iPS Cell Lines Research Articles

A human-induced pluripotent stem cell (iPSC) line (SMUSHi006-A) from an ALS patient carrying a mutation c.1126C > T in the FUS gene

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Four major genes associated with ALS—SOD1, TARDBP, FUS, and C9orf72—have been identified, with the fused in sarcoma (FUS) gene demonstrating considerable genetic heterogeneity. Our research group has previously established an induced pluripotent stem (iPS) cell line harboring the c.1562G > A mutation in the FUS gene. The objective of this study is to create another iPS cell line featuring the pathogenic c.1126C > T mutation in the FUS gene. This research aims not only to establish a disease model for ALS linked to FUS mutations but also to pave the way for potential therapeutic interventions.

Mature cardiac tissues modeling Duchenne muscular dystrophy related cardiomyopathy

Abstract Background The major cause of mortality in patients with Duchenne muscular dystrophy (DMD) is cardiomyopathy. This disease is caused by dystrophin gene mutations that result in the loss of dystrophin protein. Gene replacement therapies have been approved for skeletal muscle symptoms of DMD. However, these therapeutic modalities are not indicated for cardiomyopathy due to their low efficiency of delivery to the heart. Despite the significant unmet needs, a limitation in this area of research is the divergence of cardiomyopathy symptoms in model animals from those in patients. Alternatively, cardiomyocytes differentiated from patient-derived iPS cells have recently been analyzed. However, existing results are based on a single time point analysis and do not model disease progression or non-cardiomyocyte involvement. Purpose Our research aims to establish an in vitro model that can reproduce the pathological progression of DMD cardiomyopathy. Methods We have generated cardiac organoids and engineered heart tissues (EHTs) from patient-derived iPS cells, consisting of cardiomyocytes and epicardial-derived non-cardiomyocytes. An iPS cell line in which the dystrophin mutation was repaired by genome editing was used as a control. The cardiac organoids or EHTs were subjected to transient activation of fatty acid metabolism to promote the maturation of iPS-derived cardiomyocytes. Pathological conditions were then induced in these 3D-models by applying loads that mimicked the DMD-disease environment. Results The mature cardiac organoids displayed adult-like gene expression profiles in the components of dystrophin-associated-protein-complex and calcium handling. After continuous mechanical loading, cell death appeared in DMD-EHTs, although there was only a slight decrease in contractility and fibrosis. Elevated mitochondrial ROS production and DNA damage were observed as a part of the cell death mechanism. Furthermore, pathological stimuli in addition to mechanical loading reproduced progressive contractile dysfunction and further increase in fibrosis in DMD-EHTs. Conclusions From these observations, we concluded that our DMD-EHT model recapitulate the pathological development of DMD-cardiomyopathy. These results suggest that the key factors involved in the progression of DMD cardiomyopathy are (i) the fragility of cardiomyocytes to stress in early stages and (ii) the involvement of non-cardiomyocytes such as fibrosis. Our model provides insight into the molecular mechanisms underlying disease progression and novel therapeutic targets.

Establishment of a non-integrated iPS cell line (SDQLCHi072-A) from a patient suffered from AUTS2 syndrome

In this study, PBMCs used in reprogramming were from a boy suffered from AUTS2 syndrome confirmed by clinical and genetic diagnosis. iPSCs were established by non-integrated method, which carried AUTS2 heterozygous mutation. The established iPSCs presented similar appearance and expressed pluripotent markers in mRNA and protein level. Additionally, the iPSCs possessed favorable differentiative capacity in vivo and normal karyotype. This is an effective potential model for the study about AUTS2 syndrome and the screen about drug therapy.

Long-term engrafting multilineage hematopoietic cells differentiated from human induced pluripotent stem cells.

Hematopoietic stem cells (HSCs) derived from human induced pluripotent stem cells (iPS cells) have important biomedical applications. We identified differentiation conditions that generate HSCs defined by robust long-term multilineage engraftment in immune-deficient NOD,B6.Prkdcscid Il2rgtm1Wjl/SzJ KitW41/W41 mice. We guided differentiating iPS cells, as embryoid bodies in a defined culture medium supplemented with retinyl acetate, through HOXA-patterned mesoderm to hemogenic endothelium specified by bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF). Removal of VEGF facilitated an efficient endothelial-to-hematopoietic transition, evidenced by release into the culture medium of CD34+ blood cells, which were cryopreserved. Intravenous transplantation of two million thawed CD34+ cells differentiated from four independent iPS cell lines produced multilineage bone marrow engraftment in 25-50% of immune-deficient recipient mice. These functionally defined, multipotent CD34+ hematopoietic cells, designated iPS cell-derived HSCs (iHSCs), produced levels of engraftment similar to those achieved following umbilical cord blood transplantation. Our study provides a step toward the goal of generating HSCs for clinical translation.

Introduction of an RS1 mutation causative variant consistent with identified XLRS patient using CRISPR/Cas9 technology in normal iPSC

X-linked retinoschisis (XLRS) is a common retinal genetic disease that occurs in juvenile males and causes progressive visual impairment. this presents a schisis in the macula or peripheral retina of bilateral eyes and currently has no effective treatment. Here, we introduced the RS1 (c.C304T, p.R102W) mutation into a normal induced pluripotent stem (iPS) cell line using CRISPR/Cas9 technology. This missense mutation was consistent with that observed in the XLRS patient-derived iPS cell line (CSUASOi001-A). Conclusively, establishing a directed gene mutation cell line (CSUi007-A) provides a useful cell resource to investigate XLRS pathogenesis.

Establishment of iPS cell line (SDQLCHi080-A) from a patient with GM1 gangliosidosis due to GLB1 mutation

GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder caused by defects in the beta-galactosidase (GLB1) gene, which results in accumulation of GM1 gangliosides and related glycoconjugates in the lysosomes leading to lysosomal swelling, cellular damage, and organ dysfunction. We generated SDQLCHi080-A cell line from a patient with GM1 gangliosidosis carrying mutations of c.523C > T and c.574T > C > T in the GLB1 gene. The cell line exhibited typical iPSC morphology, expressed high levels of stemness markers, exhibited normal karyotype, and has the capability to differentiate into three germ layers. This cell line could provide a useful GM1 gangliosidosis model in vitro for further study.

Abstract Mo091: Analysis of GLA Gene Mutation Using a Female Fabry Disease Patient-derived Induced Pluripotent Stem Cells.

Introduction: Fabry disease is an inborn error of metabolism caused by mutations in the alpha-galactosidase gene ( GLA ) located on the X chromosome, resulting in intracellular accumulation of globotriaosylceramide and systemic tissue injury. In addition to enzyme replacement therapy, chaperone therapy is effective for many missense mutations. One female patient had c.547G>A mutation, which has been reported as p.G183S, and we checked the indication for migalastat, a pharmacological chaperone. However, migalastat reactivity testing using HEK293 cells transfected with mutant cDNA had not been performed for this mutation. Aims: We analyzed the c.547G>A mutation and examined the efficacy of migalastat for this mutation. Methods: Peripheral blood mononuclear cells from patients were transfected with reprogramming factors using episomal vectors to generate induced pluripotent stem (iPS) cells. Cardiomyocytes were induced from the iPS cells. The mutation site was located at the 3' end of exon 3, and splicing mutation was suspected. Thus, PCR amplification was performed between exon 2 and exon 5 of the cDNA, and the PCR product was sequenced to confirm the mRNA sequence generated in the patient's cells. Real-time PCR was performed using Taqman probe to amplify sites unaffected by the mutation, and expression levels of normal and mutant GLA were compared. iPS cells were treated with migalastat and the effect on α-galactosidase activity was examined. Results: Since the patient was female, five iPS cell lines expressing only normal GLA and four iPS cell lines expressing only mutant GLA were established due to X-chromosome inactivation. Mutant cDNA was found to have a deletion of 36 bases, part of exon 3, resulting in a deletion of 12 amino acids. Real-time PCR result showed that the GLA expression level of mutant GLA-expressing cells was about half that of normal GLA-expressing cells, which was comparable to the GLA expression level of the cells from a healthy subject. Mutant GLA-expressing iPS cells had no α-galactosidase activity, which was not increased by treatment with migalastat. Conclusions: In patient-derived iPS cells and iPS cell-derived cardiomyocytes, c.547G>A produced p.C172_G183del, not p.G183S, and migalastat was ineffective. Further therapeutic development is needed for patients with this mutation.

Establishment of a transgene-free iPS cell line (SDCHi008-A) from a young patient bearing a KCNQ2 mutation and suffering from Epilepsy

Epilepsy is a chronic neurological disease which has affected ∼ 65 million people worldwide. In this study, peripheral blood mononuclear cells were isolated from a young patient patient bearing a KCNQ2 gene mutation and suffering from Epilepsy verified by clinical and genetic diagnosis. Induced pluripotent stem cells (iPSCs) were established by a non-integrative method, using plasmids carrying OCT4, SOX2, KLF4, BCL-XL and C-MYC. The established iPSCs presented typical pluripotent cells morphology, normal karyotype, and potential to differentiate into three germ layers. Our approach offers a useful model to explore pathogenesis and therapy of Epilepsy.

GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy.

Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.

APOE4 Increases Energy Metabolism in APOE-Isogenic iPSC-Derived Neurons.

The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimer's disease (AD). In contrast, APOE2 is known to lower the AD risk, while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in the brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons have also been shown as source for APOE. However, the distinct roles of the three APOE isoforms in neuronal energy homeostasis remain poorly understood. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4, or carrying an APOE knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3, and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells, indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism.

Establishment of a transgene-free iPS cell line (SDCHi007-A) from a young patient bearing a ATP1A2 mutation and suffering from Epilepsy

Epilepsy is a chronic neurological disease. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient diagnosed as epilepsy caused by ATP1A2 gene mutation. Induced pluripotent stem cells (iPSCs) were developed using non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and C-MYC. The established iPSC line (SDCHi007-A) displayed pluripotent cell morphology, high expression levels of pluripotency markers, differentiation potential in vitro, normal karyotype, and remaining the original ATP1A2 gene mutation.

Generation of three iPSC lines with inducible systems to be used in Angelman syndrome research

The neurodevelopmental disorder Angelman syndrome (AS) has an incidence of 1:15.000 live births and is caused by absence of UBE3A protein, showing imprinted gene expression in the brain. Imprinted genes are controlled by differentially methylated regions resulting in parent-of-origin dependent gene expression. Two iPS cell lines derived from patients with AS and one healthy control iPSC line were used to integrate a 3rd generation reverse tetracycline transactivator protein (rtTA3) into the AAVS1 locus on chromosome 19. The rtTA allows tetracycline-dependent activation of an inducible promoter that can be introduced at a position of interest in the cell lines described here.

Pre-clinical evaluation of clinically relevant iPS cell derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury.

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies.

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

Generation of one induced pluripotent stem cell line JUCGRMi004-A from a Charcot-Marie-Tooth disease type 1A (CMT1A) patient with PMP22 duplication

The CMT1A variant accounts for over 60% of cases of Charcot-Marie-Tooth disease (CMT), one of the most common human neuropathies. The cause of CMT1A has been identified as the duplication of PMP22, a myelin protein expressed in Schwann cells. Yet, the pathological mechanisms have not been elucidated, and no treatment is currently available. In our study, we established an iPS cell line from a CMT1A patient with PMP22 duplication. The generated iPSCs maintain pluripotency and in vitro differentiation potency.

Abstract 141: A human induced pluripotent stem cell model of KMT2A rearranged leukemia

Abstract Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with refractoriness to therapy, early relapse, and rapid leukemia progression. KMT2A-r generates a driver fusion oncogene, most commonly KMT2A::AFF1 in iALL, which leads to epigenetic dysregulation of target gene transcription. iALL with KMT2A::AFF1 is an unusual cancer in that other somatic mutations are uncommon, there are no known genetic risk factors, and phenotypic switch from lymphoid to myeloid, mixed, or undifferentiated phenotype is common during treatment and particularly resistant to therapies. The cell of origin is thought to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Unfortunately, research into this rare but devastating disease has been hindered by a lack of appropriate, representative models. Interestingly, KMT2A-r originates in HSPCs in utero and the age of onset of iALL is a strong prognostic factor, with age younger than six months associated with the highest risk of relapse. However, little is known regarding how KMT2A-r subverts early hematopoiesis or drives the severe disease phenotype. In an effort to understand the role of the developmental state of the cell of origin in iALL, we have created a highly controlled induced pluripotent stem (iPS) cell model system of KMT2A::AFF1 leukemia. Specifically, we engineered human iPS cell lines with doxycycline regulatable expression of KMT2A::AFF1 fusion and we are employing CRISPR gene editing technology to introduce clinically identified variants of interest. Utilizing directed differentiation, we have produced functional HSPCs from iPS cells. Notably, this model recapitulates hematopoietic ontogeny, with the ability to control iALL induction at specific developmental stages. Following doxycycline treatment, we demonstrate that the cells express KMT2A::AFF1 fusion transcripts. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation of our KMT2A::AFF1 iPS cells. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment. Citation Format: Erin Guest, Meagan Vacek, Jacqelyn Nemechek, Irina Pushel, Bradley T. Thornton, Molly Leyda, Midhat S. Farooqi, John M. Perry, Jay L. Vivian. A human induced pluripotent stem cell model of KMT2A rearranged leukemia [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 141.

Generation and characterization of an iPS cell line (PUMCi006-A) from skin fibroblasts of a patient with an M239T mutation in PSEN2 gene

Presenilin-2 (PSEN2) mutation is one of the pathogenic factors of autosomal dominant early-onset Alzheimer's disease (EOAD). We generated a human induced pluripotent stem cell (iPSC) line from fibroblasts of an EOAD patient carrying PSEN2 mutation (c.716 T > C) utilizing Sendai reprogramming kit. The resulting iPSC line carried patient-specific point mutation, exhibited typical iPSC morphology, retained a normal karyotype, expressed pluripotency markers, and could form embryoid bodies. Established iPSC line serve as valuable resource for EOAD disease pathogenesis modelling and drug screening.

Establishment of a non-integrated iPSC (SDQLCHi068-A) line derived from a patient with autosomal dominant immunodeficiency-14A carrying a heterozygous mutation (c.3061G>A) in PIK3CD gene

Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (PIK3CD) gene (OMIM#602839) encodes the p110δ catalytic subunit, mainly expressed in immune cells, and is associated with autosomal dominant immunodeficiency-14A with lymphoproliferation (IMD14A, #615513). We generated a human iPS cell line from a 50-month-old boy with IMD14A carrying a heterozygous mutation (c.3061G>A, p.E1021K) in PIK3CD gene. This cell line retains the original mutation site and shows differentiation potential towards three germ layers in vitro, which can be used as a disease model for research.

Generation and characterization of the iPS cell line (SYSUSHi001-A) derived from the peripheral blood mononuclear cells (PBMCs) of a 33-year-old patient with acute myeloid leukemia (AML)

We successfully developed an induced pluripotent stem cell (iPSC) line, SYSUSHi001-A, from the peripheral blood mononuclear cells (PBMC) of a patient with Acute Myeloid Leukemia, harboring two genetic mutations (XPO1: c.591-4_591-3dupTT; PALB2: c.3296C > T; p.T1099M). This iPSC line was facilitated through the use of episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, and human miR-302. The SYSUSHi001-A iPSC line exhibited characteristic embryonic stem cell-like morphology, maintained the XPO1 and PALB2 mutations, expressed key pluripotency markers, preserved a normal karyotype (46, XY), and demonstrated the ability to differentiate into cells from all three germ layers in vitro.