Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation.

Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.

Chloroplast-targeted late embryogenesis abundant 1 increases alfalfa tolerance to drought and aluminum.

Late embryogenesis-abundant (LEA) proteins are important stress-response proteins that participate in protecting plants against abiotic stresses. Here, we investigated LEA group 3 protein MsLEA1, containing the typically disordered and α-helix structure, via overexpression and RNA interference (RNAi) approaches in alfalfa (Medicago sativa L.) under drought and aluminum (Al) stresses. MsLEA1 was highly expressed in leaves and localized in chloroplasts. Overexpressing MsLEA1 increased alfalfa tolerance to drought and Al stresses, but downregulating MsLEA1 decreased the tolerance. We observed a larger stomatal aperture and a lower water use efficiency in MsLEA1 RNAi lines compared with wild-type plants under drought stress. Photosynthetic rate, Rubisco activity, and superoxide dismutase (SOD) activity increased or decreased in MsLEA1-OE or MsLEA1-RNAi lines, respectively, under drought and Al stress. Copper/zinc SOD (Cu/Zn-SOD), iron SOD (Fe-SOD), and Rubisco large subunit proteins (Ms1770) were identified as binding partners of MsLEA1, which protected chloroplast structure and function under drought and Al stress. These results indicate that MsLEA1 recruits and protects its target proteins (SOD and Ms1770) and increases alfalfa tolerance against drought and Al stresses.

Genome-wide identification and analysis of LEA_2 gene family in alfalfa (Medicago sativa L.) under aluminum stress.

Late embryonic development abundant proteins (LEAs) are a large family of proteins commonly existing in plants. LEA_2 is the largest subfamily in the LEA, it plays an important role in plant resistance to abiotic stress. In order to explore the characteristics of LEA_2 gene family members in alfalfa (Medicago sativa L.), 155 members of LEA_2 (MsLEA_2) family were identified from alfalfa genome. Bioinformatics analysis was conducted from the aspects of phylogenetic relationship, chromosome distribution, chromosome colinearity, physical and chemical properties, motif composition, exon-intron structure, cis-element and so on. Expression profiles of MsLEA_2 gene were obtained based on Real-time fluorescent quantitative PCR (qRT-PCR) analysis and previous RNA-seq data under aluminum (Al) stress. Bioinformatics results were shown that the MsLEA_2 genes are distributed on all 32 chromosomes. Among them, 85 genes were present in the gene clusters, accounting for 54.83%, and chromosome Chr7.3 carries the largest number of MsLEA_2 (19 LEA_2 genes on Chr7.3). Chr7.3 has a unique structure of MsLEA_2 distribution, which reveals a possible special role of Chr7.3 in ensuring the function of MsLEA_2. Transcriptional structure analysis revealed that the number of exons in each gene varies from 1 to 3, and introns varies from 0 to 2. Cis-element analysis identified that the promoter region of MsLEA_2 is rich in ABRE, MBS, LTR, and MeJARE, indicating MsLEA_2 has stress resistance potential under abiotic stress. RNA-seq data and qRT-PCR analyses showed that most of the MsLEA_2 members were up-regulated when alfalfa exposed to Al stress. This study revealed that phylogenetic relationship and possible function of LEA_ 2 gene in alfalfa, which were helpful for the functional analysis of LEA_ 2 proteins in the future and provided a new theoretical basis for improving Al tolerance of alfalfa.

MsMYB741 is involved in alfalfa resistance to aluminum stress by regulating flavonoid biosynthesis.

Aluminum (Al) toxicity severely restricts plant growth in acidic soils (pH < 5.0). In this study, an R2R3-MYB transcription factor (TF) gene, MsMYB741, was cloned from alfalfa. Its function and gene regulatory pathways were studied via overexpression and RNA interference of MsMYB741 in alfalfa seedlings. Results showed that root elongation increased as a result of MsMYB741 overexpression (MsMYB741-OE) and decreased with MsMYB741 RNA interference (MsMYB741-RNAi) in alfalfa seedlings compared with the wild-type under Al stress. These were attributed to the reduced Al content in MsMYB741-OE lines, and increased Al content in MsMYB741-RNAi lines. MsMYB741 positively activated the expression of phenylalanine ammonia-lyase 1 (MsPAL1) and chalcone isomerase (MsCHI) by binding to MYB and ABRE elements in their promoters, respectively, which directly affected flavonoid accumulation in roots and secretion from root tips in plants under Al stress, eventually affecting Al accumulation in alfalfa. Additionally, MsABF2 TF directly activated the expression of MsMYB741 by binding to the ABRE element in its promoter. Taken together, our results indicate that MsMYB741 transcriptionally activates MsPAL1 and MsCHI expression to increase flavonoid accumulation in roots and secretion from root tips, leading to increased resistance of alfalfa to Al stress.

Generating functional cells through enhanced interspecies chimerism with human pluripotent stem cells.

Obtaining functional human cells through interspecies chimerism with human pluripotent stem cells (hPSCs) remains unsuccessful due to its extremely low efficiency. Here, we show that hPSCs failed to differentiate and contribute teratoma in the presence of mouse PSCs (mPSCs), while MYCN, a pro-growth factor, dramatically promotes hPSC contributions in teratoma co-formation by hPSCs/mPSCs. MYCN combined with BCL2 (M/B) greatly enhanced conventional hPSCs to integrate into pre-implantation embryos of different species, such as mice, rabbits, and pigs, and substantially contributed to mouse post-implantation chimera in embryonic and extra-embryonic tissues. Strikingly, M/B-hPSCs injected into pre-implantation Flk-1+/- mouse embryos show further enhanced chimerism that allows for obtaining live human CD34+ blood progenitor cells from chimeras through cell sorting. The chimera-derived human CD34+ cells further gave rise to various subtype blood cells in a typical colony-forming unit (CFU) assay. Thus, we provide proof of concept to obtain functional human cells through enhanced interspecies chimerism with hPSCs.

Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy.

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Wei Chang An pill regulates gastrointestinal motility in a bidirectional manner.

CONTEXT: Wei Chang An (WCA) is a commercial prescription developed for the coordination of gastrointestinal movement. OBJECTIVE: To investigate the role of WCA in the regulation of diarrhoea and constipation in rats. MATERIAL AND METHODS: The diarrhoea and constipation models were prepared by gavage of Folium senna and diphenoxylate hydrochloride. Rats were randomized equally (n = 6) into the normal group given saline daily, the positive group given Pinaverium Bromide (13.5 mg/kg) or Sennoside A (0.1 mg/kg) and three WCA-treated groups (22, 44, and 88 mg/kg) by gavage daily for 7 consecutive days. The effects of WCA were assessed by a series of faecal symptoms and histopathology. Gastrointestinal parameters were determined by ELISA. The effect of WCA on gastrointestinal tissues was evaluated by strip assay. Expression of ROCK-1 and MLCK was measured by RT-PCR and Western blotting. RESULTS: Data from Bristol stool form scale, diarrhoea index, visceral sensitivity, defaecation time, and intestinal propulsive rate showed that WCA protected rats against diarrhoea and constipation (p < 0.01). The up-regulation of Substance P and 5-hydroxytryptamine in diarrhoea rats and down-regulation of Substance P and vasoactive intestinal polypeptide in constipation rats were inhibited by WCA (p < 0.05). WCA stimulated the gastrointestinal strip contractions but inhibited ACh-induced contractions (p < 0.01). The decreased ROCK-1 and MLCK expression in diarrhoea rats and increased in constipation rats were suppressed by WCA (p < 0.01). CONCLUSIONS: WCA has both antidiarrhea and anti-constipation effects, suggesting its bidirectional role in gastrointestinal modulation, and providing evidence of WCA for irritable bowel syndrome treatment.

Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips.

A SK3 -type dehydrin MsDHN1 was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression and suppression of MsDHN1 in alfalfa seedlings or hairy roots. The results showed that MsDHN1 is a typical intrinsically disordered protein that exists in the form of monomers and homodimers in alfalfa. The plant growth rates increased as a result of MsDHN1 overexpression (MsDHN1-OE) and decreased upon MsDHN1 suppression (MsDHN1-RNAi) in seedlings or hairy roots of alfalfa compared with the wild-type or the vector line under Al stress. MsDHN1 interacting with aquaporin (AQP) MsPIP2;1 and MsTIP1;1 positively affected oxalate secretion from root tips and Al accumulation in root tips. MsABF2 was proven to be an upstream transcription factor of MsDHN1 and activated MsDHN1 expression by binding to the ABRE element of the MsDHN1 promoter. The transcriptional regulation of MsABF2 on MsDHN1 was dependent on the abscisic acid signaling pathway. These results indicate that MsDHN1 can increase alfalfa tolerance to Al stress via increasing oxalate secretion from root tips, which may involve in the interaction of MsDHN1 with two AQP.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation.

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells. A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs. Therefore, to achieve a transplantable organ in animals without rejection, creation of vascular endothelial cells derived from humans within the organ is necessary. In this study, to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals, we generated rat-mouse chimeras by injection of rat embryonic stem cells (rESCs) into mouse blastocysts with deficiency of Flk-1 protein, which is associated with endothelial and hematopoietic cell development. We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras. The whole yolk sac (YS) of Flk-1EGFP/EGFP rat-mouse chimera was full of rat blood vasculature. Rat genes related to vascular endothelial cells, arteries, and veins, blood vessels formation process, as well as hematopoietic cells, were highly expressed in the YS. Our results suggested that rat vascular endothelial cells could undergo proliferation, migration, and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells, T cells, and myeloid cells in rat-mouse chimeras, which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.

Efficient base editing for multiple genes and loci in pigs using base editors.

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.

YAP1 influences differentiation of osteoblastic MC3T3-E1 cells through the regulation of ID1.

Yes-associated protein 1 (YAP1) transcriptional coactivator has recently been identified to regulate skeletal lineage cell differentiation and bone development. However, the role and molecular mechanisms of YAP1 in the regulation of osteoblastic differentiation remains to be elucidated. In this study, we demonstrated that YAP1 expression was increased during osteogenic differentiation of rat bone mesenchymal stem cells and MC3T3-E1. YAP1 overexpression MC3T3-E1 showed increased expression of osteogenesis markers, such as runt-related transcription factor 2, osteocalcin, and osteopontin, as well as alkaline phosphatase and alizarin red staining. Conversely, YAP1 knockdown significantly suppressed MC3T3-E1 osteoblastic differentiation. Mechanistically, we found that YAP1 overexpression upregulated the mRNA and protein expression of the inhibitor of differentiation/DNA binding 1 (ID1), which was contrary to the results of YAP1-knockdown group. Moreover, the early osteogenic differentiation of MC3T3-E1 cells was enhanced by ID1 overexpression. Furthermore, transient transfection with exogenous ID1 overexpression plasmid completely recaptured the decreased effects of YAP1 knockdown on MC3T3-E1 cell differentiation. In addition, ß-catenin and AMP-activated protein kinase signaling pathways participated in YAP1 regulation processes. Taken together, our study suggests that YAP1 is a crucial modulator of osteoblast differentiation in vitro, and provides insight into the mechanism by which YAP1 regulates osteoblast differentiation.

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.

Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing.

Despite being time-consuming and costly, generating genome-edited pigs holds great promise for agricultural, biomedical, and pharmaceutical applications. To further facilitate genome editing in pigs, we report here establishment of a pig line with Cre-inducible Cas9 expression that allows a variety of ex vivo genome editing in fibroblast cells including single- and multigene modifications, chromosome rearrangements, and efficient in vivo genetic modifications. As a proof of principle, we were able to simultaneously inactivate five tumor suppressor genes (TP53, PTEN, APC, BRCA1, and BRCA2) and activate one oncogene (KRAS), achieved by delivering Cre recombinase and sgRNAs, which caused rapid lung tumor development. The efficient genome editing shown here demonstrates that these pigs can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of genome-edited pigs for disease modeling.

Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway.

Dehydrin improves plant resistance to many abiotic stresses. In this study, the expression profiles of a dehydrin gene, CdDHN4, were estimated under various stresses and abscisic acid (ABA) treatments in two bermudagrasses (Cynodon dactylon L.): Tifway (drought-tolerant) and C299 (drought-sensitive). The expression of CdDHN4 was up-regulated by high temperatures, low temperatures, drought, salt and ABA. The sensitivity of CdDHN4 to ABA and the expression of CdDHN4 under drought conditions were higher in Tifway than in C299. A 1239-bp fragment, CdDHN4-P, the partial upstream sequence of the CdDHN4 gene, was cloned by genomic walking from Tifway. Bioinformatic analysis showed that the CdDHN4-P sequence possessed features typical of a plant promoter and contained many typical cis elements, including a transcription initiation site, a TATA-box, an ABRE, an MBS, a MYC, an LTRE, a TATC-box and a GT1-motif. Transient expression in tobacco leaves demonstrated that the promoter CdDHN4-P can be activated by ABA, drought and cold. These results indicate that CdDHN4 is regulated by an ABA-dependent signal pathway and that the high sensitivity of CdDHN4 to ABA might be an important mechanism enhancing the drought tolerance of bermudagrass.

Altered expression of eNOS, prostacyclin synthase, prostaglandin G/H synthase, and thromboxane synthase in porcine aortic endothelial cells after exposure to human serum-relevance to xenotransplantation.

Under normal conditions, the activity of platelets is stringently and precisely balanced between activation and quiescent state. This guarantees rapid hemostasis and avoids uncontrolled thrombosis. However, excessive platelet activation and resulting thrombotic microangiopathy are frequently observed in pig-to-primate xenotransplantation models. Endothelium-derived inhibitory mechanisms play an important role in regulation of platelet activation. These mainly include nitric oxide (NO), prostacyclin PGI2 , and adenosine, which are synthesized by endothelial NO synthases (eNOS), prostacyclin synthase, and CD39/CD73, respectively. We investigated whether endothelium-derived regulatory mechanisms are affected in porcine aortic endothelial cells (PAECs) after exposure to human serum. In the present study, exposure of PAECs or porcine iliac arteries to human serum suppressed gene expression of eNOS and prostacyclin synthase, while induced gene expression of prostaglandin G/H synthase and thromboxane synthase. Simultaneously, exposure to human serum reduced NO and PGI2 production in PAEC culture supernatants. Thus, human serum altered the balance of endothelium-derived inhibitory mechanisms in PAECs, which may indicate a regulatory mechanism of excessive platelet activation in pig-to-primate xenotransplantation.

DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells.

Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFß pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies.

Generation of Hoxc13 knockout pigs recapitulates human ectodermal dysplasia-9.

Atrichia and sparse hair phenotype cause distress to many patients. Ectodermal dysplasia-9 (ED-9) is a congenital condition characterized by hypotrichosis and nail dystrophy without other disorders, and Hoxc13 is a pathogenic gene for ED-9. However, mice carrying Hoxc13 mutation present several other serious disorders, such as skeletal defects, progressive weight loss and low viability. Mouse models cannot faithfully mimic human ED-9. In this study, we generated an ED-9 pig model via Hoxc13 gene knockout through single-stranded oligonucleotides (c.396C > A) combined with CRISPR/Cas9 and somatic cell nuclear transfer. Eight cloned piglets with three types of biallelic mutations (five piglets with Hoxc13c.396C > A/c.396C > A, two piglets with Hoxc13c.396C > A/c.396C > A + 1 and one piglet with Hoxc13Δ40/Δ40) were obtained. Hoxc13 was not expressed in pigs with all three mutation types, and the expression levels of Hoxc13-regulated genes, namely, Foxn1, Krt85 and Krt35, were decreased. The hair follicles displayed various abnormal phenotypes, such as reduced number of follicles and disarrayed hair follicle cable without normal hair all over the body. By contrast, the skin structure, skeleton phenotype, body weight gain and growth of Hoxc13 knockout pigs were apparently normal. The phenotypes of Hoxc13 mutation in pigs were similar to those in ED-9 patients. Therefore, Hoxc13 knockout pigs could be utilized as a model for ED-9 pathogenesis and as a hairless model for hair regeneration research. Moreover, the hairless pigs without other major abnormal phenotypes generated in this study could be effective models for other dermatological research because of the similarity between pig and human skins.

Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

BACKGROUND: TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43. RESULTS: We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains. CONCLUSION: Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.