Control iPSCs Research Articles

Small and Long Non-Coding RNA Analysis for Human Trophoblast-Derived Extracellular Vesicles and Their Effect on the Transcriptome Profile of Human Neural Progenitor Cells

In mice, the fetal brain is dependent upon the placenta for factors that guide its early development. This linkage between the two organs has given rise to the term, the placenta–brain axis. A similar interrelationship between the two organs may exist in humans. We hypothesize that extracellular vesicles (EVs) released from placental trophoblast (TB) cells transport small RNA and other informational biomolecules from the placenta to the brain where their contents have pleiotropic effects. Here, EVs were isolated from the medium in which human trophoblasts (TBs) had been differentiated in vitro from induced pluripotent stem cells (iPSC) and from cultured iPSC themselves, and their small RNA content analyzed by bulk RNA-seq. EVs derived from human TB cells possess unique profiles of miRs, including hsa-miR-0149-3p, hsa-302a-5p, and many long non-coding RNAs (lncRNAs) relative to EVs isolated from parental iPSC. These miRs and their mRNA targets are enriched in neural tissue. Human neural progenitor cells (NPCs), generated from the same iPSC, were exposed to EVs from either TB or iPSC controls. Both sets of EVs were readily internalized. EVs from TB cells upregulate several transcripts in NPCs associated with forebrain formation and neurogenesis; those from control iPSC upregulated a transcriptional phenotype that resembled glial cells more closely than neurons. These results shed light on the possible workings of the placenta–brain axis. Understanding how the contents of small RNA within TB-derived EVs affect NPCs might yield new insights, possible biomarkers, and potential treatment strategies for neurobehavioral disorders that originate in utero, such as autism spectrum disorders (ASDs).

Human sensory-like neuron cultivation-An optimized protocol.

Reprogramming of human-induced pluripotent stem cells (iPSCs) and their differentiation into specific cell types, such as induced sensory-like neurons (iSNs), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, the development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving the survival and quality of iSNs. iSNs were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-β-D-arabinofuranosyl-cytosine hydrochloride (AraC) treatment, magnetic-activated cell sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively. Passaging of cells during differentiation did not increase the iSN-to-total-cell-count ratio, and MACS of immature iSNs led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10 μM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSNs shortly after seeding also helped to reduce non-iSN cells. In direct comparison with other methods, treatment with 10 μM FdU for 24 h after differentiation shows promise for improving iSN culture purity, which could benefit downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of this method and enhance its reproducibility and applicability. Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC-based research.

Generation of a control induced pluripotent stem cell line (CBRCULi014-A) derived from the lymphoblastoid cells of a pediatric individual

Lymphoblastoid cell lines serve as a readily and continuous resource for generating induced pluripotent stem cells (iPSCs), enabling the modeling of various genetic disorders in vitro. When investigating congenital and infantile diseases, age-match controls derived from pediatric individuals are typically necessary, yet they may be scarce or difficult to obtain. Here, the Sendai virus system was employed to introduce reprogramming factors into lymphoblastoid cells derived from an apparently healthy 4-year-old female. The generated iPSCs strongly expressed pluripotency cell markers and displayed robust trilineage differentiation. CBRCULi014-A is therefore a reliable control iPSC line for pediatric disease investigation.

Generation of human induced pluripotent stem cell lines from a subject with UBAP1L-associated retinal dystrophy and CRISPR/cas9-corrected isogenic iPSC lines

A Human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a patient affected with an autosomal recessive retinal dystrophy carrying the homozygous c.910-7G>A variant in UBAP1L. Three isogenic control iPSC lines derived from this affected subject line were created using CRISPR/Cas9 engineering. All iPSC lines expressing the pluripotency markers, were able to differentiate into the three germ layers, and exhibit a normal karyotype. These cellular models will provide a powerful tool to study disease mechanisms associated with the recently reported UBAP1L- associated retinal dystrophy and better understand the role of the protein in retinal physiology.

Epigenetic insights into GABAergic development in Dravet Syndrome iPSC and therapeutic implications

Dravet syndrome (DS) is a devastating early-onset refractory epilepsy syndrome caused by variants in the SCN1A gene. A disturbed GABAergic interneuron function is implicated in the progression to DS but the underlying developmental and pathophysiological mechanisms remain elusive, in particularly at the chromatin level. Induced pluripotent stem cells (iPSCs) derived from DS cases and healthy donors were used to model disease-associated epigenetic abnormalities of GABAergic development. Chromatin accessibility was assessed at multiple time points (Day 0, Day 19, Day 35, and Day 65) of GABAergic differentiation. Additionally, the effects of the commonly used anti-seizure drug valproic acid (VPA) on chromatin accessibility were elucidated in GABAergic cells. The distinct dynamics in the chromatin profile of DS iPSC predicted accelerated early GABAergic development, evident at D19, and diverged further from the pattern in control iPSC with continued differentiation, indicating a disrupted GABAergic maturation. Exposure to VPA at D65 reshaped the chromatin landscape at a variable extent in different iPSC-lines and rescued the observed dysfunctional development of some DS iPSC-GABA. The comprehensive investigation on the chromatin landscape of GABAergic differentiation in DS-patient iPSC offers valuable insights into the epigenetic dysregulations associated with interneuronal dysfunction in DS. Moreover, the detailed analysis of the chromatin changes induced by VPA in iPSC-GABA holds the potential to improve the development of personalized and targeted anti-epileptic therapies.

Epigenetic insights into GABAergic development in Dravet Syndrome iPSC and therapeutic implications.

Dravet syndrome (DS) is a devastating early-onset refractory epilepsy syndrome caused by variants in the SCN1A gene. A disturbed GABAergic interneuron function is implicated in the progression to DS but the underlying developmental and pathophysiological mechanisms remain elusive, in particularly at the chromatin level. Induced pluripotent stem cells (iPSCs) derived from DS cases and healthy donors were used to model disease-associated epigenetic abnormalities of GABAergic development. Chromatin accessibility was assessed at multiple time points (Day 0, Day 19, Day 35, and Day 65) of GABAergic differentiation. Additionally, the effects of the commonly used anti-seizure drug valproic acid (VPA) on chromatin accessibility were elucidated in GABAergic cells. The distinct dynamics in the chromatin profile of DS iPSC predicted accelerated early GABAergic development, evident at D19, and diverged further from the pattern in control iPSC with continued differentiation, indicating a disrupted GABAergic maturation. Exposure to VPA at D65 reshaped the chromatin landscape at a variable extent in different iPSC-lines and rescued the observed dysfunctional development of some DS iPSC-GABA. The comprehensive investigation on the chromatin landscape of GABAergic differentiation in DS-patient iPSC offers valuable insights into the epigenetic dysregulations associated with interneuronal dysfunction in DS. Moreover, the detailed analysis of the chromatin changes induced by VPA in iPSC-GABA holds the potential to improve the development of personalized and targeted anti-epileptic therapies.

Revealing the UV response of melanocytes in xeroderma pigmentosum group A using patient-derived induced pluripotent stem cells

BackgroundXeroderma pigmentosum (XP) is characterized by photosensitivity that causes pigmentary disorder and predisposition to skin cancers on sunlight-exposed areas due to DNA repair deficiency. Patients with XP group A (XP-A) develop freckle-like pigmented maculae and depigmented maculae within a year unless strict sun-protection is enforced. Although it is crucial to study pigment cells (melanocytes: MCs) as disease target cells, establishing MCs in primary cultures is challenging. ObjectiveElucidation of the disease pathogenesis by comparison between MCs differentiated from XP-A induced pluripotent stem cells (iPSCs) and healthy control iPSCs on the response to UV irradiation. MethodsiPSCs were established from a XP-A fibroblasts and differentiated into MCs. Differences in gene expression profiles between XP-A-iPSC-derived melanocytes (XP-A-iMCs) and Healthy control iPSC-derived MCs (HC-iMCs) were analyzed 4 and 12 h after irradiation with 30 or 150 J/m2 of UV-B using microarray analysis. ResultsXP-A-iMCs expressed SOX10, MITF, and TYR, and showed melanin synthesis. Further, XP-A-iMCs showed reduced DNA repair ability. Gene expression profile between XP-A-iMCs and HC-iMCs revealed that, numerous gene probes that were specifically upregulated or downregulated in XP-A-iMCs after 150-J/m2 of UV-B irradiation did not return to basal levels. Of note that apoptotic pathways were highly upregulated at 150 J/m2 UV exposure in XP-A-iMCs, and cytokine-related pathways were upregulated even at 30 J/m2 UV exposure. ConclusionWe revealed for the first time that cytokine-related pathways were upregulated even at low-dose UV exposure in XP-A-iMCs. Disease-specific iPSCs are useful to elucidate the disease pathogenesis and develop treatment strategies of XP.

ATP1A3 regulates protein synthesis for mitochondrial stability under heat stress.

Pathogenic variants in ATP1A3, the gene encoding the α3 subunit of the Na+/K+-ATPase, cause alternating hemiplegia of childhood (AHC) and related disorders. Impairments in Na+/K+-ATPase activity are associated with the clinical phenotype. However, it remains unclear whether additional mechanisms are involved in the exaggerated symptoms under stressed conditions in patients with AHC. We herein report that the intracellular loop (ICL) of ATP1A3 interacted with RNA-binding proteins, such as Eif4g (encoded by Eif4g1), Pabpc1 and Fmrp (encoded by Fmr1), in mouse Neuro2a cells. Both the siRNA-mediated depletion of Atp1a3 and ectopic expression of the p.R756C variant of human ATP1A3-ICL in Neuro2a cells resulted in excessive phosphorylation of ribosomal protein S6 (encoded by Rps6) and increased susceptibility to heat stress. In agreement with these findings, induced pluripotent stem cells (iPSCs) from a patient with the p.R756C variant were more vulnerable to heat stress than control iPSCs. Neurons established from the patient-derived iPSCs showed lower calcium influxes in responses to stimulation with ATP than those in control iPSCs. These data indicate that inefficient protein synthesis contributes to the progressive and deteriorating phenotypes in patients with the p.R756C variant among a variety of ATP1A3-related disorders.

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.

Reduction of Filamin C Results in Altered Proteostasis, Cardiomyopathy, and Arrhythmias.

Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood. We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib. FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

Mutual interaction of neurons and astrocytes derived from iPSCs with APP V717L mutation developed the astrocytic phenotypes of Alzheimer’s disease

BackgroundThe development of induced pluripotent stem cells (iPSCs) technology has enabled human cellular disease modeling for inaccessible cell types, such as neural cells in the brain. However, many of the iPSC-derived disease models established to date typically involve only a single cell type. These monoculture models are inadequate for accurately simulating the brain environment, where multiple cell types interact. The limited cell type diversity in monoculture models hinders the accurate recapitulation of disease phenotypes resulting from interactions between different cell types. Therefore, our goal was to create cell models that include multiple interacting cell types to better recapitulate disease phenotypes.MethodsTo establish a co-culture model of neurons and astrocytes, we individually induced neurons and astrocytes from the same iPSCs using our novel differentiation methods, and then co-cultured them. We evaluated the effects of co-culture on neurons and astrocytes using immunocytochemistry, immuno-electron microscopy, and Ca2+ imaging. We also developed a co-culture model using iPSCs from a patient with familial Alzheimer's disease (AD) patient (APPV717L mutation) to investigate whether this model would manifest disease phenotypes not seen in the monoculture models.ResultsThe co-culture of the neurons and astrocytes increased the branching of astrocyte processes, the number of GFAP-positive cells, neuronal activities, the number of synapses, and the density of presynaptic vesicles. In addition, immuno-electron microscopy confirmed the formation of a tripartite synaptic structure in the co-culture model, and inhibition of glutamate transporters increased neuronal activity. Compared to the co-culture model of the control iPSCs, the co-culture model of familial AD developed astrogliosis-like phenotype, which was not observed in the monoculture model of astrocytes.ConclusionsCo-culture of iPSC-derived neurons and astrocytes enhanced the morphological changes mimicking the in vivo condition of both cell types. The formation of the functional tripartite synaptic structures in the co-culture model suggested the mutual interaction between the cells. Furthermore, the co-culture model with the APPV717L mutation expressed in neurons exhibited an astrocytic phenotype reminiscent of AD brain pathology. These results suggest that our co-culture model is a valuable tool for disease modeling of neurodegenerative diseases.

Abstract WMP118: Utilizing Patient-Derived Induced Pluripotent Stem Cells as a Cellular Model for Studying Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a rare cerebrovascular disease that can lead to stroke. A histopathological characteristic in MMD is the thickening of the intimal layer that causes narrowing of the vessel, suggesting dysfunctions in the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The mechanism of MMD is largely unknown, due to a lack of established MMD-specific cellular or animal models. In this study, we established patient-derived cellular co-culture models using MMD iPSC-derived ECs and VSMCs. We also generated a 3D vascular organoid model to more closely mimic the MMD vasculature and function. Methods: Peripheral blood mononuclear cells (PBMCs) from MMD patients or healthy controls were used to generate iPSCs (n=4 per group). Differentiated ECs and VSMCs were validated and their functional characteristics were investigated in single and co-cultures. For co-cultures, ECs and VSMCs were labeled with Vibrant cell labeling dyes (DiO and DiD), followed by either transwell migration assay (ECs on bottom of cell culture plate and VSMCs on top Transwell insert) or in vitro angiogenic tube formation assay. Control and MMD iPSCs were used to generate 3D cellular organoids, and their cellular characteristics were examined using immunohistochemistry with antibodies labeling ECs and VSMCs. Results: While single cultures of ECs displayed similar tube formation patterns between control and MMD, co-cultures of ECs with VSMCs showed that MMD ECs failed to stabilize in vitro angiogenic tubes and formed aggregates at 24hr, when compared to control ECs. Transwell migration assays showed MMD VSMCs migrated faster than control VSMCs. Immunostaining of organoids with EC and VSMC markers showed notable differences in cellular composition, with MMD organoid showing compromised integrity of vascular networks, when compared with controls. Conclusions: We have established 3D organoid cellular models and in vitro co-cultures using MMD patient-derived iPSCs. Our co-culture studies revealed functional impairment in MMD EC/VSMC and organoid models revealed distinct characteristics in their vascular networks, possibly contributing to MMD pathology. These results shed useful insight into the cellular and molecular mechanisms driving MMD.

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

Genetic correction of induced pluripotent stem cells from a DFNA36 patient results in morphologic and functional recovery of derived hair cell-like cells

BackgroundTMC1 is one of the most common deafness genes causing DFNA36. Patient-derived human induced pluripotent stem cells (iPSCs) provide an opportunity to modelling diseases. TMC1 p.M418K mutation in human is orthologous to Beethoven mice. Here, we investigated the differentiation, morphology and electrophysiological properties of hair cell-like cells (HC-like cells) derived from DFNA36 patient.MethodsInner ear HC-like cells were induced from iPSCs derived from DFNA36 (TMC1 p.M418K) patient (M+/−), normal control (M+/+) and genetic corrected iPSCs (M+/C). Immunofluorescence, scanning electron microscopy and whole-cell patch-clamp were used to study the mechanism and influence of TMC1 p.M418K mutation.ResultsIn this study we successfully generated HC-like cells from iPSCs with three different genotypes. HC-like cells from M+/− showed defected morphology of microvilli and physiological properties compared to M+/+. HC-like cells from M+/C showed recovery in morphology of microvilli and physiological properties.ConclusionsOur results indicate that TMC1 p.M418K mutation didn’t influence inner ear hair cell differentiation but the morphology of microvilli and electrophysiological properties and gene correction induced recovery. CRISPR/Cas9 gene therapy is feasible in human patient with TMC1 p.M418K mutation.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Uncovering the Role of an ABCA7 Frameshift Deletion in African American Alzheimer’s Disease Cellular Pathology

AbstractBackgroundThe ATP Binding Cassette Subfamily A Member 7 (ABCA7) gene is a risk factor for Alzheimer’s disease (AD). While ABCA7 has been implicated as a genetic determinant of AD across populations, the risk effect is the strongest in African Americans (AAs). We previously identified a common 44 base pair deletion in ABCA7 (p.Arg578Alafs) that is predicted to truncate the protein and is significantly associated with AD in AAs (frequency in cases = 15.2%, cognitively unimpaired (CU) = 9.74%, p = 1.41×10−5). Clinically, deletion homozygotes are similar to deletion heterozygotes, supporting dominant gain‐of‐function as a putative disease‐causing mechanism. We sought to determine if the deletion transcript is stable and if the truncated protein is expressed as steps to understanding the mechanism leading to AD risk.MethodRT‐PCR analysis of heterozygotes was conducted to assess the stability of the deletion transcript. FLAG‐tagged Arg578Alafs and wildtype ABCA7 vectors were constructed then overexpressed in HEK293APPsw cells. Western blot analysis of FLAG‐Arg578Alafs was performed to determine if the deletion produces a truncated protein. Induced pluripotent stem cell (iPSC) lines from six AA individuals with AD bearing the Arg578Alafs mutation (three heterozygous; three homozygous) were created in pair with isogenic CRISPR‐corrected control lines. These iPSC lines were validated for pluripotency, genomic stability, and lack of off‐target editing. The isogenic ABCA7 deletion and CRISPR‐corrected control iPSC lines will be differentiated into microglia and neurons and functionally assessed for cell type‐specific AD phenotypes.ResultInitial results comparing AD patients to CU individuals show that patient‐derived microglia have normal rates of phagocytosis but are impaired in the uptake and clearance of fibrillar Aβ. RT‐PCR analysis of the deletion in heterozygotes demonstrates that a stable RNA transcript is expressed from the ABCA7 deletion allele. Western blot analysis of overexpressed FLAG‐Arg578Alafs shows that the truncated protein is expressed, albeit at low levels compared to the wildtype protein.ConclusionThis ABCA7 deletion produces a stable transcript which appears to be translated into low levels of protein. This supports a potential gain of function as the disease risk mechanism. Studies in isogenic lines will further elucidate the functional effects of this ABCA7 deletion on AD pathology in AAs.

Human iPSC‐microglia expressing TREM2 p.R47H risk variant for Alzheimer’s disease do not show a loss‐of‐function phenotype

AbstractBackgroundThe most common TREM2 variant associated with increased risk to develop Alzheimer’s disease (AD) is p.R47H. This variant has been classified as loss‐of‐function, mostly by comparing p.R47H with the common variant in TREM2 over‐expressing cell lines. Whether this holds true when TREM2 is expressed at physiological levels in a disease‐relevant cell type such as human microglia, has not been fully characterized.MethodWe established and thoroughly characterized human iPSC‐derived microglia endogenously expressing TREM2 to assess the effect of p.R47H on TREM2 cellular distribution, TREM2 signaling and microglial functioning. For this study microglia generated from a set of isogenic gene‐edited iPSC lines homozygous for TREM2 knockout (KO) and TREM2 p.R47H were evaluated and compared to microglia from the parental control iPSC line. In addition, microglia generated from iPSC lines obtained from several p.R47H carriers were compared to microglia from iPSC lines obtained from several healthy control non‐carriers. SNP genotyping confirmed the presence of R47H.ResultThe p.R47H and common variant TREM2 were present at similar levels at the cell surface of iPSC‐derived microglia, whereas no expression of TREM2 was detected in the TREM2 KO microglia. Microarray profiling indicated that the microglial transcriptome is very similar between the common variant and p.R47H yet distinct from TREM2 KO microglia. Biochemical evaluation of TREM2 signaling indicated no reduction in TREM2 downstream signaling activation in the p.R47H variant expressing microglia. Similarly, soluble TREM2 levels were increased in culture medium of p.R47H microglia. Finally, analysis of microglial functioning including phagocytosis of artificial and disease‐relevant substrates showed no difference between the p.R47H variant and the common variant, in contrast to TREM2 KO microglia.ConclusionWe conclude that in cultured human iPSC‐derived microglia, the p.R47H variant shows no evidence of a loss‐of‐function mechanism. Future experiments will determine whether this is also the case in microglia in their natural brain environment. Our long‐term goal is to better understand TREM2 biology to support the development of novel therapeutics for AD and other neurodegenerative diseases characterized by TREM2‐mediated neuroinflammation.

ICF1-Syndrome-Associated DNMT3B Mutations Prevent De Novo Methylation at a Subset of Imprinted Loci during iPSC Reprogramming.

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation.

CRISPR/Cas9 Strategy for Correcting ELANE Mutations and Restoring Neutrophil Differentiation in Severe Congenital Neutropenia: Insights from Patient-Derived iPSCs

Introduction The ELANE gene, encoding neutrophil elastase (NE), has been identified as the primary causative gene for more than half of patients with severe congenital neutropenia (SCN). For patients refractory to standard therapy (G-CSF), the only available option is an allogeneic hematopoietic stem cell transplant. Therefore, there is a great clinical interest in the development of novel gene therapies, including CRISPR/Cas9, which has shown promise for the correction of ELANE mutations and the restoration of neutrophil function. Here, we introduce a gene editing strategy using CRISPR/Cas9 to repair ELANE gene mutations in patient-derived induced pluripotent stem cells (iPSCs), aiming to restore proper neutrophil development and function. Methods We utilized a targeted NGS panel composed of 725 genes related to hematological disorders to sequence 282 pediatric patients suffering from chronic neutropenia. Patient-derived epithelial cells collected from urine were reprogrammed into iPSCs using the EBV-derived oriP/EBNA-1 system carrying transcription factors. CRISPR-Cas9 technology was employed to repair mutations in patient-derived iPSCs. The efficiency of transfection and gene editing was subsequently assessed by analyzing the targeted region by Sanger sequencing. Control iPSCs (healthy donors) and iPSCs from patients were differentiated into neutrophils with specific cytokine cocktail (BMP4, CHIR99021, VEGF, SCF, bFGF, SB431542, SCF, IL-3, TPO, Flt-3, G-CSF). Cells were harvested at day 25 and analyzed for surface markers resembling the distinct myeloid progenitors. Flow cytometry antibody panel (CD14, CD45, CD49d, Siglec-8, CD101, CD11b, CD35, CD16) and gating strategy allowed to identify myeloid populations such as proNeu1, preNeu, immature-Neu and mature Neu representing myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively. The morphology of myeloid precursors and mature neutrophils as well as the localization of neutrophil elastase in control iPSCs were evaluated by confocal microscopy. Results From a sequenced cohort of 282 patients, causative mutations were identified in 49 individuals, including 17 with ELANE mutations, and 9 novel variants were discovered. For this study, we selected two patients exhibiting different phenotypes for further investigation. One patient carries the NM_001972.3:c.[163T>C];[=], NP_001963:p.[(Cys55Arg)];[(=)] ELANE mutation and presents severe neutropenia (ANC=100/µL), currently undergoing G-CSF treatment. The second patient carries the NM_001972.3:c.[597+5G>A];[=], NP_001963:p.[(Val190_Phe199del)];[(=)] ELANE mutation which demonstrates in the fluctuating blood neutrophil counts characteristic for cyclic neutropenia. iPSCs were generated from both patients. Alkaline phosphatase and immunocytochemical staining confirmed that the reprogrammed cells express pluripotent stem cell markers and can differentiate into the three germ layers. Genetic defect repair was observed in 13.3% of tested cell colonies from the patient with the c.163T>C Cys55Arg ELANE mutation and in 30% of sequenced colonies from the patient with the c.597+5G>A V190_F199del ELANE mutation. Molecular cytogenetics (SNP-array) showed no significant genomic rearrangements or off-targets in iPSCs selected for differentiation. iPSC cells from healthy donors, derived from c.597+5G>A V190_F199del ELANE patient and the repaired clone were successfully differentiated into neutrophils (CD16+ CD35+) and their myeloid precursors (CD49d+ CD11b+). The percentage of immature and mature neutrophils (CD16+ and CD35+) derived from control or corrected iPSC was significantly higher compared to patient-derived neutrophils (Figure 1). Conclusions Our study demonstrates the successful use of CRISPR/Cas9 to repair ELANE gene mutations in patient-derived iPSCs, aiming to restore proper neutrophil development. Patient-derived iPSCs exhibit impaired differentiation at the myelocytes/metamyelocytes stage, as evidenced by a notably lower population of immature and mature neutrophils when compared to CRISPR/Cas9 repaired and healthy donor iPSCs. This observation aligns with the bone marrow phenotype observed in patients with SCN. Our findings indicate that iPSCs generated from patients' epithelial cells serve as a valuable cellular model for further research.

Impact of the overexpression of the tyrosine kinase receptor RET in the hematopoietic potential of induced pluripotent stem cells (iPSCs)

Previous studies have suggested that the tyrosine kinase receptor RET plays a significant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in human iPSC-derived hematopoiesis has not been tested so far. To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. The hematopoietic potential was tested using 2D cultures and evaluated regarding the phenotype and the clonogenic potential of generated cells. Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of primitive hematopoietic cells (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific activated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related to MAPK activation.