Cellular Reprogramming

Advances in regenerative nanomedicine raise a host of ethical, legal, and social questions that healthcare providers and scientists will need to consider. These questions and concerns include definitions, appropriate applications, dual use, potential risks, regulations, and access. In this chapter, we provide an overview of the questions and concerns and recommend proactive consideration and solutions.

Like breadcrumbs in the forest, cotranscriptionally acquired histone methylation acts as a memory of prior transcription. Because it can be retained through cell divisions, transcriptional memory allows cells to coordinate complex transcriptional programs during development. However, if not reprogrammed properly during cell fate transitions, it can also disrupt cellular identity. In this review, we discuss the consequences of failure to reprogram histone methylation during three crucial epigenetic reprogramming windows: maternal reprogramming at fertilization, embryonic stem cell (ESC) differentiation, and the continuous maintenance of cell identity in differentiated cells. In addition, we discuss how following the wrong breadcrumb trail of transcriptional memory provides a framework for understanding how heterozygous loss-of-function mutations in histone-modifying enzymes may cause severe neurodevelopmental disorders.

M aintenance of cellular identity relies on the expression of cell type-specific transcription factors and the underlying state of the epigenome. During somatic cell reprogramming , lineage-committed cells can be converted into iPSCs on expression of critical transcriptional regulators of embryonic stem cells; namely, OCT4, SOX2, KLF4 and MYC (OSKM) 1. This process results in a complete change in cellular identity, and involves extensive transcriptional and epigenome-wide changes 2. The state of an epigenome is determined by the activities of chromatin writer, reader and eraser proteins. Therefore, chromatin regulators have emerged as important factors of cell identity and can act as facilita-tors or barriers to reprogramming 3. Inhibition of a number of major chromatin-related pathways can facilitate reprogramming, such as DNA methylation 4 , histone deacetylation 5 , histone H3 lysine 9 methylation 6,7 , CAF-1 complex 8 and NCoR/SMRT corepressors 9. These factors safeguard cellular identity mainly by preventing the activation of pluripotency genes. However, chromatin factors also safeguard cellular identity by perpetuating active somatic-specific gene transcription programs or by preventing the silencing of such genes on OSKM expression. Recent work has indicated that loss of the 'active' histone H3 lysine 27 acet-ylation (H3K27) mark from the enhancers of somatic-specific genes is an early step in reprogramming 10. An important remaining question is which chromatin factors counteract the OSKM-mediated silencing of lineage-specific gene expression in reprogramming. We had previously identified DOT1L-mediated histone H3 lysine 79 methylation as one of the key barriers to reprogramming that counteracts the silencing of lineage-specific genes 6. DOT1L inhibition could also replace KLF4 and MYC in human iPSC generation. In subsequent studies, inhibition of DOT1L activity has been shown to increase reprogramming efficiency in a wide range of systems 11,12. Importantly, DOT1L inhibitors facilitate the derivation of chemically induced pluripotent stem cells (ciPSCs) from mouse somatic cells 13. However, the generation of ciPSCs from human cells has not been achieved so far, suggesting that additional barriers exist for human cell reprogramming. The CBP and EP300 proteins are closely related histone acetyl-transferases (HATs) that act as transcriptional coactivators 14. CBP and EP300 (also known as KAT3A and KAT3B, respectively) are large, multi-domain proteins, which in addition to their catalytic HAT domain, contain bromodomains that bind acetylated histones and are required for chromatin binding 15-17. Localization of CBP/ EP300 in the genome has been used to identify cell type-specific enhancers in mice and humans 18,19. These coactivators also occupy super-enhancer regions, which have an important role in maintaining cell identity 20,21. Both coactivators are required for early embry-onic development and proper differentiation of a wide range of cell types 22. However, the role of these two major coactivators in reprogramming remains largely unknown. In this study, we carried out a chromatin-focused chemical screen to identify epigenetic regulators that can collaborate with DOT1L inhibition in reprogramming. We discover that CBP/EP300 bromodomain inhibition enhances reprogramming to pluripotency by facilitating the silencing of the somatic gene expression program. Results A chromatin-focused chemical screen for reprogramming. We carried out a reprogramming screen in the presence of inhibitors Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity. NATURE ChEMiCAl BiOlOGy | VOL 15 | MAY 2019 | 519-528 | www.nature.com/naturechemicalbiology 519

Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD(+)) and its impact on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD(+) precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response (UPR(mt)) and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the Mdx mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs (NSCs) and melanocyte SCs (McSCs), and increased mouse lifespan. Strategies that conserve cellular NAD(+) may reprogram dysfunctional SCs and improve lifespan in mammals.

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Familial hypertrophic cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. To investigate the molecular mechanism underlying the abnormal CM functions in HCM, we derived iPSCs from an HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for 'Cell Proliferation' was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical t...

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for ...

In C. elegans, the H3K36 methyltransferase, MES-4, helps establish germ cell fate by maintaining H3K36me2/3 at germline genes between generations. Previously, we showed that the H3K4me2 demethylase, SPR-5, and the H3K9 methyltransferase, MET-2, reprogram histone methylation at fertilization to prevent the ectopic expression of germline genes in somatic tissues. Together, this indicates that SPR-5 and MET-2 maternal reprogramming may antagonize MES-4 to establish germline versus soma. Here, we show that spr-5; met-2 mutant progeny have a severe developmental delay that is associated with the ectopic maintenance of H3K36me2/3 at MES-4 targeted germline genes in somatic tissues, and the ectopic expression of these genes. We further show that the developmental delay and the ectopic expression are dependent upon MES-4. Thus, we propose that SPR-5, MET-2, and MES-4 balance inherited histone methylation to establish germline versus soma. Without this balance, the inappropriate transcription of germline genes in somatic tissues causes developmental delay.