Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway.

Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal-epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing.

BACKGROUND: Because few studies exist to describe the unique molecular network regulation behind pig pre-implantation embryonic development (PED), genetic engineering in the pig embryo is limited. Also, this lack of research has hindered derivation and application of porcine embryonic stem cells and porcine induced pluripotent stem cells (iPSCs). RESULTS: We identified and analyzed the genome wide transcriptomes of pig in vivo-derived and somatic cell nuclear transferred (SCNT) as well as mouse in vivo-derived pre-implantation embryos at different stages using mRNA deep sequencing. Comparison of the pig embryonic transcriptomes with those of mouse and human pre-implantation embryos revealed unique gene expression patterns during pig PED. Pig zygotic genome activation was confirmed to occur at the 4-cell stage via genome-wide gene expression analysis. This activation was delayed to the 8-cell stage in SCNT embryos. Specific gene expression analysis of the putative inner cell mass (ICM) and the trophectoderm (TE) revealed that pig and mouse pre-implantation embryos share regulatory networks during the first lineage segregation and primitive endoderm differentiation, but not during ectoderm commitment. Also, fatty acid metabolism appears to be a unique characteristic of pig pre-implantation embryonic development. In addition, the global gene expression patterns in the pig SCNT embryos were different from those in in vivo-derived pig embryos. CONCLUSIONS: Our results provide a resource for pluripotent stem cell engineering and for understanding pig development.

Genetic background influences pig responses to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious and economically significant virus that causes respiratory and reproductive diseases in pigs. It results in reduced productivity and increased mortality in pigs, causing substantial economic losses in the industry. Understanding the factors affecting pig responses to PRRSV is crucial to develop effective control strategies. Genetic background has emerged as a significant determinant of susceptibility and resistance to PRRSV in pigs. This review provides an overview of the basic infection process of PRRSV in pigs, associated symptoms, underlying immune mechanisms, and roles of noncoding RNA and alternative splicing in PRRSV infection. Moreover, it emphasized breed-specific variations in these aspects that may have implications for individual treatment options.

Recent Progress on Circular RNAs in the Development of Skeletal Muscle and Adipose Tissues of Farm Animals.

Circular RNAs (circRNAs) are a highly conserved and specifically expressed novel class of covalently closed non-coding RNAs. CircRNAs can function as miRNA sponges, protein scaffolds, and regulatory factors, and play various roles in development and other biological processes in mammals. With the rapid development of high-throughput sequencing technology, thousands of circRNAs have been discovered in farm animals; some reportedly play vital roles in skeletal muscle and adipose development. These are critical factors affecting meat yield and quality. In this review, we have highlighted the recent advances in circRNA-related studies of skeletal muscle and adipose in farm animals. We have also described the biogenesis, properties, and biological functions of circRNAs. Furthermore, we have comprehensively summarized the functions and regulatory mechanisms of circRNAs in skeletal muscle and adipose development in farm animals and their effects on economic traits such as meat yield and quality. Finally, we propose that circRNAs are putative novel targets to improve meat yield and quality traits during animal breeding.

Differences in histomorphology and expression of key lipid regulated genes of four adipose tissues from Tibetan pigs.

Tibetan pigs, an indigenous pig breed in China, have high overall fat deposition and flavorful and tasty meat. They are thus good models for studying adipogenesis. Few studies have been conducted focusing on expression of lipid regulated genes in different adipose tissues of Tibetan pigs. Therefore, we compared the difference of histomorphology and expression level of lipid regulated genes through qPCR and western blot in subcutaneous fat, perirenal fat, omental adipose tissue, and inguinal fat of Tibetan pigs. Our results showed that the area of subcutaneous adipocytes in Tibetan pigs was smaller, while the other three adipose tissues (perirenal fat, greater omentum fat, inguinal fat) had cell areas of similar size. The gene expression of FABP4, FASN, FABP3, and ME1 in subcutaneous fat was significantly higher than that in perirenal fat. Furthermore, the protein expression of FABP4 was significantly lower in subcutaneous fat than in perirenal fat (p < 0.05), and the expression of FASN was higher in greater omentum fat than in subcutaneous fat (p = 0.084). The difference in adipocyte cell size and expression of lipid-regulated genes in adipose tissues from the various parts of the pig body is likely due to the different cellular lipid metabolic processes. Specially, FABP4 and FASN may be involved in the regulation of fat deposition in different adipose tissues of Tibetan pigs.

PRRSV alters m6A methylation and alternative splicing to regulate immune, extracellular matrix-associated function.

The alternative splicing and N6-methyladenosine (m6A) modifications occurring during porcine reproductive and respiratory syndrome virus (PRRSV) infections remain poorly understood. Transcriptome and MeRIP-seq analyses were performed to identify the gene expression changes, splicing and m6A modifications in the lungs of PRRSV-infected pigs. In total, 1624 differentially expressed genes (DEGs) were observed between PRRSV-infected and uninfected pigs. We observed significant alterations in alternative splicing (54,367 events) and m6A modifications (2265 DASEs) in numerous genes, including LMO7, SLC25A27, ZNF185, and ECM1, during PRRSV infection. LMO7 and ZNF185 exhibited alternative splicing variants and reduced mRNA expression levels following PRRSV infection. Notably, LMO7 inhibited c-JUN, SMAD3, and FAK expression, whereas ZNF185 affected the expression of FAK, CDH1, and GSK3ß downstream. Additionally, ECM1 influenced FAK expression by targeting ITGB3 and AKT2, suggesting its involvement in extracellular matrix accumulation through the ITGB3-AKT2/FAK pathway. These changes may facilitate viral invasion and replication by modulating the expression of genes and proteins participating in crucial cellular processes associated with immunity and the extracellular matrix. We highlight the importance of these genes and their associated pathways in PRRSV infections and suggest that targeting these may be a promising therapeutic approach for treating viral infections.

Integrated lipidomics and RNA sequencing analysis reveal novel changes during 3T3-L1 cell adipogenesis.

After adipogenic differentiation, key regulators of adipogenesis are stimulated and cells begin to accumulate lipids. To identify specific changes in lipid composition and gene expression patterns during 3T3-L1 cell adipogenesis, we carried out lipidomics and RNA sequencing analysis of undifferentiated and differentiated 3T3-L1 cells. The analysis revealed significant changes in lipid content and gene expression patterns during adipogenesis. Slc2a4 was up-regulated, which may enhance glucose transport; Gpat3, Agpat2, Lipin1 and Dgat were also up-regulated, potentially to enrich intracellular triacylglycerol (TG). Increased expression levels of Pnpla2, Lipe, Acsl1 and Lpl likely increase intracellular free fatty acids, which can then be used for subsequent synthesis of other lipids, such as sphingomyelin (SM) and ceramide (Cer). Enriched intracellular diacylglycerol (DG) can also provide more raw materials for the synthesis of phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), ether-PE, and ether-PC, whereas high expression of Pla3 may enhance the formation of lysophophatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Therefore, in the process of adipogenesis of 3T3-L1 cells, a series of genes are activated, resulting in large changes in the contents of various lipid metabolites in the cells, especially TG, DG, SM, Cer, PI, PC, PE, etherPE, etherPC, LPC and LPE. These findings provide a theoretical basis for our understanding the pathophysiology of obesity.

Myostatin Alteration in Pigs Enhances the Deposition of Long-Chain Unsaturated Fatty Acids in Subcutaneous Fat.

In animals, myostatin (MSTN) is a negative regulator that inhibits muscle growth and repair. The decreased level of functional MSTN gene expression can change the amount and proportions of fats in pigs. In this study we determined the lipidomics of subcutaneous fat in MSTN single copy mutant pigs and evaluated the variations in lipid contents of the subcutaneous fat from MSTN+/- and wild type Large White (LW) pigs via ultra-performance liquid chromatography-quadrupole/Orbitrap-mass spectrometry (MS). The results showed that the quantities of glycerolipids, sphingolipids, fatty acyls and glycerophospholipids were significantly changed, particularly, the molecular diacylglycerol in glycerolipids, long-chain unsaturated fatty acids, and ceramide non-hydroxy fatty acid-sphingosine in sphingolipids were remarkably increased in the MSTN+/- group. Due to their positive bioavailability demonstrated by previous researches, these three lipids might be beneficial for human health. Further, the results of our study confirm that MSTN participates in the regulation of fat metabolism, and reduced expression of MSTN can ultimately influence the accumulation of lipid contents in the subcutaneous fat of pigs.

Viscera Characteristics of MSTN-Edited Heterozygous Pigs.

Myostatin (MSTN) is a protein that negatively regulates growth of skeletal muscle, and inactivation of MSTN improves the mass of skeletal muscle. Our previous work found that MSTN +/- pigs have higher muscle depth and lower fat depth compared to wild type without any developmental problems. Therefore, MSTN-edited pigs are most likely to appear as heterozygotes in the potential future market, but the characteristics of organs in digestive and reproductive system of pigs with MSTN gene editing remains unclear. Here, we investigated the histological of the organs in the digestive system and reproductive system in MSTN gene heterozygotes at adult stages. The length of intestine was further compared between adult heterozygous and wild type pigs. We found no significant differences in histomorphology of organs, including heart, duodenum, jejunum, ileum, cecum, colon, testis, epididymis, ovaries, oviducts and uterus, between individuals from two genotypes. Moreover, there was no significant difference in the average length of intestine in adult pigs. Our data provide a reference for further clarifying the applications of MSTN gene edited pigs.

Developmental expression of heat shock proteins 60, 70, 90, and A2 in rabbit testis.

Currently, no reports exist concerning the expression patterns and developmental changes of heat shock proteins (HSPs) in the reproductive system of the male rabbit. In the present study, the testes of rabbits were collected at post-natal months 1, 2, 3, 4, 5, and 40. HSP60, HSC70, HSP90, and HSPA2 were detected by both Western blot and immunohistochemical methods. The expression levels of HSP60 and HSC70 showed no apparent change during the developmental progress. HSP90 increased at the second month; prior to the third month, HSPA2 was expressed at a low level. Immunohistochemistry localized HSP60 in the cytoplasm of all of the cell types in the testis and in the apical pole of the spermatids. The distribution pattern of HSC70 and HSP90 was similar, both being mainly located in the spermatids of stage VII-VIII and in the cytoplasm of the spermatogonium. HSPA2 staining was mainly observed in the cytoplasm of pachytene spermatocytes and spermatids in testes of 3-, 4-, 5-, and 40-month-old rabbits. These results provide a basic reference point for studying the functions of HSPs in the male rabbit reproductive system and should be beneficial for the future determination of the mechanisms of heat shock on male rabbit fertility.

Regulation of RNA N6-methyladenosine modification and its emerging roles in skeletal muscle development.

N6-methyladenosine (m6A) is one of the most widespread and highly conserved chemical modifications in cellular RNAs of eukaryotic genomes. Owing to the development of high-throughput m6A sequencing, the functions and mechanisms of m6A modification in development and diseases have been revealed. Recent studies have shown that RNA m6A methylation plays a critical role in skeletal muscle development, which regulates myoblast proliferation and differentiation, and muscle regeneration. Exploration of the functions of m6A modification and its regulators provides a deeper understanding of the regulatory mechanisms underlying skeletal muscle development. In the present review, we aim to summarize recent breakthroughs concerning the global landscape of m6A modification in mammals and examine the biological functions and mechanisms of enzymes regulating m6A RNA methylation. We describe the interplay between m6A and other epigenetic modifications and highlight the regulatory roles of m6A in development, especially that of skeletal muscle. m6A and its regulators are expected to be targets for the treatment of human muscle-related diseases and novel epigenetic markers for animal breeding in meat production.

Validation of the Reference Genes for the Gene Expression Studies in Different Cell Lines of Pig.

Reverse transcription quantitative real-time polymerase chain reaction is one of the important methods to investigate gene expression in cells and tissues. However, if the data cannot be normalized with appropriate reference genes, the results may be unreliable. In this study, we detected the expression of 15 reference genes in three pig cell lines. The results showed that SDHA and ALDOA were the most stable reference genes in 3D4/21 cells. TOP2B, TBP, and PPIA were the most stable reference genes in PK-15 cells. SDHA and ALDOA were the most stable reference genes in IPEC-J2 cells. In addition, each cell line only needs to use two reference genes to standardize the expression of target genes. Taken together, this study provides a reference for different pig cell lines to select reference genes and also provides a theoretical basis for the use of these cell lines in related functional researches.

Integrated Microbiome and Metabolome Analysis Reveals a Positive Change in the Intestinal Environment of Myostatin Edited Large White Pigs.

Myostatin (MSTN) functional inactivation can change the proportion of lean meat and fat content in pigs. While both genotype and microbial composition are known to affect the host phenotype, so far there has been no systematic study to detect the changes in the intestinal microbial composition and metabolome of MSTN single copy mutant pigs. Here, we used 16S rDNA sequencing and metabolome analysis to investigate how MSTN gene editing affects changes in the microbial and metabolome composition in the jejunum and the cecum of Large White pigs. Our results showed that Clostridium_sensu_stricto_1, Bifidobacterium, Lachnospiraceae_UCG-007, Clostridium_sensu_stricto_6, Ruminococcaceae_UCG-002, and Ruminococcaceae_UCG-004 were significantly upregulated; while Treponema_2 and T34_unclassified were significantly downregulated in the jejunum of MSTN pigs. Similarly, Phascolarctobacterium, Ruminiclostridium_9, Succinivibrio, Longibaculum, and Candidatus_Stoquefichus were significantly upregulated, while Barnesiella was significantly downregulated in the cecum of MSTN pigs. Moreover, metabolomics analysis showed significant changes in metabolites involved in purine, sphingolipid and tryptophan metabolism in the jejunum, while those associated with glycerophospholipid and pyrimidine metabolism were changed in the cecum. Spearman correlation analysis further demonstrated that there was a significant correlation between microflora composition and metabolites. Our analyses indicated the MSTN editing affects the composition of metabolites and microbial strains in the jejunum and the cecum, which might provide more useable nutrients for the host of MSTN± Large White pigs.

Developmental stage-specific A-to-I editing pattern in the postnatal pineal gland of pigs (Sus scrofa).

BACKGROUND: RNA editing is a widespread post-transcriptional modification mechanism in mammalian genomes. Although many editing sites have been identified in domestic pigs (Sus scrofa), little is known about the characteristics and dynamic regulation of RNA editing in the pineal gland (PG), a small neuroendocrine gland that synthesizes and secretes melatonin, which is primarily responsible to modulate sleep patterns. RESULTS: This study analyzed the expression of adenosine-to-inosine (A-to-I) editing regulators and profiled the first dynamic A-to-I RNA editome during postnatal PG development. The results identified ADAR1 as the most abundantly expressed ADAR enzyme, which was down-regulated during postnatal PG development. Furthermore, 47,284 high-confidence RNA editing sites were identified, the majority of which (93.6%) were of the canonical A-to-I editing type, followed by C-to-T editing. Analysis of its characteristics showed that the A-to-I editing sites mostly localized in SINE retrotransposons PRE-1/Pre0_SS. Moreover, a strong deficiency and preference for guanine nucleotides at positions of one base upstream or downstream were found, respectively. The overall editing level at the puberty stage was higher than at both infancy and adulthood stages. Additionally, genome-wide RNA editing was found to exhibit a dynamic stage-specific fashion (postnatally). Genes that underwent developmental changes in RNA editing were associated with catabolic processes as well as protein localization and transport functions, implying that RNA editing might be responsible for the molecular machineries of the postnatal developing PG. Remarkably, RNA editing in 3'-UTRs might regulate gene expression by influencing miRNA binding during PG development. CONCLUSIONS: This study profiles the first comprehensive developmental RNA editome in the pig PG, which contributes to the understanding of the importance of post-transcriptionally mediated regulation during mammalian postnatal PG development. Moreover, this study widely extends RNA editome resources in mammals.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

The pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells.

BACKGROUND: Self-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood. METHODS: Here, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478. RESULTS: EGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G0/G1 phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs. CONCLUSIONS: Taken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.

Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency.

BACKGROUND: Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive. METHODS: We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR). RESULTS: The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8. CONCLUSIONS: In summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.

Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs) and high quality porcine induced pluripotent stem cells (piPSCs). In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC) and mouse ESC (mESC) culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs), the other resembled mESCs (here called mpiPSCs). Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

Improvement in Mouse iPSC Induction by Rab32 Reveals the Importance of Lipid Metabolism during Reprogramming.

Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages, and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency, and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.