The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry ؉ population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2 hi GFP-Bry ؉ population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2 lo GFP-Bry ؉ cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-␤͞nodal͞activin signaling simultaneously were required for the generation of the CD4-Foxa2 ؉ GFP-Bry ؉ population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2 ؉ GFP-Bry ؉ ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.gastrulation ͉ mesoderm ͉ endoderm T o be able to fully exploit the potential of embryonic stem (ES) cells in basic biology and regenerative medicine, it is essential to recapitulate the critical lineage induction events of the early embryo in this model system. One of the earliest commitment steps in embryogenesis is the formation of the primary germ layers, mesoderm, endoderm, and ectoderm, the founder populations of all somatic cell types in the body (1). Mesoderm and endoderm are formed during gastrulation, a process that involves the movement of undifferentiated epiblast cells through a structure called the primitive streak (PS). The PS can be subdivided into distinct regions, posterior, middle, and anterior, based on lineage development and gene-expression patterns. With respect to developmental potential, distinct subpopulations of mesoderm are induced in each of the different regions, whereas definitive endoderm forms from the anterior PS of the early-PS and mid-PS stages (2-5). These observations suggest that the different regions of the PS constitute different signaling environments that are responsible for the induction of specific lineages.To address questions regarding early commitment by using the ES cell model, it is essential to be able to track and isolate germ-layer populations from the differentiation cultures. By using an ES cell line with the green fluorescence protein (GFP) targeted to the PS and early mesodermal-specific gene brachyury (6) (GFPBry ES cells), it has been possible to quantify mesoderm induction and isolate and characterize different mesodermal populations (7,8). In addition, brachyury-expressing cells were found ...

When differentiated in the presence of activin A in serum-free conditions, mouse embryonic stem cells efficiently generate an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4. Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45-70%) for cells that express the alpha-fetoprotein and albumin proteins. These cells also express transcripts of Afp, Alb1, Tat, Cps1, Cyp7a1 and Cyp3a11; they secrete albumin, store glycogen, show ultrastructural characteristics of mature hepatocytes, and are able to integrate into and proliferate in injured livers in vivo and mature into hepatocytes expressing dipeptidyl peptidase IV or fumarylacetoacetate hydrolase. Together, these findings establish a developmental pathway in embryonic stem cell differentiation cultures that leads to efficient generation of cells with an immature hepatocytic phenotype.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34+CD73−CD184− fraction of day 8 embryoid bodies (EBs) and it undergoes a NOTCH-dependent EHT to generate RUNX1C+ cells with multilineage potential. Arterial and venous VE progenitors, by contrast, segregate to the CD34+CD73medCD184+ and CD34+CD73hiCD184− fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step towards the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. While several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed in order to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here we report the use of a humanized version of a single lentiviral ‘stem cell cassette’ vector in order to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either 3 or 4 reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration, that upon removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, alpha-1 antitrypsin deficiency-related emphysema, scleroderma (SSc), and sickle cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.