Reprogramming Factors Research Articles

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

TBX20 Improves Contractility and Mitochondrial Function During Direct Human Cardiac Reprogramming.

Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied. To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes. We identified TBX20 (T-box transcription factor 20) as the top cardiac gene that is unable to be activated by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133). TBX20 is required for normal heart development and cardiac function in adult cardiomyocytes, yet its role in cardiac reprogramming remains undefined. We show that the addition of TBX20 to the MGT133 cocktail (MGT+TBX20) promotes cardiac reprogramming and activates genes associated with cardiac contractility, maturation, and ventricular heart. Human induced cardiomyocytes produced with MGT+TBX20 demonstrated more frequent beating, calcium oscillation, and higher energy metabolism as evidenced by increased mitochondria numbers and mitochondrial respiration. Mechanistically, comprehensive transcriptomic, chromatin occupancy, and epigenomic studies revealed that TBX20 colocalizes with MGT reprogramming factors at cardiac gene enhancers associated with heart contraction, promotes chromatin binding and co-occupancy of MGT factors at these loci, and synergizes with MGT for more robust activation of target gene transcription. TBX20 consolidates MGT cardiac reprogramming factors to activate cardiac enhancers to promote cardiac cell fate conversion. Human induced cardiomyocytes generated with TBX20 showed enhanced cardiac function in contractility and mitochondrial respiration.

Generation of integration-free induced pluripotent stem cell line (KSCBi017-A) from peripheral blood mononuclear cells of a healthy male individual

A human induced pluripotent stem cell (hiPSC) line, KSCBi017-A, was generated from a 50-year-old male individual using non-integrating episomal vectors expressing reprogramming factors. The generated hiPSCs were integration-free, expressed pluripotency markers, exhibited the potential for differentiation into three germ layers in vivo, and maintained the normal karyotype. This cell line can be used as a control for a disease model and is available from Korea National Stem Cell Bank.

Establishment of fishing cat cell biobanking for sustainable conservation

The fishing cat (Prionailurus viverrinus) is a vulnerable wild felid that is currently under threat from habitat destruction and other human activities. The zoo provides insurance to ensure the survival of the fishing cat population. Creating a biobank of fishing cats is a critical component of recent zoo strategies for securely stocking cell samples for long-term survival. Here, our goal was to compare cell biobanking techniques (tissue collection, primary culture, and reprogramming) and tissue sources (ear skin, abdominal skin, testis) from captive (n = 6)/natural (n = 6) vs. living (n = 8)/postmortem (n = 4) fishing cats. First, we show that dermal fibroblasts from the medial border of the helix of the ear pinna and abdominal tissues of living fishing cats can be obtained, whereas postmortem animals provided far fewer fibroblasts from the ears than from the testes. Furthermore, we can extract putative adult spermatogonial stem cells from the postmortem fishing cat's testes. The main barrier to expanding adult fibroblasts was early senescence, which can be overcome by overexpressing reprogramming factors through felid-specific transfection programs, though we demonstrated that reaching iPSC state from adult fibroblasts of fishing cats was ineffective with current virus-free mammal-based induction approaches. Taken together, the success of isolating and expanding primary cells is dependent on a number of factors, including tissue sources, tissue handling, and nature of limited replicative lifespan of the adult fibroblasts. This study provides recommendations for tissue collection and culture procedures for zoological research to facilitate the preservation of cells from both postmortem and living felids.

Expression level of the reprogramming factor NeuroD1 is critical for neuronal conversion efficiency from different cell types

Several transcription factors, including NeuroD1, have been shown to act as neuronal reprogramming factors (RFs) that induce neuronal conversion from somatic cells. However, it remains unexplored whether expression levels of RFs in the original cells affect reprogramming efficiency. Here, we show that the neuronal reprogramming efficiency from two distinct glial cell types, microglia and astrocytes, is substantially dependent on the expression level of NeuroD1: low expression failed to induce neuronal reprogramming, whereas elevated NeuroD1 expression dramatically improved reprogramming efficiency in both cell types. Moreover, even under conditions where NeuroD1 expression was too low to induce effective conversion by itself, combined expression of three RFs (Ascl1, Brn2, and NeuroD1) facilitated the breaking down of cellular barriers, inducing neuronal reprogramming. Thus, our results suggest that a sufficiently high expression level of RFs, or alternatively their combinatorial expression, is the key to achieving efficient neuronal reprogramming from different cells.

Induced pluripotent stem cells of endangered avian species

The number of endangered avian-related species increase in Japan recently. The application of new technologies, such as induced pluripotent stem cells (iPSCs), may contribute to the recovery of the decreasing numbers of endangered animals and conservation of genetic resources. We established novel iPSCs from three endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl) with seven reprogramming factors (M3O, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2). The iPSCs are pluripotency markers and express pluripotency-related genes and differentiated into three germ layers in vivo and in vitro. These three endangered avian iPSCs displayed different cellular characteristics even though the same reprogramming factors use. Japanese ptarmigan-derived iPSCs have different biological characteristics from those observed in other avian-derived iPSCs. Japanese ptarmigan iPSCs contributed to chimeras part in chicken embryos. To the best of our knowledge, our findings provide the first evidence of the potential value of iPSCs as a resource for endangered avian species conservation.

Generation of induced pluripotent stem cell (iPSC) from NY-ESO-I-specific cytotoxic T cells isolated from the melanoma patient with minor HLAs: The practical pilot study for the adoptive immunotherapy for melanoma using iPSC technology.

Melanoma is one of the most severe skin cancers, derived from melanocytes. Among various therapies for melanoma, adoptive immunotherapy using tumor-infiltrating lymphocytes/chimeric antigen receptor-T cells (TCs) is advanced in recent years; however, the efficacy is still limited, and major challenges remain in terms of safety and cell supply. To solve the issues of adoptive immunotherapy, we utilized induced pluripotent stem cells (iPSCs), which have an unlimited proliferative ability and various differentiation capability. First, we monoclonally isolated CD8+ TCs specifically reactive with NY-ESO-1, one of tumor antigens, from the melanoma patient's monocytes after stimulated with NY-ESO-1 peptide by manual procedure, and cultured NY-ESO-1-specific TCs until proliferated and formed colonies. iPSCs were consequently generated from colony-forming TCs by exogenous expression of reprogramming factors using Sendai virus vector. After the RAG2 gene in TC-derived iPSCs (T-iPSCs) was knocked out for preventing T-cell receptor (TCR) rearrangement, T-iPSCs were re-differentiated into rejuvenated cytotoxic TCs. We confirmed that TCR of T-iPSC-derived TC was maintained as the same of original TCs. In conclusion, T-iPSCs have a potential to be an unlimited cell source for providing cytotoxic TCs. Our study could be a "touchstone" to develop iPSC-based adoptive immunotherapy for the treatment of melanoma for the future clinical use.

Lateral confined growth of cells activates Lef1 dependent pathways to regulate cell-state transitions

Long-term sustained mechano-chemical signals in tissue microenvironment regulate cell-state transitions. In recent work, we showed that laterally confined growth of fibroblasts induce dedifferentiation programs. However, the molecular mechanisms underlying such mechanically induced cell-state transitions are poorly understood. In this paper, we identify Lef1 as a critical somatic transcription factor for the mechanical regulation of de-differentiation pathways. Network optimization methods applied to time-lapse RNA-seq data identify Lef1 dependent signaling as potential regulators of such cell-state transitions. We show that Lef1 knockdown results in the down-regulation of fibroblast de-differentiation and that Lef1 directly interacts with the promoter regions of downstream reprogramming factors. We also evaluate the potential upstream activation pathways of Lef1, including the Smad4, Atf2, NFkB and Beta-catenin pathways, thereby identifying that Smad4 and Atf2 may be critical for Lef1 activation. Collectively, we describe an important mechanotransduction pathway, including Lef1, which upon activation, through progressive lateral cell confinement, results in fibroblast de-differentiation.

Direct Reprogramming of Mouse Subchondral Bone Osteoblasts into Chondrocyte-like Cells.

Treatment of full-thickness articular cartilage defects with exposure of subchondral bone often seen in osteoarthritic conditions has long been a great challenge, especially with a focus on the feasibility of in situ cartilage regeneration through minimally invasive procedures. Osteoblasts that situate in the subchondral bone plate may be considered a potentially vital endogenous source of cells for cartilage resurfacing through direct reprogramming into chondrocytes. Microarray-based gene expression profiles were generated to compare tissue-specific transcripts between subchondral bone and cartilage of mice and to assess age-dependent differences of chondrocytes as well. On osteoblast cell lines established from mouse proximal tibial subchondral bone, sequential screening by co-transduction of transcription factor (TF) genes that distinguish chondrocytes from osteoblasts reveals a shortlist of potential reprogramming factors exhibiting combined effects in inducing chondrogenesis of subchondral bone osteoblasts. A further combinatorial approach unexpectedly identified two 3-TF combinations containing Sox9 and Sox5 that exhibit differences in reprogramming propensity with the third TF c-Myc or Plagl1, which appeared to direct the converted chondrocytes toward either a superficial or a deeper zone phenotype. Thus, our approach demonstrates the possibility of converting osteoblasts into two major chondrocyte subpopulations with two combinations of three genes (Sox9, Sox5, and c-Myc or Plagl1). The findings may have important implications for developing novel in situ regeneration strategies for the reconstruction of full-thickness cartilage defects.

Generation of an induced pluripotent stem cell JTUi005-A from a patient with neuronal intranuclear inclusion disease

Neuronal intranuclear inclusion disease is a neurodegenerative disease caused by expansion of GGC repeats in the 5′ untranslated region (5′ UTR) of NOTCH2NLC. An induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells of a 55-year-old male patient by expressing a defined set of reprogramming factors (OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4) carried on episomal vectors, and was validated for stem cell-like pluripotency, normal karyotype and capability of in vivo differentiation into three germ layers. The NIID-iPSC line serves as a promising tool for further research into pathogenic mechanism and potential therapeutic targets.

Oxytocin promotes epicardial cell activation and heart regeneration after cardiac injury.

Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide, and frequently leads to massive heart injury and the loss of billions of cardiac muscle cells and associated vasculature. Critical work in the last 2 decades demonstrated that these lost cells can be partially regenerated by the epicardium, the outermost mesothelial layer of the heart, in a process that highly recapitulates its role in heart development. Upon cardiac injury, mature epicardial cells activate and undergo an epithelial-mesenchymal transition (EMT) to form epicardium-derived progenitor cells (EpiPCs), multipotent progenitors that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. In mammals, this process alone is insufficient for significant regeneration, but it might be possible to prime it by administering specific reprogramming factors, leading to enhanced EpiPC function. Here, we show that oxytocin (OXT), a hypothalamic neuroendocrine peptide, induces epicardial cell proliferation, EMT, and transcriptional activity in a model of human induced pluripotent stem cell (hiPSC)-derived epicardial cells. In addition, we demonstrate that OXT is produced after cardiac cryoinjury in zebrafish, and that it elicits significant epicardial activation promoting heart regeneration. Oxytocin signaling is also critical for proper epicardium development in zebrafish embryos. The above processes are significantly impaired when OXT signaling is inhibited chemically or genetically through RNA interference. RNA sequencing data suggests that the transforming growth factor beta (TGF-β) pathway is the primary mediator of OXT-induced epicardial activation. Our research reveals for the first time an evolutionary conserved brain-controlled mechanism inducing cellular reprogramming and regeneration of the injured mammalian and zebrafish heart, a finding that could contribute to translational advances for the treatment of cardiac injuries.

An Alternate Approach to Generate Induced Pluripotent Stem Cells with Precise CRISPR/Cas9 Tool.

The induced pluripotent stem cells (iPSCs) are considered powerful tools in pharmacology, biomedicine, toxicology, and cell therapy. Multiple approaches have been used to generate iPSCs with the expression of reprogramming factors. Here, we generated iPSCs by integrating the reprogramming cassette into a genomic safe harbor, CASH-1, with the use of a precise genome editing tool, CRISPR/Cas9. The integration of cassette at CASH-1 into target cells did not alter the pattern of proliferation and interleukin-6 secretion as a response to ligands of multiple signaling pathways involving tumor necrosis factor-α receptor, interleukin-1 receptor, and toll-like receptors. Moreover, doxycycline-inducible expression of OCT4, SOX2, and KLF4 reprogrammed engineered human dermal fibroblasts and human embryonic kidney cell line into iPSCs. The generated iPSCs showed their potential to make embryoid bodies and differentiate into the derivatives of all three germ layers. Collectively, our data emphasize the exploitation of CASH-1 by CRISPR/Cas9 tool for therapeutic and biotechnological applications including but not limited to reprogramming of engineered cells into iPSCs.

Generation of four iPSC lines from a family harboring a 1p36–35 haplotype linked with bipolar disorder and recurrent depressive disorder: Three-generation patients and a healthy sibling

Induced pluripotent stem cells (iPSCs) obtained from genetically characterized patients benefit the biological study of bipolar disorder (BD). Here, we present iPSC lines from three-generation patients with BD and recurrent depressive disorder (RDD) and a healthy control sibling in a family. All patients shared the specified haplotype in the 1p36–35, previously reported as the susceptibility locus of mood disorders. iPSCs were generated with the reprogramming factors OTC3/4, l-MYC, LIN28, SOX2, KLF4, and p53 shRNA through non-integrated episomal vectors. All iPSC lines strongly expressed pluripotency markers and proved the ability to differentiate into three germ lineages in vitro.

Genetic Mechanism Study of Auditory Phoenix Spheres and Transcription Factors Prediction for Direct Reprogramming by Bioinformatics.

Background: Hearing loss is the most common irreversible sensory disorder. By delivering regenerative cells into the cochlea, cell-based therapy provides a novel strategy for hearing restoration. Recently, newly-identified phoenix cells have drawn attention due to their nearly unlimited self-renewal and neural differentiation capabilities. They are a promising cell source for cell therapy and a potential substitute for induced pluripotent stem cells (iPSCs) in many in vitro applications. However, the underlying genomic mechanism of their self-renewal capabilities is largely unknown. The aim of this study was to identify hub genes and potential molecular mechanisms between differentiated and undifferentiated phoenix cells and predict transcription factors (TFs) for direct reprogramming. Material and Methods: The datasets were downloaded from the ArrayExpress database. Samples of differentiated and undifferentiated phoenix cells with three biological replicates were utilised for bioinformatic analysis. Differentially expressed genes (DEGs) were screened and the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were investigated. The gene set enrichment analysis (GSEA) was conducted to verify the enrichment of four self-defined gene set collections, followed by protein-protein interaction (PPI) network construction and subcluster analysis. The prediction of TFs for direct reprogramming was performed based on the TRANSFAC database. Results: Ten hub genes were identified to be the key candidates for self-renewal. Ten TFs were predicted as the direct reprogramming factors. This study provides a theoretical foundation for understanding phoenix cells and clues for direct reprogramming, which would stimulate further experiments and clinical applications in hearing research and treatment.

Probing cell identity hierarchies by fate titration and collision during direct reprogramming

Despite the therapeutic promise of direct reprogramming, basic principles concerning fate erasure and the mechanisms to resolve cell identity conflicts remain unclear. To tackle these fundamental questions, we established a single‐cell protocol for the simultaneous analysis of multiple cell fate conversion events based on combinatorial and traceable reprogramming factor expression: Collide‐seq. Collide‐seq revealed the lack of a common mechanism through which fibroblast‐specific gene expression loss is initiated. Moreover, we found that the transcriptome of converting cells abruptly changes when a critical level of each reprogramming factor is attained, with higher or lower levels not contributing to major changes. By simultaneously inducing multiple competing reprogramming factors, we also found a deterministic system, in which titration of fates against each other yields dominant or colliding fates. By investigating one collision in detail, we show that reprogramming factors can disturb cell identity programs independent of their ability to bind their target genes. Taken together, Collide‐seq has shed light on several fundamental principles of fate conversion that may aid in improving current reprogramming paradigms.

A natural transdifferentiation event involving mitosis is empowered by integrating signaling inputs with conserved plasticity factors.

SummaryTransdifferentiation, or direct cell reprogramming, is the conversion of one fully differentiated cell type into another. Whether core mechanisms are shared between natural transdifferentiation events when occurring with or without cell division is unclear. We have previously characterized the Y-to-PDA natural transdifferentiation in Caenorhabditis elegans, which occurs without cell division and requires orthologs of vertebrate reprogramming factors. Here, we identify a rectal-to-GABAergic transdifferentiation and show that cell division is required but not sufficient for conversion. We find shared mechanisms, including erasure of the initial identity, which requires the conserved reprogramming factors SEM-4/SALL, SOX-2, CEH-6/OCT, and EGL-5/HOX. We also find three additional and parallel roles of the Wnt signaling pathway: selection of a specific daughter, removal of the initial identity, and imposition of the precise final subtype identity. Our results support a model in which levels and antagonistic activities of SOX-2 and Wnt signaling provide a timer for the acquisition of final identity.

Graphene Nanosheets Mediate Efficient Direct Reprogramming into Induced Cardiomyocytes

In vivo cardiac reprogramming is a potential therapeutic strategy to replace cardiomyocytes in patients with myocardial infarction. However, low conversion efficiency is a limitation of In vivo cardiac reprogramming for heart failure. In this study, we showed that graphene nanosheets mediated efficient direct reprogramming into induced cardiomyocytes In vivo. We observed that the administration of graphene nanosheets led to the accumulation of H3K4me3, which resulted in direct cardiac reprogramming. Importantly, the administration of graphene nanosheets combined with cardiac reprogramming factors in a mouse model of myocardial infarction enhanced the effectiveness of directly reprogrammed cell-based cardiac repair. Collectively, our findings suggest that graphene nanosheets can be used as an excellent biomaterial to promote cardiac cell fate conversion and provide a robust reprogramming platform for cardiac regeneration in ischemic heart disease.

Tumor suppressors inhibit reprogramming of African spiny mouse ( Acomys) fibroblasts to induced pluripotent stem cells

Background: The African spiny mouse ( Acomys) is an emerging mammalian model for scar-free regeneration, and further study of Acomys could advance the field of regenerative medicine. Isolation of pluripotent stem cells from Acomys would allow for development of transgenic or chimeric animals and in vitro study of regeneration; however, the reproductive biology of Acomys is not well characterized, complicating efforts to derive embryonic stem cells. Thus, we sought to generate Acomys induced pluripotent stem cells (iPSCs) by reprogramming somatic cells back to pluripotency. Methods: To generate Acomys iPSCs, we attempted to adapt established protocols developed in Mus. We utilized a PiggyBac transposon system to genetically modify Acomys fibroblasts to overexpress the Yamanaka reprogramming factors as well as mOrange fluorescent protein under the control of a doxycycline-inducible TetON operon system. Results: Reprogramming factor overexpression caused Acomys fibroblasts to undergo apoptosis or senescence. When SV40 Large T antigen (SV40 LT) was added to the reprogramming cocktail, Acomys cells were able to dedifferentiate into pre-iPSCs. Although use of 2iL culture conditions induced formation of colonies resembling Mus PSCs, these Acomys iPS-like cells lacked pluripotency marker expression and failed to form embryoid bodies. An EOS-GiP system was unsuccessful in selecting for bona fide Acomys iPSCs; however, inclusion of Nanog in the reprogramming cocktail along with 5-azacytidine in the culture medium allowed for generation of Acomys iPSC-like cells with increased expression of several naïve pluripotency markers. Conclusions: There are significant roadblocks to reprogramming Acomys cells, necessitating future studies to determine Acomys-specific reprogramming factor and/or culture condition requirements. The requirement for SV40 LT during Acomys dedifferentiation may suggest that tumor suppressor pathways play an important role in Acomys regeneration and that Acomys may possess unreported cancer resistance.

Tumor suppressors inhibit reprogramming of African spiny mouse ( Acomys) fibroblasts to induced pluripotent stem cells.

Background: The African spiny mouse ( Acomys) is an emerging mammalian model for scar-free regeneration, and further study of Acomys could advance the field of regenerative medicine. Isolation of pluripotent stem cells from Acomys would allow for development of transgenic or chimeric animals and in vitro study of regeneration; however, the reproductive biology of Acomys is not well characterized, complicating efforts to derive embryonic stem cells. Thus, we sought to generate Acomys induced pluripotent stem cells (iPSCs) by reprogramming somatic cells back to pluripotency. Methods: To generate Acomys iPSCs, we attempted to adapt established protocols developed in Mus. We utilized a PiggyBac transposon system to genetically modify Acomys fibroblasts to overexpress the Yamanaka reprogramming factors as well as mOrange fluorescent protein under the control of a doxycycline-inducible TetON operon system. Results: Reprogramming factor overexpression caused Acomys fibroblasts to undergo apoptosis or senescence. When SV40 Large T antigen (SV40 LT) was added to the reprogramming cocktail, Acomys cells were able to dedifferentiate into pre-iPSCs. Although use of 2iL culture conditions induced formation of colonies resembling Mus PSCs, these Acomys iPS-like cells lacked pluripotency marker expression and failed to form embryoid bodies. An EOS-GiP system was unsuccessful in selecting for bona fide Acomys iPSCs; however, inclusion of Nanog in the reprogramming cocktail along with 5-azacytidine in the culture medium allowed for generation of Acomys iPSC-like cells with increased expression of several naïve pluripotency markers. Conclusions: There are significant roadblocks to reprogramming Acomys cells, necessitating future studies to determine Acomys-specific reprogramming factor and/or culture condition requirements. The requirement for SV40 LT during Acomys dedifferentiation may suggest that tumor suppressor pathways play an important role in Acomys regeneration and that Acomys may possess unreported cancer resistance.

Investigating Peripheral Regional Anesthesia Using Induced Pluripotent Stem Cell Technology: Exploring Novel Terrain.

Investigating Peripheral Regional Anesthesia Using Induced Pluripotent Stem Cell Technology: Exploring Novel Terrain.