Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

ObjectivesThe rats are crucial animal models for the basic medical researches. Rat embryonic stem cells (ESCs), which are widely studied, can self‐renew and exhibit pluripotency in long‐term culture, but the mechanism underlying how they exit pluripotency remains obscure. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome‐wide screening using a unique rat haploid system.Materials and MethodsRat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1‐GFP reporter are used for genetic screening. Genome‐wide mutations are introduced into the genomes of rat Rex1‐GFP haESCs via piggyBac transposon, and differentiation‐retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high‐throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p‐ERK1/2) is determined by western blotting.ResultsHigh‐throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p‐ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to exit from pluripotency.ConclusionsOur findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self‐renewal or pluripotency of rat ESCs.

Differentiation of pluripotent rat embryonic stem cells (ESCs) in vitro is difficult to achieve for unknown mechanisms. Rat haploid ESCs (haESCs) have been validated as a powerful tool to target unknown functional genes and pathways based on homozygous genetic screening. Xu et al. utilized Rex1‐GFP labelled‐rat haESCs to conduct genome‐scale screening of genes modulating pluripotency exiting. Validation experiments showed that Thop1 (one of the screened out genes) played very important roles in the random differentiation of rat ESCs in vitro via modulating phosphorylation of ERK.     

PINK1‐mediated mitophagy maintains pluripotency through optineurin

ObjectivesDysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self‐renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.Materials and methods Parkin^+/+ and parkin ^−/− ESCs were established from E3.5 blastocysts of parkin^+/− x parkin^+/− mating mice. The pink1 ^−/−, optn ^−/− and ndp52 ^−/− ESCs were generated by CRISPR‐Cas9. shRNAs were used for function loss assay of target genes. Mito‐Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q‐PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.ResultsPINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1 ^−/− ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub‐S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub‐binding dead OPTN mutant (D477N).ConclusionsThe mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1‐activated OPTN binding of PINK1‐phosphorylated Ubiquitin.     

Sox2 Level Is a Determinant of Cellular Reprogramming Potential

Epiblast stem cells (EpiSCs) and embryonic stem cells (ESCs) differ in their in vivo differentiation potential. While ESCs form teratomas and efficiently contribute to the development of chimeras, EpiSCs form teratomas but very rarely chimeras. In contrast to their differentiation potential, the reprogramming potential of EpiSCs has not yet been investigated. Here we demonstrate that the epiblast-derived pluripotent stem cells EpiSCs and P19 embryonal carcinoma cells (ECCs) exhibit a lower reprogramming potential than ESCs and F9 ECCs. In addition, we show that the low reprogramming ability is due to the lower levels of Sox2 in epiblast-derived stem cells. Consistent with this observation, overexpression of Sox2 enhances reprogramming efficiency. In summary, these findings suggest that a low reprogramming potential is a general feature of epiblast-derived stem cells and that the Sox2 level is a determinant of the cellular reprogramming potential.     

Effect of MiR‐100‐5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR‐100‐5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR‐100‐5p was significantly inhibited in the receptive phase (RE) rather than in the pre‐receptive phase (PE). Overexpression of miR‐100‐5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR‐100‐5p, HOXA1, was confirmed by 3′‐UTR assays. Meanwhile, the product of HOXA1 mRNA RT‐PCR increased in the RE more than that in the PE. The HOXA1‐siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR‐100‐5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ‐9110 which acted as a sponge for miR‐100‐5p reversed the relevant biological effects of miR‐100‐5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ‐9110/miR‐100‐5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR‐100‐5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR‐100‐5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR‐100‐5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ‐9110/miR‐100‐5p/HOXA1 axis in vivo.     

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH₂–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.     

An allied reprogramming,selection, expansion and differentiation platform for creating hiPSC on microcarriers

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs.     

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes

The reactivation of X‐linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X‐linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist‐inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X‐linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X‐linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.     

Enhancement of keratinocyte growth factor potential in inducing adipose‐derived stem cells differentiation into keratinocytes by collagen‐targeting

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose‐derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen‐binding domain from Vibrio mimicus metalloprotease (vibrioCBD‐KGF). KGF and vibrioCBD‐KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK‐293 and MCF‐7 cells. vibrioCBD‐KGF demonstrated enhanced collagen‐binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD‐KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen‐coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin‐10 and Involucrin), and stem cell markers (Collagen‐I and Vimentin) by real‐time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD‐KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down‐regulation of Collagen‐I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD‐KGF. The present study showed that vibrioCBD‐KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen‐based biomaterials.     

Simvastatin inhibits stem cell proliferation in human leiomyoma via TGF‐β3 and Wnt/β‐Catenin pathways

Uterine leiomyoma (UL) is the most common gynaecologic tumour, affecting an estimated 70 to 80% of women. Leiomyomas develop from the transformation of myometrial stem cells into leiomyoma stem (or tumour‐initiating) cells. These cells undergo self‐renewal and differentiation to mature cells, both are necessary for the maintenance of tumour stem cell niche and tumour growth, respectively. Wnt/β‐catenin and TGF‐β/SMAD pathways, both overactive in UL, promote stem cell self‐renewal, crosstalk between stem and mature cells, cellular proliferation, extracellular matrix (ECM) accumulation and drive overall UL growth. Recent evidence suggests that simvastatin, an antihyperlipidemic drug, may have anti‐leiomyoma properties. Herein, we investigated the effects of simvastatin on UL stem cells. We isolated leiomyoma stem cells by flow cytometry using DyeCycle Violet staining and Stro‐1/CD44 surface markers. We found that simvastatin inhibits proliferation and induces apoptosis in UL stem cells. In addition, it also suppressed the expression of the stemness markers Nanog, Oct4 and Sox2. Simvastatin significantly decreased the production of the key ECM proteins, collagen 1 and fibronectin. Finally, it inhibited genes and/or proteins expression of TGF‐β1, 2 and 3, SMAD2, SMAD4, Wnt4, β‐Catenin, LRP6, AXIN2 and Cyclin D1 in UL stem cells, all are key drivers of the TGF‐β3/SMAD2 and Wnt4/β‐Catenin pathways. Thus, we have identified a novel stem cell‐targeting anti‐leiomyoma simvastatin effect. Further studies are needed to replicate these findings in vivo.     

Study of possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in planarian regeneration processes using pharmacological inhibition analysis

Possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in the processes of regeneration and morphogenesis in freshwater planarian flatworms Schmidtea mediterranea was studied using a pharmacological inhibitor analysis. It was found that pharmacological inhibitors of these kinases significantly inhibit the regeneration of the head end of the animals and that this effect is realized due to inhibition of proliferative activity of neoblasts, planarian stem cells. It is shown that that the inhibition of the studied protein kinases in regenerating planarians markedly disturbs stem cell differentiation and morphogenesis.