Pluripotency In Somatic Cells Research Articles

Inducing Pluripotency in Somatic Cells: Historical Perspective and Recent Advances.

Inducing pluripotency in somatic cells is mediated by the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc. The resulting induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine by virtue of their ability to differentiate into different types of functional cells. Specifically, iPSCs derived directly from patients offer a powerful platform for creating in vitro disease models. This facilitates elucidation of pathological mechanisms underlying human diseases and development of new therapeutic agents mitigating disease phenotypes. Furthermore, genetically and phenotypically corrected patient-derived iPSCs by gene-editing technology or the supply of specific pharmaceutical agents can be used for preclinical and clinical trials to investigate their therapeutic potential. Despite great advances in developing reprogramming methods, the efficiency of iPSC generation remains still low and varies between donor cell types, hampering the potential application of iPSC technology. This paper reviews histological timeline showing important discoveries that have led to iPSC generation and discusses recent advances in iPSC technology by highlighting donor cell types employed for iPSC generation.

A fast chemical reprogramming system promotes cell identity transition through a diapause-like state.

Cellular reprogramming by only small molecules holds enormous potentials for regenerative medicine. However, chemical reprogramming remains a slow process and labour intensive, hindering its broad applications and the investigation of underlying molecular mechanisms. Here, through screening of over 21,000 conditions, we develop a fast chemical reprogramming (FCR) system, which significantly improves the kinetics of cell identity rewiring. We find that FCR rapidly goes through an interesting route for pluripotent reprogramming, uniquely transitioning through a developmentally diapause-like state. Furthermore, FCR critically enables comprehensive characterizations using multi-omics technologies, and has revealed unexpected important features including key regulatory factors and epigenetic dynamics. Particularly, activation of pluripotency-related endogenous retroviruses via inhibition of heterochromatin significantly enhances reprogramming. Our studies provide critical insights into how only environmental cues are sufficient to rapidly reinstate pluripotency in somatic cells, and make notable technical and conceptual advances for solving the puzzle of regeneration.

Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception

Chemistry-alone approach has recently been applied for incepting pluripotency in somatic cells, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Particularly, chemical compounds do not have specific DNA-recognition domains or transcription regulatory domains, and then how do small molecules work as a driving force for reinstating pluripotency in somatic cells? Furthermore, how to efficiently clear materials and structures of an old cell to prepare the rebuilding of a new one? Here, we show that small molecule CD3254 activates endogenous existing transcription factor RXRα to significantly promote mouse chemical reprogramming. Mechanistically, CD3254-RXRα axis can directly activate all the 11 RNA exosome component genes (Exosc1-10 and Dis3) at transcriptional level. Unexpectedly, rather than degrading mRNAs as its substrates, RNA exosome mainly modulates the degradation of transposable element (TE)-associated RNAs, particularly MMVL30, which is identified as a new barrier for cell-fate determination. In turn, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, contributing to the promotion of successful reprogramming. Collectively, our study provides conceptual advances for translating environmental cues into pluripotency inception, particularly, identifies that CD3254-RXRα-RNA exosome axis can promote chemical reprogramming, and suggests modulation of TE-mediated inflammation via CD3254-inducible RNA exosome as important opportunities for controlling cell fates and regenerative medicine.

Induced Pluripotent Stem Cells (iPSCs)-Roles in Regenerative Therapies, Disease Modelling and Drug Screening.

The discovery of induced pluripotent stem cells (iPSCs) has made an invaluable contribution to the field of regenerative medicine, paving way for identifying the true potential of human embryonic stem cells (ESCs). Since the controversy around ethicality of ESCs continue to be debated, iPSCs have been used to circumvent the process around destruction of the human embryo. The use of iPSCs have transformed biological research, wherein increasing number of studies are documenting nuclear reprogramming strategies to make them beneficial models for drug screening as well as disease modelling. The flexibility around the use of iPSCs include compatibility to non-invasive harvesting, and ability to source from patients with rare diseases. iPSCs have been widely used in cardiac disease modelling, studying inherited arrhythmias, neural disorders including Alzheimer’s disease, liver disease, and spinal cord injury. Extensive research around identifying factors that are involved in maintaining the identity of ESCs during induction of pluripotency in somatic cells is undertaken. The focus of the current review is to detail all the clinical translation research around iPSCs and the strength of its ever-growing potential in the clinical space.

Direct Differentiation of Functional Neurons from Human Pluripotent Stem Cells (hPSCs).

Somatic cell nuclear transfer and in vitro induction of pluripotency in somatic cells by defined factors provided unambiguous evidence that the epigenetic state of terminally differentiated somatic cells is not static and can be reversed to a more primitive one. Inspired by these results, stem cell biologists have identified approaches to directly convert fibroblasts into induced neuronal (iN) cells, indicating that direct lineage conversions are possible between distantly related cell types. More recently, we took advantages of pro-neurogenic capacity of iN factors and developed methods to rapidly derive functionally mature neurons directly from human pluripotent stem cells (hPSCs) through a brief induction of defined transcription factors. In this chapter, we describe the detailed methods used to attain the direct conversion from hPSCs to glutamatergic and GABAergic iN cells.

Generation of Pluripotent Stem Cells Using Somatic Cell Nuclear Transfer and Induced Pluripotent Somatic Cells from African Green Monkeys.

Patient-specific stem cells derived from somatic cell nuclear transfer (SCNT) embryos or from induced pluripotent stem cells (iPSCs) could be used to treat various diseases with minimal immune rejection. Many studies using these cells have been conducted in rats and mice; however, there exist numerous dissimilarities between the rodents and humans limiting the clinical predictive power and experimental utility of rodent experiments alone. Nonhuman primates (NHPs) share greater homology to human than rodents in all respects, including genomics, physiology, biochemistry, and the immune system. Thus, experimental data obtained from monkey studies would be more predictive for designing an effective cell replacement therapy in humans. Unfortunately, there are few iPSC lines and even fewer SCNT lines that have been derived in NHPs, hampering broader studies in regenerative medicine. One promising potential therapy would be the replacement of dopamine neurons that are lost in Parkinson's disease. After dopamine depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the African green monkey (Chlorocebus sabaeus) shows the most complete model of Parkinsonism compared with other species and brain pathology and behavioral changes are almost identical to those in humans after accidental exposure to MPTP. Therefore, we have developed a SCNT procedure to generate multiple pluripotent stem cell lines in this species for studies of possible treatment of Parkinsonism and for comparing with cells derived from iPSCs. Using 24 female monkeys as egg donors and 7 somatic cell donor monkeys, we have derived 11 SCNT embryonic stem cell lines that expressed typical stemness genes and formed all three germ layer derivatives. We also derived two iPSC lines using an episome-mediated reprogramming factor delivery system. This report describes the process for deriving these cell lines and proving their pluripotency for differentiation into various potentially therapeutic cells.

The molecular regulation of cell pluripotency in plants.

Plants have a remarkably regenerative capability to replace the damaged organs or form the new organs and individuals both in vivo and in vitro, which is fundamental for their developmental plasticity and the agricultural practices. The regenerative capacities of plants are highly dependent on the totipotency or pluripotency of somatic cells, whose fates are directed by phytohormones, wounding, and other stimuli. Recent studies have revealed that the two types of cellular reprogramming are involved in the acquisition of cell pluripotency during plant in vitro and in vivo regeneration programs. This review focuses on the recent advances of the cellular origin, molecular characteristic, and genetic and epigenetic regulations of cell pluripotency acquisition in plants, highlighting the molecular frameworks of cellular reprogramming activated by diverse stimuli and their possible potentials in regeneration-based plant biotechnologies.

Context-Dependent Impact of RAS Oncogene Expression on Cellular Reprogramming to Pluripotency.

SummaryInduction of pluripotency in somatic cells with defined genetic factors has been successfully used to investigate the mechanisms of disease initiation and progression. Cellular reprogramming and oncogenic transformation share common features; both involve undergoing a dramatic change in cell identity, and immortalization is a key step for cancer progression that enhances reprogramming. However, there are very few examples of complete successful reprogramming of tumor cells. Here we address the effect of expressing an active oncogene, RAS, on the process of reprogramming and found that, while combined expression with reprogramming factors enhanced dedifferentiation, expression within the context of neoplastic transformation impaired reprogramming. RAS induces expression changes that promote loss of cell identity and acquisition of stemness in a paracrine manner and these changes result in reprogramming when combined with reprogramming factors. When cells carry cooperating oncogenic defects, RAS drives cells into an incompatible cellular fate of malignancy.

Fusion of Reprogramming Factors Alters the Trajectory of Somatic Lineage Conversion

Simultaneous expression of Oct4, Klf4, Sox2, and cMyc induces pluripotency in somatic cells (iPSCs). Replacing Oct4 with the neuro-specific factor Brn4 leads to transdifferentiation of fibroblasts into induced neural stem cells (iNSCs). However, Brn4 was recently found to induce transient acquisition of pluripotency before establishing the neural fate. We employed genetic lineage tracing and found that induction of iNSCs with individual vectors leads to direct lineage conversion. In contrast, polycistronic expression produces a Brn4-Klf4 fusion protein that enables induction of pluripotency. Our study demonstrates that a combination of pluripotency and tissue-specific factors allows direct somatic cell transdifferentiation, bypassing the acquisition of a pluripotent state. This result has major implications for lineage conversion technologies, which hold potential for providing a safer alternative to iPSCs for clinical application both invitro and invivo.

Utility of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for an In Vitro Model of Proliferative Vitreoretinopathy.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.

An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications.

More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.

Transcriptional defects and reprogramming barriers in somatic cell nuclear reprogramming as revealed by single-embryo RNA sequencing

BackgroundNuclear reprogramming reinstates totipotency or pluripotency in somatic cells by changing their gene transcription profile. This technology is widely used in medicine, animal husbandry and other industries. However, certain deficiencies severely restrict the applications of this technology.ResultsUsing single-embryo RNA-seq, our study provides complete transcriptome blueprints of embryos generated by cumulus cell (CC) donor nuclear transfer (NT), embryos generated by mouse embryonic fibroblast (MEF) donor NT and in vivo embryos at each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst). According to the results from further analyses, NT embryos exhibit RNA processing and translation initiation defects during the zygotic genome activation (ZGA) period, and protein kinase activity and protein phosphorylation are defective during blastocyst formation. Two thousand three constant genes are not able to be reprogrammed in CCs and MEFs. Among these constant genes, 136 genes are continuously mis-transcribed throughout all developmental stages. These 136 differential genes may be reprogramming barrier genes (RBGs) and more studies are needed to identify.ConclusionsThese embryonic transcriptome blueprints provide new data for further mechanistic studies of somatic nuclear reprogramming. These findings may improve the efficiency of somatic cell nuclear transfer.

The Molecular and Functional Foundations of Conducive Somatic Cell Reprogramming to Ground State Pluripotency

The epigenetic dynamics of iPSC reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process, remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using radically efficient murine reprogramming systems. This comprehensive characterization provides single day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis identified a group of super-enhancers targeted by OSK, whose early demethylation definitively marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are predominantly regulated by Myc rather than OSK and are enriched for cell biosynthetic regulatory functions. CAPGs are distinctively regulated by multiple synergetic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to Myc regulated CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and Myc governed biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naive pluripotency.

Transflammation: Innate immune signaling in nuclear reprogramming.

Induction of pluripotency in somatic cells by retroviral overexpression of four transcription factors has revolutionized the field of stem cell biology and regenerative medicine. The efficient induction of pluripotency requires the activation of innate immune signaling in a process termed "transflammation" (Lee et al., 2012). Specifically, the stimulation of pattern recognition receptors (PRRs) causes global alterations in the expression and activity of epigenetic modifiers to favor an open chromatin configuration. Activation of toll-like receptors (TLR) or RIG-1-like receptors (RLR) (Sayed et al. 2017) trigger signaling cascades that result in NFκB or IRF-3 mediated changes in epigenetic plasticity that facilitate reprogramming. Another form of nuclear reprogramming is so-called direct reprogramming or transdifferentiation of one somatic cell to another lineage. We have shown that transdifferentiation of human fibroblasts to endothelial cells also involves transflammation (Sayed et al., 2015). Recently, we also identified reactive oxygen species (ROS) (Zhou et al. 2016) and reactive nitrogen species (RNS) (Meng et al., 2016) as mediators of innate immune signaling in nuclear reprogramming. Innate immune signaling plays a key role in nuclear reprogramming by regulating DNA accessibility (Fig. 1). Here, we review recent progress of innate immunity signaling in nuclear reprogramming and epigenetic plasticity.

Direct conversion of fibroblasts traces the way back to our first organ-the placenta.

Cell fate determination has always fascinated scientists predominantly embryologists and challenged them to decipher master genes or networks of transcriptional factors which together with cell signaling cascades act as key regulators for the cell lineage allocation. With the rediscovery of the pluripotent precursor cells in an adult body and the possibility to induce pluripotency in somatic cells (iPSCs), as sources for alternative therapeutic options in translational medicine, this field has attracted high interest in medical research and application (1). iPSC have several advantages over embryonic stem cells such as avoiding tumor formation and to circumvent ethical concerns. Meanwhile the understanding of pluripotency has broadened thanks to abundant studies on converting ESC and somatic cells into another cell fate using predominantly transfection of a bunch of lineage specific transcription factors. While many studies focused on the regulation of ESC conversion less is known about the hierarchically organized network of transcriptional factors which determine the trophoblast lineage for placenta development.

Generating human hematopoietic stem cells in vitro -exploring endothelial to hematopoietic transition as a portal for stemness acquisition.

Advances in cellular reprogramming technologies have created alternative platforms for the production of blood cells, either through inducing pluripotency in somatic cells or by way of direct conversion of nonhematopoietic cells into blood cells. However, de novo generation of hematopoietic stem cells (HSCs) with robust and sustained multilineage engraftment potential remains a significant challenge. Hemogenic endothelium (HE) has been recognized as a unique transitional stage of blood development from mesoderm at which HSCs arise in certain embryonic locations. The major aim of this review is to summarize historical perspectives and recent advances in the investigation of endothelial to hematopoietic transition (EHT) and HSC formation in the context of aiding in vitro approaches to instruct HSC fate from human pluripotent stem cells. In addition, direct conversion of somatic cells to blood and HSCs and progression of this conversion through HE stage are discussed. A thorough understanding of the intrinsic and microenvironmental regulators of EHT that lead to the acquisition of self-renewal potential by emerging blood cells is essential to advance the technologies for HSC production and expansion.

Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges

Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Stem Cells 2017;35:68-79.

Direct Comparison of DNA versus mRNA on the Efficiency of Induced Pluripotent Stem Cells Generation from Human Primary Fibroblasts

Induction of pluripotency in somatic cells has opened a brand new spectrum on patient-specific stem cell therapies, therefore several approaches seek the optimization of the existing dedifferentiation processes. DNA and mRNA have been used to induce cells using different strategies, from diverse sets of pluripotency induction genes to variations of their delivery method. In this work we present a direct comparison between circular and linear DNA, as well as mRNA, capability to dedifferentiate human primary fibroblasts.

The common or cell-specific transcription factors between oocytes and embryonic stem cells in mice

By Yamanaka and Daley group, the six genes (Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28) have been shown to induce pluripotency in somatic cells. Based on the fact that both oocytes and embryonic stem cells (ESCs) have the potential to reprogram differentiated somatic cells into pluripotent cells, we conducted the present study to compare gene expression profiles, especially transcription factors, between oocytes and ESCs for finding common or cell-specific genes as candidates of novel reprogramming factors.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells.

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.