A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker.

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript.

High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules.

Unlabelled: : This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.

Differentiation of human embryonic stem cells into mesenchymal stem cells by the "raclure" method.

Mesenchymal stem cells also called mesenchymal stromal cells (MSCs) are multipotent progenitors that can be found in many connective tissues including fat, bone, cartilage, and muscle. We report here a simple method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layers or exogenous cytokines. The cells obtained with this procedure have a normal karyotype, are morphologically similar to bone marrow MSCs, are contact-inhibited, can be grown in culture for about 20-25 passages, exhibit an immuno-phenotype similar to bone marrow MSCs (negative for CD34 and CD45, but positive for CD44, CD71, CD73, CD105, CD166, HLA ABC, and SSEA-4), and can differentiate into osteocytes and adipocytes.

Developmentally regulated extended domains of DNA hypomethylation encompass highly transcribed genes of the human beta-globin locus.

Objective: DNA methylation has long been implicated in developmental beta-globin gene regulation. However, the mechanism underlying this regulation is unclear, especially because these genes do not contain CpG islands. This has led us to propose and test the hypothesis that, just as for histone modifications, developmentally specific changes in human beta-like globin gene expression are associated with long-range changes in DNA methylation.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers.

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter.

Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells.

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch.

CG dinucleotide clustering is a species-specific property of the genome.

Cytosines at cytosine-guanine (CG) dinucleotides are the near-exclusive target of DNA methyltransferases in mammalian genomes. Spontaneous deamination of methylcytosine to thymine makes methylated cytosines unusually susceptible to mutation and consequent depletion. The loci where CG dinucleotides remain relatively enriched, presumably due to their unmethylated status during the germ cell cycle, have been referred to as CpG islands.

Large-scale production of embryonic red blood cells from human embryonic stem cells.

Objective: To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells.

Materials And Methods: Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry.

Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can be found in many connective tissues, including fat, bone, cartilage, and muscle. We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layer. The cells obtained with this procedure are morphologically similar to bone marrow MSCs, are contact-inhibited, can be grown in culture for about 20 to 25 passages, have an immunophenotype similar to bone marrow MSCs (negative for CD34 and CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, human leukocyte antigen [HLA]-ABC, and stage-specific embryonic antigen [SSEA]-4), can differentiate into osteocytes and adipocytes, and can be used as feeder cells to support the growth of undifferentiated hESCs.