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GW117 is new melatonergic antidepressant being developed to show better antidepressant action than agomelatine. The purpose of this study was to evaluate the toxicity and to determine potential target organs after oral (gavage) administration of the test article GW117 for 28 days and to assess the reversibility after a 4-week recovery phase in beagle dogs. Toxicokinetics was also evaluated. Four groups were designed in this study, including the vehicle control group and the GW117 50, 150 and 500 mg/kg/day groups, with 5 dogs/sex/group. Body weight, hematology, clinical chemistry, gross necropsy, organ weight, histopathology, and other indicators were examined. Results showed that animals dosed at ≥150 mg/kg/day showed gastrointestinal reactions (watery feces and dark green/red brown feces), with a dose-response relationship in the incidence and severity grade. Female dogs at 500 mg/kg/day had an increase in organ weight and ratios of the liver at the end of the dosing phase. Histopathology examination showed that some animals at 500 mg/kg/day, especially female animals, had minimal centrilobular hepatocyte hypertrophy in the liver, which reversed after 28-day recovery. With the exception of the above, no GW117-related abnormality was noted. Meanwhile, there were no sexual differences in drug exposure and accumulation after the first and last dosing. The no observed adverse effect dose level (NOAEL) was 150 mg/kg/day, under which mean Cmax and AUC0 â t were 583.5 and 2767.0 ng/ml*h for females and 663.2 and 4046.3 ng/ml*h for males on Day 28.
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Hígado , Masculino , Perros , Animales , Femenino , Toxicocinética , Nivel sin Efectos Adversos ObservadosRESUMEN
Handmade Cloning (HMC) is a pivotal technique for cloning pig embryos. Despite its significance, the low efficiency of this method hampers its widespread application. Although numerous factors and signaling pathways influencing embryo development have been studied, the mechanisms underlying low developmental capacity and insufficient reprogramming of cloned embryos remain elusive. In the present study, we sought to elucidate key regulatory factors involved in the development of pig HMC embryos by comparing and analyzing the gene expression profiles of HMC embryos with those of naturally fertilized (NF) embryos at the 4-cell, 8-cell, and 16-cell stages. The results showed that ZFP42 expression is markedly higher in NF embryos than in cloned counterparts. Subsequent experiments involving the injection of ZFP42 messenger RNA (mRNA) into HMC embryos showed that ZFP42 could enhance the blastocyst formation rate, upregulate pluripotent genes and metabolic pathways. This highlights the potential of ZFP42 as a critical factor in improving the development of pig HMC embryos.
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Clonación de Organismos , Técnicas de Transferencia Nuclear , Porcinos , Animales , Clonación de Organismos/métodos , Desarrollo Embrionario/fisiología , Transcriptoma , Clonación Molecular , Blastocisto/metabolismoRESUMEN
Cadmium (Cd), a prevalent environmental contaminant, has attracted widespread attention due to its serious health hazards. Ferroptosis is a form of iron-dependent oxidative cell death that contributes to the development of various kidney diseases. However, the mechanisms underlying the occurrence of ferroptosis in Cd-induced renal tubular epithelial cells (TECs) have not been fully elucidated. Hereby, both in-vitro and in-vivo experiments were established to elucidate this issue. In this study, we found that Cd elicited accumulation of lipid peroxides due to intracellular ferrous ion (Fe2+) overload and glutathione depletion, contributing to ferroptosis. Inhibition of ferroptosis via chelation of Fe2+ or reduction of lipid peroxidation can significantly mitigate Cd-induced cytotoxicity. Renal transcriptome analysis revealed that the activation of heme oxygenase 1 (HO-1) was closely related to ferroptosis in Cd-induced TECs injury. Cd-induced ferroptosis and resultant TECs injury are significantly alleviated due to HO-1 inhibition, demonstrating the crucial role of HO-1 in Cd-triggered ferroptosis. Further studies showed that accumulation of lipid peroxides due to iron overload and mitochondrial ROS (mtROS) generation was responsible for HO-1-triggered ferroptosis in Cd-induced cytotoxicity. In conclusion, the current study demonstrates that excessively upregulating HO-1 promotes iron overload and mtROS overproduction to trigger ferroptosis in Cd-induced TECs injury, highlighting that targeting HO-1-mediated ferroptosis may provide new ideas for preventing Cd-induced nephrotoxicity.
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Cadmio , Células Epiteliales , Ferroptosis , Hemo-Oxigenasa 1 , Hierro , Túbulos Renales , Mitocondrias , Especies Reactivas de Oxígeno , Ferroptosis/efectos de los fármacos , Cadmio/toxicidad , Hemo-Oxigenasa 1/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Animales , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/efectos de los fármacos , Túbulos Renales/citología , Túbulos Renales/patología , Hierro/metabolismo , Ratones , Peroxidación de Lípido/efectos de los fármacos , Línea Celular , Masculino , Humanos , Glutatión/metabolismo , Ratones Endogámicos C57BLRESUMEN
INTRODUCTION: Environmental and occupational exposure to cadmium (Cd) has been shown to cause acute kidney injury (AKI). Previous studies have demonstrated that autophagy inhibition and lysosomal dysfunction are important mechanisms of Cd-induced AKI. OBJECTIVES: Transcription factor EB (TFEB) is a critical transcription regulator that modulates autophagy-lysosome function, but its role in Cd-induced AKI is yet to be elucidated. Thus, in vivo and in vitro studies were conducted to clarify this issue. METHODS AND RESULTS: Data firstly showed that reduced TFEB expression and nuclear translocation were evident in Cd-induced AKI models, accompanied by autophagy-lysosome dysfunction. Pharmacological and genetic activation of TFEB improved Cd-induced AKI via alleviating autophagy inhibition and lysosomal dysfunction, whereas Tfeb knockdown further aggravated this phenomenon, suggesting the key role of TFEB in Cd-induced AKI by regulating autophagy. Mechanistically, Cd activated mechanistic target of rapamycin complex 1 (mTORC1) to enhance TFEB phosphorylation and thereby inhibiting TFEB nuclear translocation. Cd also activated chromosome region maintenance 1 (CRM1) to promote TFEB nuclear export. Meanwhile, Cd activated general control non-repressed protein 5 (GCN5) to enhance nuclear TFEB acetylation, resulting in the decreased TFEB transcriptional activity. Moreover, inhibition of CRM1 or GCN5 alleviated Cd-induced AKI by enhancing TFEB activity, respectively. CONCLUSION: In summary, these findings reveal that TFEB phosphorylation, nuclear export and acetylation independently suppress TFEB activity to cause Cd-induced AKI via regulating autophagy-lysosome function, suggesting that TFEB activation might be a promising treatment strategy for Cd-induced AKI.
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IMPORTANCE: Phage therapy is gaining traction as an alternative to antibiotics due to the rise of multi-drug-resistant (MDR) bacteria. This study assessed the pharmacokinetics and safety of PA_LZ7, a phage targeting MDR Pseudomonas aeruginosa, in mice. After intravenous administration, the phage showed an exponential decay in plasma and its concentration dropped significantly within 24 h for all dosage groups. Although there was a temporary increase in certain plasma cytokines and spleen weight at higher dosages, no significant toxicity was observed. Therefore, PA_LZ7 shows potential as an effective and safe candidate for future phage therapy against MDR P. aeruginosa infections.
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Bacteriófagos , Infecciones por Pseudomonas , Fagos Pseudomonas , Animales , Ratones , Fagos Pseudomonas/genética , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Infecciones por Pseudomonas/terapia , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosaRESUMEN
GW117, a novel derivate compound of agomelatine that acts as both a 5-HT2C receptor antagonist and a MT1/MT2 receptor agonist, likely underlines the potent antidepressant action with less hepatotoxicity than agomelatine. We evaluated the acute toxicity of GW117, and the genotoxicity of GW117 using bacterial reverse mutation test, mammalian chromosomal aberration test in Chinese hamster lung cells (CHL) and mouse bone marrow micronucleus test. The acute toxicity test results showed that maximum tolerated dose (MTD) of GW117 was 2000 mg/kg, under which mean Cmax and AUC0ât was 10,782 ng/mL and 81,046 ng/mL × h, respectively. The result of bacterial reverse mutation test showed that the number of bacterial colonies in each dose group of GW117 did not increase significantly compared with that in the solvent control group with or without S9 metabolic activation system. In vitro chromosome aberration test of CHL cells, the chromosome aberration rate of each dose group of GW117 did not increase with or without S9 metabolic activation system. In mouse micronucleus test, the highest dose was 2000 mg/kg, the micronucleus rate did not increase significantly. Under the conditions of this study, the MTD of a single GW117 administration was 2000 mg/kg, there was no genotoxicity effect of GW117.
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BACKGROUND: Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. A deep understanding of these phenomena may provide helpful insights for HSCs. METHODS: Here, we applied single-cell RNA-seq (scRNA-seq) to analyse the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilised peripheral blood (mPB). RESULTS: We collected over 16 000 high-quality single-cell data to construct a comprehensive inference map and characterised the HSCs under a quiescent state on the hierarchy top. Then, we compared HSCs in CB with those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with cell source (CS-SRGs) and culture time (CT-SRGs), respectively. Interestingly, CS-SRGs and CT-SRGs share genes enriched in the signalling pathways such as mRNA catabolic process, translational initiation, ribonucleoprotein complex biogenesis and cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related to purine and ATP metabolic process, which is crucial for HSC homeostasis in the stress settings. Particularly, when CT-SRGs are used as reference genes for the construction of the development trajectory of CD34+ cells, lymphoid and myeloid lineages are clearly separated after HSCs/MPPs. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs. A small molecule, cucurbitacin I, was found to efficiently expand HSCs ex vivo while maintaining its stemness. CONCLUSIONS: Our findings provide new perspectives for understanding HSCs, and the strategy to identify candidate molecules through SRGs may be applicable to study other stem cells.
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Diferenciación Celular , Sangre Fetal , Células Madre Hematopoyéticas , Humanos , Antígenos CD34/análisis , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Análisis de la Célula Individual , Perfilación de la Expresión Génica , Diferenciación Celular/genéticaRESUMEN
Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs. To date, however, it remains unexplored if human chimerism can be enhanced in animals through modifying the host embryos. Leveraging the interspecies PSC competition model, here we discovered retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, an RNA sensor, in "winner" cells plays an important role in the competitive interactions between co-cultured mouse and human PSCs. We found that genetic inactivation of Ddx58/Ifih1-Mavs-Irf7 axis compromised the "winner" status of mouse PSCs and their ability to outcompete PSCs from evolutionarily distant species during co-culture. Furthermore, by using Mavs-deficient mouse embryos we substantially improved unmodified donor human cell survival. Comparative transcriptome analyses based on species-specific sequences suggest contact-dependent human-to-mouse transfer of RNAs likely plays a part in mediating the cross-species interactions. Taken together, these findings establish a previously unrecognized role of RNA sensing and innate immunity in "winner" cells during cell competition and provides a proof-of-concept for modifying host embryos, rather than donor PSCs, to enhance interspecies chimerism.
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Deer antlers constitute a unique mammalian model for the study of both organ formation in postnatal life and annual full regeneration. Previous studies revealed that these events are achieved through the proliferation and differentiation of antlerogenic periosteum (AP) cells and pedicle periosteum (PP) cells, respectively. As the cells resident in the AP and the PP possess stem cell attributes, both antler generation and regeneration are stem cell-based processes. However, the cell composition of each tissue type and molecular events underlying antler development remain poorly characterized. Here, we took the approach of single-cell RNA sequencing (scRNA-Seq) and identified eight cell types (mainly THY1+ cells, progenitor cells, and osteochondroblasts) and three core subclusters of the THY1+ cells (SC2, SC3, and SC4). Endothelial and mural cells each are heterogeneous at transcriptional level. It was the proliferation of progenitor, mural, and endothelial cells in the activated antler-lineage-specific tissues that drove the rapid formation of the antler. We detected the differences in the initial differentiation process between antler generation and regeneration using pseudotime trajectory analysis. These may be due to the difference in the degree of stemness of the AP-THY1+ and PP-THY1+ cells. We further found that androgen-RXFP2 axis may be involved in triggering initial antler full regeneration. Fully deciphering the cell composition for these antler tissue types will open up new avenues for elucidating the mechanism underlying antler full renewal in specific and regenerative medicine in general.
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PURPOSE: To expand the mutation spectrum of patients with familial exudative vitreoretinopathy (FEVR) disease. PARTICIPANTS: 74 probands (53 families and 21 sporadic probands) with familial exudative vitreoretinopathy (FEVR) disease and their available family members (n = 188) were recruited for sequencing. METHODS: Panel-based targeted screening was performed on all subjects. Before sanger sequencing, variants of LRP5, NDP, FZD4, TSPAN12, ZNF408, KIF11, RCBTB1, JAG1, and CTNNA1 genes were verified by a series of bioinformatics tools and genotype-phenotype co-segregation analysis. RESULTS: 40.54% (30/74) of the probands were sighted to possess at least one etiological mutation of the nine FEVR-causative genes. The etiological mutation detection rate was 37.74% (20/53) in family-attainable probands while 47.62% (10/21) in sporadic cases. The diagnosis rate of patients in the early-onset subgroup (≤5 years old, 45.4%) is higher than that of the children or adolescence-onset subgroup (6-16 years old, 42.1%) and the late-onset subgroup (≥17 years old, 39.4%). A total of 36 etiological mutations were identified in this study, comprising 26 novel mutations and 10 reported mutations. LRP5 was the most prevalent mutant gene among the 36 mutation types with a percentage of 41.67% (15/36). Followed by FZD4 (10/36, 27.78%), TSPAN12 (5/36, 13.89%), NDP (4/36, 11.11%), KIF11 (1/36, 2.78%), and RCBTB1 (1/36, 2.78%). Among these mutations, 63.89% (23/36) were missense mutations, 25.00% (9/36) were frameshift mutations, 5.56% (2/36) were splicing mutations, 5.56% (2/36) were nonsense mutations. Moreover, the clinical pathogenicity of these variants was defined according to American College of Medical Genetics (ACMG) and genomics guidelines: 41.67% (15/36) were likely pathogenic variants, 27.78% (10/36) pathogenic variants, 30.55% (11/36) variants of uncertain significance. No etiological mutations discovered in the ZNF408, JAG1, and CTNNA1 genes in this FEVR cohort. CONCLUSIONS: We systematically screened nine FEVR disease-associated genes in a cohort of 74 Chinese probands with FEVR disease. With a detection rate of 40.54%, 36 etiological mutations of six genes were authenticated in 30 probands, including 26 novel mutations and 10 reported mutations. The most prevalent mutated gene is LRP5, followed by FZD4, TSPAN12, NDP, KIF11, and RCBTB1. In total, a de novo mutation was confirmed. Our study significantly clarified the mutation spectrum of variants bounded up to FEVR disease.
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Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Enfermedades de la Retina , Codón sin Sentido , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Vitreorretinopatías Exudativas Familiares/genética , Receptores Frizzled/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mutación , Linaje , Enfermedades de la Retina/genética , Tetraspaninas/genética , Factores de TranscripciónRESUMEN
Glyphosate (GLY) frequently detected in various water bodies has imposed a serious risk on fish. Head kidney of fish is an important defense organ, playing a vital part in antagonizing exogenous hazardous matter. The objective of this study was to characterize toxic mechanisms of GLY in head kidney of common carp based on transcriptome profiling. After 45-days exposure of GLY at environmentally relevant concentrations, juvenile common carp were used as experimental subjects to analyze how the head kidney responded to GLY. The transcriptome profiling identified 1381 different expressed genes (DEGs) between the control and exposure groups (5 and 50 mg/L). Functional analysis of DEGs substantiated over-representative pathways mainly involving cellular stress responses, cell proliferation and turnover, apoptosis, lipid metabolism, and innate immune processes in both treated groups compared with the control group. Predicted network of gene regulation indicated that GLY-induced tp53 played a vital role in linking a battery of signals. Furthermore, the expression of 10 candidate genes by qRT-PCR aligned with transcriptional profiling. In addition, western blotting analysis confirmed that GLY-induced apoptosis and cellular proliferation were closely involved in activating MAKP signaling pathway and lipid metabolism pathway in both treated groups. Collectively, these data demonstrate that head kidney of juvenile common carp mainly leverages upregulation of genes related to cell proliferation and turnover, apoptosis, and lipid metabolism to combat sub-chronic exposure of GLY. This study casts new understanding into the risk of GLY in aquatic animals.
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Carpas , Animales , Carpas/genética , Perfilación de la Expresión Génica , Glicina/análogos & derivados , Glicina/toxicidad , Riñón Cefálico , Transcriptoma , GlifosatoRESUMEN
Interspecies blastocyst complementation enables organ-specific enrichment of xenogeneic pluripotent stem cell (PSC) derivatives, which raises an intriguing possibility to generate functional human tissues/organs in an animal host. However, differences in embryo development between human and host species may constitute the barrier for efficient chimera formation. Here, to understand these differences we constructed a complete single-cell landscape of early embryonic development of pig, which is considered one of the best host species for human organ generation, and systematically compared its epiblast development with that of human and monkey. Our results identified a developmental coordinate of pluripotency spectrum among pigs, humans and monkeys, and revealed species-specific differences in: (1) pluripotency progression; (2) metabolic transition; (3) epigenetic and transcriptional regulations of pluripotency; (4) cell surface proteins; and (5) trophectoderm development. These differences may prevent proper recognition and communication between donor human cells and host pig embryos, resulting in low integration and survival of human cells. These results offer new insights into evolutionary conserved and divergent processes during mammalian development and may be helpful for developing effective strategies to overcome low human-pig chimerism, thereby enabling the generation of functional human organs in pigs in the future.
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Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.
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Embrión de Mamíferos/citología , Endodermo/citología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Sistemas CRISPR-Cas/genética , Linaje de la Célula , Quimera , Cadena alfa 1 del Colágeno Tipo I/genética , Embrión de Mamíferos/metabolismo , Endodermo/metabolismo , Edición Génica , Ratones , PorcinosRESUMEN
ß-thalassemia, caused by mutations in the human hemoglobin ß (HBB) gene, is one of the most common genetic diseases in the world. The HBB -28(A>G) mutation is one of the five most common mutations in Chinese patients with ß-thalassemia. However, few studies have been conducted to understand how this mutation affects the expression of pathogenesis-related genes, including globin genes, due to limited homozygote clinical materials. Therefore, we developed an efficient technique using CRISPR/Cas9 combined with asymmetric single-stranded oligodeoxynucleotides (assODNs) to generate a K562 cell model with HBB -28(A>G) named K562-28(A>G). Then, we systematically analyzed the differences between K562-28(A>G) and K562 at the transcriptome level by high-throughput RNA-seq before and after erythroid differentiation. We found that the HBB -28(A>G) mutation not only disturbed the transcription of HBB, but also decreased the expression of HBG, which may further aggravate the thalassemia phenotype and partially explain the more severe clinical outcome of ß-thalassemia patients with the HBB -28(A>G) mutation. Moreover, we found that the K562-28(A>G) cell line is more sensitive to hypoxia and shows a defective erythrogenic program compared with K562 before differentiation. Importantly, all abovementioned abnormalities in K562-28(A>G) were reversed after correction of this mutation with CRISPR/Cas9 and assODNs, confirming the specificity of these phenotypes. Overall, this is the first time to analyze the effects of the HBB -28(A>G) mutation at the whole-transcriptome level based on isogenic cell lines, providing a landscape for further investigation of the mechanism of ß-thalassemia with the HBB -28(A>G) mutation.
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Essential amino acids (EAA) of inappropriate concentration have been reported to compromise the development of embryo. This study aimed to investigate the effect of EAA on the developmental competence of porcine embryos produced by either handmade cloning (HMC) or parthenogenetic activation (PA). In experiment 1, we examined the in vitro developmental competence of PA embryos after culture in PZM-3 containing different concentrations (v/v) of EAA (0%, 1%, and 2%). The results indicated that reducing the concentration of EAA from 2% to 1% significantly improved the blastocyst formation (36% vs. 54%), while 0% would compromise the blastocyst formation rate (54% vs. 38%). In experiment 2, we further investigated the effect of EAA concentration (1% and 2%) on the in vitro developmental competence and gene expression of HMC embryos. Blastocyst rate significantly increased by reducing concentration of EAA (41% vs. 53%) and those genes upregulated were enriched in oxidative phosphorylation, PPAR signaling pathway, and metabolism-related pathways. In experiment 3, the in vivo developmental competence of HMC embryos cultured in the medium supplemented with 1% EAA was examined. Embryos derived from both non-gene-modified fetal fibroblasts (FFs) and gene-modified fetal fibroblasts (GMFFs) were transferred to recipients. The pregnancy rates were 83% and 78% separately. Out of the pregnancies, 5 (FFs) and 6 (GMFFs) were successfully developed to term. Our study indicates that supplementing EAA to embryo culture medium at a concentration of 1% can improve the in vitro developmental competence of porcine HMC embryos and the blastocyst obtained can successfully develop to term, which could be beneficial for the production of gene-modified piglets.
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Aminoácidos Esenciales/farmacología , Blastocisto/citología , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/citología , Desarrollo Embrionario/efectos de los fármacos , Oocitos/citología , Animales , Blastocisto/efectos de los fármacos , Clonación Molecular , Embrión de Mamíferos/efectos de los fármacos , Femenino , Técnicas de Transferencia Nuclear , Oocitos/efectos de los fármacos , Embarazo , PorcinosRESUMEN
Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding.
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Células de la Médula Ósea/citología , Clonación de Organismos/métodos , Embrión de Mamíferos/citología , Fibroblastos/citología , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/fisiología , Células Cultivadas , Embrión de Mamíferos/fisiología , Femenino , Fibroblastos/fisiología , Células Madre Mesenquimatosas/fisiología , PorcinosAsunto(s)
Ambystoma mexicanum/genética , Linaje de la Célula/genética , Peroxirredoxinas/genética , Regeneración/genética , Análisis de la Célula Individual/métodos , Ambystoma mexicanum/inmunología , Amputación Quirúrgica , Animales , Biomarcadores/metabolismo , Blastómeros/citología , Blastómeros/inmunología , Linaje de la Célula/inmunología , Células del Tejido Conectivo/citología , Células del Tejido Conectivo/inmunología , Células Epiteliales/citología , Células Epiteliales/inmunología , Miembro Anterior , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunidad , Peroxirredoxinas/inmunología , Regeneración/inmunología , Medicina Regenerativa/métodosRESUMEN
Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the first report of large-scale analysis of porcine cell nuclear transfer that provides important data for potential industrialization of HMC technology.