Human Pluripotent Stem Research Articles

Homology-independent targeted insertion-mediated derivation of M1-biased macrophages harbouring Megf10 and CD3ζ from human pluripotent stem cells

Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified macrophages (CAR-Ms) are challenging. Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity. These engineered cells phagocytosed cancer cells, leading to significant inhibition of cancer-cell proliferation invitro and invivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5Mζ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids. Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours. This work was supported by KRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01).

Upregulation of the sodium potassium pump current supresses ectopic activity after stem cell delivery in the human-based heart after myocardial infarction

Abstract Background Cell therapy is being explored to restore the damaged and intrinsically irreparable human heart. Whilst preclinical studies showed improvements in cardiac function after injury, they also highlighted a critical pro-arrhythmic window when delivered cells produce ectopy-induced ventricular tachycardia. Recent experiments investigating the cells in vivo maturation suggested upregulation of the inward rectifier potassium current (IK1) and knock-out of the funny current (If) and sodium calcium exchanger (INaCa) to supress the arrhythmias[1]. Purpose Whilst promising, such modifications may impact the cells’ calcium dynamics and ultimately, their efficacy. The goal of this study is to identify novel ionic targets that improve therapy safety whilst maintaining efficacy by employing the unique predictive capabilities of modelling and simulation. Methods For this, we used our established modelling and simulation framework of cell therapy in the chronically infarcted human heart with Purkinje, validated against experimental and clinical data from the action potential to the ECG. We incorporated experimentally observed gene expression of in vivo cell maturation[1] into our framework to reproduce human pluripotent stem cell-induced cardiomyocyte (hPSC-CM) electrophysiology at day 0 and day 14 after injection. To consider variability amongst experimentally reported hPSC-CMs, we also created a fast-beating hPSC-CM model by upregulating the SERCA pump current, If and INaCa. Results Our results showed that while day 0 hPSC-CMs did not cause spontaneous activity, day 14 hPSC-CMs produced ectopic beats in the ventricles every 1.5s. The rapid beating hPSC-CM phenotype at day 14 elicited tachycardia through spontaneous beating and intermittent re-entrant wavefronts (see Figure 1). Next, we demonstrated 65% upregulation of the sodium-potassium pump current (INaK) together with a 50% increase in IK1 and knock-out of If as a novel pathway to quiescence. Importantly, our simulations highlighted that compared to INaCa knock-out, an increase in INaK allowed for physiological calcium transients. Conclusion To conclude, in this work we have utilised modelling and simulation of the injured human heart to 1) capture pre-clinically observed arrhythmias after cell therapy and 2) identify INaK upregulation as a novel target to mitigate safety concerns, whilst preserving efficacy.

A differentiation protocol for generating pancreatic delta cells from human pluripotent stem cells

In this protocol, we detail a seven-stage differentiation methodology for generating pancreatic delta cells (SC-delta cells) from human pluripotent stem cells (hPSCs). In the first step, definitive endoderm is generated by activin A and CHIR99021, followed by induction of primitive gut tube and posterior foregut by treatment with FGF7, SANT1, LDN193189, PdBU, and retinoic acid (RA). The subsequent endocrine generation and directed SC-delta cell induction is achieved by a combined treatment of the FGF7 with FGF2 during stage 4 and 5, together with RA, XXI, ALK5 inhibitor II, SANT1, Betacellulin and LDN193189. The planar cultivation is converted to a suspended system after stage 5, allowing cells to aggregate into delta cell-containing spheroids. The differentiation takes approximately 4-5 weeks for delta cell generation and an additional 1-2 weeks for cell expansion and evaluation. We believe that this amenable and simplified protocol can provide a stable source of SC-delta cells from efficient differentiation, facilitating further investigation of the physiological role of delta cells as well as refinement of islet cell therapeutic strategies.

Acquisition of neurodegenerative features in isogenic OPTN(E50K) human stem cell-derived retinal ganglion cells associated with autophagy disruption and mTORC1 signaling reduction

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research, with great potential for the use of hPSC-derived RGCs for studies of human retinal development, in vitro disease modeling, drug discovery, as well as their potential use for cell replacement therapeutics. Of all these possibilities, the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention, due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies, many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus, to further advance this field of research, in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein, which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK, along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs, and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together, these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1, contributing to the degeneration of RGCs.

Lineage tracing of stem cell-derived dopamine grafts in a Parkinson's model reveals shared origin of all graft-derived cells.

Stem cell therapies for Parkinson's disease are at an exciting time of development, and several clinical trials have recently been initiated. Human pluripotent stem cells are differentiated into transplantable dopamine (DA) progenitors which are proliferative at the time of grafting and undergo terminal differentiation and maturation in vivo. While the progenitors are homogeneous at the time of transplantation, they give rise to heterogeneous grafts composed not only of therapeutic DA neurons but also of other mature cell types. The mechanisms for graft diversification are unclear. We used single-nucleus RNA-seq and ATAC-seq to profile DA progenitors before transplantation combined with molecular barcode-based tracing to determine origin and shared lineages of the mature cell types in the grafts. Our data demonstrate that astrocytes, vascular leptomeningeal cells, and DA neurons are the main component of the DAergic grafts, originating from a common progenitor that is tripotent at the time of transplantation.

Human Astrocytes Synchronize Neural Organoid Networks.

Biological neural networks exhibit synchronized activity within and across interconnected regions of the central nervous system. Understanding how these coordinated networks are established and maintained may reveal therapeutic targets for neurodegeneration and neuromodulation. Here, we tested the influence of astrocytes upon synchronous network activity using human pluripotent stem cell-derived bioengineered neural organoids. This study revealed that astrocytes significantly increase activity within individual organoids and across long distances among numerous rapidly merged organoids via influencing synapses and bioenergetics. Treatment of amyloid protein inhibited synchronous activity during neurodegeneration, yet this can be rescued by propagating activity from neighboring networks. Altogether, this study identifies critical contributions of human astrocytes to biological neural networks and delivers a rapid, reproducible, and scalable model to investigate long-range functional communication of the nervous system in healthy and disease states.

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.

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.

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.

Pristine Photopolymerizable Gelatin Hydrogels: A Low-Cost and Easily Modifiable Platform for Biomedical Applications.

The study addresses the challenge of temperature sensitivity in pristine gelatin hydrogels, widely used in biomedical applications due to their biocompatibility, low cost, and cell adhesion properties. Traditional gelatin hydrogels dissolve at physiological temperatures, limiting their utility. Here, we introduce a novel method for creating stable hydrogels at 37 °C using pristine gelatin through photopolymerization without requiring chemical modifications. This approach enhances consistency and simplifies production and functionalization of the gelatin with bioactive molecules. The stabilization mechanism involves the partial retention of the triple-helix structure of gelatin below 25 °C, which provides specific crosslinking sites. Upon activation by visible light, ruthenium (Ru) acts as a photosensitizer that generates sulphate radicals from sodium persulphate (SPS), inducing covalent bonding between tyrosine residues and "locking" the triple-helix conformation. The primary focus of this work is the characterization of the mechanical properties, swelling ratio, and biocompatibility of the photopolymerized gelatin hydrogels. Notably, these hydrogels supported better cell viability and elongation in normal human dermal fibroblasts (NHDFs) compared to GelMA, and similar performance was observed for human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). As a proof of concept for functionalization, gelatin was modified with selenous acid (GelSe), which demonstrated antioxidant and antimicrobial capacities, particularly against E. coli and S. aureus. These results suggest that pristine gelatin hydrogels, enhanced through this new photopolymerization method and functionalized with bioactive molecules, hold potential for advancing regenerative medicine and tissue engineering by providing robust, biocompatible scaffolds for cell culture and therapeutic applications.

Assembloid model to study loop circuits of the human nervous system.

Neural circuits connecting the cerebral cortex, the basal ganglia and the thalamus are fundamental networks for sensorimotor processing and their dysfunction has been consistently implicated in neuropsychiatric disorders 1-9 . These recursive, loop circuits have been investigated in animal models and by clinical neuroimaging, however, direct functional access to developing human neurons forming these networks has been limited. Here, we use human pluripotent stem cells to reconstruct an in vitro cortico-striatal-thalamic-cortical circuit by creating a four-part loop assembloid. More specifically, we generate regionalized neural organoids that resemble the key elements of the cortico-striatal-thalamic-cortical circuit, and functionally integrate them into loop assembloids using custom 3D-printed biocompatible wells. Volumetric and mesoscale calcium imaging, as well as extracellular recordings from individual parts of these assembloids reveal the emergence of synchronized patterns of neuronal activity. In addition, a multi-step rabies retrograde tracing approach demonstrate the formation of neuronal connectivity across the network in loop assembloids. Lastly, we apply this system to study heterozygous loss of ASH1L gene associated with autism spectrum disorder and Tourette syndrome and discover aberrant synchronized activity in disease model assembloids. Taken together, this human multi-cellular platform will facilitate functional investigations of the cortico-striatal-thalamic-cortical circuit in the context of early human development and in disease conditions.

GATA1-deficient human pluripotent stem cells generate neutrophils with improved antifungal immunity that is mediated by the integrin CD18.

Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal β-glucan, including an upregulation of the pentose phosphate pathway (PPP), similar to primary human neutrophils in vitro. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response.

Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

Human pluripotent stem cells (hPSCs) have the potential to differentiate into various cell types, including pancreatic insulin-producing β cells, which are crucial for developing therapies for diabetes. However, current methods for directing hPSC differentiation towards pancreatic β-like cells are often inefficient and produce cells that do not fully resemble the native counterparts. Here, we report that highly selective tankyrase inhibitors, such as WIKI4, significantly enhances pancreatic differentiation from hPSCs. Our results show that WIKI4 promotes the formation of pancreatic progenitors that give rise to islet-like cells with improved β-like cell frequencies and glucose responsiveness compared to our standard cultures. These findings not only advance our understanding of pancreatic development, but also provide a promising new tool for generating pancreatic cells for research and potential therapeutic applications.

SMYD1 modulates the proliferation of multipotent cardiac progenitor cells derived from human pluripotent stem cells during myocardial differentiation through GSK3β/β-catenin&ERK signaling.

The histone-lysine N-methyltransferase SMYD1, which is specific to striated muscle, plays a crucial role in regulating early heart development. Its deficiency has been linked to the occurrence of congenital heart disease. Nevertheless, the precise mechanism by which SMYD1 deficiency contributes to congenital heart disease remains unclear. We established a SMYD1 knockout pluripotent stem cell line and a doxycycline-inducible SMYD1 expression pluripotent stem cell line to investigate the functions of SMYD1 utilizing an in vitro-directed myocardial differentiation model. Cardiomyocytes lacking SMYD1 displayed drastically diminished differentiation efficiency, concomitant with heightened proliferation capacityof cardiac progenitor cells during the early cardiac differentiation stage. These cellular phenotypes were confirmed through experiments inducing the re-expression of SMYD1. Transcriptome sequencing and small molecule inhibitor intervention suggested that the GSK3β/β-catenin&ERK signaling pathway was involved in the proliferation of cardiac progenitor cells. Chromatin immunoprecipitation demonstrated that SMYD1 acted as a transcriptional activator of GSK3β through histone H3 lysine 4 trimethylation. Additionally, dual-luciferase analyses indicated that SMYD1 could interact with the promoter region of GSK3β, thereby augmenting its transcriptional activity. Moreover, administering insulin and Insulin-like growth factor 1 can enhance the efficacy of myocardial differentiation in SMYD1 knockout cells. Our research indicated that the participation of SMYD1 in the GSK3β/β-catenin&ERK signaling cascade modulated the proliferation of cardiac progenitor cells during myocardial differentiation. This process was partly reliant on the transcription of GSK3β. Our research provided a novel insight into the genetic modification effect of SMYD1 during early myocardial differentiation. The findings were essential to the molecular mechanism and potential interventions for congenital heart disease.

Blood-generating heart-forming organoids recapitulate co-development of the human haematopoietic system and the embryonic heart.

Despite the biomedical importance of haematopoietic stem cells and haematopoietic progenitor cells, their in vitro stabilization in a developmental context has not been achieved due to limited knowledge of signals and markers specifying the multiple haematopoietic waves as well as ethically restricted access to the human embryo. Thus, an in vitro approach resembling aspects of haematopoietic development in the context of neighbouring tissues is of interest. Our established human pluripotent stem cell-derived heart-forming organoids (HFOs) recapitulate aspects of heart, vasculature and foregut co-development. Modulating HFO differentiation, we here report the generation of blood-generating HFOs. While maintaining a functional ventricular-like heart anlagen, blood-generating HFOs comprise a mesenchyme-embedded haemogenic endothelial layer encompassing multiple haematopoietic derivatives and haematopoietic progenitor cells with erythro-myeloid and lymphoid potential, reflecting aspects of primitive and definitive haematopoiesis. The model enables the morphologically structured co-development of cardiac, endothelial and multipotent haematopoietic tissues equivalent to the intra-embryonic haematopoietic region in vivo, promoting research on haematopoiesis in vitro.

8681 Preoteogenomic Identification Of Novel Translated And Regulatory Open Reading Frames In Human Beta-Cells

Abstract Disclosure: K. Walters: None. R.C. Guiterrez: None. S. Sakhar: None. A. Baldwin: None. E.S. Nakayasu: None. H. Russ: None. N. Mukherjee: None. Preoteogenomic Identification of Novel Translated and Regulatory Open Reading Frames in Human Beta-cells The discovery of novel autoreactive T cells that target β cells during type 1 diabetes (T1D) has been challenging due to our limited knowledge in autoreactive β cell antigens. Most of these antigens are derived from canonical open reading frames (ORFs) of protein coding genes, such as preproinsulin and chromogranin A. However, a defective ribosomal product (DRiP) was recently identified from the insulin mRNA (INS-DRiP). This antigen is the result of translation from a novel or unannotated open reading frames (nuORFs). nuORFs have a range of functions from regulating translation to encoding novel small bioactive peptides to producing neoantigens. In this study, we use a transcriptome-wide approach to identify nuORFs in human β cells in an unbiased manner. We performed ribosome profiling (ribo-seq) in stem cell derived β cells (sBCs). Human pluripotent stem cells were differentiated into sBCs expressing a flag-tagged ribosomal protein (RPL22-Flag) driven by the rat insulin promoter. We performed IP-ribo-seq on sBCs enriching for RPL22-Flag containing ribosomes. These data exhibited high-resolution 3-nucleotide periodicity consistent with translating codons and identified sequencing reads consistent with translation of the INS-DRiP. We performed de novo ORF-calling using two software using complementary statistical approaches (RiboCode and ORFQuant). We identified over 12,000 canonical annotated ORFs of which ∼85% were had protein level support from matched proteomics confirming the high specificity of our approach. Of nearly 1000 nuORFs identified in sBCs, over half had protein level support in sBCs and cadaveric human islets. We found that 149 nuORFs were associated with Type-1 Diabetes (T1D) genes, including a novel upstream ORF (uORF) in TYK2, which had the strongest association with T1D and is a promising therapeutic target to halt T1D progression. Using reporter assay, we show that the novel uORF in TYK2 is a translational activator and warrants further investigation. Finally, our data represents a valuable database for the search of novel autoreactive antigens in β cells and accelerate the discovery of novel autoreactive T cells implicated in T1D pathogenesis. Presentation: 6/2/2024

7704 Generating pituitary cells from iPSCs of patients with child onset congenital hypopituitarism harboring PROP1 pathogenic allelic variants

Abstract Disclosure: J.M. Marques: None. F. Fattahi: None. L.R. Carvalho: None. Introduction: Reduced or absent levels of pituitary hormones due to genetic or organic causes is known as combined pituitary hormone deficiency (CPHD) and PROP1 is the most frequent cause of CPHD, but more than 85% of the cases are without molecular diagnosis. Novel Techniques such as induced pluripotent stem cells (iPSC), can be used to generate patient’s specific pituitary cells for disease modeling. Aim: To generate iPSC from CO-CH patients’ peripheral blood mononuclear cells (PBMC) and differentiate them into pituitary cells. Methods: Peripheral blood was collected from 2 siblings (HC1 and HC2) harboring compound heterozygous PROP1 allelic variants and their parents (HC3 and HC4). PBMCs were isolated and expanded with MNC medium prior to nucleoporation and transfection with plasmids containing pluripotency genes (OCT4 and SOX2; cMYC and KLF). After 48h, transfected cells were transferred to Geltrex coated plates and fed with E8, NaB and βFGF until pluripotent colonies were formed. iPSCs were differentiated into cranial placode cells using pituitary placode conditioned medium (PPCM) until day 15. For pituitary cell maturation, pituitary conditioned medium (PPCM without SB431542) was used until day 30. qPCR and flow cytometry using the Kit BD Stemflow - Human Pluripotent Stem Cell Transcription Factor Analysis (BD Biosciences) for stem cells and the eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization (Thermo Fisher Scientific) for the placode and pituitary markers were used. Pituitary hormonal levels from cell supernatant were measured at the Laboratory of Hormones and Molecular Genetics - HCFMUSP. Results: we generated iPSCs from patient and control blood cells. iPSCs were more than 90% positive for the pluripotency markers SOX2, NANOG, OCT3/4. Pituitary cells were generated with positivity for the placode marker SIX1 (58%) and pituitary markers, as LHX3 (56%) and PROP1 (59%), at day 15. Control cell lineages expressed higher levels of pituitary hormones at days 15 and 30 at RNA and protein levels while patient cells failed to induce hormone-producing cells properly. Pituitary hormonal release was impaired in patients pituitary cells. Conclusion: CO-CH patients’ PBMC were dedifferentiated to a pluripotent state, being able to generate pituitary hormone producing cells. Patient cells were able to mimic human phenotype. These cells can be used, in the future, to study CO-CH basis in patients specific background, to develop pituitary disease modeling as well as test possible new treatments. Presentation: 6/3/2024

Comparison of human pluripotent stem cell differentiation protocols to generate neuroblastoma tumors

Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described, the PSC were differentiated using protocols that made neural crest cells, but not specifically the trunk subtype. Here, we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP], tNCC), as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B, a marker of neuroblastoma. Transcriptionally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

Efficient generation of liver sinusoidal endothelial-like cells secreting coagulation factor VIII from human induced pluripotent stem cells

Liver sinusoidal endothelial cells (LSECs) and LSEC progenitor cells (LPCs) derived from human pluripotent stem cells (PSCs) are expected as valuable cell sources for the development of cell therapy for hemophilia A, a congenital deficiency of coagulation factor VIII (FVIII), as LSECs are responsible for FVIII production. However, there is room for improvement in the efficiency of LSECs and LPCs differentiation from PSCs. In this study, we sought to optimize the method of mesoderm differentiation induction, the initial step of LSEC differentiation from PSCs, to efficiently induce LSEC-like cells capable of secreting FVIII from human induced pluripotent stem cells (iPSCs). Following optimization of the concentration and stimulation period of CHIR99021 (glycogen synthase kinase 3β inhibitor), bone morphogenetic protein 4, fibroblast growth factor 2, and Activin A in the mesoderm induction step, approximately 65% and 54% of cells differentiated into LPCs and LSEC-like cells, respectively. Furthermore, we observed substantial FVIII protein secretion from LSEC-like cells in vitro. In conclusion, we established an efficient method for obtaining LPCs and functional LSEC-like cells from iPSCs in vitro.

Establishment and characterization of three human pluripotent stem cell lines from Charcot-Marie-Tooth disease Type 4B3 patients bearing mutations in MTMR5/Sbf1 gene

Myotubularin-Related Protein 5 (MTMR5) is an inactive, poorly characterized D3-phosphatidylinositol phosphatase. Mutations in MTMR5 have been linked to Charcot-Marie-Tooth Disease Type 4B3 (CMT4B3), a rare, early-onset, recessive peripheral neuropathy. Here, we describe the establishment and validation of three human induced pluripotent stem cell (iPSC) lines derived from unrelated CMT4B3 patients, each harboring homozygous MTMR5/Sbf1 mutations. Current MTMR5 -/- animal models do not clearly link Sbf1 mutations to severe neuropathy, so such a resource is highly desired to further elucidate the relationship between MTMR5 dysfunction and peripheral nerve degeneration.