Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells

Embryonic stem (ES) cells offer insight into early developmental fate decisions, and their controlled differentiation may yield vast regenerative potential. The molecular determinants supporting ES cell self-renewal are incompletely understood. The homeodomain proteins Nanog and Oct4 are essential for mouse ES cell self-renewal. Using a high-throughput approach, we discovered DNaseI hypersensitive sites and potential regulatory elements along a 160-kb region of the genome that includes GDF3, Dppa3, and Nanog. We analyzed gene expression, chromatin occupancy, and higher-order chromatin structure throughout this gene locus and found that expression of the reprogramming factor Oct4 is required to maintain its integrity

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with β-thalassemia major resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and β-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the β-thalassemia phenotype by genome editing. Keywords: hemoglobinopathies, BCL11a, genome editing, thalassemia, HbF reactivation, zinc finger nuclease

Integrin α6β4 Identifies Human Distal Lung Epithelial Progenitor Cells with Potential as a Cell-Based Therapy for Cystic Fibrosis Lung Disease

<div><p>To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4⁺ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4⁺ epithelial cells demonstrated key properties of stem cells <i>ex vivo</i> as compared to α6β4^- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4⁺ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4⁺ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4⁺ epithelial cells significantly rescued defects in Cl^- transport. Therefore, targeting the α6β4⁺ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease. </p> </div

NANOG-dependent function of TET1 and TET2 in establishment of pluripotency

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomaincontaining protein NANOG We expanded the NANOG interactome in mouse embryonic stem cells using an improved affinity purification and mass spectrometry (AP-MS) strategy 6,7 similar to that described previously 8 , combined with an interactomics analysis (see We then asked if ectopic TET1 expression could enhance NANOG reprogramming activity. Neural stem cells 1 rOKM were transfected with PiggyBac vectors expressing NANOG, TET1 or TET1 bearing two mutations in the catalytic domain (TET1(H1671Y, D1673A), hereafter TET1Mut) 10 ( To explore the molecular mechanism underlying the NANOG-TET1 partnership during reprogramming, we quantified global 5-hydroxymethylcytosine (5hmC) levels Given that endogenous TET2 was upregulated in the presence of NANOG and TET1Mut We compared deposited chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) data for both NANO

Human α6β4⁺ cells differentiate into both basal and Clara cells <i>ex</i><i>vivo</i>.

<p>α6⁺ cells were isolated from normal human distal lung and cultured in Matrigel for 33 days. (A, B, C) Colonies were stained with (A) basal cell marker K-5 (red) and (B) Clara cell marker CC10 (green). (C) Merged image of K-5 and CC10. (D, E, F) Immunostaining of colonies with (D) type II AEC marker SPC (red) and (E) β4 (green). (F) Merged image of SPC and β4 staining. Nuclei were stained with DAPI (blue). Scale bar=100 µm.</p

Identification and localization of α6β4⁺ cells in the human distal lung.

<p>(A) Epithelial cells were isolated from a normal human lung, and surface markers α6 and E-cadherin were labelled with Alexa Fluor-568 and Alexa Fluor-647, respectively, and analyzed by FACS. Left panel: staining with primary antibody isotype control IgG; right panel: staining with antibodies against α6 and E-cadherin. The P4 gate indicates cells that were positive for both α6 and E-cadherin, whereas the P5 gate indicates E-cadherin⁺ but α6^- cells. (B) Populations of cells from the P4 or P5 gate in (A) were isolated, cytospinned on slides, and immediately immunostained with antibodies against SPC, CC10 or K-5. Data are the quantitation of expression of SPC, CC10 and K-5 on either α6⁺ (left pie chart) or α6^- (right pie chart) cells. (C, D) Localization of α6β4⁺ cells was determined by co-immunostaining the alveolar (C) region or distal airway (D) in normal human lungs with β4 (green) and SPC (red) or K-5 (red). Nuclei were stained with DAPI (blue). (C) Upper panel, single channel images of β4. Lower panel, merged image of cells co-immunostained with β4 and SPC. Arrow indicates a β4⁺/SPC^- cell in the alveolar region. (D) Upper panel, merged image of distal airway at low magnification. Lower panels show enlarged view of dotted box. Lower left panel, single channel images of β4. Lower right panel, merged image of cells co-immunostained with β4 and K-5. Arrow indicates a β4⁺/K-5^- cell in the distal airway; arrowhead indicates a β4⁺/K-5⁺ cell in the distal airway. Scale bar= 50 µm.</p

Human α6β4+ cells express stem cell-specific transcription factor Nanog.

<p>α6⁺ (A) and α6^- (B) cells were isolated from normal human distal lung and cultured in Matrigel for one week, and then colonies were stained with Nanog (red) and α6 (green). Nuclei were stained with DAPI (blue) days. Scale bar=20 µm.</p

Specific adeno-associated viruses (AAVs) transduce α6β4⁺ cells.

<p>A series of AAVs encoding GFP were used to infect α6β4⁺ cells. (A) GFP expression in cells transduced with either 10⁷ AAV2 viral genomes/cell (Vg/cell, left panel) or Ad-5 at an MOI=100 (right panel). (B) GFP⁺ cells/colonies were quantified two weeks after infection with 10⁶ (white bars) or 10⁷ (gray bars) AAV Vg/cell or Ad-5 at MOI=10 (white bar) or MOI=100 (gray bar). CTL: no virus control infection. Data are shown as mean ± SEM; n=3 experiments from 3 independent donors. * <i>P</i><0.05 vs. AAV9 at the same MOI by ANOVA. </p