Pharmaceuticals in tap water: human health risk assessment and proposed monitoring framework in China

Background: Pharmaceuticals are known to contaminate tap water worldwide, but the relevant human health risks have not been assessed in China. Objectives: We monitored 32 pharmaceuticals in Chinese tap water and evaluated the life-long human health risks of exposure in order to provide information for future prioritization and risk management. Methods: We analyzed samples (n = 113) from 13 cities and compared detected concentrations with existing or newly-derived safety levels for assessing risk quotients (RQs) at different life stages, excluding the prenatal stage. Results: We detected 17 pharmaceuticals in 89% of samples, with most detectable concentrations (92%) at < 50 ng/L. Caffeine (median-maximum, nanograms per liter: 24.4-564), metronidazole (1.8-19.3), salicylic acid (16.6-41.2), clofibric acid (1.2-3.3), carbamazepine (1.3-6.7), and dimetridazole (6.9-14.7) were found in ≥ 20% of samples. Cities within the Yangtze River region and Guangzhou were regarded as contamination hot spots because of elevated levels and frequent positive detections. Of the 17 pharmaceuticals detected, 13 showed very low risk levels, but 4 (i.e., dimetridazole, thiamphenicol, sulfamethazine, and clarithromycin) were found to have at least one life-stage RQ ≥ 0.01, especially for the infant and child life stages, and should be considered of high priority for management. We propose an indicator-based monitoring framework for providing information for source identification, water treatment effectiveness, and water safety management in China. Conclusion: Chinese tap water is an additional route of human exposure to pharmaceuticals, particularly for dimetridazole, although the risk to human health is low based on current toxicity data. Pharmaceutical detection and application of the proposed monitoring framework can be used for water source protection and risk management in China and elsewhere

Correlating the function and phenotype of pluripotent stem cells

The unique properties of self-renewal and multilineage differentiation enables pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to be well-positioned for a range of applications. However, outcomes of these potential applications are contingent on gaining a more in-depth understanding of their biology, as well as, safety of these cells. To date the comparative safety of hESCs and iPSCs have yet to be adequately addressed. Safety of their differentiated derivatives also necessitates the development of technology which permits the reliable elimination of pluripotent cells. Current literature describing the biology of hESCs and iPSCs has utilized heterogeneous cell populations. To derive a more precise definition of what constitutes a pluripotent stem cell, defined sub-fractions were obtained via fluorescence activated cell sorting (FACS) based on the expression of surface markers CD9 (TG30) and GCTM-2. The TG30-GCTM-2 double sort yields four immunologically defined sub-fractions arbitrarily termed TG30Neg-GCTM-2Neg (P4) to TG30High-GCTM-2High (P7). This double sorting strategy was rigorously interrogated by a series of assays, including microarray analysis, colony-forming assay and teratoma assay to be a robust strategy to enrich for pluripotent cells. Having established the validity of the TG30-GCTM-2 double-sort, the equivalency of iPSCs and hESCs was then assessed by the objective comparison of corresponding sub-fractions. In agreement with current literature, initial flow cytometric and microarray analysis of both cell types displayed highly similar profiles. However, our studies subsequently demonstrated for the first time the existence of functional differences between hESCs and iPSCs. Colony-forming and teratoma formation assays, as well as, immunostaining results revealed that all four sub-fractions of iPSCs were able to give rise to stem cell colonies. In contrast, stem cell colonies were notably absent in P4 and P5 hESCs and only observed in P6 and P7 hESCs. Furthermore, flow cytometric analysis revealed spontaneous reversion of differentiated derivatives of the P4 sub-fraction of iPSCs within seven days. Gene expression studies using Q-PCR noted that iPSCs possess a marked dysregulation of a selected number of pluripotency genes. A logical progression from the findings of these initial studies was to determine the possible mechanism(s) responsible for the observed reversion phenomenon. Utilizing an additional three iPSC lines generated using different approaches and gene cocktails, it was found that whilst persistence of viral transgenes could have increased colony-forming efficiency, in isolation this factor proved to be insufficient to cause spontaneous reversion. The reversion phenomenon was also noted to be independent of the concentration of FGF-2 used to culture the two cell types. Finally, we determined that the presence of residual OCT-4+ cells in the P4 sub-fraction could not account for the reversion phenomenon. Collectively, whilst these studies have yet to definitively elucidate the exact mechanism(s) responsible for the spontaneous reversion of differentiated derivatives of iPSCs, we have at least ruled out some plausible mechanism(s). A useful tool which lends itself to the understanding the biology of PSCs, as well as, the mechanism(s) responsible for reversion and reprogramming, is a reporter cell line linked to a pluripotency gene. Such a reporter cell line would also enable the enrichment and/or elimination of pluripotent cells, with the latter addressing safety concerns regarding the clinical use of PSCs. In this regard, two pluripotency genes - GALANIN and GDF-3 were identified based on results obtained from microarray and Q-PCR analysis. GDF-3/GALANIN promoter constructs were used to create the resultant reporter cell line using a lentiviral vector approach. The GALANIN reporter cell line which appeared to be constitutively active regardless of the culture condition used was excluded from further studies. The GDF-3 reporter cell lines were then characterized using colony-forming assay, immunostaining, EB formation and karyotyping. However, karyotyping results revealed that both the GDF-3 and GALANIN cell lines possess gross chromosomal abnormalities, an outcome possibly attributed to the random insertion of viral vectors into the cells’ genome. Whilst both the GDF-3 and GALANIN reporter cell lines may not be utilized as reporter cell lines as initially desired, these constructs provide a framework for future creation of reporter cell lines

Correlating the function and phenotype of pluripotent stem cells

The unique properties of self-renewal and multilineage differentiation enables pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to be well-positioned for a range of applications. However, outcomes of these potential applications are contingent on gaining a more in-depth understanding of their biology, as well as, safety of these cells. To date the comparative safety of hESCs and iPSCs have yet to be adequately addressed. Safety of their differentiated derivatives also necessitates the development of technology which permits the reliable elimination of pluripotent cells. Current literature describing the biology of hESCs and iPSCs has utilized heterogeneous cell populations. To derive a more precise definition of what constitutes a pluripotent stem cell, defined sub-fractions were obtained via fluorescence activated cell sorting (FACS) based on the expression of surface markers CD9 (TG30) and GCTM-2. The TG30-GCTM-2 double sort yields four immunologically defined sub-fractions arbitrarily termed TG30Neg-GCTM-2Neg (P4) to TG30High-GCTM-2High (P7). This double sorting strategy was rigorously interrogated by a series of assays, including microarray analysis, colony-forming assay and teratoma assay to be a robust strategy to enrich for pluripotent cells. Having established the validity of the TG30-GCTM-2 double-sort, the equivalency of iPSCs and hESCs was then assessed by the objective comparison of corresponding sub-fractions. In agreement with current literature, initial flow cytometric and microarray analysis of both cell types displayed highly similar profiles. However, our studies subsequently demonstrated for the first time the existence of functional differences between hESCs and iPSCs. Colony-forming and teratoma formation assays, as well as, immunostaining results revealed that all four sub-fractions of iPSCs were able to give rise to stem cell colonies. In contrast, stem cell colonies were notably absent in P4 and P5 hESCs and only observed in P6 and P7 hESCs. Furthermore, flow cytometric analysis revealed spontaneous reversion of differentiated derivatives of the P4 sub-fraction of iPSCs within seven days. Gene expression studies using Q-PCR noted that iPSCs possess a marked dysregulation of a selected number of pluripotency genes. A logical progression from the findings of these initial studies was to determine the possible mechanism(s) responsible for the observed reversion phenomenon. Utilizing an additional three iPSC lines generated using different approaches and gene cocktails, it was found that whilst persistence of viral transgenes could have increased colony-forming efficiency, in isolation this factor proved to be insufficient to cause spontaneous reversion. The reversion phenomenon was also noted to be independent of the concentration of FGF-2 used to culture the two cell types. Finally, we determined that the presence of residual OCT-4+ cells in the P4 sub-fraction could not account for the reversion phenomenon. Collectively, whilst these studies have yet to definitively elucidate the exact mechanism(s) responsible for the spontaneous reversion of differentiated derivatives of iPSCs, we have at least ruled out some plausible mechanism(s). A useful tool which lends itself to the understanding the biology of PSCs, as well as, the mechanism(s) responsible for reversion and reprogramming, is a reporter cell line linked to a pluripotency gene. Such a reporter cell line would also enable the enrichment and/or elimination of pluripotent cells, with the latter addressing safety concerns regarding the clinical use of PSCs. In this regard, two pluripotency genes - GALANIN and GDF-3 were identified based on results obtained from microarray and Q-PCR analysis. GDF-3/GALANIN promoter constructs were used to create the resultant reporter cell line using a lentiviral vector approach. The GALANIN reporter cell line which appeared to be constitutively active regardless of the culture condition used was excluded from further studies. The GDF-3 reporter cell lines were then characterized using colony-forming assay, immunostaining, EB formation and karyotyping. However, karyotyping results revealed that both the GDF-3 and GALANIN cell lines possess gross chromosomal abnormalities, an outcome possibly attributed to the random insertion of viral vectors into the cells’ genome. Whilst both the GDF-3 and GALANIN reporter cell lines may not be utilized as reporter cell lines as initially desired, these constructs provide a framework for future creation of reporter cell lines

Spontaneous network activity transiently depresses synaptic transmission in the embryonic chick spinal cord

We examined the effects of spontaneous or evoked episodes of rhythmic activity on synaptic transmission in several spinal pathways of embryonic day 9-12 chick embryos. We compared the amplitude of synaptic potentials evoked by stimulation of the ventrolateral funiculus (VLF), the dorsal or ventral roots, before and after episodes of activity. With the exception of the short-latency responses evoked by dorsal root stimulation, the potentials were briefly potentiated and then reduced for several minutes after an episode of rhythmic activity. Their amplitude progressively recovered in the interval between successive episodes. The lack of post-episode depression in the short-latency component of the dorsal root evoked responses is probably attributable to the absence of firing in cut muscle afferents during an episode of activity. The post-episode depression of VLF-evoked potentials was mimicked by prolonged stimulation of the VLF, subthreshold for an episode of activity. By contrast, antidromically induced motoneuron firing and the accompanying calcium entry did not depress VLF-evoked potentials recorded from the stimulated ventral root. In addition, post-episode depression of VLF-evoked synaptic currents was observed in voltage-clamped spinal neurons. Collectively, these findings suggest that somatic postsynaptic activity and calcium entry are not required for the depression. We propose instead that the mechanism may involve a form of long-lasting activity-induced synaptic depression, possibly a combination of transmitter depletion and ligand- induced changes in the postsynaptic current accompanying transmitter release. This activity-dependent depression appears to be an important mechanism underlying the occurrence of spontaneous activity in developing spinal networks

Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme

Summary: Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation. : In this article, Carpenedo and Stanford and colleagues demonstrate a robust and reproducible single-cell seeding method for rapid induction of mesendoderm for hPSCs. Transcriptomic data indicated that the method could be applied to mesoderm and endoderm differentiation protocols, which was demonstrated experimentally. Formation of mesoderm and endoderm following pre-differentiation was further demonstrated in long-term in vitro and in vivo assays. Keywords: mesendoderm, human pluripotent stem cells, defined differentiation, human embryonic stem cells, human induced pluripotent stem cell

Identification of Unsafe Human Induced Pluripotent Stem Cell Lines Using a Robust Surrogate Assay for Pluripotency

Abstract Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify “unsafe” hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these “unsafe” hiPSC lines should be interpreted carefully.</jats:p