Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or β-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.
Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.
The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.
Replacement of mitochondria through nuclear transfer between oocytes of two different women has emerged recently as a strategy for preventing inheritance of mtDNA diseases. Although experiments in human oocytes have shown effective replacement, the consequences of small amounts of mtDNA carryover have not been studied sufficiently. Using human mitochondrial replacement stem cell lines, we show that, even though the low levels of heteroplasmy introduced into human oocytes by mitochondrial carryover during nuclear transfer often vanish, they can sometimes instead result in mtDNA genotypic drift and reversion to the original genotype. Comparison of cells with identical oocyte-derived nuclear DNA but different mtDNA shows that either mtDNA genotype is compatible with the nucleus and that drift is independent of mitochondrial function. Thus, although functional replacement of the mitochondrial genome is possible, even low levels of heteroplasmy can affect the stability of the mtDNA genotype and compromise the efficacy of mitochondrial replacement.
Background COVID-19 may be associated with adverse maternal and neonatal outcomes in pregnancy, but there is little controlled data to quantify the magnitude of these risks or to characterize the epidemiology and risk factors. Objective To quantify the associations of COVID-19 with adverse maternal and neonatal outcomes in pregnancy and to characterize the epidemiology and risk factors. Methods We performed a matched case-control study of pregnant patients with confirmed COVID-19 (cases) who delivered between 16 and 41 weeks’ gestation from March 11-June 11, 2020. Uninfected pregnant women (controls) were matched to COVID-19 cases on a 2:1 ratio based on delivery date. Maternal demographic characteristics, COVID-19 symptoms, laboratory evaluations, obstetrical and neonatal outcomes, and clinical management were chart abstracted. The primary outcomes included (i) a composite of adverse maternal outcome, defined as preeclampsia, venous thromboembolism, antepartum admission, maternal intensive care unit admission, need for mechanical ventilation, supplemental oxygen, or maternal death; and (ii) a composite of adverse neonatal outcome, defined as respiratory distress syndrome, intraventricular hemorrhage, necrotizing enterocolitis, five-minute Apgar score <5, persistent category 2 fetal heart rate tracing despite intrauterine resuscitation, or neonatal death. In order to quantify the associations between exposure to mild and severe/critical COVID-19 and adverse maternal and neonatal outcomes, unadjusted and adjusted analyses were performed using conditional logistic regression (to account for matching), with matched-pair odds ratio (OR) and 95% confidence interval (CI) based on 1000 bias-corrected bootstrap resampling as the effect measure. Associations were adjusted for potential confounders. Results 61 confirmed COVID-19 cases were enrolled during the study period (mild disease: n=54, 88.5%; severe disease: n=6, 9.8%; and critical disease: n=1, 1.6%). The odds of adverse composite maternal outcome were 3.4 times higher among cases compared to controls (18.0% versus 8.2%, adjusted OR 3.4, 95% CI 1.2-13.4). The odds of adverse composite neonatal outcome were 1.7 times higher in the case group compared to the control group (18.0% versus 13.9%, adjusted OR 1.7, 95% CI 0.8-4.8). Stratified analyses by disease severity indicated that the morbidity associated with COVID-19 in pregnancy was largely driven by the severe/critical disease phenotype. Major risk factors for associated morbidity were Black and Hispanic race, advanced maternal age, medical comorbidities, and antepartum admissions related to COVID-19. Conclusions COVID-19 during pregnancy is associated with increased risk for adverse maternal and neonatal outcomes, an association that is primarily driven by morbidity associated with severe/critical COVID-19. Black and Hispanic race, obesity, advanced maternal age, medical comorbiditi...
The exchange of the oocyte's genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cells affected in degenerative human diseases. Such cells, carrying the patient's genome, might be useful for cell replacement. Here we report that the development of human oocytes after genome exchange arrests at late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, the resultant triploid cells develop to the blastocyst stage. Stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies removal of the oocyte genome as the primary cause of developmental failure after genome exchange.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.