Investigating the pathogenicity of the recessive p.A251T variant in monogenic diabetes using iPSC-derived beta-like cells.

Monogenic diabetes, formerly called Maturity-Onset Diabetes of the Young (MODY), involves single-gene mutations, typically with dominant inheritance, and has been associated with variants in 14 genes. Among these, mutations are the most common, and their diagnosis allows the use of alternative therapies, including sulfonylureas. In an earlier study, we described a variant displaying recessive transmission, p.

Genome organization and stability in mammalian pre-implantation development.

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation.

Genome-wide screening reveals essential roles for HOX genes and imprinted genes during caudal neurogenesis of human embryonic stem cells.

Mapping the essential pathways for neuronal differentiation can uncover new therapeutics and models for neurodevelopmental disorders. We thus utilized a genome-wide loss-of-function library in haploid human embryonic stem cells, differentiated into caudal neuronal cells. We show that essential genes for caudal neurogenesis are enriched for secreted and membrane proteins and that a large group of neurological conditions, including neurodegenerative disorders, manifest early neuronal phenotypes.

Replication Stress in Activated Human NK Cells Induces Sensitivity to Apoptosis.

NK cells are innate immune effectors that kill virally infected or malignant cells. NK cell deficiency (NKD) occurs when NK cell development or function is impaired and variants in MCM4, GINS1, MCM10, and GINS4 result in NKD. Although NK cells are strongly impacted by mutational deficiencies in helicase proteins, the mechanisms underlying this specific susceptibility are poorly understood.

BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks.

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency.

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic β-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells.

Permanent neonatal diabetes-causing insulin mutations have dominant negative effects on beta cell identity.

Objective: Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM), requiring insulin therapy similar to T1D. While the negative effects on insulin processing and secretion are known, how dominant insulin mutations result in a continued decline of beta cell function after birth is not well understood.

Methods: We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations using patient-derived iPSCs and mutated hESCs.

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that both bovine and mouse cleavage stage embryos progress through S-phase in a defined pattern. Late replicating regions are associated with the nuclear lamina from the first cell cycle after fertilization, and contain few active origins, and few but long genes.

Incomplete reprogramming of DNA replication timing in induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state.

Permanent Neonatal diabetes-causing Insulin mutations have dominant negative effects on beta cell identity.

Heterozygous coding sequence mutations of the gene are a cause of permanent neonatal diabetes (PNDM) that results from beta cell failure. We explored the causes of beta cell failure in two PNDM patients with two distinct mutations. Using b and mutated hESCs, we detected accumulation of misfolded proinsulin and impaired proinsulin processing , and a dominant-negative effect of these mutations on the in vivo performance of patient-derived SC-beta cells after transplantation into NSG mice.

Incomplete Reprogramming of DNA Replication Timing in Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSC) are a widely used cell system and a foundation for cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which have the potential to affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing - a process linked to both genome regulation and genome stability - is efficiently reprogrammed to the embryonic state.

Replication stress in mammalian embryo development, differentiation, and reprogramming.

Duplicating a genome of 3 billion nucleotides is challenged by a variety of obstacles that can cause replication stress and affect the integrity of the genome. Recent studies show that replication fork slowing and stalling is prevalent in early mammalian development, resulting in genome instability and aneuploidy, and constituting a barrier to development in human reproduction. Genome instability resulting from DNA replication stress is a barrier to the cloning of animals and to the reprogramming of differentiated cells to induced pluripotent stem cells, as well as a barrier to cell transformation.

ZnT8 Loss of Function Mutation Increases Resistance of Human Embryonic Stem Cell-Derived Beta Cells to Apoptosis in Low Zinc Condition.

The rare mutation encoding a truncating p.Arg138* variant (R138X) in zinc transporter 8 (ZnT8) is associated with a 65% reduced risk for type 2 diabetes. To determine whether ZnT8 is required for beta cell development and function, we derived human pluripotent stem cells carrying the R138X mutation and differentiated them into insulin-producing cells.

Reduced calcium levels and accumulation of abnormal insulin granules in stem cell models of HNF1A deficiency.

Mutations in HNF1A cause Maturity Onset Diabetes of the Young (HNF1A-MODY). To understand mechanisms of β-cell dysfunction, we generated stem cell-derived pancreatic endocrine cells with hypomorphic mutations in HNF1A. HNF1A-deficient β-cells display impaired basal and glucose stimulated-insulin secretion, reduced intracellular calcium levels in association with a reduction in CACNA1A expression, and accumulation of abnormal insulin granules in association with SYT13 down-regulation.

Replication stress impairs chromosome segregation and preimplantation development in human embryos.

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies.

Comprehensive analysis of DNA replication timing across 184 cell lines suggests a role for MCM10 in replication timing regulation.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood.

Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of β-cells by autoreactive CD8 T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants.

Delayed DNA replication in haploid human embryonic stem cells.

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs.

The genetic architecture of DNA replication timing in human pluripotent stem cells.

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation.

Heritable human genome editing: Research progress, ethical considerations, and hurdles to clinical practice.

Our genome at conception determines much of our health as an adult. Most human diseases have a heritable component and thus may be preventable through heritable genome editing. Preventing disease from the beginning of life before irreversible damage has occurred is an admirable goal, but the path to fruition remains unclear.

Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia.

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors.

Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame.