Pluripotent Stem Cells Research Articles

Stem Cell Interventions in Neurology: From Bench to Bedside.

Stem cell therapies are progressively redefining the treatment landscape for a spectrum of neurological and age-related disorders. This review discusses the molecular and functional attributes of stem cells, emphasizing the roles of neural stem cells and mesenchymal stem cells in the context of neurological diseases such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, Parkinson's disease, and Alzheimer's disease. The review also explores the potential of stem cells in addressing the aging process. The paper analyzes stem cells' intrinsic properties of self-renewal, differentiation, and paracrine effects, alongside the importance of laboratory-modified stem cells like induced pluripotent stem cells and transgenic stem cells. Insights into disease-specific stem cell treatments are offered, reviewing both successes and challenges in the field. This includes the translational difficulties from rodent studies to human trials. The review concludes by acknowledging the uncharted territories that warrant further investigation, emphasizing the potential roles of stem cell-derived exosomes and indole-related molecules, and aiming at providing a basic understanding of stem cell therapies.

Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness

Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.

Mechanosensitive Differentiation of Human iPS Cell-Derived Podocytes.

Stem cell fate decisions, including proliferation, differentiation, morphological changes, and viability, are impacted by microenvironmental cues such as physical and biochemical signals. However, the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study, we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion, differentiation, and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically, hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering, disease modeling, and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses.

Modified mRNA-based gene editing reveals sarcomere-based regulation of gene expression in human induced-pluripotent stem cell-derived cardiomyocytes

Mutations in genes coding sarcomere components are the major causes of human inherited cardiomyopathy. Genome editing is widely applied to genetic modification of human pluripotent stem cells (hPSCs) before hPSCs were differentiated into cardiomyocytes to model cardiomyopathy. Whether genetic mutations influence the early hPSC differentiation process or solely the terminally differentiated cardiomyocytes during cardiac pathogenesis remains challenging to distinguish. To solve this problem, here we harnessed chemically modified mRNA (modRNA) and synthetic single-guide RNA to develop an efficient genome editing approach in hPSC-derived cardiomyocytes (hPSC-CMs). We showed that modRNA-based CRISPR/Cas9 mutagenesis of TNNT2, the coding gene for cardiac troponin T, results in sarcomere disassembly and contractile dysfunction in hPSC-CMs. These structural and functional phenotypes were associated with profound downregulation of oxidative phosphorylation genes and upregulation of cardiac stress markers NPPA and NPPB. These data confirmed that sarcomeres regulate gene expression in hPSC-CMs and highlighted the RNA technology as a powerful tool to achieve stage-specific genome editing during hPSC differentiation.

The restricted N-glycome of neurons is programmed during differentiation.

The protein glycome of individual cell types in the brain is unexplored, despite the critical function of these modifications in development and disease. In aggregate, the most abundant asparagine (N-) linked glycans in the adult brain are high mannose structures, and specifically Man 5 GlcNAc 2 (Man-5), which normally exits the ER for further processing in the Golgi. Mannose structures are uncommon in other organs and often overlooked or excluded in most studies. To understand cell-specific contributions to the unique brain N-glycome and its abundance of Man-5, we performed RNAseq and MALDI-MS TOF protein N-glycomics at several timepoints during differentiation of multiple cell types. To this end, homogeneous cultures of glutamatergic neurons, GABAergic neurons, and brain-specific endothelial cells were generated from monoclonal human inducible pluripotent stem cells (hiPSCs) through cellular reprogramming. Small molecule induction of stably integrated synthetic transcription units driving morphogen expression generated differentiated cells with distinct patterns mirroring intact tissue. Comparing uninduced hiPSCs for each cell type revealed identical transcriptomic and glycomic profiles before differentiation, with low quantities of Man-5. In differentiated glutamatergic and GABAergic neurons, the most abundant N-glycans became Man-5 and its immediate precursor Man-6, despite the presence of transcripts encoding enzymes for their subsequent modification. Differentiation to brain-specific endothelial cells showed an opposite effect, with the N-glycome displaying an abundance of complex N-glycans and terminal modifications of the late secretory pathway. These results confirm that the restricted N-glycome profile of brain is programmed into neuronal differentiation, with regulation independent of the transcriptome and under tight evolutionary constraint.

Influence of Microenvironmental Orchestration on Multicellular Lung Alveolar Organoid Development from Human Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have emerged as promising in vitro tools, providing a robust system for disease modelling and facilitating drug screening. Human iPSCs have been successfully differentiated into lung cells and three-dimensional lung spheroids or organoids. The lung is a multicellular complex organ that develops under the symphonic influence of the microenvironment. Here, we hypothesize that the generation of lung organoids in a controlled microenvironment (cmO) (oxygen and pressure) yields multicellular organoids with architectural complexity resembling the lung alveoli. iPSCs were differentiated into mature lung organoids following a stepwise protocol in an oxygen and pressure-controlled microenvironment. The organoids developed in the controlled microenvironment displayed complex alveolar architecture and stained for SFTPC, PDPN, and KRT5, indicating the presence of alveolar epithelial type II and type I cells, as well as basal cells. Moreover, gene and protein expression levels were also increased in the cmO. Furthermore, pathway analysis of proteomics revealed upregulation of lung development-specific pathways in the cmO compared to those growing in normal culture conditions. In summary, by using a controlled microenvironment, we established a complex multicellular lung organoid derived from iPSCs as a novel cellular model to study lung alveolar biology in both lung health and disease.

STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influences transgene silencing, and demonstrate the impact of these design features on forward programming of hiPSCs into neurons. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely-used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of both transcription factors and functional reporters. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

A stepwise approach to deriving functional β-cells from human embryonic or induced pluripotent stem cells

Abstract Our understanding of β-cell differentiation from pluripotent stem cells (PSCs) is rapidly evolving. Although progress has been made, challenges remain, particularly in achieving glucose-stimulated insulin secretion (GSIS). Human embryonic stem cells (hESCs) are valuable due to their pluripotent ability. A fixed protocol targeting master regulatory genes initiates stem cells into pancreatic lineage commitment. Due to the observations that a single stem cell can differentiate into multiple cell types depending on various factors and conditions, non-linear differentiation pathways exist. Co-expression of key factors remains essential for successful β-cell differentiation. The mature β-cell marker MAFA plays a critical role in maintaining the differentiation state and preventing dedifferentiation. Recapitulating pancreatic islet clustering enhances physiological responses, offering potential avenues for diabetes treatment. On the other hand, several enhanced differentiation protocols from induced pluripotent stem cells (iPSCs) have improved the functional insulin producing β-cells generated. These findings, with their potential to revolutionize diabetes treatment, highlight the complexity of β-cell differentiation and guide further advancements in regenerative medicine.

Phosphorylation-driven epichaperome assembly is a regulator of cellular adaptability and proliferation

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90’s interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine.

The Need for Speed; Investigating Channelopathy-Associated Epilepsy Using High Throughput Electrophysiological Approaches

Pathogenic variants in genes encoding ion channels are frequently discovered in monogenic disorders associated with epilepsy and neurodevelopmental disorders. This review covers advances in the use of automated patch clamp recording for determining the functional consequences of epilepsy-associated ion channel variants and the use of induced pluripotent stem cell (iPSC) derived neurons for in-depth investigations of the physiological consequences of such variants. The combination of these advanced technologies was a focus of the recently completed NINDS-funded Channelopathy-associated Epilepsy Research Center without Walls.

Investigating the Impact of Microgravity on Stem Cells for Cardiovascular Disease Treatment

Cardiovascular disease is a widespread cause of physical disability, and while treatment options result in reduced mortality, they are still limited in their capabilities for cardiac repair. Since their discovery, the regenerative capacities of stem cells have been hypothesized to one day reverse countless diseases. Current stem cell research in cardiovascular disease has delineated the regenerative capabilities of bone marrow–derived mesenchymal stem cells, induced pluripotent stem cells, and cardiac stem cells, but has its limitations. Additionally, current therapies reduce mortality rates and the risk of recurrent myocardial infarctions and damage, but they do not address the apparently irreversible remodeling of myocardial tissue, which is linked to frequent hospitalizations and a diminished quality of life. As researchers continue to investigate the most efficacious growth mediums for stem cells, microgravity appears to be a supportive environment for stem cell growth. Thus, this review aims to catalogue existing research on microgravity’s differing effects on various stem cell lineages and what implications it may have on the treatment of cardiovascular diseases. Cells grown under these conditions have increased expression of cardioprotective proteins and improved proliferative capacities but may have elevated tumorigenic potential and hypertrophic pathways. Additionally, the results of microgravity’s effects on stem cells are mixed. Nonetheless, microgravity’s impact on the cytoskeleton, overexpression of electrical contraction receptors, and increased proliferative and pluripotent effects make these cells promising avenues for potential improved clinical treatment options.

CRISPR-Mediated Gene Editing Tool Development for Patients with Usher Syndrome Type II

Usher syndrome, a rare genetic disease affecting hearing and vision, is an autosomal recessive condition affecting 4 to 17 per 100,000 people and accounting for about 50 percent of hereditary deaf-blindness cases. Usher syndrome type II is caused by mutations of the USH2A gene, a 15.7 kb gene encoding the protein usherin, which localizes to photoreceptor cilium and cochlear hair cells. This subtype of Usher syndrome includes hearing loss from birth and progressive loss of vision, prompting retinitis pigmentosa (RP). Prime editing is a viable option for addressing USH2A mutations and restoring usherin expression and function. To begin, a HEK293T cell line needs to be established for subsequent testing of prime editors. Since prime editing to make the cell line was unsuccessful, homology-directed repair was done and successfully created a cell population with the mutation of interest (single base-pair deletion). Now that this cell population has been created, prime editors will be tested and optimized to address the mutation in a HEK293T cell line and potentially patient-derived induced pluripotent stem cells that are differentiated into photoreceptor precursor cells.

ZMYND11 Functions in Bimodal Regulation of Latent Genes and Brain-like Splicing to Safeguard Corticogenesis.

Despite the litany of pathogenic variants linked to neurodevelopmental disorders (NDD) including autism (ASD) and intellectual disability 1,2 , our understanding of the underlying mechanisms caused by risk genes remain unclear. Here, we leveraged a human pluripotent stem cell model to uncover the neurodevelopmental consequences of mutations in ZMYND11 , a newly implicated risk gene 3,4 . ZMYND11, known for its tumor suppressor function, encodes a histone-reader that recognizes sites of transcriptional elongation and acts as a co-repressor 5,6 . Our findings reveal that ZMYND11-deficient cortical neural stem cells showed upregulation of latent developmental pathways, impairing progenitor and neuron production. In addition to its role on histones, ZMYND11 controls a brain-specific isoform switch involving the splicing regulator RBFOX2. Extending our findings to other chromatin-related ASD risk factors revealed similar developmental pathway activation and splicing dysregulation, partially rescuable through ZMYND11's regulatory functions.

A prenatal skin atlas reveals immune regulation of human skin morphogenesis.

Human prenatal skin is populated by innate immune cells, including macrophages, but whether they act solely in immunity or have additional functions in morphogenesis is unclear. Here we assembled a comprehensive multi-omics reference atlas of prenatal human skin (7-17 post-conception weeks), combining single-cell and spatial transcriptomics data, to characterize the microanatomical tissue niches of the skin. This atlas revealed that crosstalk between non-immune and immune cells underpins the formation of hair follicles, is implicated inscarless wound healing and is crucial for skin angiogenesis. We systematically compared a hair-bearing skin organoid (SkO) model derived from human embryonic stem cells and induced pluripotent stem cells to prenatal and adult skin1. The SkO model closely recapitulated in vivo skin epidermal and dermal cell types during hair follicle development and expression of genes implicated in the pathogenesis of genetic hair and skin disorders. However, the SkO model lacked immune cells and had markedly reduced endothelial cell heterogeneity and quantity. Our in vivo prenatal skin cell atlas indicated that macrophages and macrophage-derived growth factors have a role in driving endothelial development. Indeed, vascular network remodelling was enhanced following transfer of autologous macrophages derived from induced pluripotent stem cells into SkO cultures. Innate immune cells are therefore key players in skin morphogenesis beyond their conventional role in immunity, a function they achieve through crosstalk with non-immune cells.

The role and mechanism of NRG1/ErbB4 in inducing the differentiation of induced pluripotent stem cells into cardiomyocytes

BackgroundWe aimed to investigate the effect and potential mechanism of enhancing Neuregulin1 (NRG1)/v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4 (ErbB4) expression on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes.MethodsWe utilized CRISPR-CAS9 technology to knock in ErbB4 and obtained a single-cell clone IPSN-AAVS1-CMV-ErbB4 (iPSCs-ErbB4). Subsequently, we induced the differentiation of iPSCs into cardiomyocytes and quantified the number of beating embryoid bodies. Furthermore, quantitative real-time PCR assessed the expression of cardiomyocyte markers, including ANP (atrial natriuretic peptide), Nkx2.5 (NK2 transcription factor related locus 5), and GATA4 (GATA binding protein 4). On the 14th day of differentiation, we observed the α-MHC (α-myosin heavy chain)-positive area using immunofluorescent staining and conducted western blotting to detect the expression of cTnT (cardiac troponin) protein and PI3K/Akt signaling pathway-related proteins. Additionally, we intervened the iPSCs-ErbB4 + NRG1 group with the PI3K/Akt inhibitor LY294002 and observed alterations in the expression of cardiomyocyte differentiation-related genes.ResultsThe number of beating embryoid bodies increased after promoting the expression of NRG1/ErbB4 compared to the iPSCs control group. Cardiomyocyte markers ANP, Nkx2.5, and GATA4 significantly increased on day 14 of differentiation, and the positive area of α-MHC was three times that of the iPSCs control group. Moreover, there was a marked increase in cTnT protein expression. However, there was no significant difference in cardiomyocyte differentiation between the iPSCs-ErbB4 group and the iPSCs control group. Akt phosphorylation was significantly increased in the iPSCs-ErbB4 + NRG1 group. LY294002 significantly reversed the enhancing effect of NRG1/ErbB4 overexpression on Akt phosphorylation as well as the increase in α-MHC and cTnT expression.ConclusionsIn conclusion, promoting the expression of NRG1/ErbB4 induced the differentiation of iPSC into cardiomyocytes, possibly through modulation of the PI3K/Akt signaling pathway.

Multiple System Atrophy: Pathology, Pathogenesis, and Path Forward.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease characterized by autonomic failure and motor impairment. The hallmark pathologic finding in MSA is widespread oligodendroglial cytoplasmic inclusions rich in aggregated α-synuclein (αSyn). MSA is widely held to be an oligodendroglial synucleinopathy, and we outline lines of evidence to support this assertion, including the presence of early myelin loss. We consider emerging data that support the possibility of neuronal or immune dysfunction as primary drivers of MSA. These hypotheses are placed in the context of a major recent discovery that αSyn is conformationally distinct in MSA versus other synucleinopathies such as Parkinson's disease. We outline emerging techniques in epidemiology, genetics, and molecular pathology that will shed more light on this mysterious disease. We anticipate a future in which cutting-edge developments in personalized disease modeling, including with pluripotent stem cells, bridge mechanistic developments at the bench and real benefits at the bedside.

Surface tension enables induced pluripotent stem cell culture in commercially available hardware during spaceflight

Low Earth Orbit (LEO) has emerged as a unique environment for evaluating altered stem cell properties in microgravity. LEO has become increasingly accessible for research and development due to progress in private spaceflight. Axiom Mission 2 (Ax-2) was launched as the second all-private astronaut mission to the International Space Station (ISS). Frozen human induced pluripotent stem cells (hiPSCs) expressing green fluorescent protein (GFP) under the SOX2 promoter, as well as fibroblasts differentiated from SOX2-GFP hiPSCs, were sent to the ISS. Astronauts then thawed and seeded both cell types into commercially available 96-well plates, which provided surface tension that reduced fluid movement out of individual wells and showed that hiPSCs or hiPSC-derived fibroblasts could survive either in suspension or attached to a Matrigel substrate. Furthermore, both cell types could be transfected with red fluorescent protein (RFP)-expressing plasmid. We demonstrate that hiPSCs and hiPSC-fibroblasts can be thawed in microgravity in off-the-shelf, commercially-available cell culture hardware, can associate into 3D spheroids or grow adherently in Matrigel, and can be transfected with DNA. This lays the groundwork for future biomanufacturing experiments in space.

Unveiling the Immunomodulatory and regenerative potential of iPSC-derived mesenchymal stromal cells and their extracellular vesicles

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives, particularly extracellular vesicles (EVs), for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs, alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction, indicating a potential mechanism for their immunomodulatory effects. Furthermore, iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally, iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably, priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics, given their potent immunomodulatory and regenerative properties.

A cross-disease resource of living human microglia identifies disease-enriched subsets and tool compounds recapitulating microglial states.

Human microglia play a pivotal role in neurological diseases, but we still have an incomplete understanding of microglial heterogeneity, which limits the development of targeted therapies directly modulating their state or function. Here, we use single-cell RNA sequencing to profile 215,680 live human microglia from 74 donors across diverse neurological diseases and CNS regions. We observe a central divide between oxidative and heterocyclic metabolism and identify microglial subsets associated with antigen presentation, motility and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypes in situ with an RNAscope-immunofluorescence pipeline and high-dimensional MERFISH. We also leverage our dataset as a classification resource, finding that induced pluripotent stem cell model systems capture substantial in vivo heterogeneity. Finally, we identify and validate compounds that recapitulate certain subtypes in vitro, including camptothecin, which downregulates the signature of disease-enriched subtypes and upregulates a signature previously associated with Alzheimer's disease.

Establishment of a CIB1 knockout human pluripotent stem cell line via CRISPR/Cas9 genome editing technology

Calcium- and integrin-binding protein 1 (CIB1) has a diverse role in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. It is associated with cancer, cardiovascular disease and male infertility. Here, CRISPR/Cas9 genome-editing technology was employed to establish a CIB1 knockout human embryonic stem cell line, which exhibited normal pluripotency and karyotype.