Reprogramming Efficiency Research Articles

Acceleration of somatic cell reprogramming into the induced pluripotent stem cell using a mycosporine-like amino acid, Porphyra 334

Porphyra 334 (P334), a mycosporine-like amino acid (MAA), is a secondary metabolite found in diverse marine and terrestrial organisms and has several beneficial effects on fibroblast proliferation, wound healing, and antioxidant activity. Here, we report that P334 accelerates the cell reprogramming process of mouse tail-tip fibroblasts (TTFs) and human dermal papilla (HDP) cells into induced pluripotent stem cells (iPSCs). We found that P334 significantly improved the cell reprogramming efficiency by activating the tri-methylation of histone 3 lysine 4 (H3K4me3), which controls mesenchymal to epithelial transition (MET) during the reprogramming process. Thus, we found that P334 directly regulates epigenetic changes, providing an efficient approach for natural compound-based cell reprogramming.

RNA-Based Strategies for Cardiac Reprogramming of Human Mesenchymal Stromal Cells

Multipotent adult mesenchymal stromal cells (MSCs) could represent an elegant source for the generation of patient-specific cardiomyocytes needed for regenerative medicine, cardiovascular research, and pharmacological studies. However, the differentiation of adult MSC into a cardiac lineage is challenging compared to embryonic stem cells or induced pluripotent stem cells. Here we used non-integrative methods, including microRNA and mRNA, for cardiac reprogramming of adult MSC derived from bone marrow, dental follicle, and adipose tissue. We found that MSC derived from adipose tissue can partly be reprogrammed into the cardiac lineage by transient overexpression of GATA4, TBX5, MEF2C, and MESP1, while cells isolated from bone marrow, and dental follicle exhibit only weak reprogramming efficiency. qRT-PCR and transcriptomic analysis revealed activation of a cardiac-specific gene program and up-regulation of genes known to promote cardiac development. Although we did not observe the formation of fully mature cardiomyocytes, our data suggests that adult MSC have the capability to acquire a cardiac-like phenotype when treated with mRNA coding for transcription factors that regulate heart development. Yet, further optimization of the reprogramming process is mandatory to increase the reprogramming efficiency.

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos.

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10-11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.

Modeling Sporadic Alzheimer's Disease by Efficient Direct Reprogramming of the Elderly Derived Disease Dermal Fibroblasts into Neural Stem Cells.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, neuropathologically characterized by hyperphosphorylation of tau and formation of amyloid plaques. Most AD cases are sporadic with no clear cause. Cell models play an important role in understanding the pathogenesis of sporadic AD, and the cell reprogramming and epigenetic techniques have provided new avenue to model the disorder. However, since most sporadic AD patients are late-onset, it poses a challenge to reprogram elderly somatic cells into stem cells. Here, we report that combination of overexpressing a single transcription factor, hSOX, with nine small molecules, was able to directly reprogram elderly (55-75 years of age) sporadic AD and the age-matched healthy individual dermal fibroblasts into the induced neural stem cells (iNSCs). These cells possessed the typical neural stem cell properties and were able to be further differentiated into neurons and glia in vitro and in vivo. More importantly, AD iNSC-derived neurons showed hyperphosphorylation at several sites of tau and increased release of Aβ into culture medium, indicating the replication of the major neuropathological hallmarks. Thus, we described a new technique to directly convert elderly AD dermal fibroblasts into iNSCs that may serve as a useful tool for studying the pathogenesis of sporadic AD and for drug discovery to treat the disorder.

Isoform Specific Effects of Mef2C during Direct Cardiac Reprogramming.

Direct conversion of cardiac fibroblasts into induced cardiomyocytes (iCMs) by forced expression of defined factors holds great potential for regenerative medicine by offering an alternative strategy for treatment of heart disease. Successful iCM conversion can be achieved by minimally using three transcription factors, Mef2c (M), Gata4(G), and Tbx5 (T). Despite increasing interest in iCM mechanistic studies using MGT(polycistronic construct with optimal expression of M,G and T), the reprogramming efficiency varies among different laboratories. Two main Mef2c isoforms (isoform2, Mi2 and isoform4, Mi4) are present in heart and are used separately by different labs, for iCM reprogramming. It is currently unknown if differently spliced isoform of Mef2c contributes to varied reprogramming efficiency. Here, we used Mi2 and Mi4 together with Gata4 and Tbx5 in separate vectors or polycistronic vector, to convert fibroblasts to iCMs. We found that Mi2 can induce higher reprogramming efficiency than Mi4 in MEFs. Addition of Hand2 to MGT retroviral cocktail or polycistronic Mi2-GT retroviruses further enhanced the iCM conversion. Overall, this study demonstrated the isoform specific effects of Mef2c, during iCM reprogramming, clarified some discrepancy about varied efficiency among labs and might lead to future research into the role of alternative splicing and the consequent variants in cell fate determination.

A Comparative Analysis of Single-Cell Transcriptome Identifies Reprogramming Driver Factors for Efficiency Improvement

Terminally differentiated somatic cells can be reprogrammed into a totipotent state through somatic cell nuclear transfer (SCNT). The incomplete reprogramming is the major reason for developmental arrest of SCNT embryos at early stages. In our studies, we found that pathways for autophagy, endocytosis, and apoptosis were incompletely activated in nuclear transfer (NT) 2-cell arrest embryos, whereas extensively inhibited pathways for stem cell pluripotency maintenance, DNA repair, cell cycle, and autophagy may result in NT 4-cell embryos arrest. As for NT normal embryos, a significant shift in expression of developmental transcription factors (TFs) Id1, Pou6f1, Cited1, and Zscan4c was observed. Compared with pluripotent gene Ascl2 being activated only in NT 2-cell, Nanog, Dppa2, and Sall4 had major expression waves in normal development of both NT 2-cell and 4-cell embryos. Additionally, Kdm4b/4d and Kdm5b had been confirmed as key markers in NT 2-cell and 4-cell embryos, respectively. Histone acetylases Kat8, Elp6, and Eid1 were co-activated in NT 2-cell and 4-cell embryos to facilitate normal development. Gadd45a as a key driver functions with Tet1 and Tet2 to improve the efficiency of NT reprogramming. Taken together, our findings provided an important theoretical basis for elucidating the potential molecular mechanisms and identified reprogramming driver factor to improve the efficiency of SCNT reprogramming.

Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines.

Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol.

Structurally-Discovered KLF4 Variants Accelerate and Stabilize Reprogramming to Pluripotency

Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domain enable next-generation reprogramming technology with engineered reprogramming factors.

STAT3 modulates reprogramming efficiency of human somatic cells; insights from autosomal dominant Hyper IgE syndrome caused by STAT3 mutations.

ABSTRACTHuman induced pluripotent stem cell (iPSC) technology has opened exciting opportunities for stem-cell-based therapy. However, its wide adoption is precluded by several challenges including low reprogramming efficiency and potential for malignant transformation. Better understanding of the molecular mechanisms of the changes that cells undergo during reprograming is needed to improve iPSCs generation efficiency and to increase confidence for their clinical use safety. Here, we find that dominant negative mutations in STAT3 in patients with autosomal-dominant hyper IgE (Job's) syndrome (AD-HIES) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies. Analysis of normal skin fibroblasts revealed upregulation and phosphorylation of endogenous signal transducer and activator of transcription 3 (STAT3) and its binding to the NANOG promoter following transduction with OKSM factors. This coincided with upregulation of NANOG and appearance of cells expressing pluripotency markers. Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3. NANOGP8, the human-specific NANOG retrogene that is often expressed in human cancers, was also induced during reprogramming, to very low but detectable levels, in a STAT3-dependent manner. Our study revealed the critical role of endogenous STAT3 in facilitating reprogramming of human somatic cells.

Prevascularized, multiple-layered cell sheets of direct cardiac reprogrammed cells for cardiac repair.

We previously demonstrated that the efficiency of direct cardiac reprogramming from fibroblasts could be enhanced via mimicking of the in vivo cardiac microenvironment through coculture with cardiomyocytes and by providing electric cues. In the present study, we developed cell sheets using the direct cardiac reprogrammed cells and a nanothin, nanoporous poly(lactic-co-glycolic acid) membrane. Cell sheets were laid layer-by-layer and prevacularized with endothelial cells between the layers. These prevascularized, multilayered cell sheets were implanted on the epicardium of infarcted rat hearts, which led to an improvement in cardiac function and reduction in adverse cardiac remodeling post-myocardial infarction (MI). Thus, the in vivo mimicking direct cardiac reprogramming and prevascularization technique can enhance the efficiency of cell sheets in clinical applications and provide new opportunities to prevent heart failure following MI.

MRNA-Based Reprogramming Under Xeno-Free and Feeder-Free Conditions.

Reprogramming somatic cells into induced pluripotent stem cells (iPSC) has provided a gateway for many novel discoveries in the field of tissue engineering, regenerative medicine and cell therapy. The need for an efficient, less laborious and fast reprogramming protocol under xeno-free, feeder-free and chemically defined conditions has never been greater. Here we describe a novel approach to reprogramming using the StemRNA 3rd Gen Reprogramming Kit (ReproCELL) which encompasses non-modified microRNAs (NM-miRNA), non-modified E3, K3, B18R RNAs (EKB NM-RNA) and non-modified mRNAs for six crucial transcription factors (OSKMNL NM-RNA).

Efficient RNA-Based Reprogramming of Disease-Associated Primary Human Fibroblasts into Induced Pluripotent Stem Cells.

Reprogramming a patient's somatic cells into induced pluripotent stem cells (iPSCs) holds great promise for disease modeling and the development of autologous cellular therapeutics. However, it remains challenging to consistently reprogram primary human cells, as they are frequently aged, diseased, or in low abundance. Here we present a modified highly efficient and clinically relevant RNA-based method for reprogramming disease-associated and other difficult-to-reprogram human primary fibroblast lines into iPSCs. We also describe optimizations that can be employed for consistent reprogramming of these difficult-to-reprogram cells. With the provided protocol, integration-free iPSC lines can be successfully generated from a small number of primary human fibroblasts in approximately 5-7weeks.

Direct Cardiac Reprogramming - Converting Cardiac Fibroblasts to Cardiomyocytes.

Cardiovascular disease is the leading cause of death and disability worldwide. Despite advances in cardiovascular therapy, mortality in heart disease still remains high. Direct cardiac reprogramming is a promising approach for cardiac tissue repair involving in situ generation of new cardiomyocytes from endogenous cardiac fibroblasts. Although, initially, the reprogramming efficiency was low, several developments in reprogramming methods have improved the in vitro cardiac reprogramming efficiency. Subsequently, in vivo cardiac reprogramming has demonstrated improvement in cardiac function and fibrosis after myocardial infarction. Here, we review recent progress in cardiac reprogramming as a new technology for cardiac regeneration.

Very Low Efficiency of Direct Reprogramming of Astrocytes Into Neurons in the Brains of Young and Aged Mice After Cerebral Ischemia

After cerebral ischemia, the ratio between astroglial cells and neurons in the neurovascular unit is disrupted in the perilesional area. We hypothesized that restoring the balance within the neurovascular unit may lead to an improved neurorestoration after focal ischemia. Recently, an innovative technology has been invented to efficiently convert proliferating astroglial cells into neurons in the injured young brain. However, the conversion efficacy of this technology has not been explored in the post-stroke brains of the aged rodents. To this end, we used a retroviral delivery system encoding the transcription factor Ngn2 alone or in combination with the antiapoptotic factor Bcl-2 to target proliferating astrocytes in the neocortex of young and aged mice after cerebral ischemia. Successful direct in vivo reprogramming of reactive glia into neuroblasts and mature neurons was assessed by cellular phenotyping. We found that the conversion efficacy of proliferating astrocytes into neurons after cerebral ischemia in young and aged mice is disappointingly low, most likely because the therapeutic vectors carrying the conversion gene are engulfed by phagocytes shortly after intracortical administration. We conclude that other viral vectors and combinations of transcription factors should be employed to improve the efficacy of glia-to-neuron conversion after stroke in young and aged rodents.

215 Invitro evaluation of reprogramming at 20% or 5% oxygen tension in adult equine fibroblasts

The reprogramming process suffers from effects of external and internal factors, one of which is oxygen concentration, because it plays important roles in several processes of the cell. The objective of this study was to evaluate the effect of oxygen during reprogramming of equine adult fibroblasts. Fibroblasts were isolated from the skin of one male and immediately cultured under high (20%, HO group) or low (5%, LO group) oxygen tension. Generation of induced pluripotent stem cells (iPSCs) was performed by transduction with human sequences of OCT4, SOX2, KLF4, and c-MYC cDNAs. Morphology evaluation and RNA samples collection were performed at the day of transduction (Day 0), and then every 3 days (Days 3, 6, 9, 12, 15, and 18). Six days after transduction, cells were passed to mouse embryonic fibroblast (MEFs). A third group, in which the cells were cultured in high oxygen and passed to low oxygen, was formed (high to low group, HLO) and analysed. The equine (e)iPS colonies were evaluated in terms of reprogramming efficiency, morphology, detection of alkaline phosphatase, and qRT-PCR for markers of pluripotency (OCT4, REX1, NANOG, and SOX2 genes), glycolysis (PFKM and GAPDH), mitochondrial fission and fusion (DNM1L and MFN1), hypoxia (HIF1α, HIF2α, and VEGFA), and presence of the exogenous vector (HS). The values of the target genes were normalized by the average values of the housekeeping genes (HPRT1 and PPIA) and the fold changes were calculated using the 2(−ΔCT) equation. After 10, 11 and 14 days of transduction, the first colonies appeared in the LO, HO, and HLO groups. Their reprogramming efficiency was 1.67%, 0.08%, and 0.025%, respectively. The cells were primarily identified by their morphology: colonies with well-defined edges and cells with a high nuclear/cytoplasm ratio. Our results indicated that genes related to pluripotency increased at first, and then some decreased at Day 18, probably due to the time of picking the first colonies. The LO group showed more elevated levels of these genes than HO group, in accordance with the higher reprogramming efficiency found in LO. Lentiviral vector expression peaked at Day 3 and then decreased at Day 3 in LO and at Day 12 in HO. Glycolysis genes were less expressed in LO, which may suggest a successful metabolism change. The mitochondrial gene MF1 was higher in HO than in LO, and expression of DNM1L increased at Day 12 in the LO group. Expression of VEGFA, a direct target of HIF1α, behaved similarly. Expression of HIF1α and HIF2α differed from expected, being higher in LO than in HO. The expression of HIF1α and HIF2α increased when the shift from high to low oxygen occurred, suggesting that these genes were not expressed before, likely because cells were adapted to the low oxygen tension. When the shift occurs, the cells reacted with an increase of HIF1α and HIF2α. The study of these effects during reprogramming invitro is critical, and biological repetitions to reinforce this data are underway. Herein, we show that the invitro reprogramming process may be modulated by environmental oxygen changes.

218 Urine samples as a noninvasive source of induced pluripotent cells in the swine model

Noninvasive collection of cells used for generation of induced pluripotent stem cells (iPSC) generation would simplify its use for regenerative and reproductive purposes in veterinary medicine, although it is still unpublished in species other than humans. This study aimed to derive urine progenitor cells (UPCs) invitro from urine samples collected from swine, and then to reprogram them into iPSCs. For that, urine samples were collected from three females, and cells were isolated and cultured from each following the human UPCs protocol (Steichen et al. 2017 Curr. Protoc. Hum. Genet. 21, 7.1-21.7.22; https://doi.org/10.1002/cphg.26). Approximately 200mL of urine samples were collected in sterile flasks and centrifuged at 300×g; the pellet was washed in Dulbecco's PBS, resuspended, and cultured in 45% Dulbecco's modified Eagle's medium (DMEM)-high glucose, 5% fetal bovine serum (FBS), 50% Renal Epithelial Cell Growth basal medium (REBM) supplemented with 1% glutamine, 1% nonessential amino acids (NEAA), penicillin/streptomycin (P/S), and REGM supplements hEGF, insulin, hydrocortisone, GA-1000, FBS, transferrin, triiodothyronine, epinephrine (Lonza), and 10ngmL−1 basic fibroblast growth factor (bFGF). The UPCs first colonies were observed approximately 1 week after and resembled epithelial-like cells. At passage 2, one cell line was transduced with murine OCT4, SOX2, KLF4, and C-MYC cDNAs (OSKM) using a lentiviral vector. After 5 days, cells were plated onto mouse embryonic fibroblasts and cultured in knockout DMEM/F12, 20% knockout serum replacement, NEAA, L-glutamine, 2-mercaptoethanol, and P/S supplemented with 10ngmL−1 bFGF. Efficiency of reprogramming was 8.45%, measured by analysing the number of typical iPSC colonies relative to the transduced cells plated, after ~12 days. Three clonal lines (C1, C4, and C6) were maintained invitro and characterised regarding pluripotency markers for at least 30 passages. All three lines were positive for alkaline phosphatase activity in passages 15 and also 22. Immunocytochemistry analysis revealed that C6 (passage 22) was positive for the pluripotency genes OCT4 (1:100, SC), SOX2 (1:500, AB), SSEA1 (1:50, SC), TRA1- 81 (1:50, Millipore), and NANOG (1:100, AB), whereas C1 and C4 (passages 23 and 22, respectively) were positive only for OCT4, SOX2, and SSEA1. The expression of exogenous and endogenous pluripotency factors (OCT4, SOX2, and NANOG) was evaluated by qRT-PCR, comparing the three clonal lines at passages 16/17 and 21/22 and comparing different passages (10, 11, 14, 17, and 22) on C1 line. No statistical difference was observed between cell lines when compared in different passages, perhaps because of the great variation between lines. However, analysis of C1 line over time showed that pluripotency genes increased and exogenous vector expression decreased during early passages (±10 passages); however, after passage 17, OCT4 and NANOG decreased whereas SOX2 and exogenous vector expression increased. In conclusion, it was possible to reprogram UPCs into iPSCs and maintain them in culture for at least 30 passages; however, iPSCS were dependent on exogenous factors. These results represent the partial reprogramming of UPCs to iPSCs in animals for the first time, enabling the generation of invitro disease models using a noninvasive method. Financial support for this study was received from FAPESP (2019/02811-2, 2015/26818-5), CNPq (433133/2018-0), and CAPES.

16 Improvement of porcine cloned embryo developmental competence via KDM4A overexpression and H3K9me3 methyltransferase inhibitor treatment

Aberrant epigenetic reprogramming is a major reason for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. Histone H3 lysine 9 trimethylation (H3K9me3), a histone marker for transcriptional repression, is considered a key barrier to the development of cloned embryos. In the present study, we found that H3K9me3 was much higher in SCNT embryos than in IVF embryos at the 4-cell and 2-cell stages; H3K9me3 demethylase KDM4A mRNA was injected into cloned embryos 5h after activation. The intensity of H3K9me3 modification decreased obviously after microinjection of KDM4A mRNA, and the developmental efficiency of porcine cloned embryos was enhanced significantly compared with control (32.2±4.2 vs. 21.0±3.5; P<0.05). Moreover, we evaluated the effect of chaetocin, an inhibitor of histone methyltransferase suv39h1/2, on SCNT embryo development. The results showed that 10 nM chaetocin not only suppressed the H3K9me3 modification in porcine embryonic fibroblast but also downregulated the expression of SUV39H1, SUV39H2, and KDM4D. However, treatment of cloned embryos with 10 nM chaetocin efficiently decreased the H3K9me3 level. Importantly, chaetocin treatment at the 4-cell stage for 6h significantly increased the blastocyst rate (57.8±4.3 vs. 43.5±1.8; P<0.05) compared with the control group. Furthermore, because a recent study showed that a high level of H3K9me3 appears in the reprogramming resistance region of nuclear transfer embryos during zygogene activation (ZGA), we examined the expression levels of ZGA-related genes. The qPCR results showed that the expression of ZGA-related genes increased significantly in SCNT embryos with chaetocin treatment compared with the control. These results suggested that chaetocin treatment can improve the efficiency of SCNT reprogramming during ZGA. In summary, our results suggested that H3K9me3 acts as an epigenetic barrier in porcine SCNT reprogramming and that a suv39h1/2 inhibitor can effectively reduce the H3K9me3 level in the early reprogramming phase and further improve the invitro developmental competence. Due to the existence of a variety of abnormal epigenetic mechanisms during somatic cell reprogramming, the combined use of small-molecule inhibitors is required in future studies. Data analyses were performed using SPSS software. Significance was set at P<0.05 unless otherwise specified. The results are expressed as the mean±standard deviation. This work was supported by the National Natural Science Foundation of China (grant number 31601942); the Postdoctoral Science Foundation of Heilongjiang Province (grant number LBH-Z17010); and the Fund for the National Key Research and Development program of China-Stem Cell and Translational Research (2016YFA0100200).

21 The contrasting role of histone methyltransferases during nuclear reprogramming: SUV39H knockdown improves bovine somatic cell nuclear transfer, while the absence of EHMT2 hampers it

The persistence of somatic epigenetic memory is indicated as the main barrier for efficient nuclear reprogramming. The augmented levels of histone methylation on somatic nuclei have been shown as the major regulator of this aberrant remodelling. Although they occupy the same site, H3K9me2 and H3K9me3 are catalysed by different histone methyltransferases (HMTs), EHMT2 and SUV39H, respectively. However, the influence of these HMTs during nuclear reprogramming is unclear. Hence, the main goal of this project was to investigate the role of HMTs responsible for H3K9 methylation during nuclear reprogramming and its consequences on embryo development. For this, we employed a small interfering RNA (siRNA)-mediated knockdown approach targeting EHMT2, SUV39H1, and SUV39H2 in bovine fetal fibroblasts. The RT-qPCR analyses showed ~80% reduction in total RNA after siRNA treatments for the target genes when compared with the control vector. We then quantified the H3K9me2 and H3K9me3 levels by immunostaining. The analysis displayed that H3K9me2 levels were diminished ~50% compared with control, whereas the reduction of H3K9me3 levels was only ~25%. Cells transfected with siRNA targeting EHMT2, SUV39H1, and SUV39H2 (All-siRNA) or control vector (control) were used as a nuclear donor on somatic cell nuclear transfer (SCNT) in five biological replicates. The IVF embryos were used as a biological control for immunostaining analysis. Embryos at both 8- to 16-cell and blastocysts stage (n=10 from 5 replicates) were collected to evaluate the effect of HMT knockdown on H3K9me2 and H3K9me3 levels by immunostaining. We found a reduction of blastocyst rates in the treatment 28±6.3 (mean±s.e.m.) when compared with control 60±4.8 (P=0.004). The immunostaining analysis showed that the levels of H3K9me2 and H3K9me3 were higher in cloned (control) than IVF embryos (P<0.05). Moreover, the All-siRNA group displayed a reduction in H3K9me2 levels compared with the control group and IVF through developmental stages analysed (P<0.05). Differently, H3K9me3 levels were higher in the All-siRNA group at the 8- to 16-cell stage, but no difference was found between treated and control groups at the blastocyst stage. We, therefore, decided to test whether individual knockdown would display a different result. We then used cells transfected with siRNA targeting only EHMT2 (EHMT2-siRNA), targeting SUV39H1 + SUV39H2 (SUV-siRNA), or control vector (control) as a nuclear donor on SCNT in five biological replicates. Surprisingly, the SUV-siRNA group increased blastocyst production 38±4.4 when compared with the control group 29±4.4 (P=0.01), whereas the EHMT2-siRNA showed a reduction in blastocyst rates: 21±5.6 (P=0.04). Our results indicate that EHMT2 has a key role during SCNT, possibly by its crosstalk with other modifications. Even though the SUV39H knockdown induced a small reduction in H3K9me3 levels in the nuclear donor, it was enough to increase the blastocyst rates by 10%. These results will allow us to better understand the complex mechanisms involved in the persistent epigenetic memory during nuclear reprogramming.

The landscape of tiered regulation of breast cancer cell metabolism

Altered metabolism is a hallmark of cancer, but little is still known about its regulation. In this study, we measure transcriptomic, proteomic, phospho-proteomic and fluxomics data in a breast cancer cell-line (MCF7) across three different growth conditions. Integrating these multiomics data within a genome scale human metabolic model in combination with machine learning, we systematically chart the different layers of metabolic regulation in breast cancer cells, predicting which enzymes and pathways are regulated at which level. We distinguish between two types of reactions, directly and indirectly regulated. Directly-regulated reactions include those whose flux is regulated by transcriptomic alterations (~890) or via proteomic or phospho-proteomics alterations (~140) in the enzymes catalyzing them. We term the reactions that currently lack evidence for direct regulation as (putative) indirectly regulated (~930). Many metabolic pathways are predicted to be regulated at different levels, and those may change at different media conditions. Remarkably, we find that the flux of predicted indirectly regulated reactions is strongly coupled to the flux of the predicted directly regulated ones, uncovering a tiered hierarchical organization of breast cancer cell metabolism. Furthermore, the predicted indirectly regulated reactions are predominantly reversible. Taken together, this architecture may facilitate rapid and efficient metabolic reprogramming in response to the varying environmental conditions incurred by the tumor cells. The approach presented lays a conceptual and computational basis for mapping metabolic regulation in additional cancers.

CAMP/EPAC Signaling Enables ETV2 to Induce Endothelial Cells with High Angiogenesis Potential.

Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis invivo as well as invitro. Moreover, these cells could form aligned endothelium along the vascular lumen exvivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential.