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Stem Cell Research Apr 2024We used a non-integrated reprogramming approach to establish a human induced pluripotent stem cell (hiPSC) line (INNDSUi004-A) from the skin fibroblasts of a 13-year-old...
We used a non-integrated reprogramming approach to establish a human induced pluripotent stem cell (hiPSC) line (INNDSUi004-A) from the skin fibroblasts of a 13-year-old female individual with Congenital Nemaline Myopath. The cells obtained have typical characteristics of embryonic stem cells, show expression of specific pluripotency markers, and can differentiate into three germ layers in vitro. This iPSC cell line has the genetic information of the patient and is a good model for studying disease mechanisms and developing novel therapies.
PubMed: 38733812
DOI: 10.1016/j.scr.2024.103435 -
Animals : An Open Access Journal From... May 2024The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein...
The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as , , and , as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal-Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.
PubMed: 38731386
DOI: 10.3390/ani14091382 -
Stem Cell Research May 2024Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly...
Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.
PubMed: 38723411
DOI: 10.1016/j.scr.2024.103437 -
Food & Nutrition Research 2023Extensive research has been conducted to investigate the impact of capsaicin (CAP) on lipid metabolism, focusing specifically on its interaction with the vanilloid...
Extensive research has been conducted to investigate the impact of capsaicin (CAP) on lipid metabolism, focusing specifically on its interaction with the vanilloid subtype 1 (TRPV1) ion channel. Additionally, studies have illuminated the role of Akkermansia muciniphila (), a specific strain of intestinal microbiota, in lipid metabolism. In this study, a model utilizing resiniferatoxin (RTX) was employed to deactivate TRPV1 ion channels in germ-free mice, followed by the administration of A. muciniphila via gavage. Following the collection of intestinal tissues for a comprehensive analysis, employing histopathology, qPCR, and ELISA techniques, our findings revealed a significant upregulation of MUC2 and MUC3 expression induced by CAP. This upregulation resulted in the thickening of the colonic mucus layers. Notably, this effect was absent when TRPV1 was selectively inhibited. Moreover, there was no discernible impact on goblet cells. The findings strongly indicate that CAP influences the system by activating the TRPV1 ion channel, thereby enhancing the expression of mucin MUC2 and promoting an augmentation in the thickness of the mucous layer. This activation, in turn, supplies with an ample source of carbon and nitrogen. This insight potentially clarify the underlying mechanism through which CAP facilitates the increase in abundance.
PubMed: 38721112
DOI: 10.29219/fnr.v67.9990 -
Scientific Reports May 2024In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to...
In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to transcriptional, epigenetic, and functional changes. This transition can be faithfully recapitulated in vitro by the differentiation of mouse embryonic stem cells (mESCs) to epiblast-like cells (EpiLCs), that reside in naïve and formative states of pluripotency, respectively. However, the GRN that drives this conversion is not fully elucidated. Here we demonstrate that the transcription factor OCT6 is a key driver of this process. Firstly, we show that Oct6 is not expressed in mESCs but is rapidly induced as cells exit the naïve pluripotent state. By deleting Oct6 in mESCs, we find that knockout cells fail to acquire the typical morphological changes associated with the formative state when induced to differentiate. Additionally, the key naïve pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb were expressed at higher levels than in wild-type cells, indicating an incomplete dismantling of the naïve pluripotency GRN. Conversely, premature expression of Oct6 in naïve cells triggered a rapid morphological transformation mirroring differentiation, that was accompanied by the upregulation of the endogenous Oct6 as well as the formative genes Sox3, Zic2/3, Foxp1, Dnmt3A and FGF5. Strikingly, we found that OCT6 represses Nanog in a bistable manner and that this regulation is at the transcriptional level. Moreover, our findings also reveal that Oct6 is repressed by NANOG. Collectively, our results establish OCT6 as a key TF in the dissolution of the naïve pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as an important toggle mediating the transition to the formative state.
Topics: Animals; Mice; Nanog Homeobox Protein; Gene Regulatory Networks; Cell Differentiation; Mouse Embryonic Stem Cells; Pluripotent Stem Cells; Gene Expression Regulation, Developmental; Octamer Transcription Factor-3; Germ Layers; Mice, Knockout
PubMed: 38710730
DOI: 10.1038/s41598-024-59247-5 -
Frontiers in Genetics 2024Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX), the fourth...
Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX), the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene (). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions, tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study, we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC, homozygous for the *354V allele, and from a control, homozygous for the *A354 allele. Specifically, for both of these fibroblast lines we generated hiPSC, hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines, as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells, with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC, reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts, this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC.
PubMed: 38706791
DOI: 10.3389/fgene.2024.1375467 -
Stem Cell Research May 2024GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and...
GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.
PubMed: 38703669
DOI: 10.1016/j.scr.2024.103431 -
Cytotherapy Apr 2024One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC)...
Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.
One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.
PubMed: 38703154
DOI: 10.1016/j.jcyt.2024.04.004 -
Stem Cell Research Apr 2024Peripheral blood mononuclear cells (PBMCs) from a 28-year-old male patient with unipolar depression were reprogrammed with reprogramming factors by electroporation. The...
Peripheral blood mononuclear cells (PBMCs) from a 28-year-old male patient with unipolar depression were reprogrammed with reprogramming factors by electroporation. The pluripotency of transgene-free induced pluripotent stem cells (iPSCs) was verified by immunofluorescence staining for pluripotency markers, and these iPSCs were able to differentiate into the 3 germ layers in vitro. These iPSCs also showed normal karyotypes. Thus, we believe that these iPSCs could be valuable models for exploring the underlying biological mechanism of depression and the safety of antidepressants through the use of iPSCs differentiated into different kinds of neurons or brain organoids.
PubMed: 38696853
DOI: 10.1016/j.scr.2024.103428 -
FASEB Journal : Official Publication of... May 2024The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this...
The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this regionalization process is instructed by inductive signals from the adjacent mesenchyme. However, the interaction landscape between epithelium and mesenchyme during upper MD development remains largely unknown. Here, we performed single-cell transcriptomic profiling of mouse neonatal oviducts and uteri at the initiation of MD epithelial differentiation (postnatal day 3). We identified major cell types including epithelium, mesenchyme, pericytes, mesothelium, endothelium, and immune cells in both organs with established markers. Moreover, we uncovered region-specific epithelial and mesenchymal subpopulations and then deduced region-specific ligand-receptor pairs mediating mesenchymal-epithelial interactions along the craniocaudal axis. Unexpectedly, we discovered a mesenchymal subpopulation marked by neurofilaments with specific localizations at the mesometrial pole of both the neonatal oviduct and uterus. Lastly, we analyzed and revealed organ-specific signature genes of pericytes and mesothelial cells. Taken together, our study enriches our knowledge of upper MD development, and provides a manageable list of potential genes, pathways, and region-specific cell subtypes for future functional studies.
Topics: Animals; Female; Mice; Uterus; Mullerian Ducts; Oviducts; Single-Cell Analysis; Transcriptome; Gene Expression Profiling; Animals, Newborn; Cell Differentiation; Mesoderm; Epithelial Cells; Mice, Inbred C57BL; Gene Expression Regulation, Developmental
PubMed: 38686936
DOI: 10.1096/fj.202400303R