Generation of three control iPS cell lines for sickle cell disease studies by reprogramming erythroblasts from individuals without hemoglobinopathies

Paredes, Bruno Diaz; Martins, Gabriele Louise Soares; Azevedo, Carine Machado; Sampaio, Gabriela Louise de Almeida; Nonaka, Carolina Kymie Vasques; Silva, Kátia Nunes da; Soares, Milena Botelho Pereira; Santos, Ricardo Ribeiro dos; Souza, Bruno Solano de Freitas

doi:10.1016/j.scr.2019.101454

Cited by 4 publications

(6 citation statements)

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“…The donors were included in the study after confirming the SCD diagnosis by hemoglobin electrophoresis and obtaining written informed consent. PBMCs were cultured in erythroblast expansion medium and transfected with episomal vectors to generate iPSCs, as previously described [ 9 , 10 ]. Extensive characterization of the cell lines was previously performed, including pluripotency markers, karyotype, STR analysis, mutation detection by Sanger sequencing, and β S globin gene haplotype determination by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) [ 9 , 10 , 30 ].…”

Section: Methodsmentioning

confidence: 99%

“…The healthy control iPSC line CBTCi003-A [ 10 ] and SCD-derived lines CBTCi005-A, CBTCi006-A, and CBTCi007-A [ 9 ] were used for the differentiation protocols. The iPSCs were grown in 6-well plates coated with Geltrex™ LDEV-Free hESC-qualified (Thermo Fisher Scientific, Waltham, MA, USA) with StemFlex™ medium (STEMCELL Technologies, Vancouver, BC, Canada), and penicillin/streptomycin 1% (Thermo Fisher Scientific), and then incubated at 37 °C in a humidified atmosphere containing 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…The advent of somatic cell reprogramming and the generation of induced pluripotent stem cells (iPSCs) has provided new perspectives for improving disease modeling and drug screening using patient cells [ 5 , 6 , 7 , 8 ]. Several studies, including our previous work, have described the generation and characterization of iPSCs from SCD patients [ 5 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. These cells have been used in gene editing and hematopoietic differentiation studies [ 8 , 16 , 17 , 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells

Martins

Nonaka

Rossi

et al. 2023

Cells

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Background: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. Methods: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. Results: Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71+/CD235a+ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. Conclusion: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells

Martins

Nonaka

Rossi

et al. 2023

Cells

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“…We used two hiPSC lines obtained from two donors, previously obtained by integration-free reprogramming of erythroblasts with episomal vectors [ 14 ]. The cells were plated in Matrigel-coated wells (Corning; New York, NY, USA) and cultured with mTeSR1™ (Stem Cell Technologies; Vancouver, Canada).…”

Section: Methodsmentioning

confidence: 99%

A Novel High-Content Screening-Based Method for Anti-Trypanosoma cruzi Drug Discovery Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Portella

Rossi

Paredes

et al. 2021

Stem Cells International

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Chagas disease is caused by Trypanosoma cruzi infection and remains a relevant cause of chronic heart failure in Latin America. The pharmacological arsenal for Chagas disease is limited, and the available anti-T. cruzi drugs are not effective when administered during the chronic phase. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to accelerate the process of drug discovery for Chagas disease, through predictive preclinical assays in target human cells. Here, we aimed to establish a novel high-content screening- (HCS-) based method using hiPSC-CMs to simultaneously evaluate anti-T. cruzi activity and cardiotoxicity of chemical compounds. To provide proof-of-concept data, the reference drug benznidazole and three compounds with known anti-T. cruzi activity (a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B) were evaluated in the assay. hiPSC-CMs were infected with T. cruzi and incubated for 48 h with serial dilutions of the compounds for determination of EC50 and CC50 values. Automated multiparametric analyses were performed using an automated high-content imaging system. Sublethal toxicity measurements were evaluated through morphological measurements related to the integrity of the cytoskeleton by phalloidin staining, nuclear score by Hoechst 33342 staining, mitochondria score following MitoTracker staining, and quantification of NT-pro-BNP, a peptide released upon mechanical myocardial stress. The compounds showed EC50 values for anti-T. cruzi activity similar to those previously described for other cell types, and GT5B showed a pronounced trypanocidal activity in hiPSC-CMs. Sublethal changes in cytoskeletal and nucleus scores correlated with NT-pro-BNP levels in the culture supernatant. Mitochondrial score changes were associated with increased cytotoxicity. The assay was feasible and allowed rapid assessment of anti-T. cruzi action of the compounds, in addition to cardiotoxicity parameters. The utilization of hiPSC-CMs in the drug development workflow for Chagas disease may help in the identification of novel compounds.

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“…Reprogramming of erythroblasts, expanded from peripheral blood mononuclear cells (PBMCs), was performed following a protocol adapted from Paredes et al [19]. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque®-1077 (Sigma Aldrich, St. Louis, MO, USA).…”

Section: Cell Reprogrammingmentioning

confidence: 99%

Generation and characterization of human induced pluripotent stem cell lines from patients with autism spectrum disorder and SCN2A variants

Santos,

Paredes,

Adanho

et al. 2024

Preprint

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Autism spectrum disorders (ASD) comprise a group of complex neurodevelopmental disorders that affect communication and social interactions. Over a thousand genes have been associated with ASD, with SCN2A standing out due to its critical role in neuronal function and development. Induced pluripotent stem cells (iPSCs) derived from individuals with ASD have become invaluable in vitro models for investigating the cellular and molecular mechanisms underlying the disorder. In this study, we generated and characterized four iPSC clones from peripheral blood mononuclear cells (PBMCs) of two ASD patients carrying loss-of-function variants in the SCN2A gene. These iPSC lines underwent comprehensive characterization through multiple assays. Reverse transcription polymerase chain reaction (RT-PCR), flow cytometry, and immunofluorescence analyses confirmed the presence of pluripotency markers. An embryoid body formation assay demonstrated their potential to differentiate into the three germ layers. Sequencing analysis confirmed the SCN2A variants, while short tandem repeat (STR) analysis authenticated the cell lines, and karyotype analysis ensured chromosomal integrity. The iPSCs exhibited typical morphological characteristics, including large nuclei with prominent nucleoli, a high nucleus-to-cytoplasm ratio, densely packed cells, and well-defined borders. These cells maintained pluripotency markers, demonstrated the ability to differentiate into the three germ layers, and showed a normal karyotype. Furthermore, we successfully generated cerebral organoids from these cells. Our study establishes a robust platform for further exploration of the pathophysiological mechanisms of ASD, particularly those involving SCN2A.

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Generation of three control iPS cell lines for sickle cell disease studies by reprogramming erythroblasts from individuals without hemoglobinopathies

Cited by 4 publications

References 2 publications

Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells

Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells

A Novel High-Content Screening-Based Method for Anti-Trypanosoma cruzi Drug Discovery Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Generation and characterization of human induced pluripotent stem cell lines from patients with autism spectrum disorder and SCN2A variants

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