Culture Of Pluripotent Stem Cells Research Articles

Forebrain Organoids to Model the Cell Biology of Basal Radial Glia in Neurodevelopmental Disorders and Brain Evolution.

The acquisition of higher intellectual abilities that distinguish humans from their closest relatives correlates greatly with the expansion of the cerebral cortex. This expansion is a consequence of an increase in neuronal cell production driven by the higher proliferative capacity of neural progenitor cells, in particular basal radial glia (bRG). Furthermore, when the proliferation of neural progenitor cells is impaired and the final neuronal output is altered, severe neurodevelopmental disorders can arise. To effectively study the cell biology of human bRG, genetically accessible human experimental models are needed. With the pioneering success to isolate and culture pluripotent stem cells in vitro, we can now routinely investigate the developing human cerebral cortex in a dish using three-dimensional multicellular structures called organoids. Here, we will review the molecular and cell biological features of bRG that have recently been elucidated using brain organoids. We will further focus on the application of this simple model system to study in a mechanistically actionable way the molecular and cellular events in bRG that can lead to the onset of various neurodevelopmental diseases.

Towards advanced cardiac miRNA therapies through DNA-based nanostructures

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Proyecto MINECO-Ministerio de Ciencia e Innovación (Spain)- PID2019-105674RB-I00 Proyecto DGA- Diputación General de Aragón (Spain)- LMP128_21 Introduction Cardiac microRNAs (miRNAs) have been found to be dysregulated in cardiac disease and aging both in animal models and humans. Thanks to their pleiotropic effects, miRNAs are spotlighted as promising therapeutic targets to treat cardiac conditions. However, their low stability and potential off-target effects in vivo difficult the development of effective and safe miRNA therapies. Nanotechnology enables the fabrication of biocompatible carriers to perform targeted delivery of both conventional and advanced therapies. In particular, nanostructures built with DNA (DNS) have been successfully developed for miRNA therapies in cancer, but they have not been applied in cardiac therapy yet [1]. Purpose The main goal of this study is to demonstrate the capacity of DNS to deliver miRNA therapies in human cardiomyocytes. Methods Our group has identified MIR24-2 upregulated with age in the human left ventricle and miR24-2-5p to interact with genes related with the cardiac function, such as SERCA2 [2]. Different versions of DNS were designed and generated by self-assembly of DNA strands containing anti-miR24-2-5p capture segments. Physicochemical characterization of DNS was then conducted to determine their auto-assembling capacity, size, morphology and thermal stability. The characterization of the biological effect of the DNS was also conducted in vitro and in cell culture of HEK293 and human induced pluripotent stem cells (iPSC)-derived cardiomyocytes (iCM) to assess their cytotoxicity and internalization capacity. Results DNS were assembled correctly and reproducibly into carriers of around 30-50 nm. In vitro, the DNS showed thermal stability at physiological temperature, proper stability in serum and they disassembled specifically in the presence of the target miR24-2-5p sequence. In HEK293 and iCM cultures, the DNS showed lack of cytotoxicity demonstrating in vitro biocompatibility. DNS were efficiently internalized by HEK293, but the uptake capacity of DNS by iCM was remarkably lower. Conclusions We created and characterized biocompatible anti-miR-loaded DNS that effectively captured the target miR-24-2-5p in vitro. DNS internalized in iCM, but with lower efficiency than in HEK293 cells. Our results indicate that DNS are suitable candidates to carry miRNA therapies to human cardiac cells in vitro, but the observed cell-type specific differences suggest that future efforts need to be directed towards developing DNS functionalization capable of promoting efficient and specific carrier uptake in vivo by primary cardiac cells in vivo.

Recapitulating early human development with 8C-like cells.

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An invitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.

Cardiomyocytes derived from human-induced pluripotent stem cell culture in rod-shaped micropattern

Cardiomyocytes derived from human-induced pluripotent stem cells (iPS-CMs) have numerous advantages for drug screening and disease modeling but present with structural and functional immaturity features. Experiments in cardiomyocytes isolated from adult human or animal hearts are conditioned by their typical rod-shaped morphology. Here, we aimed at improving iPS-CMs architectural maturity by using a rod-shaped cell micropatterned substrate made of repeated rectangles of 120 μm-length and 30 μm-width and surrounded with a chemical cell-repellent. Cardiomyocyte differentiation was performed using a chemically defined method. Thirty-five days later the generated iPS-CMs were cultured on unpatterned vs. rod-shaped micropatterned substrates for 10 additional days. We performed molecular investigations by qPCR and immunofluorescence, and electrophysiological characterization by patch-clamp technique. The culture of micropatterned substrates resulted in the generation of rod-shaped iPS-CMs with a 4:1 cell ratio, with a more organized troponin T staining pattern exhibiting sarcomere-type striations and higher sarcomere organization score (0.25 ± 0.07 vs. 0.13 ± 0.004, P < 0.0001). The rod-shaped iPS-CMs also showed significant improvements in electrophysiological parameters with a decrease of the spontaneous beating rate and a more hyperpolarized membrane diastolic potential (−68.11 ± 6.25 vs. −59.17 ± 4.94 mV, P < 0.001) as compared to unpatterned iPS-CMs. Rod-shaped iPS-CMs displayed a more predominant ventricular-like type after analyzing action potential recordings (91% ± 1), with an increase in the percentage of late ventricular-like APs compared to unpatterned (64% vs. 29%), suggesting that rod-shaped morphology stimulates ventricular maturation. Notably, the maximum upstroke velocity (dV/dtmax) (98.09 ± 32.63 vs. 59.71 ± 50.43 V/ms, P < 0.1) and the sodium current density (INa) was increased (−89.30 ± 25.09 vs. −64.92 ± 21.63 pA/pF, P < 0.001) in rod-shaped iPS-CMs compared to unpatterned CMs. Importantly, we found no significant change in the RNA expression of the main ion channels or in major sarcomeric components. We are now assessing the Ca2 + signaling properties by confocal analysis. We developed a novel method to generate rod-shaped iPS-CMs, leading to improved functional characteristics. Preliminary results suggest that the architectural re-arrangement is the primary driver of the improved functionality observed in micropatterned iPS-CMs.

Physoxia Influences Global and Gene-Specific Methylation in Pluripotent Stem Cells.

Pluripotent stem cells (PSC) possess unlimited proliferation, self-renewal, and a differentiation capacity spanning all germ layers. Appropriate culture conditions are important for the maintenance of self-renewal, pluripotency, proliferation, differentiation, and epigenetic states. Oxygen concentrations vary across different human tissues depending on precise cell location and proximity to vascularisation. The bulk of PSC culture-based research is performed in a physiologically hyperoxic, air oxygen (21% O2) environment, with numerous reports now detailing the impact of a physiologic normoxia (physoxia), low oxygen culture in the maintenance of stemness, survival, morphology, proliferation, differentiation potential, and epigenetic profiles. Epigenetic mechanisms affect multiple cellular characteristics including gene expression during development and cell-fate determination in differentiated cells. We hypothesized that epigenetic marks are responsive to a reduced oxygen microenvironment in PSCs and their differentiation progeny. Here, we evaluated the role of physoxia in PSC culture, the regulation of DNA methylation (5mC (5-methylcytosine) and 5hmC (5-hydroxymethylcytosine)), and the expression of regulatory enzyme DNMTs and TETs. Physoxia enhanced the functional profile of PSC including proliferation, metabolic activity, and stemness attributes. PSCs cultured in physoxia revealed the significant downregulation of DNMT3B, DNMT3L, TET1, and TET3 vs. air oxygen, accompanied by significantly reduced 5mC and 5hmC levels. The downregulation of DNMT3B was associated with an increase in its promoter methylation. Coupled with the above, we also noted decreased HIF1A but increased HIF2A expression in physoxia-cultured PSCs versus air oxygen. In conclusion, PSCs display oxygen-sensitive methylation patterns that correlate with the transcriptional and translational regulation of the de novo methylase DNMT3B.

Novel RGD-containing peptides exhibited improved abilities to integrin receptor binding and cultures of human induced pluripotent stem cells

The design of novel peptide sequences and investigations of the mechanisms underlying cell adhesion is critical for the culture of human induced pluripotent stem cells (hiPSCs) on the peptide displaying surfaces. Recently, we reported that near-Asp sequences play an important role in the function of whole Arg-Gly-Asp (RGD)-containing peptides. In this study, two novel peptides with sequences of Ac-KGGTYRAYRGDVFTMP and Ac-KGGVFTMPRGDTYRAY were designed from reported peptides. Fortunately, the Ac-KGGVFTMPRGDTYRAY peptide exhibited excellent ability in sustaining cultures of hiPSCs. Moreover, we predicted the structural model of peptides by molecular dynamics simulation and successfully obtained the complex structure of peptides and αVβ3/αVβ5 integrin proteins through molecular docking. Finally, a strong affinity to the αVβ3 integrin receptor contributes to the excellent ability of peptide was confirmed. Study reveals the mechanisms by which RGD-containing peptides support the adhesion and provides a better peptide for hiPSCs culture.

Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

Differentiation of brain and retinal organoids from confluent cultures of pluripotent stem cells connected by nerve-like axonal projections of optic origin.

SummaryAdvances in the study of neurological conditions have been possible because of pluripotent stem cell technologies and organoids. Studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids are transcriptionally comparable with organoids generated by other methods. Furthermore, these organoids showed spontaneous functional network activity and proteomic analysis confirmed organoids maturity. The generation of retinal and brain organoids in close proximity enabled their mutual isolation. Suspension culture of this complex organoid system demonstrated the formation of nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of neurological diseases of the eye and brain.

Controlling neural territory patterning from pluripotency using a systems developmental biology approach

SummarySuccessful manufacture of specialized human cells requires process understanding of directed differentiation. Here, we apply high-dimensional Design of Experiments (HD-DoE) methodology to identify critical process parameters (CPPs) that govern neural territory patterning from pluripotency—the first stage toward specification of central nervous system (CNS) cell fates. Using computerized experimental design, 7 developmental signaling pathways were simultaneously perturbed in human pluripotent stem cell culture. Regionally specific genes spanning the anterior-posterior and dorsal-ventral axes of the developing embryo were measured after 3 days and mathematical models describing pathway control were developed using regression analysis. High-dimensional models revealed particular combinations of signaling inputs that induce expression profiles consistent with emerging CNS territories and defined CPPs for anterior and posterior neuroectoderm patterning. The results demonstrate the importance of combinatorial control during neural induction and challenge the use of generic neural induction strategies such as dual-SMAD inhibition, when seeking to specify particular lineages from pluripotency.

Multimodal Magnetic Resonance and Fluorescence Imaging of the Induced Pluripotent Stem Cell Transplantation in the Brain

The understanding of the engrafted cell behaviors such as the survival, growth and distribution is the prerequisite to optimize cell therapy, and a multimodal imaging at both anatomical and molecular levels is designed to achieve this goal. We constructed a lentiviral vector carrying genes of ferritin heavy chain 1 (FTH1), near-infrared fluorescent protein (iRFP) and enhanced green fluorescent protein (egfp), and established the induced pluripotent stem cells (iPSCs) culture stably expressing these three reporter genes. These iPSCs showed green and near-infrared fluorescence as well as the iron uptake capacity in vitro. After transplanted the labeled iPSCs into the rat brain, the engrafted cells could be in vivo imaged using magnetic resonance imaging (MRI) and near-infrared fluorescent imaging (NIF) up to 60 days at the anatomical level. Moreover, these cells could be detected using EGFP immunostaining and Prussian blue stain at the cellular level. The developed approach provides a novel tool to study behaviors of the transplanted cells in a multimodal way, which will be valuable for the effectiveness and safety evaluation of cell therapy.

Application of the water-insoluble, temperature-responsive block polymer poly(butyl methacrylate-block-N-isopropylacrylamide) for pluripotent stem cell culture and cell-selective detachment

Induced pluripotent stem (iPS) cells have been widely studied in regenerative medicine, pathology modeling, and drug screening. Stable mass culture of iPS cells is essential for these applications. iPS cells can spontaneously differentiate into other cells during culture, and removal of these differentiated cells is necessary. Herein, a cost-effective culture method suitable for mass culture and a detailed analysis of the selective detachment of iPS cells are presented. A simple method for coating the water-insoluble thermoresponsive polymer poly (butyl methacrylate-block-N-isopropylacrylamide) on commercially available polystyrene dishes was employed. Analysis of the effects of the polymer composition, coating thickness, and surface structure on iPS cell culture/detachment showed that a coating thickness of approximately 10-40nm using a polymer with a high poly (N-isopropylacrylamide) content was suitable for iPS cell detachment. Moreover, an interesting change in surface morphology was observed following temperature variation, thereby affecting laminin adsorption. Second, selective detachment in cocultures of iPS cells and differentiated cells enabled collection of iPS cells with more than 98% purity. Finally, long-term iPS cell culture was conducted using temperature-responsive cell detachment. Overall, long-term maintenance-free culture of iPS cells was possible without manual removal of differentiated cells.

Human 8-cell-like cells discovered.

Human naive pluripotent stem cells have the remarkable ability to generate blastoids comprising trophectoderm, epiblast, and hypoblast-like cells. In this issue, Taubenschmid-Stowers etal. (2022) show that human naive pluripotent stem cell cultures contain cells that resemble the 8-cell human embryo, providing a model to study zygotic genome activation.

In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture

Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O2 and CO2 with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that in vitro cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.

Multimodal Magnetic Resonance and Fluorescence Imaging of the Induced Pluripotent Stem Cell Transplantation in the Brain

The understanding of the engrafted cell behaviors such as the survival, growth and distribution is the prerequisite to optimize cell therapy, and a multimodal imaging at both anatomical and molecular levels is designed to achieve this goal. We constructed a lentiviral vector carrying genes of ferritin heavy chain 1 (FTH1), near-infrared fluorescent protein (iRFP) and enhanced green fluorescent protein (egfp), and established the induced pluripotent stem cells (iPSCs) culture stably expressing these three reporter genes. These iPSCs showed green and near-infrared fluorescence as well as the iron uptake capacity in vitro. After transplanted the labeled iPSCs into the rat brain, the engrafted cells could be in vivo imaged using magnetic resonance imaging (MRI) and near-infrared fluorescent imaging (NIF) up to 60 days at the anatomical level. Moreover, these cells could be detected using EGFP immunostaining and Prussian blue stain at the cellular level. The developed approach provides a novel tool to study behaviors of the transplanted cells in a multi-modal way, which will be valuable for the effectiveness and safety evaluation of cell therapy.

Matrix enrichment by black phosphorus improves ionization and reproducibility of mass spectrometry of intact cells, peptides, and amino acids

Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.

Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors.

Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into 'myeloid-cell-forming-complex' intermediates by means of a minimal cytokine cocktail. In contrast to the 'batch-like differentiation approaches' established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1-4 × 107 iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery.

Induction of Human Natural Killer Cells Under Defined Conditions by Seamless Transition from Maintenance Culture of Pluripotent Stem Cells.

The use of pluripotent stem cells (PSCs) as a source of natural killer cells (NK cells) can improve reproducibility in the analysis of the pathogenesis of NK cell-associated diseases and in the production of off-the-shelf cellular medicines. We have developed a method for the differentiation of NK cells from human PSCs under serum-free and two-dimensional condition. Our method enables the seamless transition from maintenance of PSCs to differentiation of NK cells, without the use of any techniques other than medium exchange and whole culture passage.

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation_suppl2

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation_suppl2

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation_suppl1

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation_suppl1

Induction of Human Naïve Pluripotency Using Chemical Resetting.

Human pluripotent stem cells exist in naïve and primed states that recapitulate the distinct molecular and cellular properties of pre- and post-implantation epiblast cells, respectively. Naïve pluripotent stem cells can be captured directly from blastocysts but, more commonly, the cells are reprogrammed from primed cells in a process called "resetting". Several methods to achieve resetting have been described. Chemical resetting of primed cells to a naïve pluripotent state is one such method and has come to the forefront as a simple, efficient, and transgene-free method to induce naïve pluripotency. The process involves the transient application of a histone deacetylase inhibitor to initiate resetting, followed by the emergence of nascent naïve pluripotent stem cells in supportive conditions, and finally the stabilization and expansion of naïve pluripotent stem cell cultures. Here, a detailed protocol is provided for chemical resetting starting from plating primed cells until a stable culture of naïve pluripotent stem cells is established.