Miniaturized droplet microarray platform enables maintenance of human induced pluripotent stem cell pluripotency
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8569722 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundEpigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.MethodologyHere, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illumina's Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.ConclusionsThis study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.
| S-EPMC4177110 | biostudies-literature
Project description:BackgroundHuman pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs.MethodsbFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation.ResultsOur results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs.ConclusionCollectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.
| S-EPMC6282345 | biostudies-literature
Project description:NF-?B signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-?B have been reported in mouse and human ES cells, and the role of NF-?B in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-?B signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-?B activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-?B activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-?B signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-?B signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.
| S-EPMC3577895 | biostudies-literature
Project description:Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.
| S-EPMC8329718 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation.
| S-EPMC4939377 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) are considered patient-specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c-Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical-grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non-integrating viral and non-viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.
| S-EPMC6496827 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome.
| S-EPMC3121447 | biostudies-other
Project description:Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.
| S-EPMC3683164 | biostudies-literature
Project description:ObjectivesRecently, pluripotency of induced pluripotent stem (iPS) cells has been displayed after producing adult mice, in tetraploid complementation assays. These studies lead us to the last piece of the puzzle for reprogramming somatic cells into fully pluripotent cells which function as embryonic stem cells in most applications. However, in all of previous studies, skin fibroblasts were used as the starting population for reprogramming, raising questions as to whether the pluripotency of the iPS cells was dependent on the particular starting cell type.Materials and methodsOur iPS cell lines were prepared from murine adipose stem cells (ASCs). Their multi-potency was first tested by teratoma formation in nude mice. Then, tetraploid complementation was performed to generate progeny from them.ResultsWe succeeded to the birth of viable and fertile adult mice derived entirely from reprogrammed ASC, indicating cell types other than fibroblasts can also be restored to the embryonic level of pluripotency.ConclusionsWe also directed differentiation of iPS cells into chondrocytes, thus adipose-derived iPS cells can be used as models to study chondrogenic differentiation and cartilage regeneration.
| S-EPMC6496444 | biostudies-literature
Project description:Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are characterised by an unusual and tightly regulated cell cycle that has been shown to be important for the maintenance of a pluripotent phenotype. Cyclin-dependant kinase 1 (CDK1) is a key player in cell cycle regulation and particularly mitosis; however, its role has not been studied previously in hESC and hiPSC. To investigate the impacts of CDK1 downregulation, we performed RNA interference studies which in addition to expected mitotic deficiencies revealed a large range of additional phenotypes related to maintenance of pluripotency, ability to repair double strand breaks (DSBs) and commitment to apoptosis. Downregulation of CDK1 led to the loss of typical pluripotent stem cell morphology, downregulation of pluripotency markers and upregulation of a large number of differentiation markers. In addition, human pluripotent stem cells with reduced CDK1 expression accumulated a higher number of DSBs were unable to activate CHK2 expression and could not maintain G2/M arrest upon exposure to ionising radiation. CDK1 downregulation led to the accumulation of cells with abnormal numbers of mitotic organelles, multiple chromosomal abnormalities and polyploidy. Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process. On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation. Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.
| S-EPMC4260724 | biostudies-literature