Somatic Reprogramming 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.

Abstract Mo072: Sustained Biological Pacemaker Activity Induced by AAV9-hTBX18

Background: Sinoatrial node (SAN) dysfunction is a clinical disorder characterized by the inability of the SAN to maintain an appropriate heart rate (HR). Patients with this condition often need an electronic pacemaker. Although it is the current standard of care, devices can have complications. A biological pacemaker has been created by adenovirus expressing TBX18 as an alternative. Hypothesis: However, adenoviruses are immunogenic, limiting the duration of expression. We hypothesized that an AAV9 would promote prolonged TBX18 expression, induce somatic reprogramming in vivo, and sustained pacemaker activity. Methods: AAV9 containing GFP or TBX18 was directly injected into the myocardium of eight-week-old male Sprague Dawley rats (n=70) at low (1x10 9 GC), mid (1x10 10 GC) and high (1x10 11 GC) doses. Transient AV block was induced by tail vein injection of adenosine. A permanent complete AV block was induced by ablation of the his bundle. Hearts were harvested for optical mapping and mRNA analysis. Results: Six weeks after injection, all doses of AAV9-TBX18 had increased HR (low: 104±4, p=0.04; mid: 106±3.4, p=0.008; high: 103±4.1, p=0.04; vs GFP: 89±3 bpm) compared to AAV9-GFP when we induced AV block using adenosine. AAV9-GFP and -TBX18 mid dose injected rats underwent surgery to induce complete AV block. TBX18 group presented superior HR over the 24 hours monitored. 6-lead EKG analysis of the QRS axis in the frontal plane revealed that the ventricular ectopic beats from the AAV9-TBX18-injected group originated from the injection site. Activation maps further confirmed that the injection site originated those beats in TBX18-injected rats only. Successful reprogramming of adult ventricular myocytes into SAN-like cells was confirmed by transcriptional profiles with increased expression of TBX18 (2.2±0.5 FC, p<0.0001) and Hcn2 (25.2±1.1 FC, p=0.02), and reduced Gja1 (-10.8±0.7 FC, p=0.005), Scn5a (-1.1±0.8 FC, p=0.01) and Nkx2.5 (-2.0±0.6 FC, p=0.004) relative to GFP. Conclusion: Collectively, these data indicate that the AAV-based somatic reprogramming induced by TBX18 leads to sustained biological pacemaker activity in rats with AV block. Long-term studies are needed to characterize the long-term effects of sustained TBX18 expression.

Abstract Or118: Transcription Factor Gene Therapy for Treatment of Junctional Bradycardia in a Porcine Model of Complete Atrioventricular Block Following His Bundle Ablation

Background: Pacemaker implantation is standard treatment for patients with complete atrioventricular (AV) block. However, contraindications for device implantations create a need for alternative non-hardware cardiac pacing solutions. We developed a porcine model of complete heart block with junctional bradycardia to test a novel transcription factor-based gene therapy consisting of Tbx18, Shox2 and Tbx3 (TST). Somatic reprogramming of ventricular cardiomyocytes by TST induces pacemaker-like activity and provides biological pacing in vitro . Methods: Eight domestic swine (Yorkshire/Landrace) received catheter ablation of the AV node/His Bundle resulting in complete AV block, confirmed by dissociation of P and R waves on ECG. An implanted device pacemaker (VVI mode) maintained cardiac output during occurrences of junctional bradycardia below 50 bpm. Telemetric 3-lead ECG was recorded continuously. Subjects received TST gene therapy or control gene (GFP) via catheter-based intramuscular injection into the high right ventricular septum. Pacemaker usage and ECG were continuously monitored for 8 weeks. Results: Following AV node ablation, junctional escape rhythm decelerated into junctional bradycardia. After 3 weeks of AV block, 49.9 ± 13.1% of heartbeats were initiated by the device pacemaker. Upon reaching pacemaker dependence of 95% in an average of 27.9 ± 5.5 days, subjects received TST gene therapy or control injection. In subjects receiving TST therapy, daily pacemaker reliance decreased to 50% dependency in an average of 14.7 ± 2.3 days, while in GFP controls, daily pacemaker dependency at 50 bpm remained unchanged at >98% throughout follow-up. In TST-treated subjects, reductions in pacemaker dependence were associated with increases in ventricular rates exceeding 70 bpm. Efficacy of gene therapy waned after about one month; an improvement of >10% pacemaker dependency over two consecutive days was still observed on average 29.0 ± 4.1 days after TST injection. Conclusion: In a porcine model of complete AV block, ventricular junctional escape rhythm decelerated into bradycardia in less than one month. Subsequent gene therapy by TST injection into the high ventricular septum alleviated pacemaker dependency and enhanced heart rate for 29.0 ± 4.1 days. Somatic reprogramming of cardiomyocytes by TST gene delivery may provide a biological pacemaker alternative to hardware device implantation for therapeutic cardiac pacing.

Reprogramming fibroblast into human iBlastoids.

The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human 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.

Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

Generating Retinas through Guided Pluripotent Stem Cell Differentiation and Direct Somatic Cell Reprogramming.

Retinal degeneration diseases affect millions of people worldwide but are among the most difficult eye diseases to cure. Studying the mechanisms and developing new therapies for these blinding diseases requires researchers to have access to many retinal cells. In recent years there has been substantial advances in the field of biotechnology in generating retinal cells and even tissues in vitro, either through programmed sequential stem cell differentiation or direct somatic cell lineage reprogramming. The resemblance of these in vitro-generated retinal cells to native cells has been increasingly utilized by researchers. With the help of these in vitro retinal models, we now have a better understanding of human retinas and retinal diseases. Furthermore, these in vitro-generated retinal cells can be used as donor cells which solves a major hurdle in the development of cell replacement therapy for retinal degeneration diseases, while providing a promising option for patients suffering from these diseases. In this review, we summarize the development of pluripotent stem cell-to-retinal cell differentiation methods, the recent advances in generating retinal cells through direct somatic cell reprogramming, and the translational applications of retinal cells generated in vitro. Finally, we discuss the limitations of the current protocols and possible future directions for improvement.

Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies.

Somatic cell reprogramming allows the generation of human induced pluripotent stem cells (iPSCs) from patient's cells. The derived iPSCs provide an unlimited source of patient-specific cells that can be virtually differentiated in any cell of the human body. The generation of iPSCs has important implications for all human medicine fields, as they can be used for drug discovery, regenerative medicine, and developmental studies. Klinefelter Syndrome (KS) is the most common chromosome aneuploidy in males. KS is typically characterized by a 47,XXY karyotype, representing 80-90% of KS patients. In rare cases, high-grade sex chromosome aneuploidies (SCAs), 48,XXXY; 48,XXYY; 49,XXXXY, are also observed in males. Since the advent of the reprogramming technique, a few KS-iPSCs have been described. Here, we detail the methodology for generating primary fibroblasts from patients' skin biopsies and the subsequent derivation of iPSCs using an efficient integrative-free mRNA-based somatic reprogramming approach.

SIRT6 Knockdown in Buffalo Fetal Fibroblasts Exacerbates Premature Senescence Caused by DNA and Telomere Damage.

As a gene with antiaging functions, sirtuin6 (SIRT6) belonging to the sirtuin family plays a vital role in DNA repair, telomerase function, and cellular senescence, as well as maintains epigenomic stability and promotes longevity. However, its role in cell senescence in large animals, such as buffaloes, remains unknown. Fibroblasts are commonly used for somatic reprogramming, and their physiological characteristics affect the efficiency of this process. We aimed to elucidate the role of SIRT6 in cellular senescence and proliferation and analyze its effect on the biological function of buffalo fibroblasts to help improve the efficiency of buffalo somatic cell reprogramming. The expression of SIRT6 and related DNA damage was measured in buffalo fibroblasts obtained at different developmental stages (in the fetus and at 3 and 10 years of age), and the effect of SIRT6 knockdown on the senescence of buffalo fetal fibroblast was investigated. An inverse relationship was observed between SIRT6 expression and senescence in buffalo fibroblasts obtained from animals of various ages. This was accompanied by decreased cell growth, viability, and increased DNA damage. Short hairpin RNA-mediated SIRT6 knockdown accelerated the senescence of buffalo fetal fibroblasts. It blocked the cell cycle during in vitro cell culture, which further enhanced DNA damage, particularly with respect to the telomeres. Collectively, our findings suggest that SIRT6 expression was closely associated with buffalo senescence in fibroblasts. These findings serve as a foundation to better understand the cellular functions of SIRT6 and also aid in selecting donor cells for buffalo somatic cell reprogramming.

Profiling A-to-I RNA editing during mouse somatic reprogramming at the single-cell level

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells through a highly heterogeneous process regulated by numerous biological factors, including adenosine-to-inosine (A-to-I) RNA editing. In this study, we analyzed A-to-I RNA editing sites using a single-cell RNA sequencing (scRNA-seq) dataset with high-depth and full-length coverage. Our method revealed that A-to-I RNA editing frequency varied widely at the single-cell level and underwent dynamic changes. We also found that A-to-I RNA editing level was correlated with the expression of the RNA editing enzyme ADAR1. The analysis combined with gene ontology (GO) enrichment revealed that ADAR1-dependent A-to-I editing may downregulate the expression levels of Igtp, Irgm2, Mndal, Ifi202b, and Tapbp in the early stage, to inhibit the pathways of cellular response to interferon-beta and regulation of protein complex stability to promote mesenchymal-epithelial transition (MET). Notably, we identified a negative correlation between A-to-I RNA editing frequency and the expression of certain genes, such as Nras, Ube2l6, Zfp987, and Adsl.

TGFβ inhibition and mesenchymal to epithelial transition initiation by Xenopus egg extract: first steps towards early reprogramming in fish somatic cell

Xenopus egg extract is a powerful material to modify cultured cells fate and to induce cellular reprogramming in mammals. In this study, the response of goldfish fin cells to in vitro exposure to Xenopus egg extract, and subsequent culture, was studied using a cDNA microarray approach, gene ontology and KEGG pathways analyses, and qPCR validation. We observed that several actors of the TGFβ and Wnt/β-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove cultured fin cells towards a mesenchymal-epithelial transition. This indicates that Xenopus egg extract treatment relieved some barriers of somatic reprogramming in fish cells. However, the lack of re-expression of pou2 and nanog pluripotency markers, the absence of DNA methylation remodeling of their promoter region, and the strong decrease in de novo lipid biosynthesis metabolism, indicate that reprogramming was only partial. The observed changes may render these treated cells more suitable for studies on in vivo reprogramming after somatic cell nuclear transfer.

Mitochondrial dynamics and cristae shape changes during metabolic reprogramming.

The architecture of the mitochondrial network and cristae critically impact cell differentiation and identity. Cells undergoing metabolic reprogramming to aerobic glycolysis (Warburg effect), such as immune cells, stem cells, and cancer cells, go through controlled modifications in mitochondrial architecture, which is critical for achieving the resulting cellular phenotype. Recent studies in immunometabolism have shown that the manipulation of mitochondrial network dynamics and cristae shape directly affects T cell phenotype and macrophage polarization through altering energy metabolism. Similar manipulations also alter the specific metabolic phenotypes that accompany somatic reprogramming, stem cell differentiation, and cancer cells. The modulation of OXPHOS activity, accompanied by changes in metabolite signaling, ROS generation, and ATP levels is the shared underlying mechanism. The plasticity of mitochondrial architecture is particularly vital for metabolic reprogramming. Consequently, failure to adapt the appropriate mitochondrial morphology often compromises the differentiation and identity of the cell. Immune, stem, and tumor cells exhibit striking similarities in their coordination of mitochondrial morphology with metabolic pathways. However, although many general unifying principles can be observed, their validity is not absolute, and the mechanistic links thus need to be further explored. Better knowledge of the molecular mechanisms involved and their relationships to both mitochondrial network and cristae morphology will not only further deepen our understanding of energy metabolism but may also contribute to improved therapeutic manipulation of cell viability, differentiation, proliferation, and identity in many different cell types.

The NuRD complex cooperates with SALL4 to orchestrate reprogramming

Cell fate decision involves rewiring of the genome, but remains poorly understood at the chromatin level. Here, we report that chromatin remodeling complex NuRD participates in closing open chromatin in the early phase of somatic reprogramming. Sall4, Jdp2, Glis1 and Esrrb can reprogram MEFs to iPSCs efficiently, but only Sall4 is indispensable capable of recruiting endogenous components of NuRD. Yet knocking down NuRD components only reduces reprogramming modestly, in contrast to disrupting the known Sall4-NuRD interaction by mutating or deleting the NuRD interacting motif at its N-terminus that renders Sall4 inept to reprogram. Remarkably, these defects can be partially rescured by grafting NuRD interacting motif onto Jdp2. Further analysis of chromatin accessibility dynamics demonstrates that the Sall4-NuRD axis plays a critical role in closing the open chromatin in the early phase of reprogramming. Among the chromatin loci closed by Sall4-NuRD encode genes resistant to reprogramming. These results identify a previously unrecognized role of NuRD in reprogramming, and may further illuminate chromatin closing as a critical step in cell fate control.

Cloning by SCNT: Integrating Technical and Biology-Driven Advances.

Somatic cell nuclear transfer (SCNT) into enucleated oocytes initiates nuclear reprogramming of lineage-committed cells to totipotency. Pioneer SCNT work culminated with cloned amphibians from tadpoles, while technical and biology-driven advances led to cloned mammals from adult animals. Cloning technology has been addressing fundamental questions in biology, propagating desired genomes, and contributing to the generation of transgenic animals or patient-specific stem cells. Nonetheless, SCNT remains technically complex and cloning efficiency relatively low. Genome-wide technologies revealed barriers to nuclear reprogramming, such as persistent epigenetic marks of somatic origin and reprogramming resistant regions of the genome. To decipher the rare reprogramming events that are compatible with full-term cloned development, it will likely require technical advances for large-scale production of SCNT embryos alongside extensive profiling by single-cell multi-omics. Altogether, cloning by SCNT remains a versatile technology, while further advances should continuously refresh the excitement of its applications.

Abstract 14996: Single Cell Rnaseq Analysis Reveals a Two-Step Mechanism for the Reprogramming of Ventricular Myocytes to Pacemaker Cells by Tbx18

Background: We have demonstrated that an embryonic transcription factor, TBX18, suffices to reprogram ventricular myocytes (VMs) to induced pacemaker cells (iPMs). Here, we sought to gain a mechanistic understanding of the reprogramming process using single cell (sc) RNAseq analysis. Methods: Cells from the ventricles of the neonatal rat heart were plated as monolayers, which consisted of both cardiomyocytes (CMs) and nonmyocytes, and were transduced with Adeno-GFP and Adeno-TBX18 in vitro. scRNAseq was performed at days (d) 3, 6, and 14. Result: TBX18- CMs exhibited repression of chamber CM genes at d3, including those involved in cardiac muscle contraction and action potential. Markers of CM dedifferentiation ( Acta2 and Dab2 ) were upregulated, while markers of chamber CM differentiation ( Mef2a and Rbm20 ) were downregulated. Nodal pacemaker cell gene expression followed the CM dedifferentiation in TBX18-CMs, with the proportion of Hcn4 + CMs increasing gradually from 20.9% (d3) to 34.0% (d14), accompanied by increased expression of Tbx3 and Tbx18 . The proportion of iPMs ( Hcn4 + , Gja1 low , Nkx2-5 low , Tnni3 high , and Actn2 high ) in TBX18-CMs also increased from 3.6% at d3 to 6.4% at d14. Both TBX18-CMs and GFP-CMs exhibited a subpopulation of Acta2 high dedifferentiated CMs (deCMs), which were represented higher in TBX18-CMs compared to control. Among TBX18-CM subpopulations, the proportion of iPMs was the highest in deCMs (6.4% at d3 and 7.3% at d14). TBX18-deCMs were distinct from other TBX18-CM subpopulations in that extracellular matrix organization and Tgfβ signaling were enriched as well as higher expression of SP1 transcription factor which directly activates Hcn4 channel expression. Conclusion: Dedifferentiation and loss of CM function precedes nodal pacemaker cell gain of function during TBX18-induced somatic reprogramming of VMs to iPMs. Tgfβ signaling appears to figure prominently during this process.

Glis1 and oxaloacetate in nucleus pulposus stromal cell somatic reprogramming and survival.

Regenerative medicine aims to repair degenerate tissue through cell refurbishment with minimally invasive procedures. Adipose tissue (FAT)-derived stem or stromal cells are a convenient autologous choice for many regenerative cell therapy approaches. The intervertebral disc (IVD) is a suitable target. Comprised of an inner nucleus pulposus (NP) and an outer annulus fibrosus (AF), the degeneration of the IVD through trauma or aging presents a substantial socio-economic burden worldwide. The avascular nature of the mature NP forces cells to reside in a unique environment with increased lactate levels, conditions that pose a challenge to cell-based therapies. We assessed adipose and IVD tissue-derived stromal cells through in vitro transcriptome analysis in 2D and 3D culture and suggested that the transcription factor Glis1 and metabolite oxaloacetic acid (OAA) could provide NP cells with survival tools for the harsh niche conditions in the IVD.

GLH-1/Vasa represses neuropeptide expression and drives spermiogenesis in the C. elegans germline

Germ granules harbor processes that maintain germline integrity and germline stem cell capacity. Depleting core germ granule components in C. elegans leads to the reprogramming of germ cells, causing them to express markers of somatic differentiation in day-two adults. Somatic reprogramming is associated with complete sterility at this stage. The resulting germ cell atrophy and other pleiotropic defects complicate our understanding of the initiation of reprogramming and how processes within germ granules safeguard the totipotency and immortal potential of germline stem cells. To better understand the initial events of somatic reprogramming, we examined total mRNA (transcriptome) and polysome-associated mRNA (translatome) changes in a precision full-length deletion of glh-1, which encodes a homolog of the germline-specific Vasa/DDX4 DEAD-box RNA helicase. Fertile animals at a permissive temperature were analyzed as young adults, a stage that precedes by 24 ​h the previously determined onset of somatic reporter-gene expression in the germline. Two significant changes are observed at this early stage. First, the majority of neuropeptide-encoding transcripts increase in both the total and polysomal mRNA fractions, suggesting that GLH-1 or its effectors suppress this expression. Second, there is a significant decrease in Major Sperm Protein (MSP)-domain mRNAs when glh-1 is deleted. We find that the presence of GLH-1 helps repress spermatogenic expression during oogenesis, but boosts MSP expression to drive spermiogenesis and sperm motility. These insights define an early role for GLH-1 in repressing somatic reprogramming to maintain germline integrity.

Modeling human pregastrulation development by 3D culture of blastoids generated from primed-to-naïve transitioning intermediates.

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.

Lysine demethylase 1B (Kdm1b) enhances somatic reprogramming through inducing pluripotent gene expression and promoting cell proliferation

Lysine demethylase 1B (Kdm1b) is known as an epigenetic modifier with demethylase activity against H3K4 and H3K9 histones and plays an important role in tumor progression and tumor stem cell enrichment. In this study, we attempted to elucidate the role of Kdm1b in somatic cell reprogramming. We found that exogenous expression of Kdm1b in human dermal fibroblasts (HDFs) can influence the epigenetic modifications of histones. Subsequent analysis further suggests that the overexpression of Kdm1b can promote cell proliferation, reprogram metabolism and inhibit cell apoptosis. In addition, a series of multipotent factors including Sox2 and Nanog, and several epigenetic factors that may reduce epigenetic barriers were upregulated to varying degrees. More importantly, HDFs transfected with the combination of Oct4 (POU5F1), Sox2, Klf4 and c-Myc and Kdm1b (OSKMK) achieved higher reprogramming efficiency. Therefore, we suggest that Kdm1b is an important epigenetic factor associated with pluripotency.

The Methylation Game: Epigenetic and Epitranscriptomic Dynamics of 5-Methylcytosine.

DNA and RNA methylation dynamics have been linked to a variety of cellular processes such as development, differentiation, and the maintenance of genome integrity. The correct deposition and removal of methylated cytosine and its oxidized analogues is pivotal for cellular homeostasis, rapid responses to exogenous stimuli, and regulated gene expression. Uncoordinated expression of DNA/RNA methyltransferases and demethylase enzymes has been linked to genome instability and consequently to cancer progression. Furthermore, accumulating evidence indicates that post-transcriptional DNA/RNA modifications are important features in DNA/RNA function, regulating the timely recruitment of modification-specific reader proteins. Understanding the biological processes that lead to tumorigenesis or somatic reprogramming has attracted a lot of attention from the scientific community. This work has revealed extensive crosstalk between epigenetic and epitranscriptomic pathways, adding a new layer of complexity to our understanding of cellular programming and responses to environmental cues. One of the key modifications, m5C, has been identified as a contributor to regulation of the DNA damage response (DDR). However, the various mechanisms of dynamic m5C deposition and removal, and the role m5C plays within the cell, remains to be fully understood.