotent stem cell (hPSC) technologies have prompted the emergence of new research fields and applications for human neurons and brain organoids. Brain organoids have gained attention as an in vitro model system that recapitulates the higher structure, cellular diversity and function of the brain to explore brain development, disease modeling, drug screening, and regenerative medicine. This progress has been accelerated by abundant interactions of brain organoid technology with various research fields. A cross-disciplinary approach with human brain organoid technology offers a higher-ordered advance for more accurately understanding the human brain. In this review, we summarize the status of neural induction in two- and three-dimensional culture systems from hPSCs and the modeling of neurodegenerative diseases using brain organoids. We also highlight the latest bioengineered technologies for the assembly of spatially higher-ordered neural tissues and prospects of brain organoid technology toward the understanding of the potential and abilities of the human brain.

A brief chronicle of research on human pluripotent stem cells.

Today, human pluripotent stem cell technologies find widespread application across biomedical research, as models for early human development, as platforms for functional human genomics, as tools for the study of disease, drug screening and toxicology, and as a renewable source of cellular therapeutics for a range of intractable diseases. The foundations of this human pluripotent stem cell revolution rest on advances in a wide range of disciplines, including cancer biology, assisted reproduction, cell culture and organoid technology, somatic cell nuclear transfer, primate embryology, single-cell biology, and gene editing. This review surveys the slow emergence of the study of human pluripotency and the exponential growth of the field during the past several decades.

Three-dimensional cardiac models: a pre-clinical testing platform.

Major advancements in human pluripotent stem cell (hPSC) technology over recent years have yielded valuable tools for cardiovascular research. Multi-cell type 3-dimensional (3D) cardiac models in particular, are providing complementary approaches to animal studies that are better representatives than simple 2-dimensional (2D) cultures of differentiated hPSCs. These human 3D cardiac models can be broadly divided into two categories; namely those generated through aggregating pre-differentiated cells and those that form self-organizing structures during their in vitro differentiation from hPSCs. These models can either replicate aspects of cardiac development or enable the examination of interactions among constituent cell types, with some of these models showing increased maturity compared with 2D systems. Both groups have already emerged as physiologically relevant pre-clinical platforms for studying heart disease mechanisms, exhibiting key functional attributes of the human heart. In this review, we describe the different cardiac organoid models derived from hPSCs, their generation methods, applications in cardiovascular disease research and use in drug screening. We also address their current limitations and challenges as pre-clinical testing platforms and propose potential improvements to enhance their efficacy in cardiac drug discovery.

Compact lens-free imager using a thin-film transistor for long-term quantitative monitoring of stem cell culture and cardiomyocyte production.

With advancements in human induced pluripotent stem cell (hiPSC) technology, there is an increasing demand for quality control techniques to manage the long-term process of target cell production effectively. While monitoring systems designed for use within incubators are promising for assessing culture quality, existing systems still face challenges in terms of compactness, throughput, and available metrics. To address these limitations, we have developed a compact and high-throughput lens-free imaging device named INSPCTOR. The device is as small as a standard culture plate, which allows for the installation of multiple units within an incubator. INSPCTOR utilises a large thin-film transistor image sensor, enabling simultaneous observation of six independent culture environments, each approximately 1 cm2. With this device, we successfully monitored the confluency of hiPSC cultures and identified the onset timing of epithelial-to-mesenchymal transition during mesodermal induction. Additionally, we quantified the beating frequency and conduction of hiPSC-derived cardiomyocytes by using high-speed imaging modes. This enabled us to identify the onset of spontaneous beating during differentiation and assess chronotropic responses in drug evaluations. Moreover, by tracking beating frequency over 10 days of cardiomyocyte maturation, we identified week-scale and daily-scale fluctuations, the latter of which correlated with cellular metabolic activity. The metrics derived from this device would enhance the reproducibility and quality of target cell production.

Combining Off-flow, a Nextflow-coded program, and whole genome sequencing reveals unintended genetic variation in CRISPR/Cas-edited iPSCs

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.

Abstract 18445: Rigorous Mechanics in Engineered Heart Tissues

Introduction: Heart failure can develop or be exacerbated by mechanical stressors, including increased diastolic strain (preload) and increased systolic impedance (afterload). Recent advances in human induced pluripotent stem cell (iPSC) technology and tissue engineering have enabled long term culture and maturation of engineered heart tissues (EHTs). Most current platforms do not allow for mechanical assessment under a range of loading conditions. Here, we develop a technique to engineer cardiac tissues that allow for non-destructive rigorous phenotyping of tissue mechanics. Methods: Custom tissue anchors were 3D printed using Boston Micro Fabrication HTL Resin and placed within polydimethylsiloxane tissue casting molds. iPS cardiomyocytes and ventricular fibroblasts were mixed 5:1 in 2 mg/mL collagen and cast in rectangular molds. Tissues were compacted around custom tissue anchors over 14 days. Tissues were dismounted from the molds and fixed for immunostaining or hooked onto a force transducer for length-tension, force-frequency, and work-loop analysis. Results: 3D printed tissue anchors accommodated EHT molding and compaction. EHTs spontaneously contracted after 3 days in culture, and compacted to 15.9±0.8% their initial tissue area by 14 days of culture. Engineered tissues were cell dense and mature, with bands of aligned sarcomeres. During isometric testing, tissues display a physiologic length-tension relationship, with linear increases in force with stretch between L 0 (slack length) and L max (length at maximum active force generation, 4.3±0.7 mN/mm 2 ; r 2 =0.96). These are coupled with increases in contraction and relaxation velocities. EHTs were capable of mimicking physiologic work loops. Conclusions: In summary, we have shown the ability to grow and mature three-dimensional human engineered heart tissues capable of rigorous mechanical assessment, enabling future studies on load-dependent cardiac remodeling.

An Improved Protocol for Targeted Differentiation of Primed Human Induced Pluripotent Stem Cells into HLA-G-Expressing Trophoblasts to Enable the Modeling of Placenta-Related Disorders.

Abnormalities at any stage of trophoblast development may result in pregnancy-related complications. Many of these adverse outcomes are discovered later in pregnancy, but the underlying pathomechanisms are constituted during the first trimester. Acquiring developmentally relevant material to elucidate the disease mechanisms is difficult. Human pluripotent stem cell (hPSC) technology can provide a renewable source of relevant cells. BMP4, A83-01, and PD173074 (BAP) treatment drives trophoblast commitment of hPSCs toward syncytiotrophoblast (STB), but lacks extravillous trophoblast (EVT) cells. EVTs mediate key functions during placentation, remodeling of uterine spiral arteries, and maintenance of immunological tolerance. We optimized the protocol for a more efficient generation of HLA-Gpos EVT-like trophoblasts from primed hiPSCs. Increasing the concentrations of A83-01 and PD173074, while decreasing bulk cell density resulted in an increase in HLA-G of up to 71%. Gene expression profiling supports the advancements of our treatment regarding the generation of trophoblast cells. The reported differentiation protocol will allow for an on-demand access to human trophoblast cells enriched for HLA-Gpos EVT-like cells, allowing for the elucidation of placenta-related disorders and investigating the immunological tolerance toward the fetus, overcoming the difficulties in obtaining primary EVTs without the need for a complex differentiation pathway via naïve pluripotent or trophoblast stem cells.

Human induced pluripotent stem cell-derived liver-on-a-chip for studying drug metabolism: the challenge of the cytochrome P450 family.

The liver is the primary organ responsible for the detoxification and metabolism of drugs. To date, a lack of preclinical models that accurately emulate drug metabolism by the human liver presents a significant challenge in the drug development pipeline, particularly for predicting drug efficacy and toxicity. In recent years, emerging microfluidic-based organ-on-a-chip (OoC) technologies, combined with human induced pluripotent stem cell (hiPSC) technology, present a promising avenue for the complete recapitulation of human organ biology in a patient-specific manner. However, hiPSC-derived organoids and liver-on-a-chip models have so far failed to sufficiently express cytochrome P450 monooxygenase (CYP450) enzymes, the key enzymes involved in first-pass metabolism, which limits the effectiveness and translatability of these models in drug metabolism studies. This review explores the potential of innovative organoid and OoC technologies for studying drug metabolism and discusses their existing drawbacks, such as low expression of CYP450 genes. Finally, we postulate potential approaches for enhancing CYP450 expression in the hope of paving the way toward developing novel, fully representative liver drug-metabolism models.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

Ca2+-Mediated Signaling Pathways: A Promising Target for the Successful Generation of Mature and Functional Stem Cell-Derived Pancreatic Beta Cells In Vitro.

Diabetes mellitus is a chronic disease affecting over 500 million adults globally and is mainly categorized as type 1 diabetes mellitus (T1DM), where pancreatic beta cells are destroyed, and type 2 diabetes mellitus (T2DM), characterized by beta cell dysfunction. This review highlights the importance of the divalent cation calcium (Ca2+) and its associated signaling pathways in the proper functioning of beta cells and underlines the effects of Ca2+ dysfunction on beta cell function and its implications for the onset of diabetes. Great interest and promise are held by human pluripotent stem cell (hPSC) technology to generate functional pancreatic beta cells from diabetic patient-derived stem cells to replace the dysfunctional cells, thereby compensating for insulin deficiency and reducing the comorbidities of the disease and its associated financial and social burden on the patient and society. Beta-like cells generated by most current differentiation protocols have blunted functionality compared to their adult human counterparts. The Ca2+ dynamics in stem cell-derived beta-like cells and adult beta cells are summarized in this review, revealing the importance of proper Ca2+ homeostasis in beta-cell function. Consequently, the importance of targeting Ca2+ function in differentiation protocols is suggested to improve current strategies to use hPSCs to generate mature and functional beta-like cells with a comparable glucose-stimulated insulin secretion (GSIS) profile to adult beta cells.

Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning.

Sensory neurons (SNs) detect a wide range of information from the body and the environment that is critical for homeostasis. There are three main subtypes of SNs: nociceptors, mechanoreceptors, and proprioceptors, which express different membrane proteins, such as TRKA, TRKB, or TRKC, respectively. Human pluripotent stem cell technology provides an ideal platform to study development and diseases of SNs, however there is not a viable method to isolate individual SN subtype for downstream analysis available. Here, we employ the method immunopanning to isolate each SN subtype. This method is very gentle and allows proper survival after the isolation. We use antibodies against TRKA, TRKB, and TRKC to isolate nociceptors, mechanoreceptors, and proprioceptors, respectively. We show that our cultures are enriched for each subtype and express their respective subtype markers. Furthermore, we show that the immunopanned SNs are electrically active and respond to specific stimuli. Thus, our method can be used to purify viable neuronal subtypes using respective membrane proteins for downstream studies.

Neuromuscular junction disorders: Experimental models and pathophysiological mechanisms.

Located between skeletal muscle fibers and motoneurons, the neuromuscular junction is a chemical synapse essential for the transmission of information from nervous system to skeletal muscle. There are many diseases related to neuromuscular junction dysfunction, including myasthenia gravis, Lambert‑Eaton myasthenic syndrome, congenital myasthenic syndromes, amyotrophic lateral sclerosis, and spinal muscular atrophy. The pathophysiological mechanisms of these diseases have been investigated using many animal models. Among them, mouse models are the most commonly used and have provided the majority of current data. Moreover, advances in human induced pluripotent stem cell technology has resulted in new opportunities to study neuromuscular junction disorders from both patients and healthy individuals. Currently, patient‑specific induced pluripotent stem cells derived from motor neurons have begun to be studied. These studies will help us achieve a more comprehensive understanding of diseases related to neuromuscular junction disorders. We will describe the research models of neuromuscular junction disorders and provide an overview of recent key findings.

Modeling Human Brain Tumors and the Microenvironment Using Induced Pluripotent Stem Cells

Brain cancer is a group of diverse and rapidly growing malignancies that originate in the central nervous system (CNS) and have a poor prognosis. The complexity of brain structure and function makes brain cancer modeling extremely difficult, limiting pathological studies and therapeutic developments. Advancements in human pluripotent stem cell technology have opened a window of opportunity for brain cancer modeling, providing a wealth of customizable methods to simulate the disease in vitro. This is achieved with the advent of genome editing and genetic engineering technologies that can simulate germline and somatic mutations found in human brain tumors. This review investigates induced pluripotent stem cell (iPSC)-based approaches to model human brain cancer. The applications of iPSCs as renewable sources of individual brain cell types, brain organoids, blood-brain barrier (BBB), and brain tumor models are discussed. The brain tumor models reviewed are glioblastoma and medulloblastoma. The iPSC-derived isogenic cells and three-dimensional (3D) brain cancer organoids combined with patient-derived xenografts will enhance future compound screening and drug development for these deadly human brain cancers.

Harshening stem cell research and precision medicine: The states of human pluripotent cells stem cell repository diversity, and racial and sex differences in transcriptomes.

In vitro investigation on human development, disease modeling, and drug discovery has been empowered by human induced pluripotent stem cell (hiPSC) technologies that form the foundation of precision medicine. Race and sex genetic backgrounds have become a major focus of many diseases modeling and drug response evaluation in the pharmaceutical industry. Here, we gathered data from major stem cell repositories to analyze the diversity with respect to ethnicity, sex, and disease types; and we also analyzed public datasets to unravel transcriptomics differences between samples of different ethnicities and sexes. We found a lack of diversity despite the large sample size of human induced pluripotent stem cells. In the ethnic comparison, the White group made up the majority of the banked hiPSCs. Similarly, for the organ/disease type and sex comparisons, the neural and male hiPSCs accounted for the majority of currently available hiPSCs. Bulk RNA-seq and single-cell transcriptomic analysis coupled with Machine Learning and Network Analysis revealed panels of gene features differently expressed in healthy hiPSCs and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of different races and sexes. The data highlights the current ethnic and sex inequality in stem cell research and demonstrates the molecular biological diversity of hiPSCs and cardiomyocytes from different races and genders. We postulate that future efforts in stem cell biology, regenerative and precision medicine should be guided towards an inclusive, diverse repository reflecting the prevalence of diseases across racial and ethnic groups and the sexes, important for both common and rare disease modeling, drug screening, and cell therapeutics.

Blastocyst complementation and interspecies chimeras in gene edited pigs.

The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.

Stem Cell-Based Therapeutic Strategies for Premature Ovarian Insufficiency and Infertility: A Focus on Aging

Reproductive aging is on the rise globally and inseparable from the entire aging process. An extreme form of reproductive aging is premature ovarian insufficiency (POI), which to date has mostly been of idiopathic etiology, thus hampering further clinical applications and associated with enormous socioeconomic and personal costs. In the field of reproduction, the important functional role of inflammation-induced ovarian deterioration and therapeutic strategies to prevent ovarian aging and increase its function are current research hotspots. This review discusses the general pathophysiology and relative causes of POI and comprehensively describes the association between the aging features of POI and infertility. Next, various preclinical studies of stem cell therapies with potential for POI treatment and their molecular mechanisms are described, with particular emphasis on the use of human induced pluripotent stem cell (hiPSC) technology in the current scenario. Finally, the progress made in the development of hiPSC technology as a POI research tool for engineering more mature and functional organoids suitable as an alternative therapy to restore infertility provides new insights into therapeutic vulnerability, and perspectives on this exciting research on stem cells and the derived exosomes towards more effective POI diagnosis and treatment are also discussed.

Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies.

Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack human in vitro models for airway cholinergic neurons. Here, we aimed to develop a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca2+ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated. The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. Neurons formed contacts with airway smooth muscle cells cultured in the axonal compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.

Induced Pluripotent Stem Cells, a Stepping Stone to In Vitro Human Models of Hearing Loss.

Hearing loss is the most prevalent sensorineural impairment in humans. Yet despite very active research, no effective therapy other than the cochlear implant has reached the clinic. Main reasons for this failure are the multifactorial nature of the disorder, its heterogeneity, and a late onset that hinders the identification of etiological factors. Another problem is the lack of human samples such that practically all the work has been conducted on animals. Although highly valuable data have been obtained from such models, there is the risk that inter-species differences exist that may compromise the relevance of the gathered data. Human-based models are therefore direly needed. The irruption of human induced pluripotent stem cell technologies in the field of hearing research offers the possibility to generate an array of otic cell models of human origin; these may enable the identification of guiding signalling cues during inner ear development and of the mechanisms that lead from genetic alterations to pathology. These models will also be extremely valuable when conducting ototoxicity analyses and when exploring new avenues towards regeneration in the inner ear. This review summarises some of the work that has already been conducted with these cells and contemplates future possibilities.

Genome Editing and Human Pluripotent Stem Cell Technologies for in vitro Monogenic Diabetes Modeling.

Diabetes is a metabolic disease characterized by chronic hyperglycemia. Polygenic diabetes, which encompasses type-1 and type-2 diabetes, is the most prevalent kind of diabetes and is caused by a combination of different genetic and environmental factors, whereas rare phenotype monogenic diabetes is caused by a single gene mutation. Monogenic diabetes includes Neonatal diabetes mellitus and Maturity-onset diabetes of the young. The majority of our current knowledge about the pathogenesis of diabetes stems from studies done on animal models. However, the genetic difference between these creatures and humans makes it difficult to mimic human clinical pathophysiology, limiting their value in modeling key aspects of human disease. Human pluripotent stem cell technologies combined with genome editing techniques have been shown to be better alternatives for creating in vitro models that can provide crucial knowledge about disease etiology. This review paper addresses genome editing and human pluripotent stem cell technologies for in vitro monogenic diabetes modeling.