Wild-type iPSCs Research Articles

Abstract 5244: SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models

Abstract The CD47-SIRPα axis is a critical checkpoint that prevents SIRPα-positive macrophages from phagocytosing CD47-expressing solid tumors. Several agents aiming to block this axis have recently entered early clinical trials including anti-CD47 and anti-SIRPα monoclonal antibodies (mAb). These checkpoint inhibitors (CPI) aim to modulate the phagocytotic activity of endogenous tumor associated macrophages (TAMs). However, the adoptive transfer of macrophages resistant to CD47-based inhibition in the tumor microenvironment (TME) could also increase clinical efficacy while avoiding side effects linked to the use of anti-CD47 mAb. Cell therapy based on autologous macrophages have gained increasing attention for cancer treatment due to their ability to infiltrate into the immunosuppressive TME and their unique immunomodulatory characteristics. However, due to required intricate genetic manipulation of autologous macrophage cell product for each patient, optimization and consistency of the cell product remains challenging. In contrast, the use of induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) facilitates the introduction of genetic modifications to further optimize the iMAC cell product and limits the need for combination therapies. The knockout (KO) of the SIRPα gene in iMACs is a promising genetic modification that results in a potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells. A SIRPα KO was introduced in a fully characterized GMP iPSC line that was then differentiated to iMACs using Evotec’s 3D differentiation protocol. The SIRPα KO iMACs were then evaluated for their antibody-dependent cellular phagocytosis (ADCP) capacity in comparison to antibody-loaded wildtype (WT) iMACs when co-cultured with CD47-expressing tumor cells. SIRPα KO iPSCs showed differentiation potential comparable to WT iPSC and resulted in iMACs expressing typical markers of fully differentiated macrophages. SIRPα KO iMACs exhibited increased phagocytic potency and killing capacity compared to WT iMACs when both cell types were exposed to CD47-positive tumor cells and loaded with the same tumor-targeting mAb. This increased phagocytosis of tumor cells by antibody-loaded SIRPα KO iMACs was also comparable to ADCP observed for WT iMACs in the presence of an anti-CD47 blocking antibody. Using Evotec’s gene editing platform we were able to efficiently generate SIRPα-deficient iPSCs that serve as the starting material to manufacture highly pure, genetically modified iMACs that lack SIRPα expression rendering them resistant to CD47-dependent inhibition of phagocytosis. This novel allogeneic off-the-shelf iMAC cell product overcomes the need to combine this cell therapeutic with CD47-SIRPα axis CPIs and provides the basis to develop innovative treatments for solid tumors. Citation Format: Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Tanja Schneider, Michael Esquerré, Audrey Holtzinger, Michael Epstein, Daniel Sommermeyer, Michael Paillasse, Andreas Scheel, Markus Dangl, Monika Braun, Nadja Wagner. SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5244.

The fate of iPSCs-derived low immunogenic dopaminergic neuron precursors after transplantation into the striatum of rats with 6-OHDA-induced parkinsonism

The fate of iPSCs-derived low immunogenic dopaminergic neuron precursors after transplantation into the striatum of rats with 6-OHDA-induced parkinsonism

Human iPSC-Derived NK Cells with Knock-in of the BCL2 G101V Mutation Are Resistant to Venetoclax and Demonstrate Improved Anti-AML Activity In Vivo

Treatment options for acute myeloid leukemia (AML) patients who are unfit for intensive chemotherapy are limited. Both targeted therapy and cellular therapy have been investigated, typically as separate treatments. For patients with AML unfit for intensive induction chemotherapy, venetoclax, a BCL-2 inhibitor, containing regimens have significantly improved treatment outcomes. However, patients treated long-term with venetoclax can develop resistance. Based on the characterization of the BCL2 G101V mutation that mediates resistance to venetoclax, we investigated the ability of homozygous BCL2 G101V knock-in in induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells to promote resistance to venetoclax in these effector cells to enable concurrent treatment with venetoclax and the engineered iPSC-derived NK cells that we hypothesize will provide improved NK cell-mediated killing of AML. Our group has pioneered the production of genetically modified NK cells from human iPSCs. iPSCs provide an advantage of a stable platform for genome engineering and previous studies demonstrate we can engineer iPSCs to express or delete genes of interest to derive genetically modified iPSC-NK cells with improved anti-tumor activity. To generate iPSC-derived NK cells with resistance to venetoclax, we employed CRISPR-Cas9 technology to knock-in the BCL2 G101V mutation (BCL2 G101V) in iPSCs. iPSCs homozygous for BCL2 G101V were selected and differentiated to NK cells. Homozygous BCL2 G101V engineered iPSC-NK cells demonstrated resistance to venetoclax compared to wildtype (WT) iPSC-NK cells in vitro. BCL2 G101V iPSC-NK cells were 94-fold more resistant to venetoclax compared to wildtype iPSC NK cells with an EC50 of 6018 nM, above the serum levels of patients receiving venetoclax. Analysis of cell surface proteins demonstrated that both sets of iPSC-NK cells had a typical NK cell phenotype with no differences in expression of receptors analyzed. There was no difference seen in cytotoxicity against K562 tumor cells between BCL2 G101V iPSC-NK cells and WT-iPSC NK cells. Additional functional analyses demonstrated that activity of the BCL2 G101V iPSC NK cells was preserved upon exposure to venetoclax. Cytotoxic activity, as measured by CD107a expression on WT iPSC-NK cells stimulated by MOLM13 AML tumors cells, was reduced nearly 3-fold upon addition of venetoclax. In contrast, activation of BCL2 G101V iPSC NK cells was not significantly affected by addition of venetoclax. This resistance to venetoclax was confirmed in longer term 36-hour Incucyte cytotoxicity assays where BCL2 G101V iPSC-NK cells demonstrated more than 10-fold increase in anti-AML activity compared to the WT-iPSC-NK cells. We then conducted in vivo studies to test the WT and engineered NK cells with and without addition of venetoclax in an AML xenograft model using NSG mice (Figure Panels A-D). These studies used MOLM13 AML cells made resistant to venetoclax and demonstrated that without venetoclax treatment, both the WT and the BCL2 G101V iPSC NK cells mediate effective anti-AML killing. With addition of venetoclax, the WT NK cells were unable to effectively kill the AML cells while the BCL2 G101V iPSC cells mediated potent anti-AML activity and demonstrated significantly improved survival when the NK cells are given in combination with venetoclax. Specifically, in this system, the mice treated with WT iPSC-NK cells had a median survival of 22.8 days while the median survival of mice treated with the BCL2 G101V iPSC NK cells was not reached (p<0.01) Intriguingly, BCL2 G101V iPSC NK cells also demonstrated improved activity against MOLM13 cells resistant to venetoclax. Together our results demonstrate that iPSC-NK cells can be engineered to generate venetoclax-resistance for use in combination with concurrent venetoclax therapy to markedly improve treatment of AML. Furthermore, this work demonstrates that novel drug resistance mechanisms can be introduced via genome engineering into iPSC-derived NK cells as a new strategy to produce improved cell products for “off-the-shelf” therapy.

In depth characterization of midbrain organoids derived from wild type iPSC lines.

The ability to model human neurological tissues in vitro has been a major hurdle to effective drug development for neurological disorders. iPSC-derived brain organoids have emerged as a compelling solution to this problem as they have the potential to relevantly model the protein expression pattern and physiology of specific brain regions. Although many protocols now exist for the production of brain organoids, few attempts have been made to do an in-depth kinetic evaluation of expression of mature regiospecific markers of brain organoids. To address this, we differentiated midbrain-specific brain organoids from iPSC-lines derived from three apparently healthy individuals using a matrix-free, bioreactor method. We monitored the expression of midbrain-specific neuronal markers from 7 to 90-days using immunofluorescence and immunohistology. The organoids were further characterized using electron microscopy and RNA-seq. In addition to serving as a potential benchmark for the future evaluation of other differentiation protocols, the markers observed in this study can be useful as control parameters to identify and evaluate the disease phenotypes in midbrain organoid derived from patient iPSC-lines with genetic neurological disorders.

An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.

The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

Generation of a Syngeneic Heterozygous ACVRL1(wt/mut) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Pathophysiological Investigation of Skeletal Deformities of Musculocontractural Ehlers-Danlos Syndrome Using Induced Pluripotent Stem Cells.

Musculocontractural Ehlers-Danlos syndrome caused by mutations in the carbohydrate sulfotransferase 14 gene (mcEDS-CHST14) is a heritable connective tissue disorder characterized by multiple congenital malformations and progressive connective tissue fragility-related manifestations in the cutaneous, skeletal, cardiovascular, visceral, and ocular systems. Progressive skeletal deformities are among the most frequent and serious complications affecting the quality of life and activities of daily living in patients. After establishing induced pluripotent stem cells (iPSCs) from cultured skin fibroblasts of three patients with mcEDS-CHST14, we generated a patient iPSC-based human osteogenesis model and performed an in vitro assessment of the phenotype and pathophysiology of skeletal deformities. Patient-derived iPSCs presented with remarkable downregulation of osteogenic-specific gene expression, less alizarin red staining, and reduced calcium deposition compared with wild-type iPSCs at each stage of osteogenic differentiation, including osteoprogenitor cells, osteoblasts, and osteocytes. These findings indicated that osteogenesis was impaired in mcEDS-CHST14 iPSCs. Moreover, the decrease in decorin (DCN) expression and increase in collagen (COL12A1) expression in patient-derived iPSCs elucidated the contribution of CHST14 dysfunction to skeletal deformities in mcEDS-CHST14. In conclusion, this disease-in-a-dish model provides new insight into the pathophysiology of EDS and may have the potential for personalized gene or drug therapy.

Development of porcine induced pluripotent stem cells with a CD163 reporter system

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Differentiation of human induced pluripotent stem cells into cortical neural stem cells.

Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Prime Editing Strategy to Install the PRPH2 c.828+1G>A Mutation.

Mutations in peripherin 2 (PRPH2) are associated with a spectrum of inherited retinal diseases (IRDs) including retinitis pigmentosa (RP) and macular degeneration. As PRPH2 is localized to cone and rod outer segments, mutations in PRPH2 lead the disorganization or absence of photoreceptor outer segments. Here, we report on a patient with PRPH2-linked RP who exhibited widespread RPE atrophy with a central area of macular atrophy sparing the fovea. In future studies, we plan to model the pathobiology of PRPH2-based RP using induced pluripotent stem cell (iPSC)-derived retinal organoids. To effectively model rare mutations using iPSC-derived retinal organoids, we first require a strategy that can install the desired mutation in healthy wild-type iPSC, which can efficiently generate well-laminated retinal organoids. In this study, we developed an efficient prime editing strategy for the installation of the pathogenic PRPH2 c.828+1 G>A splice-site mutation underlying our patient's disease.

Targeted Genomic Insertion of Cars in iPSC-Derived Macrophages Leads to Improved Expression and Anti-Tumor Activity

Expression of chimeric antigen receptors (CARs) provides an important strategy to improve the anti-tumor activity of immune cells. T cells equipped with CARs specific to tumor antigens have been very effective for treatment of B cell malignancies. However, CAR-T cell-mediated treatment had limited success in solid tumors. Since macrophages are capable of infiltrating into tumor microenvironment, detecting phagocytic targets and engulfing them, as well as stimulating other endogenous immune cells, macrophages engineered with CAR constructs have been gaining interest for cancer immunotherapy. We have successfully generated CAR-expressing induced pluripotent stem cells (iPSCs) through either PiggyBac transposon-mediated transfection or lentiviral transduction. Subsequently, the CAR-expressing iPSCs were differentiated into macrophages (iPSC-CARMACs). The CAR expression in undifferentiated iPSCs were high and stable, but gradually downregulated during the differentiation into macrophages. Due to the loss of CAR expression, these iPSC-CARMACs only modestly improved phagocytic activity compared to wild-type iPSC-derived macrophages (iPSC-MACs) in vitro. Additionally, we did not find a significant benefit for in vivo anti-tumor activity of the iPSC-CARMACs compared to iPSC-MACs. We hypothesized the deficiency of anti-tumor activity might be due to the loss of CAR-expression that can become silenced after random integration into the genome. Therefore, to test this hypothesis as well as improve and stabilize the CAR expression, we engineered human iPSCs using CRISPR-Cas9 mediated CAR insertion into AAVS1 locus, a known safe harbor locus that is less prone to genetic silencing. We engineered a series of CAR constructs containing the same anti-Mesothelin (Meso) extracellular single-chain antibody variable fragment, combined with different intracellular signaling domains including Bai1, MegF10, MerTK and CD3ζ. To determine the phagocytic activity of the anti-Meso CARs expressing these different intracellular domains, we initially introduced CAR constructs into THP-1 cells. We tested these CAR-expressing THP-1-derived macrophages in phagocytosis assay with Meso-expressing A1847 ovarian cancer cells. We found Bai1 and MegF10 triggered higher levels of engulfment of tumor cells than other CAR constructs either by themselves or when combined with anti-CD47 antibody (Figure A). Given the potential anti-tumor activity of anti-Meso-Bai1 and anti-Meso-MegF10 CARs, we next generated anti-Meso-Bai1 and anti-Meso-MegF10 CAR-expressing iPSC-CARMACs by targeting at AAVS1 locus to improve CAR expression compared to the previous iPSC-CARMACs made by transposon transfection or lentiviral transduction. We successfully obtained iPSCs expressing anti-Meso-Bai1 CAR integrated into AAVS1 site (AAVS1-Meso-Bai1-iPSCs). We have differentiated the AAVS1-Meso-Bai1-iPSCs to macrophages (Meso-Bai1-CARMACs) and demonstrated Meso-Bai1-CARMACs significantly improved phagocytic activity compared to iPSC-MACs with or without the addition of anti-CD47 antibody (Figure B). Notably, CAR expression was stably maintained at a high level on the mature macrophages generated from the AAVS1-Meso-Bai1 iPSCs compared to those either from transposon or lentiviral vector mediated CAR expressing iPSCs (Figure C). We isolated two homozygous clones of AAVS1-Meso-Bai1 iPSCs that have even stronger CAR expression than bulk population of AAVS1-Meso-Bai1 iPSCs. The differentiation of these single clones, as well as the evaluation of their in vitro activity are in progress. We are also testing the in vivo function of these new AAVS1-iPSC-CARMACs in an ovarian tumor model. Together, these studies demonstrate that we can successfully produce CAR-expressing iPSC derived macrophages, which exhibit improved anti-tumor activity compared to wild-type iPSC derived macrophages. CAR expression can be best maintained on macrophages by AAVS1-locus targeting insertion. Importantly, these studies also demonstrate that strategies used to genetically modify iPSC-derived cells may need to be modified or optimized depending on which mature cell type of interest is being produced. That is, while randomly integrated CARs seem to work well in iPSC-derived NK cells and T cells, this targeted insertion approach is likely needed for derivation of more functional iPSC-derived CAR-macrophages. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Knockout of integrin β1 in induced pluripotent stem cells accelerates skin-wound healing by promoting cell migration in extracellular matrix

BackgroundInduced pluripotent stem cells (iPSCs) have the potential to promote wound healing; however, their adhesion to the extracellular matrix (ECM) might decrease iPSC migration, thereby limiting their therapeutic potential. Integrin β1 (Itgb1) is the major integrin subunit that mediates iPSC-ECM adhesion, suggesting that knocking out Itgb1 might be an effective method for enhancing the therapeutic efficacy of iPSCs.MethodsWe knocked out Itgb1 in mouse iPSCs and evaluated its effects on the therapeutic potential of topically applied iPSCs, as well as their underlying in vivo and in vitro mechanisms.ResultsThe Itgb1-knockout (Itgb1-KO) did not change iPSC pluripotency, function, or survival in the absence of embedding in an ECM gel but did accelerate wound healing, angiogenesis, blood perfusion, and survival in skin-wound lesions. However, embedding in an ECM gel inhibited the in vivo effects of wild-type iPSCs but not those of Itgb1-knockout iPSCs. Additionally, in vitro results showed that Itgb1-knockout decreased iPSC-ECM adhesion while increasing ECM-crossing migration. Moreover, ECM coating on the culture surface did not change cell survival, regardless of Itgb1 status; however, the in vivo and in vitro functions of both Itgb1-knockout and wild-type iPSCs were not affected by the presence of agarose gel, which does not contain integrin-binding sites. Knockout of Integrin α4 (Itga4) did not change the above-mentioned cellular and therapeutic functions of iPSCs.ConclusionsItgb1-knockout increased iPSCs migration and the wound-healing-promoting effect of topically applied iPSCs. These findings suggest the inhibition of Itgb1 expression is a possible strategy for increasing the efficacy of iPSC therapies.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Generation of a MIR5004 knockout cell line from human induced pluripotent stem cells by CRISPR/Cas9 gene editing

MIR5004 is located in the intronic region of SYNGAP1, a genetic risk factor for Autism Spectrum Disorders (ASD), and co-expressed with SYNGAP1 in brain tissue, which indicates that MIR5004 may play an important role in ASD pathogenesis through the regulation of SYNGAP1. Here, we generated a MIR5004 knockout human induced pluripotent stem cell (iPSC) line SHCDCLi001-B. SHCDCLi001-B shows expression of pluripotent markers, three lineage differentiation capacity, normal morphology and karyotypes, the same DNA origin with wild type iPSC (iPSC-WT) and no off-target effects, making it as a valuable tool for studying the interplay between MIR5004 and SYNGAP1 in ASD pathogenesis.

Stem cell conversion to the cardiac lineage requires nucleotide signalling from apoptosing cells.

Pluripotent stem cells can be driven by manipulation of Wnt signaling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that initiation of differentiation in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) triggers widespread apoptosis, followed by a synchronous epithelial-mesenchymal transition (EMT). Apoptosis is caused by absence of bFGF from the differentiation medium. EMT requires induction of transcription factors SNAI1/SNAI2 downstream of MESP1 expression, and double knock-out of SNAI1/2, or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis chemically or by ablation of pro-apoptotic genes also completely prevents the EMT, suppressing even the earliest events in mesoderm conversion, including BRA/T, TBX6, and MESP1 induction. Conditioned medium from WNT-activated WT iPSCs overcomes the block to EMT by cells incapable of apoptosis (Apop-), suggesting involvement of soluble factors from apoptotic cells in mesoderm conversion. Knockout of the PANX1 channel blocked EMT, while treatment with a purinergic P2 receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signaling. ATP and/or UTP was sufficient to induce a partial EMT in Apop- cells treated with WNT activator. Notably, knockout of the ATP/UTP-specific P2Y2 receptor blocked EMT and mesoderm induction. We conclude that nucleotides, in addition to acting as chemo-attractants for clearance of apoptotic cells can function as essential paracrine signals that, with WNT signaling, create a logical AND gate for mesoderm specification.

Single cell transcriptomic profiling of tau pathophysiology in a novel 3D neural-glial coculture model.

Research into neurodegeneration has been hampered by a lack of systems that rapidly and accurately recapitulate neurodegenerative processes. We report development of AstAD, a model combining human iPSC derived neurons (hiNCs) and astrocytes (hiACs) in self-aggregating 3D organoids that reproducibly develop a robust range of tau pathology and neurodegeneration. AstAD is a scalable and highly manipulatable model of neurodegeneration and critical understudied glial responses. Additionally, the AstAD model induces pathology without reliance on genetic overexpression. AstAD represents an important technological advance in modeling Alzheimer's Disease (AD), and is a promising tool for identifying disease modifying treatments. The AstAD system was developed by combining advances in 3D cell modeling and tau propagation. We perform directed differentiation of wild-type iPSC to hiNCs and hiACs, and hiNCs are selectively exposed to toxic aggregations to induce pathology. Cells are combined in microwells to induce self-aggregation, and pathology develops over the course of three weeks. Tau pathogenesis in the AstAD model was correlated with human tauopathy progression by immunopathology and single cell transcriptomics, and the application of a selective inhibitor PU-H71 demonstrated the efficacy of the model to explore disease modifying treatment. The AstAD model consistently displays a robust range of tau pathology within three weeks, including progressive development of hyperphosphorylated tau at positions T181, S262, and S396/404, misfolded tau (MC1, TOMA2), and fibrillar tau (THIOS). Neurodegeneration (FlouroJadeB, LDH) is accompanied by reactive astrogliosis (GFAP), which single cell transcriptomics reveals to be driven by heat shock proteins (HSPs). The observed tau pathology and transcriptional responses were reduced by treatment with PU-H71, which eliminates dysfunctional HSP epichaperome. Together this evidence demonstrates a rapid and reversible recapitulation of tau pathogenesis. The novel AstAD model consistently recapitulates tau-induced neurodegeneration in a human system, and allows for the elucidation of neuronal and astrocytic responses. Additionally, we have demonstrated the power of AstAD to model the reduction of tau pathology by drug treatment. Continued expansion of the AstAD model will include integration of microglia. The AstAD model represents a technological advance that will allow for the rapid recapitulation of neurodegeneration in a manipulatable system that promises to accelerate drug development for AD.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia

AbstractX-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Customized Induced Pluripotent Stem Cell-Derived Red Cell Reagents

Red blood cell transfusion is an essential therapy for patients with sickle cell disease (SCD). A major problem is the complexity of antibodies found in patients with SCD, in large part due to RH gene variation. Rh antibodies are common despite provision of serologic Rh-matched transfusions. The antibodies may be mistaken for autoantibodies when in fact they are alloantibodies to high prevalence Rh antigens that are absent or have altered Rh epitopes on the patient's RBCs. Finding compatible red cell units is often complicated by a lack of reagent red cells to properly identify the fine antibody specificity. This can delay patient care, increase costs, and make transfusion therapy impossible for some patients. We hypothesized that human induced pluripotent stem cells (iPSCs) could be reprogrammed from rare donors or genetically engineered to produce standard and reliable red cell reagents to aid identification for patients with complex antibodies. However, most human iPSC-derived red blood cells (iRBCs) are developmentally primitive red cells that differ from donor-derived red cells with high levels of embryonic globin expression, are larger in size and fail to enucleate in culture. We aimed to improve iPSC hematopoietic differentiation protocols to generate iRBCs compatible with standard blood bank assays.We designed a panel of customized iPSCs that included 1) Rh null cells, 2) cells lacking the high prevalence Rh antigen termed hrS, 3) cells expressing a partial C antigen and lacking the high prevalence Rh antigen termed hrB and, 4) cells expressing the low prevalence Rh antigens V, VS and lacking hrB. For the Rh null line, we identified wild type iPSC lines that were group O, RhD- by homozygous RHD deletion. We used CRISPR/Cas9 genetic engineering to disrupt the RHCE alleles either by a large deletion from exon 1 to exon 2 and/or via insertion-deletion mutations resulting in a frameshift and early stop codon. For lines lacking high prevalence Rh antigens, we reprogrammed group O donor cells whose genotypes were heterozygous for RHD*DAU0/RHCE*ceMO and RHD*DOL/RHCE*ceBI, homozygous for RHD*DIIIa-CE(4-7)-D/RHCE*ceS, and homozygous for RHD/RHCE*ce733G, respectively. At least 2 independent clones from each customized line were characterized for pluripotency by standard flow cytometry and gene expression markers, and a normal karyotype confirmed.We performed hematopoietic differentiation of customized iPSCs by a 3-step embryoid body protocol in defined, serum-free media with combinations of hematopoietic cytokines. We obtained CD41+235+ primitive hematopoietic progenitors on day 8 and cultured these cells in liquid culture with erythroid specific cytokines to generate iRBCs. We assessed erythroid cell maturation by morphology on cytospin preparations and by flow cytometry. We found an almost pure population of CD71+235+ iRBCs with erythroblast morphology by day 6 of culture, and progressive maturation to Band 3 high, alpha integrin low iRBCs that were smaller and had condensed nuclei that resemble erythroid cultures of primary CD34+ cells. Comparison of total Rh protein expression by flow cytometry on untargeted group O, D+ iRBCs compared to donor-derived RBCs demonstrated comparable cell surface Rh expression. As proof of principal, we assessed RBC agglutination by gel card assay of untargeted group O, D+, E-, e+ and the genetically engineered Rh null iRBCs using standard anti-D, -E, and -e reagents for Rh typing (Ortho Bioclone). As expected, the untargeted O, D+ iRBCs agglutinated with anti-D and anti-e while the engineered Rh-null iRBCs showed no agglutination with all 3 antibodies. Only 0.5 e6 iRBCs were required per gel assay.We have designed a panel of customized iPSCs reprogrammed from rare donors or genetically engineered to express rare blood group antigen phenotypes or combinations that are difficult or impossible to find as donor red cells. Any number of combinations not found in natural populations can be produced and generated in quantities sufficient for reagents. iRBC produced from these customized iPSCs can be used with standard blood bank assays and potentially provide the means to streamline and standardize antibody identification in alloimmunized patients with complex antibody specificities. In the future, when technology for scale-up is available, Rh null iRBCs could be used as “universal” donor cells for future therapeutic applications. DisclosuresNo relevant conflicts of interest to declare.

Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

Jazf1 acts as a regulator of insulin-producing β-cell differentiation in induced pluripotent stem cells and glucose homeostasis in mice.

Genetic susceptibility of type 2 diabetes and Juxtaposed with another zinc finger protein 1 (Jazf1) has been reported; however, the precise role of Jazf1 in metabolic processes remains elusive. In this study, using Jazf1-knockout (KO)-induced pluripotent stem cells (iPSC), pancreatic beta cell line MIN6 cells, and Jazf-1 heterozygous KO (Jazf1+/- ) mice, the effect of Jazf1 on gradual differentiation was investigated. We checked the alterations of the genes related with β-cell specification, maturation, and insulin release against glucose treatment by the gain and loss of the Jazf1 gene in the MIN6 cells. Because undifferentiated Jazf1-KO iPSC were not significantly different from wild-type (WT) iPSC, the size and endoderm marker expression after embryoid body (EB) and teratoma formation were investigated. Compared to EB and teratomas formed with WT iPSC, the EB and teratomas from with Jazf1-KO iPSC were smaller, and in teratomas, the expression of proliferation markers was reduced. Moreover, the expression of the gene sets for β-cell differentiation and the levels of insulin and C-peptide secreted by insulin precursor cells were notably reduced in β-cells differentiated from Jazf1-KO iPSC compared with those differentiated from WT iPSC. A comparison of Jazf1+/- and WT mice showed that Jazf1+/- mice had lower levels of serum insulin, pancreatic insulin expression, and decreased pancreatic β-cell size, which resulted in defects in the glucose homeostasis. These findings suggest that Jazf1 plays a pivotal role in the differentiation of β-cells and glucose homeostasis.