Generation of iPSCs Using Sendai Virus Vectors.

Induced pluripotent stem cells (iPSCs) are in vitro-derived cells capable of giving rise to several different cell types. The generation of iPSCs holds great promise for regenerative medicine and drug discovery research because it allows mature cells to be reprogrammed into a state of pluripotency. These highly versatile cells can then be induced to produce a variety of cell lineages and tissues by activating specific regulatory genes that drive their differentiation along distinct lineages. The great potential of these cells was recognized by Shinya Yamanaka who was awarded the 2012 Nobel Prize for the discovery of iPSCs. Following their discovery, various methods have now been developed for generating iPSCs. Here, we describe a method for deriving iPSCs from human dental pulp using Sendai virus vectors.

Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.

Human-induced pluripotent stem cell (hiPSC) technology has enabled comprehensive human cell-based disease modeling in vitro. Due to limited accessibility of primary human neurons as well as species-specific divergence between human and rodent brain tissues, hiPSC-derived neurons have become a popular tool for studying neuronal biology in a dish. Here, we provide methods for transcription factor-driven directed differentiation of neurons from hiPSCs via a neural progenitor cell (NPC) intermediate. Doxycycline-inducible expression of neuron fate-determining transcription factors neurogenin 2 (NGN2) and achaete-scute homolog 1 (ASCL1) enables rapid and controllable differentiation of human neurons for disease modeling applications. The provided method is also designed to improve the reproducibility of human neuron differentiation by reducing the batch-to-batch variation of NPC differentiation and lentiviral transduction.

Generating Homogeneous Brain Organoids from Human iPSCs.

There is a high demand for the development of in vitro models for human brain development and diseases due to the inaccessibility of human brain tissues. The human iPSC-derived brain organoids provide a promising in vitro model for studying human brain development and disorders. However, it is challenging to generate a large number of brain organoids with high consistency for modeling human neurological diseases. Here, we describe a method for generating high-yield brain organoids with high consistency by combining large-scale embryoid body (EB) generation and incorporating a quality control screening step during differentiation. The method described in this chapter provides a robust way to generate brain organoids for studying human brain development and modeling neurological diseases.

Laminin Alpha 2 Enhances the Protective Effect of Exosomes on Human iPSC-Derived Cardiomyocytes in an In Vitro Ischemia-Reoxygenation Model.

Ischemic heart disease, a leading cause of death worldwide, manifests clinically as myocardial infarction. Contemporary therapies using mesenchymal stromal cells (MSCs) and their derivative (exosomes, EXOs) were developed to decrease the progression of cell damage during ischemic injury. Laminin alpha 2 (LAMA2) is an important extracellular matrix protein of the heart. Here, we generated MSC-derived exosomes cultivated under LAMA2 coating to enhance human-induced pluripotent stem cell (hiPSC)-cardiomyocyte recognition of LAMA2-EXOs, thus, increasing cell protection during ischemia reoxygenation. We mapped the mRNA content of LAMA2 and gelatin-EXOs and identified 798 genes that were differentially expressed, including genes associated with cardiac muscle development and extracellular matrix organization. Cells were treated with LAMA2-EXOs 2 h before a 4 h ischemia period (1% O2, 5% CO2, glucose-free media). LAMA2-EXOs had a two-fold protective effect compared to non-treatment on plasma membrane integrity and the apoptosis activation pathway; after a 1.5 h recovery period (20% O2, 5% CO2, cardiomyocyte-enriched media), cardiomyocytes treated with LAMA2-EXOs showed faster recovery than did the control group. Although EXOs had a protective effect on endothelial cells, there was no LAMA2-enhanced protection on these cells. This is the first report of LAMA2-EXOs used to treat cardiomyocytes that underwent ischemia-reoxygenation injury. Overall, we showed that membrane-specific EXOs may help improve cardiomyocyte survival in treating ischemic cardiovascular disease.

Stem Cell Therapy against Ischemic Heart Disease.

Ischemic heart disease, which is one of the top killers worldwide, encompasses a series of heart problems stemming from a compromised coronary blood supply to the myocardium. The severity of the disease ranges from an unstable manifestation of ischemic symptoms, such as unstable angina, to myocardial death, that is, the immediate life-threatening condition of myocardial infarction. Even though patients may survive myocardial infarction, the resulting ischemia-reperfusion injury triggers a cascade of inflammatory reactions and oxidative stress that poses a significant threat to myocardial function following successful revascularization. Moreover, despite evidence suggesting the presence of cardiac stem cells, the fact that cardiomyocytes are terminally differentiated and cannot significantly regenerate after injury accounts for the subsequent progression to ischemic cardiomyopathy and ischemic heart failure, despite the current advancements in cardiac medicine. In the last two decades, researchers have realized the possibility of utilizing stem cell plasticity for therapeutic purposes. Indeed, stem cells of different origin, such as bone-marrow- and adipose-derived mesenchymal stem cells, circulation-derived progenitor cells, and induced pluripotent stem cells, have all been shown to play therapeutic roles in ischemic heart disease. In addition, the discovery of stem-cell-associated paracrine effects has triggered intense investigations into the actions of exosomes. Notwithstanding the seemingly promising outcomes from both experimental and clinical studies regarding the therapeutic use of stem cells against ischemic heart disease, positive results from fraud or false data interpretation need to be taken into consideration. The current review is aimed at overviewing the therapeutic application of stem cells in different categories of ischemic heart disease, including relevant experimental and clinical outcomes, as well as the proposed mechanisms underpinning such observations.

The Role of Stem Cells in the Treatment of Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of death and include several vascular and cardiac disorders, such as atherosclerosis, coronary artery disease, cardiomyopathies, and heart failure. Multiple treatment strategies exist for CVDs, but there is a need for regenerative treatment of damaged heart. Stem cells are a broad variety of cells with a great differentiation potential that have regenerative and immunomodulatory properties. Multiple studies have evaluated the efficacy of stem cells in CVDs, such as mesenchymal stem cells and induced pluripotent stem cell-derived cardiomyocytes. These studies have demonstrated that stem cells can improve the left ventricle ejection fraction, reduce fibrosis, and decrease infarct size. Other studies have investigated potential methods to improve the survival, engraftment, and functionality of stem cells in the treatment of CVDs. The aim of the present review is to summarize the current evidence on the role of stem cells in the treatment of CVDs, and how to improve their efficacy.

C9ORF72 patient-derived endothelial cells drive blood-brain barrier disruption and contribute to neurotoxicity.

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.

Cockayne Syndrome Patient iPSC-Derived Brain Organoids and Neurospheres Show Early Transcriptional Dysregulation of Biological Processes Associated with Brain Development and Metabolism.

Cockayne syndrome (CS) is a rare hereditary autosomal recessive disorder primarily caused by mutations in Cockayne syndrome protein A (CSA) or B (CSB). While many of the functions of CSB have been at least partially elucidated, little is known about the actual developmental dysregulation in this devasting disorder. Of particular interest is the regulation of cerebral development as the most debilitating symptoms are of neurological nature. We generated neurospheres and cerebral organoids utilizing Cockayne syndrome B protein (CSB)-deficient induced pluripotent stem cells derived from two patients with distinct severity levels of CS and healthy controls. The transcriptome of both developmental timepoints was explored using RNA-Seq and bioinformatic analysis to identify dysregulated biological processes common to both patients with CS in comparison to the control. CSB-deficient neurospheres displayed upregulation of the VEGFA-VEGFR2 signalling pathway, vesicle-mediated transport and head development. CSB-deficient cerebral organoids exhibited downregulation of brain development, neuron projection development and synaptic signalling. We further identified the upregulation of steroid biosynthesis as common to both timepoints, in particular the upregulation of the cholesterol biosynthesis branch. Our results provide insights into the neurodevelopmental dysregulation in patients with CS and strengthen the theory that CS is not only a neurodegenerative but also a neurodevelopmental disorder.

Challenges and Considerations of Preclinical Development for iPSC-Based Myogenic Cell Therapy.

Cell therapies derived from induced pluripotent stem cells (iPSCs) offer a promising avenue in the field of regenerative medicine due to iPSCs' expandability, immune compatibility, and pluripotent potential. An increasing number of preclinical and clinical trials have been carried out, exploring the application of iPSC-based therapies for challenging diseases, such as muscular dystrophies. The unique syncytial nature of skeletal muscle allows stem/progenitor cells to integrate, forming new myonuclei and restoring the expression of genes affected by myopathies. This characteristic makes genome-editing techniques especially attractive in these therapies. With genetic modification and iPSC lineage specification methodologies, immune-compatible healthy iPSC-derived muscle cells can be manufactured to reverse the progression of muscle diseases or facilitate tissue regeneration. Despite this exciting advancement, much of the development of iPSC-based therapies for muscle diseases and tissue regeneration is limited to academic settings, with no successful clinical translation reported. The unknown differentiation process in vivo, potential tumorigenicity, and epigenetic abnormality of transplanted cells are preventing their clinical application. In this review, we give an overview on preclinical development of iPSC-derived myogenic cell transplantation therapies including processes related to iPSC-derived myogenic cells such as differentiation, scaling-up, delivery, and cGMP compliance. And we discuss the potential challenges of each step of clinical translation. Additionally, preclinical model systems for testing myogenic cells intended for clinical applications are described.

Unlocking the Future: Pluripotent Stem Cell-Based Lung Repair.

The human respiratory system is susceptible to a variety of diseases, ranging from chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis to acute respiratory distress syndrome (ARDS). Today, lung diseases represent one of the major challenges to the health care sector and represent one of the leading causes of death worldwide. Current treatment options often focus on managing symptoms rather than addressing the underlying cause of the disease. The limitations of conventional therapies highlight the urgent clinical need for innovative solutions capable of repairing damaged lung tissue at a fundamental level. Pluripotent stem cell technologies have now reached clinical maturity and hold immense potential to revolutionize the landscape of lung repair and regenerative medicine. Meanwhile, human embryonic (HESCs) and human-induced pluripotent stem cells (hiPSCs) can be coaxed to differentiate into lung-specific cell types such as bronchial and alveolar epithelial cells, or pulmonary endothelial cells. This holds the promise of regenerating damaged lung tissue and restoring normal respiratory function. While methods for targeted genetic engineering of hPSCs and lung cell differentiation have substantially advanced, the required GMP-grade clinical-scale production technologies as well as the development of suitable preclinical animal models and cell application strategies are less advanced. This review provides an overview of current perspectives on PSC-based therapies for lung repair, explores key advances, and envisions future directions in this dynamic field.

Development of a high throughput system to screen compounds that revert the activated hepatic stellate cells to a quiescent-like state.

Chronic liver injury induces fibrosis that often proceeds to cirrhosis and hepatocellular carcinoma, indicating that prevention and/or resolution of fibrosis is a promising therapeutic target. Hepatic stellate cells (HSCs) are the major driver of fibrosis by expressing extracellular matrices (ECM). HSCs, in the normal liver, are quiescent and activated by liver injury to become myofibroblasts that proliferate and produce ECM. It has been shown that activated HSCs (aHSCs) become a "quiescent-like" state by removal of liver insults. Therefore, deactivation agents can be a therapeutic drug for advanced liver fibrosis. Using aHSCs prepared from human induced pluripotent stem cells, we found that aHSCs were reverted to a quiescent-like state by a combination of chemical compounds that either inhibit or activate a signaling pathway, Lanifibranor, SB431542, Dorsomorphin, retinoic acid, palmitic acid and Y27632, in vitro. Based on these results, we established a high throughput system to screen agents that induce deactivation and demonstrate that a single chemical compound can induce deactivation.

Midbrain organoids for Parkinson's disease (PD) - A powerful tool to understand the disease pathogenesis.

Brain Organiods (BOs) are a promising technique for researching disease progression in the human brain. These organoids, which are produced from human induced pluripotent stem cells (HiPSCs), can construct themselves into structured frameworks. In the context of Parkinson's disease (PD), recent advancements have been made in the development of Midbrain organoids (MBOs) models that consider key pathophysiological mechanisms such as alpha-synuclein (α-Syn), Lewy bodies, dopamine loss, and microglia activation. However, there are limitations to the current use of BOs in disease modelling and drug discovery, such as the lack of vascularization, long-term differentiation, and absence of glial cells. To address these limitations, researchers have proposed the use of spinning bioreactors to improve oxygen and nutrient perfusion. Modelling PD utilising modern experimental in vitro models is a valuable tool for studying disease mechanisms and elucidating previously unknown features of PD. In this paper, we exclusively review the unique methods available for cultivating MBOs using a pumping system that mimics the circulatory system. This mechanism may aid in delivering the required amount of oxygen and nutrients to all areas of the organoids, preventing cell death, and allowing for long-term culture and using co-culturing techniques for developing glial cell in BOs. Furthermore, we emphasise some of the significant discoveries about the BOs and the potential challenges of using BOs will be discussed.

Protein-truncating variants in BSN are associated with severe adult-onset obesity, type 2 diabetes and fatty liver disease.

Obesity is a major risk factor for many common diseases and has a substantial heritable component. To identify new genetic determinants, we performed exome-sequence analyses for adult body mass index (BMI) in up to 587,027 individuals. We identified rare loss-of-function variants in two genes (BSN and APBA1) with effects substantially larger than those of well-established obesity genes such as MC4R. In contrast to most other obesity-related genes, rare variants in BSN and APBA1 were not associated with normal variation in childhood adiposity. Furthermore, BSN protein-truncating variants (PTVs) magnified the influence of common genetic variants associated with BMI, with a common variant polygenic score exhibiting an effect twice as large in BSN PTV carriers than in noncarriers. Finally, we explored the plasma proteomic signatures of BSN PTV carriers as well as the functional consequences of BSN deletion in human induced pluripotent stem cell-derived hypothalamic neurons. Collectively, our findings implicate degenerative processes in synaptic function in the etiology of adult-onset obesity.

The use of human iPSC-derived alveolar organoids to explore SARS-CoV-2 variant infections and host responses.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system. Physiologically relevant human lung models are indispensable to investigate virus-induced host response and disease pathogenesis. In this study, we generated human induced pluripotent stem cell (iPSC)-derived alveolar organoids (AOs) using an established protocol that recapitulates the sequential steps of in vivo lung development. AOs express alveolar epithelial type II cell protein markers including pro-surfactant protein C and ATP binding cassette subfamily A member 3. Compared to primary human alveolar type II cells, AOs expressed higher mRNA levels of SARS-CoV-2 entry factors, angiotensin-converting enzyme 2 (ACE2), asialoglycoprotein receptor 1 (ASGR1) and basigin (CD147). Considering the localization of ACE2 on the apical side in AOs, we used three AO models, apical-in, sheared and apical-out for SARS-CoV-2 infection. All three models of AOs were robustly infected with the SARS-CoV-2 irrespective of ACE2 accessibility. Antibody blocking experiment revealed that ASGR1 was the main receptor for SARS-CoV2 entry from the basolateral in apical-in AOs. AOs supported the replication of SARS-CoV-2 variants WA1, Alpha, Beta, Delta, and Zeta and Omicron to a variable degree with WA1 being the highest and Omicron being the least. Transcriptomic profiling of infected AOs revealed the induction of inflammatory and interferon-related pathways with NF-κB signaling being the predominant host response. In summary, iPSC-derived AOs can serve as excellent human lung models to investigate infection of SARS-CoV-2 variants and host responses from both apical and basolateral sides.

Comparative Study of Basement-membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

SARS-CoV-2 causes dysfunction in human iPSC-derived brain microvascular endothelial cells potentially by modulating the Wnt signaling pathway.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is associated with various neurological symptoms, including nausea, dizziness, headache, encephalitis, and epileptic seizures. SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood-brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the detailed mechanism has not been fully elucidated. METHODS: Using the original strain of SARS-CoV-2, the infection in BMECs was confirmed by a detection of intracellular RNA copy number and localization of viral particles. BMEC functions were evaluated by measuring transendothelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics, and expression levels of proinflammatory genes. BMEC signaling pathway was examined by comprehensive RNA-seq analysis. RESULTS: We observed that iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs) were infected with SARS-CoV-2. SARS-CoV-2 infection resulted in decreased TEER. In addition, SARS-CoV-2 infection decreased expression levels of tight junction markers CLDN3 and CLDN11. SARS-CoV-2 infection also increased expression levels of proinflammatory genes, which are known to be elevated in patients with COVID-19. Furthermore, RNA-seq analysis revealed that SARS-CoV-2 dysregulated the canonical Wnt signaling pathway in iPSC-BMELCs. Modulation of the Wnt signaling by CHIR99021 partially inhibited the infection and the subsequent inflammatory responses. CONCLUSION: These findings suggest that SARS-CoV-2 infection causes BBB dysfunction via Wnt signaling. Thus, iPSC-BMELCs are a useful in vitro model for elucidating COVID-19 neuropathology and drug development.

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

Insights for disease modeling from single-cell transcriptomics of iPSC-derived Ngn2-induced neurons and astrocytes across differentiation time and co-culture.

BACKGROUND: Trans-differentiation of human-induced pluripotent stem cells into neurons via Ngn2-induction (hiPSC-N) has become an efficient system to quickly generate neurons a likely significant advance for disease modeling and in vitro assay development. Recent single-cell interrogation of Ngn2-induced neurons, however, has revealed some similarities to unexpected neuronal lineages. Similarly, a straightforward method to generate hiPSC-derived astrocytes (hiPSC-A) for the study of neuropsychiatric disorders has also been described. RESULTS: Here, we examine the homogeneity and similarity of hiPSC-N and hiPSC-A to their in vivo counterparts, the impact of different lengths of time post Ngn2 induction on hiPSC-N (15 or 21 days), and the impact of hiPSC-N/hiPSC-A co-culture. Leveraging the wealth of existing public single-cell RNA-seq (scRNA-seq) data in Ngn2-induced neurons and in vivo data from the developing brain, we provide perspectives on the lineage origins and maturation of hiPSC-N and hiPSC-A. While induction protocols in different labs produce consistent cell type profiles, both hiPSC-N and hiPSC-A show significant heterogeneity and similarity to multiple in vivo cell fates, and both more precisely approximate their in vivo counterparts when co-cultured. Gene expression data from the hiPSC-N show enrichment of genes linked to schizophrenia (SZ) and autism spectrum disorders (ASD) as has been previously shown for neural stem cells and neurons. These overrepresentations of disease genes are strongest in our system at early times (day 15) in Ngn2-induction/maturation of neurons, when we also observe the greatest similarity to early in vivo excitatory neurons. We have assembled this new scRNA-seq data along with the public data explored here as an integrated biologist-friendly web-resource for researchers seeking to understand this system more deeply: https://nemoanalytics.org/p?l=DasEtAlNGN2&g=NES . CONCLUSIONS: While overall we support the use of the investigated cellular models for the study of neuropsychiatric disease, we also identify important limitations. We hope that this work will contribute to understanding and optimizing cellular modeling for complex brain disorders.

Profiling the role of m6A effectors in the regulation of pluripotent reprogramming.

The N6-methyladenosine (m6A) RNA modification plays essential roles in multiple biological processes, including stem cell fate determination. To explore the role of the m6A modification in pluripotent reprogramming, we used RNA-seq to map m6A effectors in human iPSCs, fibroblasts, and H9 ESCs, as well as in mouse ESCs and fibroblasts. By integrating the human and mouse RNA-seq data, we found that 19 m6A effectors were significantly upregulated in reprogramming. Notably, IGF2BPs, particularly IGF2BP1, were among the most upregulated genes in pluripotent cells, while YTHDF3 had high levels of expression in fibroblasts. Using quantitative PCR and Western blot, we validated the pluripotency-associated elevation of IGF2BPs. Knockdown of IGF2BP1 induced the downregulation of stemness genes and exit from pluripotency. Proteome analysis of cells collected at both the beginning and terminal states of the reprogramming process revealed that the IGF2BP1 protein was positively correlated with stemness markers SOX2 and OCT4. The eCLIP-seq target analysis showed that IGF2BP1 interacted with the coding sequence (CDS) and 3'UTR regions of the SOX2 transcripts, in agreement with the location of m6A modifications. This study identifies IGF2BP1 as a vital pluripotency-associated m6A effector, providing new insight into the interplay between m6A epigenetic modifications and pluripotent reprogramming.

Genetically engineered macrophages derived from iPSCs for self-regulating delivery of anti-inflammatory biologic drugs.

In rheumatoid arthritis, dysregulated cytokine signaling has been implicated as a primary factor in chronic inflammation. Many antirheumatic and biological therapies are used to suppress joint inflammation, but despite these advances, effectiveness is not universal, and delivery is often at high doses, which can predispose patients to significant off-target effects. During chronic inflammation, the inappropriate regulation of signaling factors by macrophages accelerates progression of disease by driving an imbalance of inflammatory cytokines, making macrophages an ideal cellular target. To develop a macrophage-based therapy to treat chronic inflammation, we engineered a novel induced pluripotent stem cell (iPSC)-derived macrophage capable of delivering soluble TNF receptor 1 (TNFR1), an anti-inflammatory biologic inhibitor of tumor necrosis factor alpha (TNF-α), in an auto-regulated manner in response to TNF-α. Murine iPSCs were differentiated into macrophages (iMACs) over a 17-day optimized protocol with continued successful differentiation confirmed at key timepoints. Varying inflammatory and immunomodulatory stimuli demonstrated traditional macrophage function and phenotypes. In response to TNF-α, therapeutic iMACs produced high levels of sTNFR1 in an autoregulated manner, which inhibited inflammatory signaling. This self-regulating iMAC system demonstrated the potential for macrophage-based drug delivery as a novel therapeutic approach for a variety of chronic inflammatory diseases.