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1.
Stem Cell Reports ; 19(4): 562-578, 2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38552632

ABSTRACT

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, because cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but their functional consequences in differentiating cells are scarcely described. In this work, we show that the loss of chromosome 18q impairs neuroectoderm commitment and that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for this failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, and transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, suggesting that these cells are in an altered state of pluripotency.


Subject(s)
Human Embryonic Stem Cells , Pluripotent Stem Cells , Humans , Cell Differentiation/genetics , Pluripotent Stem Cells/metabolism , Chromosomes
2.
Nat Commun ; 15(1): 1232, 2024 Feb 09.
Article in English | MEDLINE | ID: mdl-38336715

ABSTRACT

Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.


Subject(s)
Infant, Premature , Premature Birth , Pregnancy , Infant, Newborn , Female , Humans , Pregnancy Outcome , Pregnancy, Multiple , Premature Birth/epidemiology , Birth Weight , Mitochondria/genetics , DNA, Mitochondrial/genetics
3.
Hum Mol Genet ; 31(21): 3629-3642, 2022 10 28.
Article in English | MEDLINE | ID: mdl-35285472

ABSTRACT

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.


Subject(s)
DNA, Mitochondrial , Embryonic Development , Adult , Humans , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Cell Lineage/genetics , Embryonic Development/genetics , Oocytes/metabolism , Mitochondria/genetics , Mosaicism
4.
Biol Open ; 11(1)2022 01 15.
Article in English | MEDLINE | ID: mdl-35019138

ABSTRACT

Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.


Subject(s)
Myotonic Dystrophy , Embryonic Stem Cells/metabolism , Humans , Methylation , Muscle Development/genetics , Muscle, Skeletal/metabolism , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Myotonin-Protein Kinase/genetics , Myotonin-Protein Kinase/metabolism , RNA/metabolism
5.
Sci Rep ; 11(1): 6137, 2021 03 17.
Article in English | MEDLINE | ID: mdl-33731744

ABSTRACT

Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.


Subject(s)
Endoderm , Gene Expression Regulation, Developmental , Human Embryonic Stem Cells , Wnt Signaling Pathway , Cell Differentiation , Cell Line , Endoderm/cytology , Endoderm/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans
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