Expression Of Pluripotency Markers Research Articles

Generation of a human induced pluripotent stem cell (iPSC) line from a patient with a CDKL5 gene mutation

CDKL5(Cyclin-dependent kinase-like 5) encodes a kinase that may influence components of molecular pathways associated with MeCP2 and regulates axon outgrowth, dendritic morphogenesis, and synapse formation early in life. Here, we report an induced pluripotent stem cell (iPSC) line generated from an epilepsy patient harboring the CDKL5 c.2684C > T (p.P895L) variant. Peripheral blood mononuclear cells from the patient were successfully reprogrammed into iPSCs that expressed pluripotency markers, a normal karyotype, and the capacity to differentiate into the three germ layers in vivo. This iPSC line is a potential resource for studying the pathogenic mechanisms of epilepsy.

Generation of human induced pluripotent stem cell (DMSCi001-A) line from hematopoietic stem cells of a healthy female donor

Hematopoietic stem cell isolated from a healthy 39-year-old woman were successfully reprogrammed and transformed into induced pluripotent stem cell (iPSCs) by using the integration-free episomal vector included OCT3/4/shp53, Sox2/KLF4, L-MYC/LIN28 and EBNA-1 reprogramming factors. The transformed iPSC lines were cultured and expanded under feeder-free condition. They demonstrated the normal karyotype, expressed pluripotency markers and differentiated into cells derived from the three germ layers. These iPSCs are capable of differentiating into numerous cell subtypes for the purposes of drug discovery and mechanism investigation.

Generation of CRISPR/Cas9 modified human iPSC line with correction of heterozygous mutation in exon 6 of the CaSR gene.

The calcium-sensing receptor (CaSR) gene encodes a cell membrane G protein-coupled receptor (GPCR) which has a key role in maintaining the extracellular Ca2+ homeostasis. We aimed at correcting the compound heterozygous mutation in the 6th [c.1656delA, p.I554SfsX73] and 7th [c.2217T > A, p.C739X] exons of the CASR gene which the original patient-derived iPSC line had. The mutation is associated with neonatal severe primary hyperparathyroidism of the patient. We generated and characterized a CRISP/Cas9-edited hiPSC line with the restored sequence in the sixth exon of the CASR gene, bearing only heterozygous mutation in the 7th exon. The results showed that the new genetically modified cell line has karyotype without abnormalities, typical hiPSCs morphology, characteristic expression of pluripotency markers, and ability to develop into three germ layers, and differentiates in chondrogenic, adipogenic, osteogenic directions. This new cell line will complement the existing pool of CaSR-mutated cell lines, a valuable resource for in-depth understanding of neonatal severe primary hyperparathyroidism. This will allow further exploration of the application of pharmacological drugs in the context of personalized medicine to correct Ca-homeostasis disorders.

Generation of an induced pluripotent stem cell line (HMSCATi004-A) from an early onset Parkinson’s disease patient with PRKN gene mutation

Parkin (PRKN) is recognized as causative gene in early-onset Parkinson’s disease (PD). Induced pluripotent stem cells (iPSCs) were derived from a 29-year-old PD patient carrying a heterozygous c.823C > T (p.R275W) variant and an exon 2–4 deletion in PRKN. The Generated iPSCs maintain a normal karyotype, express pluripotency markers, and retain the ability to differentiate into the three germ layers. This iPSC line serves as a valuable cellular model for investigating the pathogenesis of early-onset PD and development of potential therapeutic interventions.

Genetically engineered human induced pluripotent stem cells for the production of brain-targeting extracellular vesicles

BackgroundExtracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising, natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture, human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However, hiPSC-EVs only have a moderate ability for brain delivery. Herein, we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor.MethodsAn RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging.ResultsHere we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype, express pluripotency markers, and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways, compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency.ConclusionTaken together, we have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain-targeted drug delivery.

Targeting CREB-binding protein (CBP) abrogates colorectal cancer stemness through epigenetic regulation of C-MYC.

Colorectal cancer (CRC) is a common cancer worldwide with an increasing annual incidence. Cancer stem cells (CSCs) play important roles in the occurrence, development, recurrence, and metastasis of CRC. The molecular mechanism regulating the development of colorectal CSCs remains unclear. The discovery of human induced pluripotent stem cells (hiPSCs) through somatic cell reprogramming has revolutionized the fields of stem cell biology and translational medicine. In the present study, we converted hiPSCs into cancer stem-like cells by culture with conditioned medium (CM) from CRC cells. These transformed cells, termed hiPSC-CSCs, displayed cancer stem-like properties, including a spheroid morphology and the expression of both pluripotency and CSC markers. HiPSC-CSCs showed tumorigenic and metastatic abilities in mouse models. The epithelial-mesenchymal transition phenotype was observed in hiPSC-CSCs, which promoted their migration and angiogenesis. Interestingly, upregulation of C-MYC was observed during the differentiation of hiPSC-CSCs. Mechanistically, CREB binding protein (CBP) bound to the C-MYC promoter, while histone deacetylase 1 and 3 (HDAC1/3) dissociated from the promoter, ultimately leading to an increase in histone acetylation and C-MYC transcriptional activation during the differentiation of hiPSC-CSCs. Pharmacological treatment with a CBP inhibitor or abrogation of CBP expression with a CRISPR/Cas9-based strategy reduced the stemness of hiPSC-CSCs. This study demonstrates for the first time that colorectal CSCs can be generated from hiPSCs. The upregulation of C-MYC via histone acetylation plays a crucial role during the conversion process. Inhibition of CBP is a potential strategy for attenuating the stemness of colorectal CSCs.

Generation of heterozygous KCNA2 knockout induced pluripotent stem cell line

The KCNA2 gene is the voltage-gated ion channel from both functional and structural perspectives. KCNA2 is involved in diverse functions including the regulation of neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. To investigate the relevant pathophysiological mechanisms, we generated heterozygous KCNA2 knockout human induced pluripotent stem cell (iPSC) line via CRISPR/Cas9 gene editing. The generated iPSCs had a normal karyotype, were free of genetically integrated epitaxial plasmids, expressed pluripotency markers, and maintained trilineage differentiation potential.

Generation of patient-derived induced pluripotent stem cell line UJSi004-A from ultra-treatment-resistant schizophrenia

Schizophrenia is a severe mental illness with disabling effects. Induced pluripotent stem cells (iPSCs) are generated by reprogramming peripheral blood mononuclear cells (PBMCs) from patients with ultra-treatment-resistant schizophrenia (UTRS). iPSCs have normal karyotype, express pluripotency markers and differentiate into three germ layers in vivo. This iPSC cell line carries the genetic information of the patient and is a good model for studying disease mechanisms and developing new therapies.

Generation of an integration-free induced pluripotent stem cell line, FJMAi001-A, from a Marfan syndrome patient with a heterozygous mutation c.2777G > A (p.Cys926Tyr) in FBN1

Marfan syndrome (MFS) is a heritable dominant disorder of fibrous connective tissue, caused by mutations in the gene encoding fibrillin-1 on chromosome 15. Here, we report an induced pluripotent stem cell (iPSC) line generated from a patient with MFS who carries a heterozygous mutation of c.2777G > A(p.Cys926Tyr) in the FBN1 gene using an episomal method. The hiPS-MFS cell line has normal karyotype, expresses pluripotency markers, and has the ability to form three germ layers in vivo.This MFS-specific iPSC line can be used as a cell disease model to study the pathogenesis of Marfan syndrome.

Establishment of a human induced pluripotent stem cell (iPSC) line (JUCTCi018-A) from a patient with Charcot-Marie-Tooth disease type 2EE (CMT2EE) due to a homozygous c.122G > A p.(Arg41Gln) mutation in the MPV17 gene

(Charcot-Marie-Tooth disease (CMT) is a genetic disorder affecting peripheral nerves. The human induced pluripotent stem cell (iPSC) line JUCTCi018-A was created using dermal fibroblasts from a Charcot-Marie-Tooth disease type 2EE (CMT2EE) patient with a homozygous missense mutation in the MPV17 gene (c. 122G > A, p.Arg41Gln). These fibroblasts were reprogrammed using Sendai viruses that encoded OCT4, SOX2, KLF4, and c-MYC reprogramming factors. The iPSCs demonstrated normal morphology and karyotype, expressed pluripotency markers, and the ability to differentiate into the three germ layers. This iPSC line is valuable for investigating the mechanisms underlying CMT2EE.

Generation and characterization of two isogenic induced pluripotent stem cell lines from a young female with microcephaly carrying a compound heterozygous mutation in BUB1 gene

Mutations in the Budding uninhibited by benzimidazoles (BUB1) gene were recently associated with neurodevelopmental disorders (Carvalhal et al., 2022). Here, we describe the generation and characterization of two induced pluripotent stem cells (iPSC) clones from a young female with microcephaly. The patient carried two variants in the BUB1 gene (OMIM # 602452), one (c.[2197dupG]; p.[D732fs*11]) paternally inherited and one (c.[2625+1G>A]; p.[V822_L875del] maternally inherited. The generated clones exhibit a normal karyotype (UALGi003-A) and trisomy 8 (UALGi003-B), express pluripotency markers, and differentiate into trilineage cells in vitro. These cell lines can be used to study neurodevelopment and the processes of chromosome segregation.

Generation of an induced pluripotent stem cell line from a type 1 neurofibromatosis patient with NF1 mutation

A human induced pluripotent stem cell (iPSC) line was generated from patient with type 1 neurofibromatosis (NF-1), carrying heterozygous mutation in NF1 gene. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating delivery of KFL4, OCT4, SOX2, BCL-XL and c-MYC. The iPSC line expresses pluripotency markers, displays a normal karyotype, and is able to differentiate into three germ layers in vitro. This iPSC line represents a valuable cell model for NF1 in humans.

Generation of RAB4A homozygous knockout induced pluripotent stem cell (iPSC) line

The RAB4A gene is a member of the largest group in the Ras superfamily of small GTPases, which regulate membrane trafficking. The encoded protein is associated with early endosomes and is involved in sorting and recycling. In this study, we generated induced pluripotent stem cells (iPSC) from a healthy individual by electroporation of peripheral blood mononuclear cells. We generated a RAB4A homozygous knockout human iPSC line via CRISPR/Cas9 gene editing. The iPSCs-RAB4A−/− had a normal karyotype, expressed pluripotency markers, and maintained trilineage differentiation potential.

Mesenchymal Stromal/Stem Cells Isolated by Explant Culture Method from Wharton’s Jelly and Subamnion Possess Similar Biological Characteristics

Human umbilical cord (UC) is an attractive source of mesenchymal stromal/stem cells (MSCs) for tissue engineering and regenerative medicine due to its easy availability, non-invasive procedure of collection, and no ethical concerns. The aim of this study was to isolate MSCs from the Wharton’s jelly (WJ) and subamnion (SA) from the same umbilical cord by an optimized explant method, and to compare the morphology, proliferation, and stemness properties of the MSCs from both sources. Cells from the WJ and SA of six umbilical cords were characterized by flow cytometry, differentiation capacity and proliferation assays, immunofluorescence staining, and RT-PCR. The optimized explant method was successfully used to isolate WJ-MSCs and SA-MSCs. The MSCs from both sources showed similar patterns of growth kinetics, adipogenic and osteogenic potential, and the expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA-4). The current findings support the usage of the optimized explant method to generate a relatively homogenous population of MSCs from Wharton’s jelly and subamnion, which can facilitate the reproducibility of the results from experimental and practical applications of the obtained cells.

Refined home-brew media for cost-effective, weekend-free hiPSC culture and genetic engineering

Background Cost-effective, practical, and reproducible culture of human induced pluripotent stem cells (hiPSCs) is required for both basic and translational research. This is especially crucial for large-scale expansion of hiPSCs for cell therapy, which should be made accessible to many patients regardless of their socioeconomic background. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hiPSC culture. However, homebrewing of some recombinant growth factors for B8 can be a bottleneck towards both access and reproducibility of this technology. Moreover, we found the published B8 formulation to be suboptimal in normoxic hiPSC culture, which is widely used. Lastly, the suitability of B8 for applications such as genome editing or organoid differentiation remains to be assessed. Methods We formulated B8 with commercially available, animal-free growth factors, refined its composition to support normoxic culture of the widely-used WTC11 hiPSC line, and compared it to commercial Essential 8 (E8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle with flow cytometry, and investigated the transcriptional profiles by bulk RNA sequencing. We also assessed the efficiency of gene editing, single-cell sorting, and cardiac differentiation in both monolayer and organoids. Results hE8 performed similarly to commercial E8 in all the assays. Despite morphological changes, cells in B8+, our optimised variant of B8, expressed the pluripotency marker NANOG at the highest level. At the same time, cells grown in B8+ were primed towards a mesendodermal fate. B8+ outperformed other media with regard to genome editing via homology directed recombination, and was on par with other media in other assays. Conclusions Overall, optimised weekend-free media formulations promise to democratise the generation of engineered cells for a wide range of applications.

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as β-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Autologous hGMSC-Derived iPS: A New Proposal for Tissue Regeneration.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers' markers' expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

Generation of human induced pluripotent stem cell lines from patients with a RYR2 gene variant c.14201A>G (p.Y4734C): Implications for idiopathic ventricular fibrillation and catecholaminergic polymorphic ventricular tachycardia

Human induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells (PBMCs) isolated from two related patients diagnosed with either idiopathic ventricular fibrillation or catecholaminergic polymorphic ventricular tachycardia, carrying an unknown variant in the RYR2 gene, c.14201A>G (p.Y4734C) and one healthy related individual. Reprogramming was done using a commercially available Epi5 Reprogramming Kit. The pluripotency of the iPSC lines was verified by the expression of pluripotency markers and by their capacity to differentiate into all three embryonic germ layers in vitro. These iPSC lines are available for functional analysis and in vitro studies of RYR2 channelopathy.

Arachidonic acid regulates pluripotency by modulating cellular energetics via fatty acid synthesis and mitochondrial fission

Arachidonic acid (AA) is an important omega-6 fatty acid that can be metabolised into an impressive spectrum of biologically active mediators participating in various cellular functions. Studies have shown that fatty acid synthesis is enhanced in embryonic stem cells (ESCs), and it is crucial for the cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Fatty acid synthesis increases the cellular lipid contents and, in turn, promotes mitochondrial fission and cellular reprogramming. AA was found to induce acetyl-CoA carboxylase 1 (ACC1) expression, a major enzyme in fatty acid synthesis. In this study, we have investigated the regulation of pluripotency, fatty acid synthesis and mitochondrial activities of the human induced pluripotent stem cells (hiPSCs) and the human embryonal carcinoma (hEC) NTERA-2 cells upon treatment with varying concentrations of AA. Our results indicate that a lower concentration of AA can increase pluripotency, as evidenced by an increased expression of pluripotency markers, increased fatty acid synthesis as evidenced by lipid estimation and modulated mitochondrial fission, as evidenced by mitotracker staining for fissioned mitochondria. Moreover, higher concentrations of AA-induced the opposite effect, leading to pluripotent stem cell differentiation. Molecular docking simulations predicted the possible interactions between AA and its metabolites with fatty acid synthesis regulators ACC1 and CREB1 (Cyclic adenosine monophosphate Response Element Binding Protein 1) as a mechanism for AA regulating pluripotency.

Generation of hereditary spastic paraplegia patient-derived induced pluripotent stem cell line UJSi003-A

Hereditary spastic paraplegia (HSP) is a rare neurodegenerative disorder with the predominant clinical manifestation of spasticity in the lower extremities. Patients with HSP experience spastic paralysis in both lower limbs, leading to progressive walking difficulties, increased reflexes, spasms, and extensor plantar responses. We successfully generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with HSP. The iPSCs exhibited a normal karyotype, expressed pluripotency markers, and differentiated into the three germ layers in vitro.