Copper-induced sublethal effects in Bufo gargarizans tadpoles: growth, intestinal histology and microbial alternations.

Copper (Cu) is one of the environmental contaminations which can pose significant risks for organisms. The current study explores the effects of Cu exposure on the growth, intestinal histology and microbial ecology in Bufo gargarizans. The results revealed that 0.5-1 µM Cu exposure induced growth retardation (including reduction of total body length and wet weight) and intestinal histological injury (including disordered enterocyte, changes in the villi and vacuoles) of tadpoles. Also, high-throughput sequencing analysis showed that Cu exposure caused changes in richness, diversity and structure of intestinal microbiota. Moreover, the composition of intestinal microbiota was altered in tadpoles exposed to different concentrations of Cu. At the phylum level, we observed the abundance of proteobacteria was increased, while the abundance of fusobacteria was decreased in the intestinal microbiota of tadpoles exposed to 1 µM Cu. At the genus level, a reduced abundance of kluyvera and aeromonas was observed in the intestinal microbiota of tadpoles under the exposure of 0-0.5 µM Cu. Finally, functional predictions revealed that tadpoles exposed to copper may be at a higher risk of developing metabolic disorders or diseases. Above all, our results will develop a comprehensive view of the Cu exposure in amphibians and will yield a new consideration for sublethal effects of Cu on aquatic organisms.

Effects of temperature on growth, development and the leptin signaling pathway of Bufo gargarizans.

Climate change is one of the most important causes of the decline in amphibians. Changes in temperature have an important effect on the growth and development and energy metabolism of amphibians. The aim of this study is to unravel the effects of temperature on the leptin signaling pathway of Bufo gargarizans and its molecular mechanisms. Our results showed that high temperature accelerated the development rate of tadpoles, but reduced body size and mass, while low temperature deferred the development of tadpoles, but increased size and mass. Both high temperature and low temperature exposure caused pathological damage of the liver in B. gargarizans. The results of RT-qPCR revealed that the high temperature treatment significantly upregulated the transcript levels of genes related to thyroid hormone (DIO2 (D2), Thyroid Hormone Receptor-α (TRα)) and the leptin signaling pathway (Leptin Receptor (LepR), Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Tyrosine kinase 2 (TYK2), Signal Transducer And Activator Of Transcription 3 (STAT3), Signal Transducer And Activator Of Transcription 3.1 (STAT3.1), and Signal Transducer And Activator Of Transcription 6 (STAT6)), while there was a decrease of mRNA expression of these genes (TRα, Thyroid Hormone Receptor-Beta (TRß), LepR, JAK1, and TYK2) in the liver of tadpoles exposed to high temperature compared with the intermediate temperature treatment. Therefore, our results suggested that temperature extremes might interfere with the thyroid and leptin signaling pathways and affect the growth and development of B. gargarizans. Furthermore, tissue injury of the liver could occur due to exposure to temperature extremes. This work promotes public awareness of environmental protection and species conservation needs, also provides valuable experimental data and a theoretical basis for the protection of amphibians.

Effects of sodium perchlorate and exogenous L-thyroxine on growth, development and leptin signaling pathway of Bufo gargarizans tadpoles during metamorphosis.

Sodium perchlorate (NaClO4) and exogenous L-thyroxine (T4), two kinds of endocrine-disrupting chemicals (EDCs), mainly affect the circulating thyroid hormones, which regulate the initiation and rate of metamorphosis in amphibian. The aim of this study is to evaluate the potential role of EDCs in regulating the development of tadpoles and leptin signaling pathway of liver during the metamorphosis of Bufo gargarizans. There was completely opposite result of average development stage of tadpoles and morphological parameters between the NaClO4 and T4 exposure groups. Histological analysis revealed that NaClO4 and T4 exposure both caused liver injury, such as the decreased size of hepatocytes, atrophy of nucleus, increased melanomacrophage centres and disappearance of hepatocyte membranes. In addition, the results of RT-qPCR revealed that NaClO4 treatment significantly inhibited the transcript levels of genes related to thyroid hormone (D2, TRα and TRß) and leptin signaling pathway (LepR, JAK1, JAK2, and TYK2), while there was an increase of mRNA expression of these genes in the liver of tadpoles administrated with T4 compared with control. This work lays an important foundation for assessing the risk of EDCs in relation to amphibian development during metamorphosis.

Response Patterns of Biomarkers as Tools to Identify Toxic Effects of Cadmium and Lead on Bufo gargarizans Embryo.

Molecular biomarkers play an increasing crucial role in evaluating and predicting toxicity of metals. Expressions patterns of genes related to oxidative stress, apoptosis, immune and inflammation response in the Bufo gargarizans embryo exhibited a development dependent manner. The genes related to oxidative stress (HSP, GPx and SOD) are the first response in the development of embryo, followed by the apoptosis (Bax, BCLAF1 and TRAIL) and inflammation and immune response (SOCS3, IL-27 and IL-17D), respectively. Then, we have verified the HSP, Bax and SOCS3 IL-27 (expressed highest in their respective processes) exhibited the most significant changes in Cd-Pb mixed group compared with control. In addition, we found exposure of Cd-Pb mixed metals causes greater adverse effects than Cd, Pb alone on development and morphology of embryo. Overall, our results provide a useful tool to use the sensitive molecular biomarkers as indicators of developmental toxicity in amphibian embryo.

The morphological changes and molecular biomarker responses in the liver of fluoride-exposed Bufo gargarizans larvae.

The goal of the current study was to evaluate the negative influences of fluoride on liver of Bufo gargarizans larvae. B. gargarizans larvae were treated with 42.4mgF-/L for 0, 24, 48 and 72h at Gosner stage 37. The morphological changes and responses of molecular biomarkers involved in lipid metabolism, oxidative stress and apoptosis were examined in liver. Disappearance of cell boundaries, degeneration of hepatic parenchyma cells and significant increase in the number of melanomacrophage centres and the quantity of lipid droplets were found in the liver treated with 42.4mgF-/L for 72h. In addition, in the relative expression of acetyl CoA carboxylase 1 (ACC-1), fatty acid elongase 1 (FAE-1), sterol carrier protein 2 (SCP-2), and carnitine palmitoyltransferase-1 (CPT-1), decrease was observed after 24, 48 and 72h of 42.4mgF-/L exposure. Furthermore, the transcript levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were downregulated in tadpoles exposed for 24, 48 and 72h to 42.4mgF-/L, while the transcript level of heat shock protein 90 (HSP90) was upregulated at 42.4mgF-/L for 72h. Also, mRNA expression of Bcl-2-associated transcription factor 1(BCLAF1) and thyroid hormone receptors (TRα and TRß) was significantly upregulated in tadpoles treated with 42.4mgF-/L for 72h. Therefore, our results suggested that the liver injury induced by fluoride might result from disruption of lipid metabolism, oxidative damage and apoptosis.

The effect of cadmium exposure on diversity of intestinal microbial community of Rana chensinensis tadpoles.

Cadmium is a natural and widely distributed toxicant, and can be commonly found in environment. Intestinal microbiota plays a very important role in maintaining its host's health. The effects of cadmium on the intestinal microbiota composition and stability of amphibians are little known. We exposed Rana chensinensis (R. chensinensis) embryos to different concentrations of cadmium (0, 112 and 448 µg Cd L-1) until they reached Gosner stage 38, and analyzed their microbial communities using 16S rRNA amplicon sequencing. By measures of both alpha and beta diversity, intestinal microbial communities were significantly differentiated in 448 µg Cd L-1 exposure groups. Cadmium exposure significantly altered the intestinal microflora diversity and composition of R. chensinensis. At the phylum level, it is worth noting that Fusobacteria and Spirochaetae were not detected in 448 µg Cd L-1 exposure groups. Firmicutes rapidly decreased in 448 µg Cd L-1 exposure group. At the genus level, Succinispira (Firmicutes), Desulfovibrio (Proteobacteria) and Fusobacterium (Fusobacteria) vanished in 448 µg Cd L-1 exposure groups. Our results demonstrate that cadmium exposure changed the composition and decreased the community diversity of intestinal microbiota of R. chensinensis tadpoles. Our study may provide a new framework based on intestinal microbiota to evaluate the response of amphibians to environmental chemicals pollution.

Effects of perchlorate and exogenous T4 exposures on development, metamorphosis and endochondral ossification in Bufo gargarizans larvae.

Several endocrine-disrupting chemicals (EDCs) have been proven to interfere with the physiological function of thyroid hormone (TH), which affected growth and development. However, few studies have investigated the effects of EDCs on TH axis with consequence for skeletal development in amphibians. This study thus examined the potential role of perchlorate and T4 in growth, development and endochondral ossification during metamorphosis of Bufo gargarizans. Our studies showed that NaClO4 treatment caused weight gain and delayed the developmental stage in B. gargarizans tadpoles, while T4 decreased body size and survival rate, accelerated metamorphic duration and increased the risk of early death. Histological sections suggested that NaClO4 and T4 treatments caused damages to thyroid tissue, such as decreased thyroid gland size, follicle size, colloid area, the height of follicular epithelial cells and the number of follicles. In addition, the double skeletal staining and RT-qPCR showed that NaClO4 and T4 treatments inhibited the endochondral ossification by regulating TH synthesis (TRs, Dios) and endochondral ossification-related genes (MMPs, Runxs, VEGFs and VEGFRs) expression levels, which might affect terrestrial locomotion and terrestrial life. Altogether, these thyroid injury and gene expression changes as caused by NaClO4 and T4 may have an influence on development and endochondral ossification during the metamorphosis of amphibians.

Intestinal response characteristic and potential microbial dysbiosis in digestive tract of Bufo gargarizans after exposure to cadmium and lead, alone or combined.

The gastrointestinal tract is the largest immune organ in the body and meanwhile, accommodates a large number of microorganisms. Heavy metals could disturb the intestinal homeostasis and change the gut microbial composition. However, the information regarding the links between dysbiosis of gut microbiota and imbalance of host intestinal homeostasis induced by the mixture of heavy metals is insufficient. The present study investigates the effects of Cd/Pb, both single and combination exposure, on the growth performance, intestinal histology, digestive enzymes activity, oxidative stress and immune parameters, and intestinal microbiota in Bufo gargarizans tadpoles. Our results revealed that co-exposure of Cd-Pb induced more severe impacts not only on the host, but the intestinal microbiota. On the one hand, co-exposure of Cd-Pb significantly induced growth retardation, intestinal histological injury, decreased activities of digestive enzymes. On the other hand, Cd and Pb exposure, especially in mixed form, changed the diversity and richness, structure of microbiota. Also, the intestinal microbial composition was altered by Cd/Pb exposure (alone and combination) both at the different levels. Proteobacteria, act as front-line responder, was significantly increased in tadpoles under the exposure of metals. Finally, the functional prediction revealed that the disorders of metabolism and immune responses of intestinal microbiota was increased in tadpoles exposed to Cd/Pb (especially the mixture of Cd and Pb). Our research complements the understanding of links between changes in host fitness loss and intestinal microbiota and will add a new dimension of knowledge to the ecological risks of mixed heavy metals in amphibian.

Comparison of the characteristics of intestinal microbiota response in Bufo gargarizans tadpoles: Exposure to the different environmental chemicals (Cu, Cr, Cd and NO3-N).

Environmental pollutants could change the intestinal microbiota communities, while data concerning the dynamics of the intestinal microbiota in response to different environmental chemicals in amphibian are lacking. We compared the effects of Cu, Cd, Cr and NO3-N on intestinal microbiota of B. gargarizans tadpoles by using high-throughput 16S rRNA sequencing technology. Our results revealed that responses of intestinal microbiota to three metals and NO3-N showed different characteristics. At the phylum level, the most 100 OTUs were predominantly colonized by Proteobacteria, and meanwhile, expansion of Proteobacteria was observed in Cu 64 µg/L, Cd (100 µg/L and 200 µg/L) and NO3-N100 mg/L treatment groups. In addition, the abundance of Bacteroidetes significantly increased in the gut administrate with Cu, Cd, Cr, NO3-N 20 mg/L exposures, while declined abundance of Fusobacteria was observed in Cu64 µg/L Cd100 µg/L Cd200µg/L-exposed groups. At the genus level, several genera exhibited increased prevalence of abundance such as Shewanella, Azospira and Flavobacterium. The functional prediction revealed that exposures of three metals and NO3-N increase the risks of metabolic disorders and diseases. Our research could be an important step toward an assessment of the ecological risks of different chemicals to aquatic organisms using intestinal microbiota.

Effects of fluoride on the histology, lipid metabolism, and bile acid secretion in liver of Bufo gargarizans larvae.

In our study, Bufo gargarizans (B. gargarizans) larvae were exposed to control, 0.5, 5, 10 and 50 mg/L of NaF from Gs 26 to 42. At Gs 42, we evaluated the changes of liver histology and the mRNA levels of target genes in liver. In addition, we also examined the composition and content of fatty acids. Histological analysis revealed that fluoride caused liver injury, such as the increase of number of melanomacrophage centres, atrophy of nucleus, dilation of bile canaliculus, and decrease of quantity, degradation and deposition of lipid droplets. The results of RT-qPCR indicated that exposure to 5, 10 and 50 mg/L of NaF significantly decreased the transcript levels of genes related to fatty acid synthesis (FASN, FAE, MECR, KAR and TECR) in liver. Besides, mRNA expression of genes involved in fatty acid ß-oxidation (ECHS1, HADHA, SCP2, CPT2, ACAA1 and ACAA2) and oxidative stress (SOD, GPx, MICU1 and HSP90) was significantly downregulated in 0.5, 5, 10 and 50 mg/L of NaF treatment groups. Also, in the relative expression of genes associated with synthesis and secretion of bile acid, BSEP significantly increased at 0.5, 5 and 50 mg/L of NaF while HSD3B7 significantly reduced in 0.5, 5, 10 and 50 mg/L of NaF. Finally, the fatty acid extraction and GC-MS analysis showed that the content of saturated fatty acids (SFAs) was decreased and the content of polyunsaturated fatty acids (PUFAs) was increased in all fluoride treatment groups. Taken together, the present results indicated that fluoride-induced the histological alterations of liver might be linked to the disorder of lipid metabolism, oxidative damage.

Transcriptome analyses reveal molecular mechanisms that regulate endochondral ossification in amphibian Bufo gargarizans during metamorphosis.

BACKGROUND: A developmental transition from aquatic to terrestrial existence is one of the most important events in the evolution of terrestrial vertebrates. Amphibian metamorphosis is a classic model to study this transition. The development of the vertebrate skeleton can reflect its evolutionary history. Endochondral ossification serves a vital role in skeletal development. Thus, we sought to unravel molecular mechanisms that regulate endochondral ossification during Bufo gargarizans metamorphosis. METHODS: The alizarin red-alcian blue double staining method was used to visualize the skeletal development of B. gargarizans during metamorphosis. RNA sequencing (RNA-seq) was used to explore the transcriptome of B. gargarizans in four key developmental stages during metamorphosis. Real-time quantitative PCR (RT-qPCR) was used to validate the expression patterns of endochondral ossification related genes. RESULTS: Endochondral ossification increased gradually in skeletal system of B. gargarizans during metamorphosis. A total of 137,264 unigenes were assembled and 44,035 unigenes were annotated. 10,352 differentially expressed genes (DEGs) were further extracted among four key developmental stages. In addition, 28 endochondral ossification related genes were found by searching for DEG libraries in B. gargarizans. Of the 28 genes, 10 genes were validated using RT-qPCR. CONCLUSIONS: The exquisite coordination of the 28 genes is essential for regulation of endochondral ossification during B. gargarizans metamorphosis. GENERAL SIGNIFICANCE: The present study will not only provide an invaluable genomic resource and background for further research of endochondral ossification in amphibians but will also aid in enhancing our understanding of the evolution of terrestrial vertebrates.

Determining threatened species distributions in the face of limited data: Spatial conservation prioritization for the Chinese giant salamander (Andrias davidianus).

The purpose of this study was to determine whether limited occurrence data for highly threatened species can provide useful spatial information to inform conservation. The study was conducted across central and southern China. We developed a habitat suitability model for the Critically Endangered Chinese giant salamander (Andrias davidianus) based on one biotic and three abiotic parameters from single-site locality records, which represent the only relevant environmental data available for this species. We then validated model quality by testing whether increased percentage of predicted suitable habitat at the county level correlated with independent data on giant salamander presence. We randomly selected 48 counties containing historical records which were distinct from, and independent of, the single-site records used to develop the model, and 47 additional counties containing >50% predicted suitable habitat. We interviewed 2,812 respondents near potential giant salamander habitat across these counties and tested for differences in respondent giant salamander reports between counties selected using each method. Our model predicts that suitable giant salamander habitat is found widely across central and southern China, with counties containing ≥50% predicted suitable habitat distributed in 13 provinces. Counties with historical records contain significantly more predicted suitable habitat than counties without historical records. There are no statistical differences in any patterns of respondent giant salamander reports in surveyed counties selected from our model compared with the areas of known historical giant salamander distribution. A Chinese giant salamander habitat suitability model with strong predictive power can be derived from the restricted range of environmental variables associated with limited available presence-only occurrence records, constituting a cost-effective strategy to guide spatial allocation of conservation planning. Few reported sightings were recent, however, with most being over 20 years old, so that identification of areas of suitable habitat does not necessarily indicate continued survival of the species at these locations.

Imminent extinction in the wild of the world's largest amphibian.

Species with large geographic ranges are considered resilient to global decline [1]. However, human pressures on biodiversity affect increasingly large areas, in particular across Asia, where market forces drive overexploitation of species [2]. Range-wide threat assessments are often costly and thus extrapolated from non-representative local studies [3]. The Chinese giant salamander (Andrias davidianus), the world's largest amphibian, is thought to occur across much of China, but populations are harvested for farming as luxury food [4]. Between 2013 and 2016, we conducted field surveys and 2,872 interviews in possibly the largest wildlife survey conducted in China. This extensive effort revealed that populations of this once-widespread species are now critically depleted or extirpated across all surveyed areas of their range, and illegal poaching is widespread.

The Chinese giant salamander exemplifies the hidden extinction of cryptic species.

Overexploitation, habitat destruction, human-driven climate change and disease spread are resulting in the extinction of innumerable species, with amphibians being hit harder than most other groups [1]. Few species of amphibians are widespread, and those that are often represent complexes of multiple cryptic species. This is especially true for range-restricted salamanders [2]. Here, we used the widespread and critically endangered Chinese giant salamander (Andrias davidianus) to show how genetically uninformed management efforts can negatively affect species conservation. We find that this salamander consists of at least five species-level lineages. However, the extensive recent translocation of individuals between farms, where the vast majority of extant salamanders now live, has resulted in genetic homogenization. Mitochondrial DNA (mtDNA) haplotypes from northern China now predominate in farms. Unfortunately, hybrid offspring are being released back into the wild under well-intentioned, but misguided, conservation management. Our findings emphasize the necessity of genetic assessments for seemingly well-known, widespread species in conservation initiatives. Species serve as the primary unit for protection and management in conservation actions [3], so determining the taxonomic status of threatened species is a major concern, especially for amphibians. The level of threat to amphibians may be underestimated, and existing conservation strategies may be inadvertently harmful if conducted without genetic assessment.

Effects of nitrate on metamorphosis, thyroid and iodothyronine deiodinases expression in Bufo gargarizans larvae.

Chinese toad (Bufo gargarizans) tadpoles were exposed to nitrate (10, 50 and 100mg/L NO3-N) from the beginning of the larval period through metamorphic climax. We examined the effects of chronic nitrate exposure on metamorphosis, mortality, body size and thyroid gland. In addition, thyroid hormone (TH) levels, type II iodothyronine deiodinase (Dio2) and type III iodothyronine deiodinase (Dio3) mRNA levels were also measured to assess disruption of TH synthesis. Results showed that significant metamorphic delay and mortality increased were caused in larvae exposed to 100mg/L NO3-N. The larvae exposed to 100mg/L NO3-N clearly exhibited a greater reduction in thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels. Moreover, treatment with NO3-N induced down-regulation of Dio2 mRNA levels and up-regulation of Dio3 mRNA levels, reflecting the disruption of thyroid endocrine. It seems that increased mass and body size may be correlated with prolonged metamorphosis. Interestingly, we observed an exception that exposure to 100mg/L NO3-N did not exhibit remarkable alterations of thyroid gland size. Compared with control groups, 100mg/L NO3-N caused partial colloid depletion in the thyroid gland follicles. These results suggest that nitrate can act as a chemical stressor inducing retardation in development and metamorphosis. Therefore, we concluded that the presence of high concentrations nitrate can influence the growth, decline the survival, impair TH synthesis and induce metamorphosis retardation of B. gargarizans larvae.

Chronic toxicity of copper on embryo development in Chinese toad, Bufo gargarizans.

This study examined the effects of copper exposure on embryonic development of Chinese toad, Bufo gargarizans. Firstly, the LC(50) values from 24 to 96 h of exposure were 3.61×10(-6) M, by means of a 4 d toxicity test with B. gargarizans embryos. Secondly, Chinese toad embryos were exposed to 10(-9)-10(-6) M copper from mid gastrula stage to operculum completion stage. Measurements included mortality, tadpole weight, tadpole total length, growth retardation, duration of different embryo stages and malformation. Embryonic survival was not affected by copper. Relative to control tadpoles, significantly decreased weight and total length were found at 10(-9)-10(-6) M reduced percentage of the embryos in right operculum stage after 10 d exposure to copper and reduced percentage of embryos in operculum completion stage after 12 d exposure to copper were also observed. Moreover, the duration of embryonic development increased at neural, circulation and operculum development stage in copper-treated groups. For the scanning microscope and histological observation, the abnormalities were malformation of wavy dorsal fin, flexural tail, curvature body axis, yolk sac oedema and reduced pigmentation in the yolk sac. Histopathological changes in olfactory, retinal epithelium and skin were also observed. DNA strand breaks exposed to the copper were analyzed by DNA ladder. In conclusion, copper induced toxic effects on B. gargarizans embryos. The present study indicated chronic toxicity tests may provide more accurate way in formulating the "safe levels" of heavy metals to amphibian.

WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived CD133+ cells in vitro.

Cord blood-derived CD133+ cells have a degree of non-hematopoietic potential and express transcripts of pluripotency markers including Oct-4, Sox-2, Rex-1, and leukemia inhibitory factor (LIF) receptor, as well as markers of progenitor cells, such as HoxB4, brachyury, and nestin. Having shown by transcriptome analysis that the mouse embryonic fibroblast (MEF) cells routinely used to maintain pluripotent embryonic stem cells express transcripts of the WNT/BMP families of signaling factors, we have assessed the effects on proliferation and differentiation of CD133+ cells of medium conditioned (CM) by MEF, by NIH3T3, and by NIH3T3 cells stably expressing WNT1, WNT3a, WNT4, WNT5a, and WNT11. Cultivation of CD133+ cells in MEF-CM led to a significant increase in cell number after 7 days of culture, while WNT-1, WNT3a-, and WNT11-CM increased the cell number significantly by 14 days of culture. During this period, WNT3a-CM increased the proportion of nestin-expressing cells and increased the ratio of blast-like cells to macrophages, suggesting that these signaling molecules contribute to the maintenance of an undifferentiated, blast-like phenotype. The number of cells expressing the endothelial-related marker CD31+ was significantly increased following culture in WNT5a- and WNT11-CM, whereas the number of cells positive for von Willebrand (vW) factor was maintained during 14 days of culture only in the presence of WNT4-CM. In addition, WNT5a-CM led to increased beta-catenin mRNA levels and the presence of beta-catenin protein in the cytoplasm and nucleus, consistent with the activation of the WNT signaling pathway. We conclude that in vitro conditioning of CD133+ cells by media containing specific WNT signaling factors influences the non-hematopoietic potential of CD133+ cells and dynamically alters the expression of the neural stem/progenitor cell marker nestin and the endothelial-related cell surface markers CD31 and vW factor.

Duplex polymerase chain reaction quantification of human cells in a murine background.

Studies of the regenerative potential of human stem cells commonly involve their transplantation into immune-deficient mice or in vitro coculture with mouse cells. The optimal use of such models requires the detection and quantification of relatively low numbers of human cells in a murine background. We report here a duplex polymerase chain reaction (PCR) approach involving the coamplification of human-and mouse-specific repetitive sequences. The determination of product ratios compensates against variations in sample quality and enables quantitation from >50% down to 0.01% human-in-mouse from a single reaction. Product ratios are determined by standard electrophoresis of end-stage PCR reactions followed by image analysis techniques using freely available software, with no requirement for real-time PCR. The approach has been used to analyze tissue from mice transplanted with human cells and cocultures between differentiating mouse embryonal stem cells and human umbilical cord blood cells.