Since the production of the first sheep by somatic cell nuclear transfer a great deal of effort has been made to improve efficiency and to understand nuclear reprogramming mechanisms. Unfortunately efficiency remains low, and nuclear reprogramming mechanisms remain uncharacterized. The objectives of this research were to identify factors associated with somatic cell nuclear transfer efficiency and to analyze the transcriptome of blastocyst-stage clone and control embryos and cotyledonary tissue in an effort to elucidate mechanisms responsible for the low developmental efficiency and high post-implantation losses. The experiments reported here identify factors including oocyte source and timing of activation following nuclear transfer that yield improved efficiencies. It was determined the use of cow oocytes for somatic cell nuclear transfer results in improved in vitro development and increased pregnancy rates. These data further indicate prolonged exposure of the donor nucleus to pre-activated oocyte cytoplasm results in increased nuclear fragmentation and reduced developmental efficiency in vitro. Several aberrantly expressed genes were identified in nuclear transfer blastocysts and cotyledons that could impact cloning efficiency. Major histocompatibility complex I and down-regulator of transcription 1 were overexpressed in nuclear transfer blastocysts, and retinol binding protein 1 was overexpressed in nuclear transfer cotyledons. The functions of these genes in immune response, transcriptional regulation, and retinol binding and transport make them attractive candidates for further nuclear transfer research. Expression levels of six developmentally important genes were analyzed in various stages of preimplantation nuclear transfer embryos by real-time polymerase chain reaction to determine the timing of nuclear reprogramming following nuclear transfer. Five of the six genes were aberrantly expressed multiple developmental stages, however by the blastocyst stage only one gene was aberrantly expressed. These data indicate reprogramming is delayed in nuclear transfer embryos resulting in over- or under-expression of developmentally important genes during early embryogenesis. These experiments report factors associated with improved nuclear transfer efficiency; provide insight into potential mechanisms for low developmental rates, abnormal placentation, and fetal loss of clones; and characterize the timing of nuclear reprogramming following somatic cell nuclear transfer.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20092号 / 農博第2199号 / 新制||農||1046(附属図書館) / 学位論文||H29||N5026(農学部図書室) / 33208 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 松井 徹, 教授 久米 新一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21647号 / 医博第4453号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 藤渕 航, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Somatic Cell Nuclear Transfer (SCNT), commonly known as cloning, is the transfer of a somatic nucleus into an enucleated oocyte to produce a clone. The chromatin structure of somatic cells permits the expression of certain genes, while silencing the rest of the genome. The cytoplasm of oocytes can reprogram a somatic nucleus by reactivating the genes necessary for embryonic development and silencing the somatic genes. However, the low efficiency of SCNT indicates that successful nuclear reprogramming is a rare event. The objectives of this study were determine the extent of transcriptional reprogramming in bovine blastocysts produced by serial rounds of chromatin transfer (from first and fourth generations), using blastocysts produced by in vitro fertilization (IVF) as controls, to identify cumulative errors in the transcriptome profile. Differentially expressed genes were studied further to determine their function in embryonic development. We identified a set of transcripts consistently misregulated in cloned blastocyst, some of which had a more marked misregulation in the embryos produced by 4 successive rounds of cloning. Among the genes significantly upregulated in both CT groups compared to IVF blastocysts were both de novo DNA methylation enzymes DNMT3A and DNMT3B. Expression patterns, structural and functional analyses were performed for DNA methyltransferases. A high structural and functional conservation was observed for DNA methyltransferases among human, mouse, and bovine species. A set of genes that participate in early embryonic development, chromatin remodeling and DNA methylation were differentially regulated in cloned embryos and had not been fully annotated at the time of the analysis. We annotated those genes and submitted them to the Bovine Genome Sequencing Consortium database. These results have important implications for the selection of models for the study of DNA methylation during early development. The present study provides a valuable data set for identifying possible cumulative errors in somatic cell chromatin transfer that could hinder nuclear reprogramming shedding light on the epigenetic role in reprogramming and cell plasticity.

Skeletal muscle can perceive cellular energy status and substrate availability and demonstrates remarkable plasticity in response to environmental changes. Nonetheless, how skeletal muscle and its resident stem cells (satellite cells; SCs) sense and respond to nutrient flux remains largely undefined. The dynamic post-translational modification O-GlcNAcylation has been shown to serve as a cellular nutrient sensor in a wide range of cells and tissues, yet its role in skeletal muscle and SCs remains unexplored. Here, we ablated skeletal muscle O-GlcNAc transferase (OGT), and thus O-GlcNAcylation, and found the knockout mice exhibited enhanced glucose uptake, insulin sensitivity, and resistance to high-fat diet induced obesity. Additionally, mKO mice had a 3-fold increase in circulating levels of interleukin-15 (IL-15), a potent anti-obesity cytokine, potentially through epigenetic regulation of Il15 by OGT. To further investigate if there was a causal relationship between OGT ablation and the lean phenotype, we generated muscle specific OGT and interleukin-15 receptor alpha (IL-15ra) double knockout mice (mDKO). As a result, mDKO mice had blunted IL-15 secretion and minimal protection against HFD-induced obesity. Together, these data indicate the skeletal muscle OGT-IL15 axis plays an essential role in the maintenance of skeletal muscle and whole-body metabolic homeostasis. As satellite cells (SCs) play an indispensable role in postnatal muscle growth and adult regenerative myogenesis, we investigated the role of O-GlcNAcylation in SC function. To this end, we conditionally ablated OGT in SCs (cKO) and found cKO mice had impaired SC proliferation, in vivo cycling properties, population stability, metabolic regulation, and adult regenerative myogenesis. Together these findings show that SCs require O-GlcNAcylation, presumably to gauge nutritional signals, for proper function and metabolic homeostasis. Another critical yet often neglected player in myogenesis are mitochondria. Traditionally depicted as a power plant in cells, mitochondria are critical for numerous nonconventional, energy-independent cellular process. To investigate the role of both mitochondrial energy production and alternative mitochondrial functions in myogenic regulation, we ablated ATP synthase subunit beta (ATP5b) and ubiquinol-cytochrome c reductase (UQCRFS1) in C2C12 myoblasts to disrupt mitochondrial ATP production and mitochondrial membrane potential, respectively. Ablation of UQCRFS1, but not ATP5b, impaired myoblast proliferation, although lack of either gene compromised myoblast fusion. Interestingly, addition of the potent myogenic stimulator IGF-1 rescued ATP5b fusion but could not override UQCRFS1 knockout effects on proliferation or differentiation. These data demonstrate mitochondrial ATP production is not the "metabolic switch" that governs myogenic progression but rather an alternative mitochondrial function. In summary, skeletal muscle and their resident stem cell population (SCs) both use O-GlcNAcylation, feasibly to sense and respond to nutritional cues, for the maintenance of metabolic homeostasis and normal physiology. A deeper understand of both muscle and SC metabolic regulation may provide therapeutic targets to improve global metabolism and muscle growth. / Doctor of Philosophy / Skeletal muscle is responsible for approximately 20% of basal energy expenditure and 70-90% of insulin-mediated glucose disposal, and as such changes in skeletal muscle metabolism and insulin sensitivity have profound impacts on whole body metabolism. Skeletal muscle is a plastic tissue that can perceive nutrient availability, which permits metabolic adaptations to environmental changes. Deletion of the nutrient sensing pathway O-GlcNAcylation in skeletal muscle (mKO) protected mice from high-fat diet induced obesity and ameliorates whole-body insulin sensitivity. Skeletal muscle can secrete myokines to elicit endocrine effects on other tissues in the body, and as such, we proposed perturbation of this nutrient sensing pathway in skeletal muscle alters myokine secretion to elicit responses in other metabolically active tissues to support its energy requirements. Indeed, circulating levels of interleukin-15, a potent anti-obesity myokine, increased 3-fold in mKO mice. To determine the contribution of IL-15 to the mKO phenotype, we used a genetic approach to blunt IL-15 secretion from skeletal muscle (mDKO), which partially negated the lean mKO phenotype. Our findings show the ability of skeletal muscle to "sense" changes in nutrients through O-GlcNAcylation is necessary for proper muscle and whole-body metabolism. Moreover, this nutrient sensing mechanism is also important for proper muscle stem cell function, also known as satellite cells (SCs). Loss of O-GlcNAcylation in SCs impairs their ability to regenerate muscle after injury, which can be attributed to a reduced capacity to proliferate and an inability to maintain a healthy SC population. Interestingly, SCs lacking O-GlcNAcylation have a greater mitochondrial content. Using a myoblast cell line, we investigated the contribution of mitochondria to myogenesis, the formation of muscle, and found mitochondrial energy production is dispensable in the myogenic process. Our studies show skeletal muscle and SCs rely on highly integrated signaling cascades that sense and respond to intrinsic metabolic changes and extrinsic nutritional cues to function properly.

Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases. / Master of Science / Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts (the stage of development just prior to implantation), or can be made by dedifferentiating, or reprogramming, adult cells into stem cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient’s own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create cell lines in the laboratory with which to study regenerative medicine. First, we knocked down the expression of myogenin, a key factor in muscle cell development, to induce the dedifferentiation of mouse myotubes (adult muscle cells) into myoblasts (progenitor cells). Various methods of analysis supported successful dedifferentiation into myoblasts, but the inability to completely separate myotubes and myoblasts prior to myogenin knockdown confounded the results. With better separation of the cells, this system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing. Second, we used a viral vector encoding reprogramming factors to create both mouse and pig induced pluripotent stem cells (iPSCs) from skin cells. Pluripotent cells have the ability to differentiate into any cell type in the body, except for the placenta. Multiple pluripotency assays indicated that both the mouse and pig iPSCs were truly pluripotent. Additionally, we were able to differentiate the iPSCs into adult cells, then reprogram those back into “secondary” iPSCs. The production of secondary iPSCs is much more efficient compared to the initial creation of the primary iPSCs, which increases the usefulness of these cells for future experiments. Unfortunately, the porcine iPSCs were dependent on the reprogramming vector to maintain pluripotency. This indicates that these cells are not fully reprogrammed. Despite this, the system can still be used in the future to study the process of reprogramming, to develop cellular differentiation protocols, and to model diseases.

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Nous avons montré, aussi bien in vitro que in vivo, l’impossibilité de la reprogrammation directe des macrophages en cellules béta, et ce en en utilisant les facteurs publiés dans la littérature actuelle comme étant déterminants et importants pour la différenciation en cellules béta du pancréas. Les macrophages préalablement transduits par des virus, ont été cultivés dans des conditions spécifiques visant à mimer le microenvironnement pancréatique, ou injectés dans le pancréas des souris afin de faciliter la reprogrammation. De plus, l’échec dans la reprogrammation des lignages, la culture des macrophages en présence de composés régulant la différentiation des cellules béta induit la formation d’organoïdes. Ces “clusters” de cellules montrent une forte inhibition du potentiel prolifératif comme a été démontré par l’arrêt du cycle cellulaire. Le criblage de différents composés a permis l’identification de la nicotinamide (vitamine B3) comme étant la molécule responsable de l’inhibition de la progression du cycle cellulaire. Les analyses transcriptomiques ont montré l’augmentation de l’expression des inhibiteurs du cycle cellulaire ainsi que la réduction des niveaux d’expression des régulateurs positifs, confirmant que la nicotinamide régule négativement le cycle cellulaire. De manière intéressante, nous avons montré que ce phénomène est réversible, étant donné que le retrait de la nicotinamide résulte en un réengagement dans le cycle cellulaire et la formation de colonies. Ces résultats indiquent le possible rôle de la nicotinamide dans la régulation du potentiel prolifératif des macrophages Maf-DKO. / We have shown here that direct reprogramming of macrophages into beta cells was not possible, both in vitro and in vivo, using pancreatic fate determinants already reported in current literature. Transduced macrophages were cultured in specific conditions or injected into the murine pancreas, aiming to mimic the pancreatic microenvironment as a reprogramming inducer. Besides this failure in lineage reprogramming, culturing macrophages with compounds regulating beta cell differentiation induced the formation of organoids. These cell clusters showed strong inhibition of the proliferative potential, as demonstrated by the arrest of their cell cycle. Screening of the different compounds, allowed the identification of nicotinamide as the responsible molecule for the inhibition of cell cycle progression. Transcriptomic analysis depicted an increased expression of cell cycle inhibitors along with reduced levels of positive regulators, confirming the nicotinamide-induced negative regulation of cell cycle. Most importantly, we have demonstrated that this phenomenon was a reversible proliferation inhibition, since withdrawal of nicotinamide resulted in cell cycle re-entry and rescue of the colony formation phenotype. Together these results indicate a possible role of nicotinamide (Vitamin B3) in the regulation the Maf-DKO macrophages proliferative ability. Further investigations are needed to assess a role for Nicotinamide in controlling the biology of wild-type macrophages, and determine the molecular pathways controlling this transient phenomenon.

Técnicas de reprodução assistida (TRAs) são utilizadas tanto na medicina humana quanto na medicina veterinária com o objetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática (TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamente idênticos, permitindo a produção de rebanhos homogêneos de alto mérito genético e servindo como modelo de estudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é considerada responsável pelo aumento na geração de conceptos portadores de alterações durante e após o desenvolvimento embrionário e fetal. A provável causa principal é a alteração na regulação da reprogramação epigenética devido à manipulação de gametas e embriões no período inicial do desenvolvimento, levando a alterações na regulação epigenética de genes imprinted. O presente estudo teve como objetivo principal avaliar marcas epigenéticas e expressão de genes imprinted no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial (IA). Para tal, foram coletadas amostras de tecido muscular e membranas corioalantoideana e amniótica de animais na fase pré natal (fetal) e tecidos muscular, nervoso e hepático na fase pós natal (animais nascidos saudáveis adultos ou não) de animais derivados de IA ou TNCS. Foi analisada a expressão dos genes imprinted H19, IGF2, IGF2R e Airn quando possível, assim como a metilação do DNA no locus H19/IGF2 na fase pós natal. Foi observado que na fase pré natal não foi detectada expressão do IGF2, enquanto que a expressão de H19 é aumentada em relação ao IGF2R, porém, sem diferenças entre os grupos nos tecidos estudados. Na fase pós natal, o padrão de expressão dos genes IGF2, H19 e IGF2R indica diminuição da expressão gênica relativa no fígado de animais TNCS e no aumento da expressão gênica do H19 na musculatura de animais adultos (saudáveis) bovinos produzidos por TNCS, apesar de o padrão de metilação dos genes imprinted IGF2/H19 não ser diferente entre organismos considerados saudáveis e não saudáveis. Os resultados deste projeto contribuem para o entendimento dos mecanismos epigenéticos relacionados ao desenvolvimento embrionário e fetal, em especial aqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico / Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicine aiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) is of particular importance in veterinary as it enables the generation of genetically identical organisms, allowing the production of homogeneous genetically improved herds, and also serving as a model for reprogramming studies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmental-related abnormalities in the conceptuses. Such phenotypes are probably caused by a disruption during the epigenetic reprogramming due to the manipulation of gametes and embryos during the early development period, and therefore leading to disturbances in the epigenetic regulation of imprinted genes. The present study aimed to evaluate epigenetic marks and expression of imprinted genes in different developmental periods of cattle generated by SCNT or artificial insemination (AI). For that, corionic/alantoic and amniotic membranes from fetuses and muscular, nervous and hepatic tissues from born animals, healthy (adult) or not, produced by SCNT or AI were collected. The expression of the imprinted genes H19, IGF2, IGF2R and Airn was analyzed as well as the DNA methylation at locus H19/IGF2 in post-natal period. It was observed that IGF2 was not detected during pre-natal period, whereas H19 expression is increased when compared to IGF2R in the groups studied herein. At post-natal period the IGF2, H19 and IGF2R expression patterns infers the decrease of relative gene expression in the liver and the increase of H19 expression in the muscle of SCNT adult animals. The methylation pattern of IGF2/H19 locus, however, did not differ between healthy or not animals. The results described herein may contribute to the understanding of the epigenetic mechanisms related to embryonic and fetal development, and in special, to those related to the epigenetic dynamics during genomic imprinting

A geração de células-tronco pluripotentes induzidas (iPS) a partir de células somáticas demonstrou que células adultas de mamíferos podem ser reprogramadas a um estágio de pluripotência através da inserção de fatores de transcrição embrionários. Esta descoberta tem levantado questões fundamentais sobre os mecanismos, que através destes fatores de transcrição, influenciam epigeneticamente as células e seus potenciais de diferenciação após a reprogramação e um normal desenvolvimento. Componentes lipídicos e lipoprotéicos afetam vários aspectos no comportamento celular durante sua manutenção e diferenciação, podendo afetar diretamente fatores essenciais em processos de reprogramação celular, manutenção da pluripotência e perfil epigenético das células. Nesse sentido, esta tese propôs o estudo da composição lipídica com diferentes abordagens entre células iPS, células-tronco embrionárias (H1) e células fibroblastos (BJ). Foram produzidas três linhagens de células pluripotentes induzidas no modelo humano que foram caracterizadas quanto 1a sua pluripotência e utilizadas, juntamente às linhagens H1 e BJ como modelos para o estudo da composição lipídica proposto. Foram identificadas e estudadas um total de 44 espécies lipídicas das classes PC, PE, PI, SM e PS, e discutidas frente a reprogramação celular e manutenção da pluripotência. Foi identificado um padrão de composição fosfolipídica distinta entre células pluripotentes e não pluripotentes, e especulamos que a presença dessas espécies parecem ter um envolvimento fundamental para a manutenção da pluripotência. Este padrão, mostrou pela análise de componente principal, que durante o processo de reprogramação, alterações na composição lipídica ocorrem de forma com que a pluripotência surge durante a reprogramação, evidenciando alterações lipídicas particulares do estádio da pluripotência, sugerindo uma ligação entre estas alterações na composição lipídica com as alterações metabólicas da própria reprogramação celular. O estudo da quantificação de fosfolipídios entre linhagens celulares pluripotentes e não pluripotentes evidenciaram que existe uma diferença fosfolipídica entre estas linhagens, observamos que as linhagens iPS e H1, do ponto de vista das classes observadas e os fosfolipídios quantificados, são similares entre si e diferentes de células não pluripotentes. É evidente que estas moléculas lipídicas, individualmente, não são capazes de modular processos como a reprogramação celular, entretanto, é de extrema importância o entendimento das mesmas dentro da reprogramação celular e manutenção da pluripotência. Nossos dados sugerem que a composição lipídica de células pluripotentes tem importante papel para o desenvolvimento e evolução do processo de reprogramação celular e o entendimento da manutenção da pluripotência / The generation of induced pluripotent stem cells (iPS) from adult somatic cells has shown that mammalian cells can be reprogrammed to a pluripotent state by the insertion of embryonic transcription factors. This finding has raised questions about the fundamental mechanisms through which these transcription factors epigenetically influence cells, their potential of differentiation after reprogramming and normal development. Lipid and lipoprotein components affect numerous aspects of cell behavior during its maintenance and differentiation, which can directly affect main factors in cell reprogramming processes, maintenance of pluripotency and epigenetic profile of the cells. Thus, this thesis proposed to study, with different approaches, the lipid composition of iPS cells, embryonic stem cells (H1) and fibroblast cells (BJ). Three induced pluripotent cell lines were produced in the human model. They were characterized regarding their pluripotency and used along with H1 and BJ cell lines, as models for the proposed lipid composition study. A total of 44 species of lipid from the classes PC, PE, PI, PS and SM have been identified, studied and discussed regarding cellular reprogramming and maintenance of pluripotency. A different phospholipid composition pattern was observed between pluripotent and non-pluripotent cells, and it is speculated that the presence of these species appears to have a major involvement on the maintenance of pluripotency. This array showed, by the principal component analysis, that during the reprogramming process changes in the lipid composition occur, so that pluripotency takes place during reprogramming, highlighting lipid changes particular of the pluripotency state, suggesting a connection between these changes in lipid composition and the metabolic changes of cell reprogramming. The study of the quantitation of phospholipids from pluripotent and non-pluripotent cell lines indicated a phospholipid difference between these cell lines when considering the observed classes and quantified phospholipid. It was eminent that iPS lines and H1 are similar and differ from non-pluripotent cells. It is clear that these lipid molecules are not individually capable of modulating processes such as cell reprogramming, however, it is extremely important to understand them within cellular reprogramming and maintenance of pluripotency. Our data suggests that the lipid composition of pluripotent cells has important role in the development and evolution of cellular reprogramming process and the understanding the maintenance of pluripotency