Pluripotent Stem Cell State Research Articles

Induction of Pluripotent Stem Cells from Muscle Cells of Large Yellow Croaker (Larimichthys Crocea) Via Electrotransfection.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

Crosstalk between Signaling Pathways and Energy Metabolism in Pluripotency.

The sequential change from totipotency to multipotency occurs during early mammalian embryo development. However, due to the lack of cellular models to recapitulate the distinct potency of stem cells at each stage, their molecular and cellular characteristics remain ambiguous. The establishment of isogenic naïve and primed pluripotent stem cells to represent the pluripotency in the inner cell mass of the pre-implantation blastocyst and in the epiblast from the post-implantation embryo allows the understanding of the distinctive characteristics of two different states of pluripotent stem cells. This review discusses the prominent disparities between naïve and primed pluripotency, including signaling pathways, metabolism, and epigenetic status, ultimately facilitating a comprehensive understanding of their significance during early mammalian embryonic development.

Dynamic nucleolar phase separation influenced by non-canonical function of LIN28A instructs pluripotent stem cell fate decisions

LIN28A is important in somatic reprogramming and pluripotency regulation. Although previous studies addressed that LIN28A can repress let-7 microRNA maturation in the cytoplasm, few focused on its role within the nucleus. Here, we show that the nucleolus-localized LIN28A protein undergoes liquid-liquid phase separation (LLPS) in mouse embryonic stem cells (mESCs) and in vitro. The RNA binding domains (RBD) and intrinsically disordered regions (IDR) of LIN28A contribute to LIN28A and the other nucleolar proteins’ phase-separated condensate establishment. S120A, S200A and R192G mutations in the IDR result in subcellular mislocalization of LIN28A and abnormal nucleolar phase separation. Moreover, we find that the naive-to-primed pluripotency state conversion and the reprogramming are associated with dynamic nucleolar remodeling, which depends on LIN28A’s phase separation capacity, because the LIN28A IDR point mutations abolish its role in regulating nucleolus and in these cell fate decision processes, and an exogenous IDR rescues it. These findings shed light on the nucleolar function in pluripotent stem cell states and on a non-canonical RNA-independent role of LIN28A in phase separation and cell fate decisions.

A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

Long-term in vivo chimeric cells tracking in non-human primate.

Non-human primates (NHPs) are increasingly used in preclinical trials to test the safety and efficacy of biotechnology therapies. Nonetheless, given the ethical issues and costs associated with this model, it would be highly advantageous to use NHP cellular models in clinical studies. However, developing and maintaining the naïve state of primate pluripotent stem cells (PSCs) remains difficult as does in vivo detection of PSCs, thus limiting biotechnology application in the cynomolgus monkey. Here, we report a chemically defined, xeno-free culture system for culturing and deriving monkey PSCs in vitro. The cells display global gene expression and genome-wide hypomethylation patterns distinct from monkey-primed cells. We also found expression of signaling pathways components that may increase the potential for chimera formation. Crucially for biomedical applications, we were also able to integrate bioluminescent reporter genes into monkey PSCs and track them in chimeric embryos in vivo and in vitro. The engineered cells retained embryonic and extra-embryonic developmental potential. Meanwhile, we generated a chimeric monkey carrying bioluminescent cells, which were able to track chimeric cells for more than 2 years in living animals. Our study could have broad utility in primate stem cell engineering and in utilizing chimeric monkey models for clinical studies.

Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures.

Introduction: With the increase in aging populations around the world, the development of in vitro human cell models to study neurodegenerative disease is crucial. A major limitation in using induced pluripotent stem cell (hiPSC) technology to model diseases of aging is that reprogramming fibroblasts to a pluripotent stem cell state erases age-associated features. The resulting cells show behaviors of an embryonic stage exhibiting longer telomeres, reduced oxidative stress, and mitochondrial rejuvenation, as well as epigenetic modifications, loss of abnormal nuclear morphologies, and age-associated features. Methods: We have developed a protocol utilizing stable, non-immunogenic chemically modified mRNA (cmRNA) to convert adult human dermal fibroblasts (HDFs) to human induced dorsal forebrain precursor (hiDFP) cells, which can subsequently be differentiated into cortical neurons. Analyzing an array of aging biomarkers, we demonstrate for the first time the effect of direct-to-hiDFP reprogramming on cellular age. Results: We confirm direct-to-hiDFP reprogramming does not affect telomere length or the expression of key aging markers. However, while direct-to-hiDFP reprogramming does not affect senescence-associated β-galactosidase activity, it enhances the level of mitochondrial reactive oxygen species and the amount of DNA methylation compared to HDFs. Interestingly, following neuronal differentiation of hiDFPs we observed an increase in cell soma size as well as neurite number, length, and branching with increasing donor age suggesting that neuronal morphology is altered with age. Discussion: We propose direct-to-hiDFP reprogramming provides a strategy for modeling age-associated neurodegenerative diseases allowing the persistence of age-associated signatures not seen in hiPSC-derived cultures, thereby facilitating our understanding of neurodegenerative disease and identification of therapeutic targets.

Binary Colloidal Crystals Promote Cardiac Differentiation of Human Pluripotent Stem Cells via Nuclear Accumulation of SETDB1

Biophysical cues can facilitate the cardiac differentiation of human pluripotent stem cells (hPSCs), yet the mechanism is far from established. One of the binary colloidal crystals, composed of 5 μm Si and 400 nm poly(methyl methacrylate) particles named 5PM, has been applied as a substrate for hPSCs cultivation and cardiac differentiation. In this study, cell nucleus, cytoskeleton, and epigenetic states of human induced pluripotent stem cells on the 5PM were analyzed using atomic force microscopy, molecular biology assays, and the assay for transposase-accessible chromatin sequencing (ATAC-seq). Cells were more spherical with stiffer cell nuclei on the 5PM compared to the flat control. ATAC-seq revealed that chromatin accessibility decreased on the 5PM, caused by the increased entry of histone lysine methyltransferase SETDB1 into the cell nuclei and the amplified level of histone H3K9me3 modification. Reducing cytoskeleton tension using a ROCK inhibitor attenuated the nuclear accumulation of SETDB1 on the 5PM, indicating that the effect is cytoskeleton-dependent. In addition, the knockdown of SETDB1 reversed the promotive effects of the 5PM on cardiac differentiation, demonstrating that biophysical cue-induced cytoskeletal tension, cell nucleus deformation, and then SETDB1 accumulation are critical outside-in signal transformations in cardiac differentiation. Human embryonic stem cells showed similar results, indicating that the biophysical impact of the 5PM surfaces on cardiac differentiation could be universal. These findings contribute to our understanding of material-assistant hPSC differentiation, which benefits materiobiology and stem cell bioengineering.

PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

Derivation and Primordial Germ Cell Induction of Intermediate Pluripotent Stem Cells.

Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of the pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. By modulating the FGF, TGF-β, and WNT pathways, we have derived intermediate PSCs (FTW-PSCs) that are permissive for direct primordial germ cell-like cell (PGC-LC) induction in vitro. Here, we describe the method for derivation and maintenance of mouse and human FTW-PSCs, as well as PGC-LC induction from FTW-PSCs.

Podocalyxin-Like Protein 1 Regulates Pluripotency through the Cholesterol Biosynthesis Pathway.

Deciphering signaling mechanisms critical for the extended pluripotent stem cell (EPSC) state and primed pluripotency is necessary for understanding embryonic development. Here, a membrane protein, podocalyxin-like protein 1 (PODXL) as being essential for extended and primed pluripotency, is identified. Alteration of PODXL expression levels affects self-renewal, protein expression of c-MYC and telomerase, and induced pluripotent stem cell (iPSC) and EPSC colony formation. PODXL is the first membrane protein reported to regulate de novo cholesterol biosynthesis, and human pluripotent stem cells (hPSCs) are more sensitive to cholesterol depletion than fibroblasts. The addition of exogenous cholesterol fully restores PODXL knockdown-mediated loss of pluripotency. PODXL affects lipid raft dynamics via the regulation of cholesterol. PODXL recruits the RAC1/CDC42/actin network to regulate SREBP1 and SREBP2 maturation and lipid raft dynamics. Single-cell RNA sequencing reveals PODXL overexpression enhanced chimerism between human cells in mouse host embryos (hEPSCs 57%). Interestingly, in the human-mouse chimeras, laminin and collagen signaling-related pathways are dominant in PODXL overexpressing cells. It is concluded that cholesterol regulation via PODXL signaling is critical for ESC/EPSC.

Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells.

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.

Dynamic cytosolic foci of DPPA4 in human pluripotent stem cells

DPPA4 is essential for the pluripotent stem cell state, yet its function is poorly understood. DPPA4 is localized in the nucleus, where it is associated with active chromatin. We now report that it is also present in the cytosol, where it appears as diffused clouds, distinct foci and sometimes as spaghetti-like structures. This cytosolic localization is dynamic and DPPA4 shuttles between the cytosol and the nucleus. Its presence is almost abolished from the nucleus upon differentiation. Co-immunoprecipitation studies highlighted novel protein interactors, many of which are also found in the cytosol and are implicated in mRNA processing and RNA and protein transport between the cytosol and the nucleus. Finally, the depletion of DPPA4 resulted in cytosolic accumulation of vesicles. The cytosolic presence of DPPA4 highlights unexplored research directions that could significantly advance the understanding of DPPA4 in pluripotent stem cells and in cancer.

Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection

Stem cells intrinsically express a subset of genes which are normally associated with interferon stimulation and the innate immune response. However, the expression of these interferon‐stimulated genes (ISG) in stem cells is independent from external stimuli such as viral infection. Here, we show that the interferon regulatory factor 1, Irf1, is directly controlled by the murine formative pluripotency gene regulatory network and transiently upregulated during the transition from naive to formative pluripotency. IRF1 binds to regulatory regions of a conserved set of ISGs and is required for their faithful expression upon exit from naive pluripotency. We show that in the absence of IRF1, cells exiting the naive pluripotent stem cell state are more susceptible to viral infection. Irf1 therefore acts as a link between the formative pluripotency network, regulation of innate immunity genes, and defense against viral infections during formative pluripotency.

Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp.

In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of a man’s life that travels through time reversing the aging process; as the tale progresses, the frail old man becomes a vigorous, vivacious young man, then man becomes boy and boy becomes baby. The reality of cellular time travel, however, is far more wondrous: we now have the ability to both reverse and then forward time on mature cells. Four proteins were found to rewind the molecular clock of adult cells back to their embryonic, “blank canvas” pluripotent stem cell state, allowing these pluripotent stem cells to then be differentiated to fast forward their molecular clocks to the desired adult specialist cell types. These four proteins – the “Yamanaka factors” – form critical elements of this cellular time travel, which deservedly won Shinya Yamanaka the Nobel Prize for his lab’s work discovering them. Human induced pluripotent stem cells (hiPSCs) hold much promise in our understanding of physiology and medicine. They encapsulate the signaling pathways of the desired cell types, such as cardiomyocytes or neurons, and thus act as model cells for defining the critical ion channel activity in healthy and disease states. Since hiPSCs can be derived from any patient, highly specific, personalized (or stratified) physiology, and/or pathophysiology can be defined, leading to exciting developments in personalized medicines and interventions. As such, hiPSC married with high throughput automated patch clamp (APC) ion channel recording platforms provide a foundation for significant physiological, medical and drug discovery advances. This review aims to summarize the current state of affairs of hiPSC and APC: the background and recent advances made; and the pros, cons and challenges of these technologies. Whilst the authors have yet to finalize a fully functional time traveling machine, they will endeavor to provide plausible future projections on where hiPSC and APC are likely to carry us. One future projection the authors are confident in making is the increasing necessity and adoption of these technologies in the discovery of the next blockbuster, this time a life-enhancing ion channel drug, not a fantastical movie.

Role of heat shock protein 60 in primed and naïve states of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) exist in at least two distinct states in mammals: naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. To identify and characterize the surface molecules that are necessary for the maintenance of naïve hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve state of hPSCs. Flow cytometry showed that N1-A4, one of the MAbs, bound to naïve hPSCs but not to primed hPSCs. Cell surface biotinylation and immunoprecipitation analysis identified that N1-A4 recognized heat shock protein 60 (HSP60) expressed on the surface of naïve hPSCs. Quantitative polymerase chain reaction (qPCR) analysis showed that HSP60 expression was rapidly downregulated during the embryoid body (EB) differentiation of primed hPSCs. HSP60 knockdown led to a decrease in the expression of pluripotency genes in primed hPSCs. HSP60 depletion also led to a decrease in the expression of pluripotency genes and representative naïve-state-specific genes in naïve hPSCs. Taken together, the results suggest that HSP60 is downregulated during differentiation of hPSCs and is required for the maintenance of pluripotency genes in both primed and naïve hPSCs, suggesting that HSP60 is a regulator of hPSC pluripotency and differentiation.

Modulation of PKM1/2 Levels by Steric Blocking Morpholinos Alters the Metabolic and Pluripotent State of Murine Pluripotent Stem Cells.

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state.

Reprogramming Stars #7: Dynamic Pluripotent Stem Cell States and Their Applications-An Interview with Dr. Jun Wu.

Reprogramming Stars #7: Dynamic Pluripotent Stem Cell States and Their Applications-An Interview with Dr. Jun Wu.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2.

The pluripotent state of embryonic stem cells (ESCs) is regulated by a sophisticated network of transcription factors. High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs (hESCs). However, the functional role of KLF17 in naive state is not clear. Here, by employing various gain and loss-of-function approaches, we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in hESCs. Mechanistically, we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17. Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency. Furthermore, we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species. Finally, we show that Klf17 may be essential for early embryo development in mouse. These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.

Functional redundancy among Polycomb complexes in maintaining the pluripotent state of embryonic stem cells.

SummaryPolycomb group proteins assemble into multi-protein complexes, known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), that guide cell fate decisions during embryonic development. PRC1 forms an array of biochemically distinct canonical PRC1 (cPRC1) or non-canonical PRC1 (ncPRC1) complexes characterized by the mutually exclusive presence of PCGF (PCGF1-PCGF6) paralog subunit; however, whether each one of these subcomplexes fulfills a distinct role remains largely controversial. Here, by performing a CRISPR-based loss-of-function screen in embryonic stem cells (ESCs), we uncovered a previously unappreciated functional redundancy among PRC1 subcomplexes. Disruption of ncPRC1, but not cPRC1, displayed severe defects in ESC pluripotency. Remarkably, coablation of non-canonical and canonical PRC1 in ESCs resulted in exacerbation of the phenotype observed in the non-canonical PRC1-null ESCs, highlighting the importance of functional redundancy among PRC1 subcomplexes. Together, our studies demonstrate that PRC1 subcomplexes act redundantly to silence lineage-specific genes and ensure robust maintenance of ESC identity.