Cell Reprogramming: The Many Roads to Success.

Cellular reprogramming experiments from somatic cell types have demonstrated the plasticity of terminally differentiated cell states. Recent efforts in understanding the mechanisms of cellular reprogramming have begun to elucidate the differentiation trajectories along the reprogramming processes. In this review, we focus mainly on direct reprogramming strategies by transcription factors and highlight the variables that contribute to cell fate conversion outcomes. We review key studies that shed light on the cellular and molecular mechanisms by investigating differentiation trajectories and alternative cell states as well as transcription factor regulatory activities during cell fate reprogramming. Finally, we highlight a few concepts that we believe require attention, particularly when measuring the success of cell reprogramming experiments.

In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming.

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

Modulation of host cell biology by plant pathogenic microbes.

Plant-pathogen interactions can result in dramatic visual changes in the host, such as galls, phyllody, pseudoflowers, and altered root-system architecture, indicating that the invading microbe has perturbed normal plant growth and development. These effects occur on a cellular level but range up to the organ scale, and they commonly involve attenuation of hormone homeostasis and deployment of effector proteins with varying activities to modify host cell processes. This review focuses on the cellular-reprogramming mechanisms of filamentous and bacterial plant pathogens that exhibit a biotrophic lifestyle for part, if not all, of their lifecycle in association with the host. We also highlight strategies for exploiting our growing knowledge of microbial host reprogramming to study plant processes other than immunity and to explore alternative strategies for durable plant resistance.

Zygotic genome activation during the maternal-to-zygotic transition.

Embryogenesis depends on a highly coordinated cascade of genetically encoded events. In animals, maternal factors contributed by the egg cytoplasm initially control development, whereas the zygotic nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized, and maternal factors are cleared. This transfer of developmental control is called the maternal-to-zygotic transition (MZT). In this review, we discuss recent advances toward understanding the scope, timing, and mechanisms that underlie zygotic genome activation at the MZT in animals. We describe high-throughput techniques to measure the embryonic transcriptome and explore how regulation of the cell cycle, chromatin, and transcription factors together elicits specific patterns of embryonic gene expression. Finally, we illustrate the interplay between zygotic transcription and maternal clearance and show how these two activities combine to reprogram two terminally differentiated gametes into a totipotent embryo.

Cohesin controls X chromosome structure remodeling and X-reactivation during mouse iPSC-reprogramming.

Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming.

Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.

Neuronal identities derived by misexpression of the POU IV sensory determinant in a protovertebrate.

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin, to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants.

ZNF281 enhances cardiac reprogramming by modulating cardiac and inflammatory gene expression.

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.

Stage II oesophageal carcinoma: peril in disguise associated with cellular reprogramming and oncogenesis regulated by pseudogenes.

INTRODUCTION: Pseudogenes have been implicated for their role in regulating cellular differentiation and organismal development. However, their role in promoting cancer-associated differentiation has not been well-studied. This study explores the tumour landscape of oesophageal carcinoma to identify pseudogenes that may regulate events of differentiation to promote oncogenic transformation. MATERIALS AND METHOD: De-regulated differentiation-associated pseudogenes were identified using DeSeq2 followed by 'InteractiVenn' analysis to identify their expression pattern. Gene expression dependent and independent enrichment analyses were performed with GSEA and ShinyGO, respectively, followed by quantification of cellular reprogramming, extent of differentiation and pleiotropy using three unique metrics. Stage-specific gene regulatory networks using Bayesian Network Splitting Average were generated, followed by network topology analysis. MEME, STREME and Tomtom were employed to identify transcription factors and miRNAs that play a regulatory role downstream of pseudogenes to initiate cellular reprogramming and further promote oncogenic transformation. The patient samples were stratified based on the expression pattern of pseudogenes, followed by GSEA, mutation analysis and survival analysis using GSEA, MAF and 'survminer', respectively. RESULTS: Pseudogenes display a unique stage-wise expression pattern that characterizes stage II (SII) ESCA with a high rate of cellular reprogramming, degree of differentiation and pleiotropy. Gene regulatory network and associated topology indicate high robustness, thus validating high pleiotropy observed for SII. Pseudogene-regulated expression of SOX2, FEV, PRRX1 and TFAP2A in SII may modulate cellular reprogramming and promote oncogenesis. Additionally, patient stratification-based mutational analysis in SII signifies APOBEC3A (A3A) as a potential hallmark of homeostatic mutational events of reprogrammed cells which in addition to de-regulated APOBEC3G leads to distinct events of hypermutations. Further enrichment analysis for both cohorts revealed the critical role of combinatorial expression of pseudogenes in cellular reprogramming. Finally, survival analysis reveals distinct genes that promote poor prognosis in SII ESCA and patient-stratified cohorts, thus providing valuable prognostic bio-markers along with markers of differentiation and oncogenesis for distinct landscapes of pseudogene expression. CONCLUSION: Pseudogenes associated with the events of differentiation potentially aid in the initiation of cellular reprogramming to facilitate oncogenic transformation, especially during SII ESCA. Despite a better overall survival of SII, patient stratification reveals combinatorial de-regulation of pseudogenes as a notable marker for a high degree of cellular differentiation with a unique mutational landscape.

An induced thymic epithelial cell-based high throughput screen for thymus extracellular matrix mimetics.

Thymic epithelial cells (TECs) are key effectors of the thymic stroma and are critically required for T-cell development. TECs comprise a diverse set of related but functionally distinct cell types that are scarce and difficult to isolate and handle. This has precluded TEC-based screening assays. We previously described induced thymic epithelial cells (iTECs), an artificial cell type produced in vitro by direct reprogramming, raising the possibility that iTECs might provide the basis for functional screens related to TEC biology. Here, we present an iTEC-based three-stage medium/high-throughput in vitro assay for synthetic polymer mimics of thymic extracellular matrix (ECM). Using this assay, we identified, from a complex library, four polymers that bind iTEC as well as or better than gelatin but do not bind mesenchymal cells. We show that these four polymers also bind and maintain native mouse fetal TECs and native human fetal TECs. Finally, we show that the selected polymers do not interfere with iTEC function or T-cell development. Collectively, our data establish that iTECs can be used to screen for TEC-relevant compounds in at least some medium/high-throughput assays and identify synthetic polymer ECM mimics that can replace gelatin or ECM components in TEC culture protocols.

A Youthful Touch: Reversal of Aging Hallmarks by Cell Reprogramming.

BACKGROUND: With the elderly population projected to double by 2050, there is an urgent need to address the increasing prevalence of age-related debilitating diseases and ultimately minimize discrepancies between the rising lifespan and stagnant health span. Cellular reprogramming by overexpression of Oct3/4, Klf4, Sox2, and cMyc (OKSM) transcription factors is gaining attention in this context thanks to demonstrated rejuvenating effects in human cell cultures and live mice, many of which can be uncoupled from dedifferentiation and loss of cell identity. SUMMARY: Here, we review current evidence of the impact of cell reprogramming on established aging hallmarks and the underlying mechanisms that mediate these effects. We also provide a critical assessment of the challenges in translating these findings and, overall, cell reprogramming technologies into clinically translatable antiaging interventions. KEY MESSAGES: Cellular reprogramming has the potential to reverse at least partially some key hallmarks of aging. However, further research is necessary to determine the biological significance and duration of such changes and to ensure the safety of cell reprogramming as a rejuvenation approach. With this review, we hope to stimulate new research directions in the quest to extend health span effectively.

H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase.

AIMS: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. METHODS: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. RESULTS: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. CONCLUSION: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

Cellular reprogramming of fibroblasts in heart regeneration.

Myocardial infarction causes the loss of cardiomyocytes and the formation of cardiac fibrosis due to the activation of cardiac fibroblasts, leading to cardiac dysfunction and heart failure. Unfortunately, current therapeutic interventions can only slow the disease progression. Furthermore, they cannot fully restore cardiac function, likely because the adult human heart lacks sufficient capacity to regenerate cardiomyocytes. Therefore, intensive efforts have focused on developing therapeutics to regenerate the damaged heart. Several strategies have been intensively investigated, including stimulation of cardiomyocyte proliferation, transplantation of stem cell-derived cardiomyocytes, and conversion of fibroblasts into cardiac cells. Resident cardiac fibroblasts are critical in the maintenance of the structure and contractility of the heart. Fibroblast plasticity makes this type of cells be reprogrammed into many cell types, including but not limited to induced pluripotent stem cells, induced cardiac progenitor cells, and induced cardiomyocytes. Fibroblasts have become a therapeutic target due to their critical roles in cardiac pathogenesis. This review summarizes the reprogramming of fibroblasts into induced pluripotent stem cell-derived cardiomyocytes, induced cardiac progenitor cells, and induced cardiomyocytes to repair a damaged heart, outlines recent findings in utilizing fibroblast-derived cells for heart regeneration, and discusses the limitations and challenges.

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

BACKGROUND: 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. METHODS: To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes. RESULTS: 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. CONCLUSIONS: 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.

Metabolic Changes Associated With Cardiomyocyte Dedifferentiation Enable Adult Mammalian Cardiac Regeneration.

BACKGROUND: Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. METHODS: We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo. RESULTS: Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. CONCLUSIONS: Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

Derivation and Differentiation of Human Pluripotent Stem Cells in Microfluidic Devices.

An integrative approach based on microfluidic design and stem cell biology enables capture of the spatial-temporal environmental evolution underpinning epigenetic remodeling and the morphogenetic process. We examine the body of literature that encompasses microfluidic applications where human induced pluripotent stem cells are derived starting from human somatic cells and where human pluripotent stem cells are differentiated into different cell types. We focus on recent studies where the intrinsic features of microfluidics have been exploited to control the reprogramming and differentiation trajectory at the microscale, including the capability of manipulating the fluid velocity field, mass transport regime, and controllable composition within micro- to nanoliter volumes in space and time. We also discuss studies of emerging microfluidic technologies and applications. Finally, we critically discuss perspectives and challenges in the field and how these could be instrumental for bringing about significant biological advances in the field of stem cell engineering.

Involvement of trained immunity during autoimmune responses.

Recently, it has been described that innate immune cells such as monocytes, macrophages, and natural killer cells can develop a non-specific immune response induced by different stimuli, including lipopolysaccharides, Mycobacterium bovis Bacillus Calmette-Guérin, and oxidized low-density lipoprotein. This non-specific immune response has been named "trained immunity," whose mechanism is essential for host defense and vaccine response, promoting better infection control. However, limited information about trained immunity in other non-infectious diseases, such as autoimmune illness, has been reported. The complexity of autoimmune pathology arises from dysfunctions in the innate and adaptive immune systems, triggering different clinical outcomes depending on the disease. Nevertheless, T and B cell function dysregulation is the most common characteristic associated with autoimmunity by promoting the escape from central and peripheral tolerance. Despite the importance of adaptative immunity to autoimmune diseases, the innate immune system also plays a prominent and understudied role in these pathologies. Accordingly, epigenetic and metabolic changes associated with innate immune cells that undergo a trained process are possible new therapeutic targets for autoimmune diseases. Even so, trained immunity can be beneficial or harmful in autoimmune diseases depending on several factors associated with the stimuli. Here, we reviewed the role of trained immunity over the innate immune system and the possible role of these changes in common autoimmune diseases, including Systemic Lupus Erythematosus, Rheumatoid Arthritis, Multiple Sclerosis, and Type 1 Diabetes.

Stem cell programming - prospects for perinatal medicine.

Recreating human cell and organ systems in vitro has tremendous potential for disease modeling, drug discovery and regenerative medicine. The aim of this short overview is to recapitulate the impressive progress that has been made in the fast-developing field of cell programming during the past years, to illuminate the advantages and limitations of the various cell programming technologies for addressing nervous system disorders and to gauge their impact for perinatal medicine.

Effects of extremely low-frequency magnetic fields on human MDA-MB-231 breast cancer cells: proteomic characterization.

Extremely low-frequency electromagnetic fields (ELF-MF) can modify the cell viability and regulatory processes of some cell types, including breast cancer cells. Breast cancer is a multifactorial disease where a role for ELF-MF cannot be excluded. ELF-MF may influence the biological properties of breast cells through molecular mechanisms and signaling pathways that are still unclear. This study analyzed the changes in the cell viability, cellular morphology, oxidative stress response and alteration of proteomic profile in breast cancer cells (MDA-MB-231) exposed to ELF-MF (50 Hz, 1 mT for 4 h). Non-tumorigenic human breast cells (MCF-10A) were used as control cells. Exposed MDA-MB-231 breast cancer cells increased their viability and live cell number and showed a higher density and length of filopodia compared with the unexposed cells. In addition, ELF-MF induced an increase of the mitochondrial ROS levels and an alteration of mitochondrial morphology. Proteomic data analysis showed that ELF-MF altered the expression of 328 proteins in MDA-MB-231 cells and of 242 proteins in MCF-10A cells. Gene Ontology term enrichment analysis demonstrated that in both cell lines ELF-MF exposure up-regulated the genes enriched in "focal adhesion" and "mitochondrion". The ELF-MF exposure decreased the adhesive properties of MDA-MB-231 cells and increased the migration and invasion cell abilities. At the same time, proteomic analysis, confirmed by Real Time PCR, revealed that transcription factors associated with cellular reprogramming were upregulated in MDA-MB-231 cells and downregulated in MCF-10A cells after ELF-MF exposure. MDA-MB-231 breast cancer cells exposed to 1 mT 50 Hz ELF-MF showed modifications in proteomic profile together with changes in cell viability, cellular morphology, oxidative stress response, adhesion, migration and invasion cell abilities. The main signaling pathways involved were relative to focal adhesion, mitochondrion and cellular reprogramming.

Hypoxia-induced reprogrammed myoblasts enhance the formation of neuromuscular junctions: A pioneer study.

We previously reported that muscle cells could reprogram into progenitors after traumatic injuries. These injury-induced muscle stem cells (iMuSCs) have increased migration and differentiation capacities, including neuronal differentiation. Recent studies in our laboratory suggest that the hypoxia-induced by tissue injury plays an essential role in the reprogramming process of muscle cells. We hypothesize that muscle cells reprogrammed with hypoxia have increased neuronal differentiation potentials and the neuronal differentiation extends into the formation of neuromuscular junction (NMJ)-like structures. In this study, C2C12 myoblasts were cultured under hypoxic conditions and subsequently in neural differentiation media to generate neurospheres, and then with muscle differentiation media to induce NMJ-like structure formation. Hypoxia-induced muscle cells also produced more robust NMJs compared to controls after intramuscular cell transplantation. Our results suggest hypoxia plays a role in the reprogramming of muscle stem cells, which may have the potential to form neuromuscular junctions and ultimately contribute to functional muscle healing.