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Stem Cell Research & Therapy Jun 2024Cartilage, an important connective tissue, provides structural support to other body tissues, and serves as a cushion against impacts throughout the body. Found at the... (Review)
Review
Cartilage, an important connective tissue, provides structural support to other body tissues, and serves as a cushion against impacts throughout the body. Found at the end of the bones, cartilage decreases friction and averts bone-on-bone contact during joint movement. Therefore, defects of cartilage can result from natural wear and tear, or from traumatic events, such as injuries or sudden changes in direction during sports activities. Overtime, these cartilage defects which do not always produce immediate symptoms, could lead to severe clinical pathologies. The emergence of induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine, providing a promising platform for generating various cell types for therapeutic applications. Thus, chondrocytes differentiated from iPSCs become a promising avenue for non-invasive clinical interventions for cartilage injuries and diseases. In this review, we aim to highlight the current strategies used for in vitro chondrogenic differentiation of iPSCs and to explore their multifaceted applications in disease modeling, drug screening, and personalized regenerative medicine. Achieving abundant functional iPSC-derived chondrocytes requires optimization of culture conditions, incorporating specific growth factors, and precise temporal control. Continual improvements in differentiation methods and integration of emerging genome editing, organoids, and 3D bioprinting technologies will enhance the translational applications of iPSC-derived chondrocytes. Finally, to unlock the benefits for patients suffering from cartilage diseases through iPSCs-derived technologies in chondrogenesis, automatic cell therapy manufacturing systems will not only reduce human intervention and ensure sterile processes within isolator-like platforms to minimize contamination risks, but also provide customized production processes with enhanced scalability and efficiency.
Topics: Humans; Induced Pluripotent Stem Cells; Regenerative Medicine; Cell Differentiation; Chondrogenesis; Precision Medicine; Chondrocytes; Animals
PubMed: 38926793
DOI: 10.1186/s13287-024-03794-1 -
Scientific Reports Jun 2024Ischemic heart diseases are a major global cause of death, and despite timely revascularization, heart failure due to ischemia-hypoxia reperfusion (IH/R) injury remains...
Ischemic heart diseases are a major global cause of death, and despite timely revascularization, heart failure due to ischemia-hypoxia reperfusion (IH/R) injury remains a concern. The study focused on the role of Early Growth Response 1 (EGR1) in IH/R-induced apoptosis in human cardiomyocytes (CMs). Human induced pluripotent stem cell (hiPSC)-derived CMs were cultured under IH/R conditions, revealing higher EGR1 expression in the IH/R group through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). Immunofluorescence analysis (IFA) showed an increased ratio of cleaved Caspase-3-positive apoptotic cells in the IH/R group. Using siRNA for EGR1 successfully downregulated EGR1, suppressing cleaved Caspase-3-positive apoptotic cell ratio. Bioinformatic analysis indicated that EGR1 is a plausible target of miR-124-3p under IH/R conditions. The miR-124-3p mimic, predicted to antagonize EGR1 mRNA, downregulated EGR1 under IH/R conditions in qRT-PCR and WB, as confirmed by IFA. The suppression of EGR1 by the miR-124-3p mimic subsequently reduced CM apoptosis. The study suggests that treatment with miR-124-3p targeting EGR1 could be a potential novel therapeutic approach for cardioprotection in ischemic heart diseases in the future.
Topics: MicroRNAs; Early Growth Response Protein 1; Humans; Myocytes, Cardiac; Induced Pluripotent Stem Cells; Apoptosis; Down-Regulation; Myocardial Reperfusion Injury
PubMed: 38926457
DOI: 10.1038/s41598-024-65373-x -
Nature Communications Jun 2024Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and...
Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.
Topics: Induced Pluripotent Stem Cells; Humans; Myocytes, Cardiac; Myocardial Contraction; Software; Cell Differentiation; Single-Cell Analysis; Cells, Cultured
PubMed: 38926342
DOI: 10.1038/s41467-024-49755-3 -
Osteoarthritis and Cartilage Jun 2024Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a...
OBJECTIVE
Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process.
DESIGN
The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway.
RESULTS
We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9.
CONCLUSIONS
This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported.
PubMed: 38925474
DOI: 10.1016/j.joca.2024.06.009 -
The Journal of Biological Chemistry Jun 2024The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and...
The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect hPSC-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide ChIP enrichment and mechanistic studies show that p38 MAPK-mediated CEBPD upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in MSCs exerts a negative regulatory effect on osteogenesis through a similar mechanism. ChIP-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.
PubMed: 38925326
DOI: 10.1016/j.jbc.2024.107494 -
Stem Cell Research Jun 2024The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We...
The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.
PubMed: 38924973
DOI: 10.1016/j.scr.2024.103484 -
Biomimetics (Basel, Switzerland) Jun 2024The ability of bone biomaterials to promote osteogenic differentiation is crucial for the repair and regeneration of osseous tissue. The development of a temporary bone...
The ability of bone biomaterials to promote osteogenic differentiation is crucial for the repair and regeneration of osseous tissue. The development of a temporary bone substitute is of major importance in enhancing the growth and differentiation of human-derived stem cells into an osteogenic lineage. In this study, nanocomposite hydrogels composed of gelatin methacryloyl (GelMA), bioactive glass (BG), and multiwall carbon nanotubes (MWCNT) were developed to create a bone biomaterial that mimics the structural and electrically conductive nature of bone that can promote the differentiation of human-derived stem cells. GelMA-BG-MWCNT nanocomposite hydrogels supported mesenchymal stem cells derived from human induced pluripotent stem cells, hereinafter named iMSCs. Cell adhesion was improved upon coating nanocomposite hydrogels with fibronectin and was further enhanced when seeding pre-differentiated iMSCs. Osteogenic differentiation and mature mineralization were promoted in GelMA-BG-MWCNT nanocomposite hydrogels and were most evidently observed in the 70-30-2 hydrogels, which could be due to the stiff topography characteristic from the addition of MWCNT. Overall, the results of this study showed that GelMA-BG-MWCNT nanocomposite hydrogels coated with fibronectin possessed a favorable environment in which pre-differentiated iMSCs could better attach, proliferate, and further mature into an osteogenic lineage, which was crucial for the repair and regeneration of bone.
PubMed: 38921218
DOI: 10.3390/biomimetics9060338 -
Cells Jun 2024Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs)....
Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of , the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in cells by transduction with gene replacement (ShH10-CMV-) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.
Topics: Humans; Adaptor Proteins, Signal Transducing; Autophagy; Choroideremia; Induced Pluripotent Stem Cells; Mechanistic Target of Rapamycin Complex 1; Models, Biological; rab GTP-Binding Proteins; Retinal Pigment Epithelium; Signal Transduction
PubMed: 38920696
DOI: 10.3390/cells13121068 -
Cells Jun 2024Human induced pluripotent stem cell (iPSC) and CRISPR-Cas9 gene-editing technologies have become powerful tools in disease modeling and treatment. By harnessing recent... (Review)
Review
Human induced pluripotent stem cell (iPSC) and CRISPR-Cas9 gene-editing technologies have become powerful tools in disease modeling and treatment. By harnessing recent biotechnological advancements, this review aims to equip researchers and clinicians with a comprehensive and updated understanding of the evolving treatment landscape for metabolic and genetic disorders, highlighting how iPSCs provide a unique platform for detailed pathological modeling and pharmacological testing, driving forward precision medicine and drug discovery. Concurrently, CRISPR-Cas9 offers unprecedented precision in gene correction, presenting potential curative therapies that move beyond symptomatic treatment. Therefore, this review examines the transformative role of iPSC technology and CRISPR-Cas9 gene editing in addressing metabolic and genetic disorders such as alpha-1 antitrypsin deficiency (A1AD) and glycogen storage disease (GSD), which significantly impact liver and pulmonary health and pose substantial challenges in clinical management. In addition, this review discusses significant achievements alongside persistent challenges such as technical limitations, ethical concerns, and regulatory hurdles. Future directions, including innovations in gene-editing accuracy and therapeutic delivery systems, are emphasized for next-generation therapies that leverage the full potential of iPSC and CRISPR-Cas9 technologies.
Topics: Humans; alpha 1-Antitrypsin Deficiency; Induced Pluripotent Stem Cells; CRISPR-Cas Systems; Glycogen Storage Disease; Gene Editing; Genetic Therapy; Animals
PubMed: 38920680
DOI: 10.3390/cells13121052 -
Cells Jun 2024Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac...
Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac differentiation using human pluripotent stem cells (hPSCs) has been used to uncover the intricate gene-network regulation involved in the proper formation and function of the human heart. Here, we searched for uncharacterized cardiac-development genes by combining a temporal evaluation of human cardiac specification in vitro with an analysis of gene expression in fetal and adult heart tissue. We discovered that (CARdiac DEvelopment Long non-coding RNA; LINC00890; SERTM2) expression coincides with the commitment to the cardiac lineage. knockout hPSCs differentiated poorly into cardiac cells, and hPSC-derived cardiomyocytes showed faster beating rates after controlled overexpression of during differentiation. Altogether, we provide physiological and molecular evidence that expression contributes to sculpting the cardiac program during cell-fate commitment.
Topics: Humans; RNA, Long Noncoding; Cell Differentiation; Heart; Homeostasis; Myocytes, Cardiac; Gene Expression Regulation, Developmental; Pluripotent Stem Cells; Cell Lineage; Organogenesis
PubMed: 38920678
DOI: 10.3390/cells13121050