The tumorigenic potential of pluripotent stem cells: What can we do to minimize it?

Human pluripotent stem cells (hPSCs) have the potential to fundamentally change the way that we go about treating and understanding human disease. Despite this extraordinary potential, these cells also have an innate capability to form tumors in immunocompromised individuals when they are introduced in their pluripotent state. Although current therapeutic strategies involve transplantation of only differentiated hPSC derivatives, there is still a concern that transplanted cell populations could contain a small percentage of cells that are not fully differentiated. In addition, these cells have been frequently reported to acquire genetic alterations that, in some cases, are associated with certain types of human cancers. Here, we try to separate the panic from reality and rationally evaluate the true tumorigenic potential of these cells. We also discuss a recent study examining the effect of culture conditions on the genetic integrity of hPSCs. Finally, we present a set of sensible guidelines for minimizing the tumorigenic potential of hPSC-derived cells. © 2016 The Authors. Inside the Cell published by Wiley Periodicals, Inc.

Genomic instability in pluripotent stem cells: implications for clinical applications.

Human pluripotent stem cells (hPSCs) are known to acquire genomic changes as they proliferate and differentiate. Despite concerns that these changes will compromise the safety of hPSC-derived cell therapy, there is currently scant evidence linking the known hPSC genomic abnormalities with malignancy. For the successful use of hPSCs for clinical applications, we will need to learn to distinguish between innocuous genomic aberrations and those that may cause tumors. To minimize any effects of acquired mutations on cell therapy, we strongly recommend that cells destined for transplant be monitored throughout their preparation using a high-resolution method such as SNP genotyping.

Matched miRNA and mRNA signatures from an hESC-based in vitro model of pancreatic differentiation reveal novel regulatory interactions.

The differentiation of human pluripotent stem cells (hPSCs) to insulin-expressing beta islet-like cells is a promising in vitro model system for studying the molecular signaling pathways underlying beta cell differentiation, as well as a potential source of cells for the treatment of type 1 diabetes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate many biological processes, including cellular differentiation. We studied the miRNA and mRNA expression profiles of hPSCs at five stages of in vitro differentiation along the pancreatic beta cell lineage (definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor and hormone-expressing endocrine cells) in the context of samples of primary human fetal pancreas and purified adult islet cells using microarray analysis. Bioinformatic analysis of the resulting data identified a unique miRNA signature in differentiated beta islet cells, and predicted the effects of key miRNAs on mRNA expression. Many of the predicted miRNA-mRNA interactions involved mRNAs known to play key roles in the epithelial-mesenchymal transition process and pancreatic differentiation. We validated a subset of the predictions using qRT-PCR, luciferase reporter assays and western blotting, including the known interaction between miR-200 and ZEB2 (involved in epithelial-mesenchymal transition) and the novel interaction between miR-200 and SOX17 (a key transcription factor in specification of definitive endoderm). In addition, we found that miR-30d and let-7e, two miRNAs induced during differentiation, regulated the expression of RFX6, a transcription factor that directs pancreatic islet formation. These findings suggest that precise control of target mRNA expression by miRNAs ensures proper lineage specification during pancreatic development.

Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells.

5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at base resolution. Implementing this approach, termed "TAB-array", we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular progenitors and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we identified a large number of novel dynamically methylated genomic regions that are implicated in the development of these lineages. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease.

Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development.

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.

The Promoting Equity in Stem Cell Genomics Survey.

Aim: This study aimed to determine knowledge and attitudes toward induced pluripotent stem cell technology and biobanking. Methods: A survey instrument was developed to determine individuals' knowledge of and attitudes toward these technologies. Results: Results from 276 ethnically diverse participants who took the online survey demonstrated significant associations (p ≤ 0. 05) in knowledge by ethnicity and race regarding properties of stem cells, different types of stem cells and previous sample donation behavior. Significantly more Whites 39% (n = 53) compared with Blacks or African-Americans 19.2% (n = 14) had previous knowledge of induced pluripotent stem cells (χ2 = 8.544; p = 0.003) Conclusion: Overall, White race was associated with greater knowledge about stem cells and biobanks and greater willingness to donate samples for future research.

Stem cell biobanks have few samples from minorities for genomic studies. We conducted an online survey to understand knowledge and attitudes toward stem cell biobanks and technologies. Overall, we learned that White race was associated with the greatest knowledge about stem cell biobanks and willingness to contribute tissue samples for biobanks. More education is required so that minorities are willing to contribute tissue samples toward stem cell biobanks. This will help researchers study the genomic bases of disease and pursue translational research toward addressing health inequities.

EXIT procedure: technique and indications with prenatal imaging parameters for assessment of airway patency.

Successful management of fetal conditions in which airway obstruction is anticipated is now possible because of advances in prenatal imaging and the development of innovative techniques to secure the fetal airway before complete separation of the fetus from the maternal circulation. Fetal ultrasonography and fetal magnetic resonance imaging are complementary imaging modalities in the assessment of fetuses with potential airway obstruction. The ex utero intrapartum therapy (EXIT) procedure is used to secure the fetal airway before complete delivery of the fetus. However, successful intrapartum treatment of fetuses who may need prolonged placental support depends on a multidisciplinary assessment in which the benefits of the EXIT procedure for the fetus are weighed against the risk of maternal complications that may occur during prolongation of the intrapartum period to secure the fetal airway. This multidisciplinary approach requires an understanding of the types of lesions in which intrapartum fetal airway access would be beneficial, a knowledge of the prenatal images that would best delineate the anatomic defect and thus help guide the best approach to securing the airway, and consensus and coordination among medical ethicists, radiologists, obstetric anesthesiologists and obstetricians, pediatric surgeons and anesthesiologists, and neonatologists.

Development of a prenatal clinical care pathway for uncomplicated gastroschisis and literature review.

BACKGROUND: Gastroschisis is an abdominal wall defect wherein the bowel is herniated into the amniotic fluid. Controversy exists regarding optimal prenatal surveillance strategies that predict fetal well-being and help guide timing of delivery. Our objective was to develop a clinical care pathway for prenatal management of uncomplicated gastroschisis at our institution. METHODS: We performed a review of literature from January 1996 to May 2017 to evaluate prenatal ultrasound (US) markers and surveillance strategies that help determine timing of delivery and optimize outcomes in fetal gastroschisis. RESULTS: A total 63 relevant articles were identified. We found that among the US markers, intraabdominal bowel dilatation, polyhydramnios, and gastric dilatation are potentially associated with postnatal complications. Prenatal surveillance strategy with monthly US starting at 28weeks of gestational age (wGA) and twice weekly non-stress testing beginning at 32wGA is recommended to optimize fetal wellbeing. Timing of delivery should be based on obstetric indications and elective preterm delivery prior to 37wGA is not indicated. CONCLUSIONS: Close prenatal surveillance of fetal gastroschisis is necessary due to the high risk for adverse outcomes including intrauterine fetal demise in the third trimester. Decisions regarding the timing of delivery should take into consideration the additional prematurity-associated morbidity.

Rewinding Extinction in the Northern White Rhinoceros: Genetically Diverse Induced Pluripotent Stem Cell Bank for Genetic Rescue.

Extinction rates are rising, and current conservation technologies may not be adequate for reducing species losses. Future conservation efforts may be aided by the generation of induced pluripotent stem cells (iPSCs) from highly endangered species. Generation of a set of iPSCs from multiple members of a species can capture some of the dwindling genetic diversity of a disappearing species. We generated iPSCs from fibroblasts cryopreserved in the Frozen Zoo®: nine genetically diverse individuals of the functionally extinct northern white rhinoceros (Ceratotherium simum cottoni) and two from the closely related southern white rhinoceros (Ceratotherium simum simum). We used a nonintegrating Sendai virus reprogramming method and developed analyses to confirm the cells' pluripotency and differentiation potential. This work is the first step of a long-term interdisciplinary plan to apply assisted reproduction techniques to the conservation of this highly endangered species. Advances in iPSC differentiation may enable generation of gametes in vitro from deceased and nonreproductive individuals that could be used to repopulate the species.

Agricultural-related chemical exposures, season of conception, and risk of gastroschisis in Washington State.

OBJECTIVE: We sought to determine if periconceptional exposure to agrichemicals was associated with the development of gastroschisis. STUDY DESIGN: We conducted a retrospective, case-controlled study using Washington State Birth Certificate and US Geological Survey databases. Cases included all live-born singleton infants with gastroschisis. Distance between a woman's residence and site of elevated exposure to agrichemicals was calculated. Multivariate regression was used to estimate the association between surface water concentrations of agrichemicals and the risk of gastroschisis. RESULTS: Eight hundred five cases and 3616 control subjects were identified. Gastroschisis occurred more frequently among those who resided <25 km from a site of high atrazine concentration (odds ratio, 1.6). Risk was related inversely to the distance between the maternal residence and the closest toxic atrazine site. In multivariate analysis, nulliparity, tobacco use, and spring conception remained significant predictive factors for gastroschisis. CONCLUSION: Maternal exposure to surface water atrazine is associated with fetal gastroschisis, particularly in spring conceptions.

Delayed interval delivery in the setting of placental abruption: a case report.

BACKGROUND: Attempts at delayed interval deliveries in multifetal gestations have become more common. However, selection criteria are imperative to success, and placental abruption is generally considered a contraindication. CASE: A woman with a diamniotic-dichorionic twin gestation at 23 weeks presented after a motor vehicle accident with placental abruption, hypofibrinogenemia an intrauterine fetal demise of twin A. She was expectantly managed, and the hypofibrinogenemia was nonprogressive. One week later, after delivery of twin A, a delayed interval delivery was attempted with tocolysis and antibiotics. Prolongation of the pregnancy allowed the delivery of a viable neonate. CONCLUSION: Delayed interval delivery can be a reasonable option in the setting of placental abruption if maternal hemodynamic status is closely monitored and the patient is thoroughly counseled.

Author Correction: Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Opioid prescribing trends in postpartum women: a multicenter study.

BACKGROUND: The postpartum period can be a particularly vulnerable time for exposure to opioid medications, and there are currently no consensus guidelines for physicians to follow regarding opioid prescribing during this period. OBJECTIVE: The purpose of this study was to evaluate inter- and intrahospital variability in opioid prescribing patterns in postpartum women and better understand the role of clinical variables in prescribing. STUDY DESIGN: Data were extracted from electronic medical records on 4248 patients who delivered at 6 hospitals across the United States from January 2016 through March 2016. The primary outcome of the study was postpartum opioid prescription at the time of hospital discharge. Age, parity, route of delivery, and hospital were analyzed individually and with multivariate analyses to minimize confounding factors. Statistical methods included χ2 to analyze frequency of opioid prescription by hospital, parity, tobacco use, delivery method, and laceration type. An analysis of variance was used to analyze morphine equivalent dose by hospital. RESULTS: The percentage of women prescribed postpartum opioids varied significantly by hospital, ranging from 27.6% to 70.9% (P <0.001). Oxycodone-acetaminophen was the most commonly prescribed medication (50.3%) with each hospital having its preferred opioid type. Median number of tablets prescribed ranged from 20 to 40 (P < .0001). Primiparous women were more likely to receive opioids than multiparous women when broken down by a parity of 1, 2, 3, 4, and ≥5 (52.8%, 48.0%, 47.6%, 40.1%, and 45.8%, respectively, P = .0005). Among women who had vaginal deliveries, opioid prescription rates were higher in women who experienced either a second-degree laceration (35.5%, P = .0002) or a third-/fourth-degree laceration (59.3%, P < .001). CONCLUSION: Postpartum opioid prescription rates vary widely among hospitals, but providers within the same hospital tend to follow similar prescribing trends. The variation in prescribing found in our study illustrates the need for clear consensus guidelines for postpartum pain management.

First-trimester pregnancy-associated plasma protein A and subsequent abnormalities of fetal growth.

OBJECTIVE: The purpose of this study was to describe the relationship between first-trimester pregnancy-associated plasma protein A (PAPP-A) and birthweight along its continuum and at its extremes. STUDY DESIGN: This was a retrospective cohort of 1371 women who underwent first-trimester screening for fetal aneuploidy and who delivered at our hospital. RESULTS: First-trimester PAPP-A has a positive relationship with birthweight. As PAPP-A decreases, the risk of small-for-gestational-age (SGA) infants increases. PAPP-A of <10%, <5%, and <1% were associated with an increasing adjusted odds ratio for SGA infants (2.0 [95% CI, 1.2-3.5; P = .012]; 2.4 [95% CI, 1.2-4.7; P = .015]; 9.3 [95% CI, 3.4-25.5; P = .001], respectively). PAPP-A levels of >90% were associated with an adjusted odds ratio for birthweight of >4500 g of 2.9 (95% CI, 1.02-8.17; P = 0.046). CONCLUSION: First-trimester PAPP-A is a marker of placental function that correlates with birthweight along its continuum and at its extremes. The strong association between low PAPP-A and SGA warrants further investigation of its usefulness as a screening tool.

Chromosomal mosaicism in neural stem cells.

Neural stem and progenitor cells (referred to here as NSCs), located in the proliferative zones of embryonic brains, can be seen undergoing mitosis at the ventricular surface. Mitotic NSCs can be arrested in metaphase and chromosome "spreads" produced to reveal their chromosomal complement. Studies in mice and humans have revealed a prominent developmental presence of aneuploid NSCs, whereas other chromosomal defects, such as interchromosomal translocations and partial chromosomal deletions/insertions, are extremely rare (1,2). Aneuploidy is defined as the loss or gain of whole chromosomes, resulting in cells that deviate from the normal diploid number of chromosomes (46 in humans, 40 in mice). In NSCs, aneuploidy can occur as a result of mis-segregation during mitosis, through events such as lagging chromosomes, supernumerary centrosomes, and nondisjunction events (3). The percentage of aneuploid NSCs can be altered by in vivo and in vitro growth conditions as well as through genetic deletion of genes involved in DNA surveillance and repair (1,4). Aneuploidy can be detected by classical cytogenetic methods such as counting the number of chromosomes visualized by DNA dyes (e.g., 4,6-diamidino-2-phenylindole) by using standard light or fluorescence microscopy. Precise chromosome identification is much more difficult: classical methods using banding patterns or size to assign identity are very time consuming even under ideal conditions, and they are notoriously difficult in mice, which often have ambiguous banding patterns and acrocentric chromosomes. A comparatively new technique that allows the unambiguous identification of chromosomes in mice and humans is "spectral karyotyping" or SKY, developed by Ried et al. at the National Institutes of Health for the study of cancer cells (5). This technique uses chromosomal "paints" that are hybridized to chromosome spreads to produce a distinct spectral output for each chromosome. SKY offers superior speed and sensitivity in its ability to detect many types of chromosomal defects, including deletions, insertions, translocations, and aneuploidy.

30 mL Single- versus 80 mL double-balloon catheter for pre-induction cervical ripening: a randomized controlled trial.

OBJECTIVE: To compare the efficacy of an 80 mL double-balloon catheter versus a 30 mL single-balloon catheter for pre-induction cervical ripening. METHODS: We performed a randomized controlled trial of women ≥18 years with a singleton, vertex pregnancy, a reactive non-stress test, and a Bishop score ≤5 comparing an 80 mL double- versus a 30 mL single-balloon catheters for cervical ripening. Women were randomly assigned to the two catheter types, stratified 1:1 by nulliparity or multiparity. The primary outcome was achieving a Bishop score ≥6 at the time of catheter expulsion or removal assessed by chi-squared, stratified by parity. RESULTS: A total of 98 women were included in the analysis (50 in the 80 mL double and 48 in the 30 mL single-balloon catheter groups). Among nulliparous women, a greater proportion of those randomized to the 80 mL double achieved a Bishop score ≥6 at time of catheter removal (88.0% versus 28.0%; p ≤ 0.001) and delivered vaginally (60.0% versus 32.0%; p = 0.047) compared to those with the 30 mL single-balloon catheter. We found no difference by catheter type in achieving a Bishop score ≥6 or vaginal delivery among multiparous women. CONCLUSIONS: These findings suggest the 80 mL double-balloon catheter is more effective than the 30 mL single-balloon catheter for pre-induction cervical ripening and achieving a vaginal delivery in nulliparous women.

The 'sweet' spot of cellular pluripotency: protein glycosylation in human pluripotent stem cells and its applications in regenerative medicine.

INTRODUCTION: Human pluripotent stem cells (hPSCs) promise for the future of regenerative medicine. The structural and biochemical diversity associated with glycans makes them a unique type of macromolecule modification that is involved in the regulation of a vast array of biochemical events and cellular activities including pluripotency in hPSCs. The primary focus of this review article is to highlight recent advances in stem cell research from a glycobiological perspective. We also discuss how our understanding of glycans and glycosylation may help overcome barriers hindering the clinical application of hPSC-derived cells. AREAS COVERED: A literature survey using NCBI-PubMed and Google Scholar was performed in 2014. EXPERT OPINION: Regenerative medicine hopes to provide novel strategies to combat human disease and tissue injury that currently lack effective therapies. Although progress in this field is accelerating, many critical issues remain to be addressed in order for cell-based therapy to become a practical and safe treatment option. Emerging evidence suggests that protein glycosylation may significantly influence the regulation of cellular pluripotency, and that the exploitation of protein glycosylation in hPSCs and their differentiated derivatives may lead to transformative and translational discoveries for regenerative medicine. In addition, hPSCs represent a novel research platform for investigating glycosylation-related disease.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells.

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.

Protein post-translational modifications and regulation of pluripotency in human stem cells.

Post-translational modifications (PTMs) are known to be essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Defects in PTMs have been linked to numerous developmental disorders and human diseases, highlighting the importance of PTMs in maintaining normal cellular states. Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are capable of self-renewal and differentiation into a variety of functional somatic cells; these cells hold a great promise for the advancement of biomedical research and clinical therapy. The mechanisms underlying cellular pluripotency in human cells have been extensively explored in the past decade. In addition to the vast amount of knowledge obtained from the genetic and transcriptional research in hPSCs, there is a rapidly growing interest in the stem cell biology field to examine pluripotency at the protein and PTM level. This review addresses recent progress toward understanding the role of PTMs (glycosylation, phosphorylation, acetylation and methylation) in the regulation of cellular pluripotency.