It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.

We show that knockdown of KLF1 in human and mouse adult erythroid progenitors markedly reduces BCL11A levels and increases human gamma-globin/beta-globin expression ratios. These results suggest that KLF1 controls globin gene switching by directly activating beta-globin and indirectly repressing gamma-globin gene expression. Controlled knockdown of KLF1 in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with beta-thalassemia or sickle cell disease.

Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients.

We present an investigation of molecular permeation of gases through nanoporous graphene membranes via molecular dynamics simulations; four different gases are investigated, namely helium, hydrogen, nitrogen, and methane. We show that in addition to the direct (gas-kinetic) flux of molecules crossing from the bulk phase on one side of the graphene to the bulk phase on the other side, for gases that adsorb onto the graphene, significant contribution to the flux across the membrane comes from a surface mechanism by which molecules cross after being adsorbed onto the graphene surface. Our results quantify the relative contribution of the bulk and surface mechanisms and show that the direct flux can be described reasonably accurately using kinetic theory, provided the latter is appropriately modified assuming steric molecule-pore interactions, with gas molecules behaving as hard spheres of known kinetic diameters. The surface flux is negligible for gases that do not adsorb onto graphene (e.g., He and H2), while for gases that adsorb (e.g., CH4 and N2) it can be on the order of the direct flux or larger. Our results identify a nanopore geometry that is permeable to hydrogen and helium, is significantly less permeable to nitrogen, and is essentially impermeable to methane, thus validating previous suggestions that nanoporous graphene membranes can be used for gas separation. We also show that molecular permeation is strongly affected by pore functionalization; this observation may be sufficient to explain the large discrepancy between simulated and experimentally measured transport rates through nanoporous graphene membranes.

Gas transport through intrinsic defects and tears is a critical yet poorly understood phenomenon in graphene membranes for gas separation. We report that independent stacking of graphene layers on a porous support exponentially decreases flow through defects. On the basis of experimental results, we develop a gas transport model that elucidates the separate contributions of tears and intrinsic defects on gas leakage through these membranes. The model shows that the pore size of the porous support and its permeance critically affect the separation behavior, and reveals the parameter space where gas separation can be achieved regardless of the presence of nonselective defects, even for single-layer membranes. The results provide a framework for understanding gas transport in graphene membranes and guide the design of practical, selectively permeable graphene membranes for gas separation.

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress–inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress–induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative death by ATF4 deletion was associated with decreased consumption of the antioxidant glutathione. Forced expression of ATF4 was sufficient to promote cell death and loss of glutathione. In ATF4−/− neurons, restoration of ATF4 protein expression reinstated sensitivity to oxidative death. In addition, ATF4−/− mice experienced significantly smaller infarcts and improved behavioral recovery as compared with wild-type mice subjected to the same reductions in blood flow in a rodent model of ischemic stroke. Collectively, these findings establish ATF4 as a redox-regulated, prodeath transcriptional activator in the nervous system that propagates death responses to oxidative stress in vitro and to stroke in vivo.

Heme-regulated eIF2␣ kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of ␤-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in ␤-thalassemia increases eIF2␣ phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2␣ phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hriactivated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri Ϫ/Ϫ erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri Ϫ/Ϫ mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri Ϫ/Ϫ fetal liver erythroid progenitors. Importantly, the HrieIF2␣P-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2␣P-Atf4 signaling with chemical compounds as pharmaceutical therapies for ␤-thalassemia. IntroductionHeme-regulated eIF2␣ kinase (Hri) was the first discovered member of the family of eIF2␣ kinases, which control translational initiation under diverse stress conditions by phosphorylation of the ␣-subunit of eIF2 (eIF2␣P). 1,2 Hri is necessary to coordinate translation of globin mRNAs with the availability of heme for the production of large amounts of hemoglobin during erythroid maturation. 3 In Hri deficiency, excessive globins synthesized during heme deficiency precipitate and form inclusions causing proteotoxicity. 3,4 Beyond heme deficiency, Hri is also activated by oxidative stress and denatured proteins, 5 both of which occur in thalassemia. 6 Indeed, Hri is required to reduce the phenotypic severity of the Hbb Ϫ/Ϫ mouse model of ␤-thalassemia intermedia lacking both copies of ␤-globin major. 4 Besides Hri, there are 3 additional mammalian eIF2␣ kinases: the double-stranded RNA-dependent eIF2␣ kinase (Pkr), the Gcn2 protein kinase, and the Pkr-like ER resident kinase (Perk). 1 Each kinase elicits a major physiologic response in vivo: Pkr responds to viral infection; Gcn2 senses nutrient starvations; Perk is activated by endoplasmic reticulum (ER) stress; and Hri is regulated by heme. In addition to inhibition of global protein synthesis, the second function of eIF2␣ phosphorylation is to reprogram translation and transcription necessary for adaptation to stress 7 as illustrated in Figure 7A. In mammalian cells, translation of activating transcriptional factor 4 (Atf4) is selectively increas...

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3 0 long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic ''hit and run'' vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.