Pluripotent Stem Cells in Clinical Setting-New Developments and Overview of Current Status.

The number of clinical trials using human pluripotent stem cells (hPSC)-both embryonic and induced pluripotent stem cells (hESC/iPSC)-has expanded in the last several years beyond expectations. By the end of 2021, a total of 90 trials had been registered in 13 countries with more than 3000 participants. However, only US, Japan, China, and the UK are conducting both hESC- and hiPSC-based trials. Together US, Japan, and China have registered 78% (70 out of 90) of all trials worldwide. More than half of all trials (51%) are focused on the treatment of degenerative eye diseases and malignancies, enrolling nearly 2/3 of all participants in hPSC-based trials. Although no serious adverse events resulting in death or morbidity due to hPSC-based cellular therapy received have been reported, information about safety and clinical efficacy are still very limited. With the availability of novel technologies for precise genome editing, a new trend in the development of hPSC-based cellular therapies seems to be emerging. Engineering universal donor hPSC lines has become a holy grail in the field. Indeed, because of its effectiveness and simplicity nanomedicine and in vivo delivery of gene therapy could become more advantageous than cellular therapies for the treatment of multiple diseases. In the future, for the best outcome, hPSC-based cellular therapy might be combined with other technological advancements, such as biomimetic epidural electrical stimulation that can restore trunk and leg motor functions after complete spinal injury.

A novel non-invasive prenatal sickle cell disease test for all at-risk pregnancies.

Sickle cell disease (SCD) is the most common genetic haematological disorder. The availability of non-invasive prenatal diagnosis (NIPD) is predicted to increase uptake of prenatal diagnosis for SCD, as it has no perceived procedure-related miscarriage risk. We report the development of a targeted massively parallel sequencing (MPS) assay for the NIPD of fetal SCD using fetal cell-free (cf)DNA from maternal plasma, with no requirement for paternal or proband samples. In all, 64 plasma samples from pregnant women were analysed: 42 from SCD carriers, 15 from women with homozygous (Hb SS) SCD and seven from women with compound heterozygous (Hb SC) SCD. Our assay incorporated a relative mutation dosage assay for maternal carriers and a wild type allele detection assay for affected women (Hb SS/Hb SC). Selective analysis of only smaller cfDNA fragments and modifications to DNA fragment hybridisation capture improved diagnostic accuracy. Clinical sensitivity was 100% and clinical specificity was 100%. One sample with a fetal fraction of <4% was correctly called as 'unaffected', but with a discordant genotype (Hb AA rather than Hb AS). Six samples gave inconclusive results, of which two had a fetal fraction of <4%. This study demonstrates that NIPD for SCD is approaching clinical utility.

Effects of thyroid hormone on mitochondria and metabolism of human preimplantation embryos.

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10-7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.

Assisted reproductive technologies to prevent human mitochondrial disease transmission.

Mitochondria are essential cytoplasmic organelles that generate energy (ATP) by oxidative phosphorylation and mediate key cellular processes such as apoptosis. They are maternally inherited and in humans contain a 16,569-base-pair circular genome (mtDNA) encoding 37 genes required for oxidative phosphorylation. Mutations in mtDNA cause a range of pathologies, commonly affecting energy-demanding tissues such as muscle and brain. Because mitochondrial diseases are incurable, attention has focused on limiting the inheritance of pathogenic mtDNA by mitochondrial replacement therapy (MRT). MRT aims to avoid pathogenic mtDNA transmission between generations by maternal spindle transfer, pronuclear transfer or polar body transfer: all involve the transfer of nuclear DNA from an egg or zygote containing defective mitochondria to a corresponding egg or zygote with normal mitochondria. Here we review recent developments in animal and human models of MRT and the underlying biology. These have led to potential clinical applications; we identify challenges to their technical refinement.

Human embryos from induced pluripotent stem cell-derived gametes: ethical and quality considerations.

Protocols for successful differentiation of male and female gametes from induced pluripotent stem cells have been published. Although culture of precursor cells in a natural microenvironment remains necessary to achieve terminal differentiation, the creation of human preimplantation embryos from induced pluripotent stem cell-derived gametes is technically feasible. Such embryos could provide a solution to the scarcity of human cleavage-stage embryos donated for research. Here, we discuss current technology, major research-related ethical concerns and propose the norms that would assure the quality and reliability of such embryos.

Efficient and cost-effective genetic analysis of products of conception and fetal tissues using a QF-PCR/array CGH strategy; five years of data.

BACKGROUND: Traditional testing of miscarriage products involved culture of tissue followed by G-banded chromosome analysis; this approach has a high failure rate, is labour intensive and has a resolution of around 10 Mb. G-banded chromosome analysis has been replaced by molecular techniques in some laboratories; we previously introduced a QF-PCR/MLPA testing strategy in 2007. To improve diagnostic yield and efficiency we have now updated our testing strategy to a more comprehensive QF-PCR assay followed by array CGH. Here we describe the results from the last 5 years of service. METHODS: Fetal tissue samples and products of conception were tested using QF-PCR which will detect aneuploidy for chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. Samples that were normal were then tested by aCGH and all imbalance >1Mb and fully penetrant clinically significant imbalance <1Mb was reported. RESULTS: QF-PCR analysis identified aneuploidy/triploidy in 25.6% of samples. aCGH analysis detected imbalance in a further 9.6% of samples; this included 1.8% with submicroscopic imbalance and 0.5% of uncertain clinical significance. This approach has a failure rate of 1.4%, compared to 30% for G-banded chromosome analysis. CONCLUSIONS: This efficient QF-PCR/aCGH strategy has a lower failure rate and higher diagnostic yield than karyotype or MLPA strategies; both findings are welcome developments for couples with recurrent miscarriage.

Concise Review: Human Embryonic Stem Cells-What Have We Done? What Are We Doing? Where Are We Going?

Human pluripotent stem cells possess remarkable proliferative and developmental capacity and thus have great potential for advancement of cellular therapy, disease modeling, and drug discovery. Twelve years have passed since the first reported isolation of human embryonic stem cell lines (hESC), followed in October 2010 by the first treatment of a patient with hESC-based cellular therapy at the Shepherd Center in Atlanta. Despite seemingly insurmountable challenges and obstacles in the early days, hESC clinical potential reached application in an extraordinarily short time. Eight currently ongoing clinical trials are yielding encouraging results, and these are likely to lead to new trials for other diseases. However, with the discovery of induced pluripotent stem cells (iPSC), disease-specific hESC lines derived from patients undergoing preimplantation genetic diagnosis for single gene disorders fell short of expectations. Lack of ethical controversy made human iPSC (hiPSC) with specific genotypes/phenotypes more appealing than hESC for drug discovery and toxicology-related studies, and in time, lines from HLA-homologous hiPSC banks are likely to take over from hESC in clinical applications. Currently, hESC are indispensable; the results of hESC-based clinical trials will set a gold standard for future iPSC-based cellular therapy. Stem Cells 2017;35:17-25.

Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

BACKGROUND: Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. OBJECTIVE AND RATIONALE: Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. SEARCH METHODS: A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. OUTCOMES: A very limited number of studies have been conducted in humans and non-human primates. The published material, especially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical, but also for research, purposes. WIDER IMPLICATIONS: The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human embryos.

Chromosomal microarray testing in adults with intellectual disability presenting with comorbid psychiatric disorders.

Chromosomal copy-number variations (CNVs) are a class of genetic variants highly implicated in the aetiology of neurodevelopmental disorders, including intellectual disabilities (ID), schizophrenia and autism spectrum disorders (ASD). Yet the majority of adults with idiopathic ID presenting to psychiatric services have not been tested for CNVs. We undertook genome-wide chromosomal microarray analysis (CMA) of 202 adults with idiopathic ID recruited from community and in-patient ID psychiatry services across England. CNV pathogenicity was assessed using standard clinical diagnostic methods and participants underwent comprehensive medical and psychiatric phenotyping. We found an 11% yield of likely pathogenic CNVs (22/202). CNVs at recurrent loci, including the 15q11-q13 and 16p11.2-p13.11 regions were most frequently observed. We observed an increased frequency of 16p11.2 duplications compared with those reported in single-disorder cohorts. CNVs were also identified in genes known to effect neurodevelopment, namely NRXN1 and GRIN2B. Furthermore deletions at 2q13, 12q21.2-21.31 and 19q13.32, and duplications at 4p16.3, 13q32.3-33.3 and Xq24-25 were observed. Routine CMA in ID psychiatry could uncover ~11% new genetic diagnoses with potential implications for patient management. We advocate greater consideration of CMA in the assessment of adults with idiopathic ID presenting to psychiatry services.

Human Embryos Created by Embryo Splitting Secrete Significantly Lower Levels of miRNA-30c.

Studies reporting term pregnancy and the production of genetically identical offspring from isolated blastomeres of early stage embryos have been carried out in small and large animals. However, very little is known about the effects of embryo splitting on the development and reproductive competency of human embryos. In this study, we investigated the effects of embryo splitting on profile of microRNAs (miRNAs) detected in their spent blastocyst medium (SBM) by comparative analysis of miRNA profiles in SBM of human twin embryos created by blastomere biopsy and SBM of blastocysts that resulted in a healthy pregnancy and live birth following embryo transfer. The profile of miRNA secretion in in vitro culture media consistently distinguishes twin from control embryos. We found that six miRNAs are significantly more abundant in SBM from twin embryos, while nine are significantly more abundant in SBM from euploid implanted blastocysts. These nine include miRNA-30c, a previously reported marker of blastocyst implantation potential. Furthermore, 22.9% of miRNAs secreted by twin embryos were never detected in SBM from normal reproductively competent blastocysts, or from trophectoderm (TE) samples from normal blastocysts donated for the research. The miRNA profile, unique to twin blastocysts, might be a result of differential lineage commitment in these embryos.

Gestation related karyotype, QF-PCR and CGH-array failure rates in diagnostic amniocentesis.

BACKGROUND: Few data exist describing laboratory related failure rates in prenatal diagnosis. The aim of this study is to assess the laboratory associated failure rate for karyotype, QF-PCR and CGH-array following amniocentesis in relation to gestation. METHODS: Retrospective database study of amniocenteses performed 2004-2014 comparing laboratory failure rate for karyotype, QF-PCR and CGH-array between 16 + 0 and 40 + 0 weeks' gestation. RESULTS: A total of 10 484 amniotic fluid test results were collected in three databases. Karyotype failed in 41/1797 (2.3%) tests; failure rate was significantly greater with advancing gestation reaching 43% at 36-40 weeks. QF-PCR failed in 132/5715 tests (2.3%) and was significantly greater with advancing gestation reaching 7% at 36-40 weeks. For CGH-array, 10/298 tests (3.4%) failed analysis. In one case, no result was obtainable by any technique. CONCLUSIONS: These data provide gestation specific laboratory failure rates for amniocentesis enabling informed decisions about the timing and laboratory technique most applicable to the clinical situation. Before 20 weeks, karyotype is least likely to fail of the three techniques. However, in the late third trimester, QF-PCR and, in particular, karyotyping are more likely to fail than CGH-array. Although there is some overlap between the three different tests, they may be preferentially offered in different clinical scenarios. © 2016 John Wiley & Sons, Ltd.

Parental Origin of Interstitial Duplications at 15q11.2-q13.3 in Schizophrenia and Neurodevelopmental Disorders.

Duplications at 15q11.2-q13.3 overlapping the Prader-Willi/Angelman syndrome (PWS/AS) region have been associated with developmental delay (DD), autism spectrum disorder (ASD) and schizophrenia (SZ). Due to presence of imprinted genes within the region, the parental origin of these duplications may be key to the pathogenicity. Duplications of maternal origin are associated with disease, whereas the pathogenicity of paternal ones is unclear. To clarify the role of maternal and paternal duplications, we conducted the largest and most detailed study to date of parental origin of 15q11.2-q13.3 interstitial duplications in DD, ASD and SZ cohorts. We show, for the first time, that paternal duplications lead to an increased risk of developing DD/ASD/multiple congenital anomalies (MCA), but do not appear to increase risk for SZ. The importance of the epigenetic status of 15q11.2-q13.3 duplications was further underlined by analysis of a number of families, in which the duplication was paternally derived in the mother, who was unaffected, whereas her offspring, who inherited a maternally derived duplication, suffered from psychotic illness. Interestingly, the most consistent clinical characteristics of SZ patients with 15q11.2-q13.3 duplications were learning or developmental problems, found in 76% of carriers. Despite their lower pathogenicity, paternal duplications are less frequent in the general population with a general population prevalence of 0.0033% compared to 0.0069% for maternal duplications. This may be due to lower fecundity of male carriers and differential survival of embryos, something echoed in the findings that both types of duplications are de novo in just over 50% of cases. Isodicentric chromosome 15 (idic15) or interstitial triplications were not observed in SZ patients or in controls. Overall, this study refines the distinct roles of maternal and paternal interstitial duplications at 15q11.2-q13.3, underlining the critical importance of maternally expressed imprinted genes in the contribution of Copy Number Variants (CNVs) at this interval to the incidence of psychotic illness. This work will have tangible benefits for patients with 15q11.2-q13.3 duplications by aiding genetic counseling.

Microduplications at the pseudoautosomal SHOX locus in autism spectrum disorders and related neurodevelopmental conditions.

BACKGROUND: The pseudoautosomal short stature homeobox-containing (SHOX) gene encodes a homeodomain transcription factor involved in cell-cycle and growth regulation. SHOX/SHOX enhancers deletions cause short stature and skeletal abnormalities in a female-dominant fashion; duplications appear to be rare. Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASDs), are complex disorders with high heritability and skewed sex ratio; several rare (<1% frequency) CNVs have been implicated in risk. METHODS: We analysed data from a discovery series of 90 adult ASD cases, who underwent clinical genetic testing by array-comparative genomic hybridisation (CGH). Twenty-seven individuals harboured CNV abnormalities, including two unrelated females with microduplications affecting SHOX. To determine the prevalence of SHOX duplications and delineate their associated phenotypic spectrum, we subsequently examined array-CGH data from a follow-up sample of 26 574 patients, including 18 857 with NDD (3541 with ASD). RESULTS: We found a significant enrichment of SHOX microduplications in the NDD cases (p=0.00036; OR 2.21) and, particularly, in those with ASD (p=9.18×10(-7); OR 3.63) compared with 12 594 population-based controls. SHOX duplications affecting the upstream or downstream enhancers were enriched only in females with NDD (p=0.0043; OR 2.69/p=0.00020; OR 7.20), but not in males (p=0.404; OR 1.38/p=0.096; OR 2.21). CONCLUSIONS: Microduplications at the SHOX locus are a low penetrance risk factor for ASD/NDD, with increased risk in both sexes. However, a concomitant duplication of SHOX enhancers may be required to trigger a NDD in females. Since specific SHOX isoforms are exclusively expressed in the developing foetal brain, this may reflect the pathogenic effect of altered SHOX protein dosage on neurodevelopment.

Autism and chromosome abnormalities-A review.

The neuro-behavioral disorder of autism was first described in the 1940s and was predicted to have a biological basis. Since that time, with the growth of genetic investigations particularly in the area of pediatric development, an increasing number of children with autism and related disorders (autistic spectrum disorders, ASD) have been the subject of genetic studies both in the clinical setting and in the wider research environment. However, a full understanding of the biological basis of ASDs has yet to be achieved. Early observations of children with chromosomal abnormalities detected by G-banded chromosome analysis (karyotyping) and in situ hybridization revealed, in some cases, ASD associated with other features arising from such an abnormality. The introduction of higher resolution techniques for whole genome screening, such as array comparative genome hybridization (aCGH), allowed smaller imbalances to be detected, some of which are now considered to represent autism susceptibility loci. In this review, we describe some of the work underpinning the conclusion that ASDs have a genetic basis; a brief history of the developments in genetic analysis tools over the last 50 years; and the most common chromosome abnormalities found in association with ASDs. Introduction of next generation sequencing (NGS) into the clinical diagnostic setting is likely to provide further insights into this complex field but will not be covered in this review. Clin. Anat. 29:620-627, 2016. © 2016 Wiley Periodicals, Inc.

Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study.

Discordant growth is a common complication of monochorionic/diamniotic pregnancies; in approximately 50% of cases, the cause is unknown. The case presented here suggests that discordant growth of monozygotic twins could start during preimplantation development. Two inner cell masses (ICMs) within the same blastocyst may originate in uneven splitting of a single "parental" ICM, or the two ICMs may be formed independently de novo. We studied the transcriptomes of two morphologically distinct ICMs within a single blastocyst using high-resolution RNA sequencing. The data indicated that the two ICM were at different stages of development; one was in the earliest stages of lineage commitment, while the other had already differentiated into epiblast and primitive endoderm. IGF1-mediated signaling is likely to play a key role in ICM growth and to be the major driver behind these differences.

Developmental clock compromises human twin model created by embryo splitting.

STUDY QUESTION: Is the quality of the human embryos generated by twinning in vitro comparable to the quality of the embryos created by fertilization? SUMMARY ANSWER: Our data suggest that the human twin embryos created in vitro are unsuitable not only for clinical use but also for research purposes. WHAT IS KNOWN ALREADY: Pregnancy from in vitro generated monozygotic twins by embryo splitting or twinning leads to live birth of healthy animals. Similar strategies, however, have been less successful in primates. Recent reports suggest that the splitting of human embryos might result in viable, morphologically adequate blastocysts, although the qualitative analyses of the embryos created in such a way have been very limited. STUDY DESIGN, SIZE, DURATION: This study was a comparative analysis of embryos generated by twinning in vitro and the embryos created by in vitro fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analysed morphokinetics and developmental competence of 176 twin embryos created by splitting of 88 human embryos from either early (2-5 blastomeres, n = 43) or late (6-10 blastomeres, n = 45) cleavage stages. We compared the data with morphometrics of embryos created by in vitro fertilization and resulting in pregnancy and live birth upon single blastocyst transfer (n = 42). MAIN RESULTS AND THE ROLE OF CHANCE: The morphokinetic data suggested that the human preimplantation development is subjected to a strict temporal control. Due to a 'developmental clock', the size of twin embryos was proportionate to the number of cells used for their creation. Furthermore, the first fate decision was somewhat delayed; the inner cell mass (ICM) became distinguishable later in the twin than in the normal blastocysts obtained through fertilization. If an ICM was present at all, it was small and of poor quality. The majority of the cells in the twin embryos expressed ICM and trophectoderm markers simultaneously. LIMITATIONS, REASONS FOR CAUTION: We created monozygotic twins by blastomere separation from cleavage stage embryos. Embryo twinning by blastocyst bisection may circumvent limitations set by the developmental clock. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our data suggest that the human twin embryos created in vitro are unsuitable not only for clinical use but also for research purposes.

Microdeletions of ELP4 Are Associated with Language Impairment, Autism Spectrum Disorder, and Mental Retardation.

Copy-number variations (CNVs) are important in the aetiology of neurodevelopmental disorders and show broad phenotypic manifestations. We compared the presence of small CNVs disrupting the ELP4-PAX6 locus in 4,092 UK individuals with a range of neurodevelopmental conditions, clinically referred for array comparative genomic hybridization, with WTCCC controls (n = 4,783). The phenotypic analysis was then extended using the DECIPHER database. We followed up association using an autism patient cohort (n = 3,143) compared with six additional control groups (n = 6,469). In the clinical discovery series, we identified eight cases with ELP4 deletions, and one with a partial duplication of ELP4 and PAX6. These cases were referred for neurological phenotypes including language impairment, developmental delay, autism, and epilepsy. Six further cases with a primary diagnosis of autism spectrum disorder (ASD) and similar secondary phenotypes were identified with ELP4 deletions, as well as another six (out of nine) with neurodevelopmental phenotypes from DECIPHER. CNVs at ELP4 were only present in 1/11,252 controls. We found a significant excess of CNVs in discovery cases compared with controls, P = 7.5 × 10(-3) , as well as for autism, P = 2.7 × 10(-3) . Our results suggest that ELP4 deletions are highly likely to be pathogenic, predisposing to a range of neurodevelopmental phenotypes from ASD to language impairment and epilepsy.