The Effect of Long-Term Passage on Porcine SMCs' Function and the Improvement of TGF-ß1 on Porcine SMCs' Secretory Function in Late Passage.

Cultured meat is one of the meat substitutes produced through tissue engineering and other technologies. Large-scale cell culture is the key for cultured meat products to enter the market. Therefore, this study is aimed to explore the effect of long-term passage in vitro on smooth muscle cells (SMCs) and the effect of transforming growth factor-ß1 (TGF-ß1) on SMCs in the late passage. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage. Transcriptome results showed that the ECM pathway and aging-related signaling pathways were significantly up-regulated in the late passage period. TGF-ß1 did not promote SMCs of late passage proliferation at the proliferation stage but promoted the gene and protein expression of collagen as the main protein of the extracellular matrix proteins at the differentiation stage. In addition, proteomic analysis revealed that TGF-ß1 promoted the expression of cell adhesion molecules which activate the Hippo signaling pathway and the HIF-1 signaling pathway and further promoted the production of collagen-containing extracellular matrix proteins. This could provide ideas for large-scale production of cultured meat products using SMCs.

Lonicera japonica polysaccharides alleviate D-galactose-induced oxidative stress and restore gut microbiota in ICR mice.

Lonicera japonica polysaccharides (LJPs) exhibit anti-aging effect in nematodes. Here, we further studied the function of LJPs on aging-related disorders in D-galactose (D-gal)-induced ICR mice. Four groups of mice including the control group, the D-gal-treated group, the intervening groups with low and high dose of LJPs (50 and 100 mg/kg/day) were raised for 8 weeks. The results showed that intragastric administration with LJPs improved the organ indexes of D-gal-treated mice. Moreover, LJPs improved the activity of superoxide dismutase (SOD), catalase (CAT) as well as glutathione peroxidase (GSH-Px) and decreased the malondialdehyde (MDA) level in serum, liver and brain. Meanwhile, LJPs restored the content of acetylcholinesterase (AChE) in the brain. Further, LJPs reversed the liver tissue damages in aging mice. Mechanistically, LJPs alleviate oxidative stress at least partially through regulating Nrf2 signaling. Additionally, LJPs restored the gut microbiota composition of D-gal-treated mice by adjusting the Firmicutes/Bacteroidetes ratio at the phylum level and upregulating the relative abundances of Lactobacillaceae and Bifidobacteriacesa. Notably, the KEGG pathways involved in hazardous substances degradation and flavone and flavonol biosynthesis were significantly enhanced by LJPs treatment. Overall, our study uncovers the role of LJPs in modulating oxidative stress and gut microbiota in the D-gal-induced aging mice.

Critically short telomeres derepress retrotransposons to promote genome instability in embryonic stem cells.

Telomeres, at the ends of chromosomes, protect chromosomes from fusion and preserve genomic stability. However, the molecular mechanisms underlying telomere attrition-induced genome instability remain to be understood. We systematically analyzed the expression of retrotransposons and performed genomic sequencing of different cell and tissue types with telomeres of varying lengths due to telomerase deficiency. We found that critically short telomeres altered retrotransposon activity to promote genomic instability in mouse embryonic stem cells, as evidenced by elevated numbers of single nucleotide variants, indels and copy number variations (CNVs). Transpositions of retrotransposons such as LINE1 resulting from the short telomeres can also be found in these genomes with elevated number of mutations and CNVs. Retrotransposon activation is linked to increased chromatin accessibility, and reduced heterochromatin abundance correlates with short telomeres. Re-elongation of telomeres upon recovery of telomerase partly represses retrotransposons and heterochromatin accumulation. Together, our findings suggest a potential mechanism by which telomeres maintain genomic stability by suppressing chromatin accessibility and retrotransposon activity.

Static Magnetic Fields Promote Generation of Muscle Lineage Cells from Pluripotent Stem Cells and Myoblasts.

Static magnetic fields (SMFs) exhibit numerous biological effects and regulate the proliferation and differentiation of several adult stem cells. However, the role of SMFs in the self-renewal maintenance and developmental potential of pluripotent embryonic stem cells (ESCs) remains largely uninvestigated. Here, we show that SMFs promote the expression of the core pluripotent markers Sox2 and SSEA-1. Furthermore, SMFs facilitate the differentiation of ESCs into cardiomyocytes and skeletal muscle cells. Consistently, transcriptome analysis reveals that muscle lineage differentiation and skeletal system specification of ESCs are remarkably strengthened by SMF stimuli. Additionally, when treated with SMFs, C2C12 myoblasts exhibit an increased proliferation rate, improved expression of skeletal muscle markers and elevated myogenic differentiation capacity compared with control cells. Together, our data show that SMFs effectively promote muscle cell generation from pluripotent stem cells and myoblasts. The noninvasive and convenient physical stimuli can be used to increase the production of muscle cells in regenerative medicine and the manufacture of cultured meat in cellular agriculture.

Snap29 Is Dispensable for Self-Renewal Maintenance but Required for Proper Differentiation of Mouse Embryonic Stem Cells.

Pluripotent embryonic stem cells (ESCs) can self-renew indefinitely and are able to differentiate into all three embryonic germ layers. Synaptosomal-associated protein 29 (Snap29) is implicated in numerous intracellular membrane trafficking pathways, including autophagy, which is involved in the maintenance of ESC pluripotency. However, the function of Snap29 in the self-renewal and differentiation of ESCs remains elusive. Here, we show that Snap29 depletion via CRISPR/Cas does not impair the self-renewal and expression of pluripotency-associated factors in mouse ESCs. However, Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Furthermore, transcriptome analysis reveals that Snap29 depletion significantly decreased the expression of numerous genes required for germ layer differentiation. Interestingly, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our data show that Snap29 is dispensable for self-renewal maintenance, but required for the proper differentiation of mouse ESCs.

Lonicera japonica polysaccharides improve longevity and fitness of Caenorhabditis elegans by activating DAF-16.

Polysaccharide is one of the main active ingredients in Lonicera japonica Thunb. (L. japonica). In this study, we examined the anti-aging activities of L.japonica polysaccharides (LJPs) and further explored the mechanisms. Polysaccharides from L.japonica including the crude LJP (CLJP) and the purified fraction (LJP-2-1) were characterized. The molecular weights of CLJP and LJP-2-1 were 1450 kDa and 1280 kDa, respectively. Meanwhile, CLJP was mainly composed of galacturonic acid (23.57 %), galactose (23.45 %) and arabinose (23.45 %). LJP-2-1 was mainly composed of galacturonic acid (51.25 %) and arabinose (22.89 %). In Caenorhabditis elegans (C. elegans), LJPs maximally prolonged mean lifespan by 13.97 %, promoted fitness with increased motility by 40.92 % and pharyngeal pumping by 25.72 %, and decreased lipofuscin accumulation by 38.9 % with intact body length and fecundity. Moreover, CLJP extended the mean lifespan of nematodes under oxidative and heat stress by 16.76 % and 14.05 % respectively by activating stress-related genes and the antioxidant system. Further, CLJP required DAF-16 to prolong the lifespan of nematodes. CLJP upregulated the expression of daf-16 and its targeted downstream genes, including sod-3, gst-4 and hsp-16.2. Moreover, nuclear accumulation of DAF-16 was promoted upon CLJP treatment. Together, our data uncover the role of LJPs in extending lifespan and healthspan through DAF-16.

Production of cultured meat from pig muscle stem cells.

Cultured meat is meat for consumption produced in a more sustainable way. It involves cell harvesting and expansion, differentiation into myotubes, construction into muscle fibres and meat structuring. We isolated 5.3 × 104 porcine muscle stem cells from 1 g of neonatal pig muscle tissue. According to calculations, we need to expand muscle stem cells 106-107 times to produce 100 g or 1 kg of cultured meat. However, the cells gradually lost the ability to express stemness and mature muscle cell markers (PAX7, MyHC). To tackle this critical issue and maintain cell function during cell expansion, we found that long-term culture with (100 µM) l-Ascorbic acid 2-phosphate (Asc-2P) accelerated cell proliferation while preserving the muscle cell differentiation. We further optimized a scalable PDMS mold. Porcine muscle stem cells formed structurally-organized myotubes similar to muscle fibres in the mold. Asc-2P enhanced porcine muscle cells grown as 3D tissue networks that can produce a relatively large 3D tissue networks as cultured meat building blocks, which showed improved texture and amino acid content. These results established a realistic workflow for the production of cultured meat that mimics the pork meat structurally and is potentially scalable for industry.

Zscan4 Contributes to Telomere Maintenance in Telomerase-Deficient Late Generation Mouse ESCs and Human ALT Cancer Cells.

Proper telomere length is essential for indefinite self-renewal of embryonic stem (ES) cells and cancer cells. Telomerase-deficient late generation mouse ES cells and human ALT cancer cells are able to propagate for numerous passages, suggesting telomerase-independent mechanisms responding for telomere maintenance. However, the underlying mechanisms ensuring the telomere length maintenance are unclear. Here, using late generation telomerase KO (G4 Terc-/-) ESCs as a model, we show that Zscan4, highly upregulated in G4 Terc-/- ESCs, is responsible for the prolonged culture of these cells with stably short telomeres. Mechanistically, G4 Terc-/- ESCs showed reduced levels of DNA methylation and H3K9me3 at Zscan4 promoter and subtelomeres, which relieved the expression of Zscan4. Similarly, human ZSCAN4 was also derepressed by reduced H3K9me3 at its promoter in ALT U2 OS cells, and depletion of ZSCAN4 significantly shortened telomeres. Our results define a similar conserved pathway contributing to the telomere maintenance in telomerase-deficient late generation mESCs and human ALT U2OS cancer cells.

YAP Promotes Cell Proliferation and Stemness Maintenance of Porcine Muscle Stem Cells under High-Density Condition.

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.

Gene Manipulation Protocols in Autophagy.

Macroautophagy (referred to as autophagy hereafter) is a highly conserved catabolic process in eukaryotic cells. Autophagy is essential for cellular homeostasis through elimination and recycling of large cytoplasmic components, such as abnormal protein aggregates and damaged organelles, via lysosomal degradation. Since being originally identified by genetic screening in yeast, autophagy-related (ATG) genes have played a central role in autophagy research in different organisms, including plants, worms, flies, and mammals. Mouse models for monitoring autophagic activity or clarifying its biological functions have also been established. These mice are powerful tools to investigate roles of autophagy in vivo. Owing to the rapid technological advances in molecular biology, it is ever more efficient and simpler to manipulate autophagy-associated genes. Herein, we will introduce some commonly used approaches of gene silencing in mammalian cells, including CRIPSR/Cas9-mediated gene knockout and siRNA- and shRNA-mediated gene knockdown. We also summarized the common mouse models used for assessing autophagy. We hope to bring the researchers some useful information as they study autophagy.

[Prokaryotic expression, protein purification and functional verification of human homotypic fusion and vacuole protein sorting complex subunit].

Homotypic fusion and vacuole protein sorting(HOPS) is a protein complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39, VPS41 and regulates membrane transport in vivo through membrane fusion mechanisms. The evidence suggests that HOPS complex as a fusion factor, facilitates autophagosome-lysosome fusion. To determine whether the HOPS complex directly interacts with the autophagic SNARE protein STX17 in vitro, the coding sequence of the six genes were amplified from the existing plasmids by PCR, and then ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA. After identification through colony PCR and DNA sequencing, 6 recombinant plasmids were constructed and transferred into Escherichia coli BL21 (DE3). The recombinant proteins were purified by glutathione sepharose 4B and nickel column. We used the tobacco etch virus protease to cut off the GST-tag or His-NusA-tag, to obtain HA-VPS11 protein of about 105 kDa, Flag-VPS16 protein of about 97 kDa, HA-VPS18 protein of about 108 kDa, Flag-VPS33 protein of about 70 kDa, HA-VPS39 protein of about 97 kDa, and Flag-VPS41 protein of about 98 kDa. The function of the purified proteins was verified by in vitro glutathione S-transferases pull-down assay, confirming that autophagic SNARE protein STX17 interacted directly with HOPS components. Our findings provide experimental basis to further study the function and mechanism of HOPS complex in the process of autophagosome-lysosome fusion.

Telomere dysfunction impairs epidermal stem cell specification and differentiation by disrupting BMP/pSmad/P63 signaling.

Telomere shortening is associated with aging and age-associated diseases. Additionally, telomere dysfunction resulting from telomerase gene mutation can lead to premature aging, such as apparent skin atrophy and hair loss. However, the molecular signaling linking telomere dysfunction to skin atrophy remains elusive. Here we show that dysfunctional telomere disrupts BMP/pSmad/P63 signaling, impairing epidermal stem cell specification and differentiation of skin and hair follicles. We find that telomere shortening mediated by Terc loss up-regulates Follistatin (Fst), inhibiting pSmad signaling and down-regulating P63 and epidermal keratins in an ESC differentiation model as well as in adult development of telomere-shortened mice. Mechanistically, short telomeres disrupt PRC2/H3K27me3-mediated repression of Fst. Our findings reveal that skin atrophy due to telomere dysfunction is caused by a previously unappreciated link with Fst and BMP signaling that could be explored in the development of therapies.

Zscan4c activates endogenous retrovirus MERVL and cleavage embryo genes.

Endogenous retroviruses (ERVs) contribute to ∼10 percent of the mouse genome. They are often silenced in differentiated somatic cells but differentially expressed at various embryonic developmental stages. A minority of mouse embryonic stem cells (ESCs), like 2-cell cleavage embryos, highly express ERV MERVL. However, the role of ERVs and mechanism of their activation in these cells are still poorly understood. In this study, we investigated the regulation and function of the stage-specific expressed ERVs, with a particular focus on the totipotency marker MT2/MERVL. We show that the transcription factor Zscan4c functions as an activator of MT2/MERVL and 2-cell/4-cell embryo genes. Zinc finger domains of Zscan4c play an important role in this process. In addition, Zscan4c interacts with MT2 and regulates MT2-nearby 2-cell/4-cell genes through promoting enhancer activity of MT2. Furthermore, MT2 activation is accompanied by enhanced H3K4me1, H3K27ac, and H3K14ac deposition on MT2. Zscan4c also interacts with GBAF chromatin remodelling complex through SCAN domain to further activate MT2 enhancer activity. Taken together, we delineate a previously unrecognized regulatory axis that Zscan4c interacts with and activates MT2/MERVL loci and their nearby genes through epigenetic regulation.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline Alters Autophagosome Maturation, Cellular Lipidomic Profiles, and Expression of Core Pluripotent Factors.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant heterocyclic aromatic amines (HAAs) found in the human diet, is primarily produced during high-temperature meat or fish cooking. While MeIQx has been investigated as a potential carcinogen, the cytotoxicity and related molecular mechanisms remain unclear. Here, we demonstrate that autophagosome maturation is blocked by MeIQx. Mechanistically, MeIQx inhibits acidification of lysosomes rather than prevents autophagosome-lysosome fusion. Moreover, cellular lipid profiles are altered by MeIQx treatment. Notably, many phospholipids and sphingolipids are significantly upregulated after exposure to MeIQx. Furthermore, MeIQx decreases expression of pluripotency-associated proteins in mouse embryonic stem cells (ESCs). Together, MeIQx blocks autophagosome maturation through inhibiting acidification of lysosomes, alters lipid metabolism, and decreases expression of pluripotent factors. Our studies provide more cytotoxic evidence and elucidate related mechanisms on the risk of HAA exposure and are expected to promote supervision of food safety and human health.

Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells.

Feeder cells like mouse embryonic fibroblasts (MEFs) have been widely applied for culture of pluripotent stem cells, but their roles remain elusive. Noticeably, ESCs cultured on the feeders display transcriptional heterogeneity. We investigated roles of feeder cells by examining the telomere maintenance. Here we show that telomere is longer in mESCs cultured with than without the feeders. mESC cultures without MEF feeders exhibit telomere loss, chromosomal fusion, and aneuploidy with increasing passages. Notably, feeders facilitate heterogeneous transcription of 2-cell genes including Zscan4 and telomere elongation. Moreover, feeders produce Fstl1 that together with BMP4 periodically activate Zscan4. Interestingly, Zscan4 is repressed in mESCs cultured in 2i (inhibitors of Mek and Gsk3ß signaling) media, associated with shorter telomeres and increased chromosome instability. These data suggest the important role of feeders in maintaining telomeres for long-term stable self-renewal and developmental pluripotency of mESCs.

IFITM1 suppresses expression of human endogenous retroviruses in human embryonic stem cells.

Interferon-induced transmembrane protein 1 (IFITM1), a member of the IFITM protein family, is a component of a multimeric complex involved in the transduction of antiproliferation and cell adhesion signals. IFITM1 is thought to play a role in antiproliferation and immune surveillance, and has been shown to restrict infection by numerous viruses. It is highly expressed in human embryonic stem cells (hESCs) but its role in hESCs remains to be elucidated. In this study, knockout of IFITM1 mediated by CRISPR/Cas9 in hESCs did not affect self-renewal, pluripotency, telomerase activity or telomeres. However expression of human endogenous retroviruses (HERVs) was higher than in wild-type hESCs, and there was also a reduced level of trimethylation of histone H3 on lysine 9 at HERV loci. These data show that IFITM1 suppresses HERVs in hESCs by regulating epigenetic modifications.

Tet Enzymes Regulate Telomere Maintenance and Chromosomal Stability of Mouse ESCs.

Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3ß inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.

Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells.

Mouse embryonic stem cell (ESC) cultures exhibit a heterogeneous mixture of metastable cells sporadically entering the 2-cell (2C)-embryo-like state, critical for ESC potency. One of 2-cell genes, Zscan4, has been shown to be responsible for telomere maintenance, genomic stability and pluripotency of mouse ESCs. Functions of other 2C-genes in ESCs remain elusive. Here we show that 2C-genes Tcstv1 and Tcstv3 play a role in regulation of telomere lengths. Overexpression or knockdown Tcstv1 and Tcstv3 does not immediately affect proliferation, pluripotency and differentiation in vitro of ESCs. However, ectopic expression of Tcstv1 or Tcstv3 results in telomere elongation, whereas Tcstv1/3 knockdown shortens telomeres of ESCs. Overexpression of Tcstv1 or Tcstv3 does not alter telomere stability. Furthermore, Tcstv1 can increase Zscan4 protein levels and telomere recombination by telomere sister chromatid exchange (T-SCE). Depletion of Tcstv1/3 reduces Zscan4 protein levels. Together, Tcstv1 and Tcstv3 are involved in telomere maintenance that is required for long-term self-renewal of mouse ESCs. Our data also suggests that Tcstv1/3 may co-operate and stabilize Zscan4 protein but the molecular bases remain to be determined.

Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells.

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs.