Jizhong Zou

Gene Targeting of a Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem Cells

We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%-0.24% in both cell types transfected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that induce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by > 1400-fold without detectable alterations in stem cell karyotypes or pluripotency. Efficient HR-mediated insertional mutagenesis was also achieved at the endogenous PIG-A locus, with a > 200-fold enhancement by ZFNs targeted to that gene. Clonal PIG-A null hESCs and iPSCs with normal karyotypes were readily obtained. The phenotypic and biological defects were rescued by PIG-A transgene expression. Our study provides the first demonstration of HR-mediated gene targeting in hiPSCs and shows the power of ZFNs for inducing specific genetic modifications in hiPSCs, as well as hESCs.

Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15‐ to 51‐fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100‐ to 200‐fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome‐wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency‐associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming. STEM CELLS 2010;28:713–72028:713–720

Improved Efficiency and Pace of Generating Induced Pluripotent Stem Cells from Human Adult and Fetal Fibroblasts

It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23–70‐fold from both human adult and fetal fibroblasts. Discernible hES‐like colonies also emerged 1–2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES‐like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hES‐like clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES‐like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle.

Notch Signaling Activation in Human Embryonic Stem Cells Is Required for Embryonic, but Not Trophoblastic, Lineage Commitment

The Notch signaling pathway plays important roles in cell-fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell-fate choices in human embryonic stem cells (hESCs). Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hESC lines. We report here that activation of Notch signaling is required for undifferentiated hESCs to form the progeny of all three embryonic germ layers, but not trophoblast cells. In addition, transient Notch signaling pathway activation enhanced generation of hematopoietic cells from committed hESCs. These new insights into the roles of Notch in hESC-fate determination may help to efficiently direct hESC differentiation into therapeutically relevant cell types.

Trophoblast Differentiation Defect in Human Embryonic Stem Cells Lacking PIG-A and GPI-Anchored Cell-Surface Proteins

Pluripotent human embryonic stem (hES) cells can differentiate into various cell types derived from the three embryonic germ layers and extraembryonic tissues such as trophoblasts. The mechanisms governing lineage choices of hES cells are largely unknown. Here, we report that we established two independent hES cell clones lacking a group of cell surface molecules, glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs). The GPI-AP deficiency in these two hES clones is due to the deficiency in the gene expression of PIG-A (phosphatidyl-inositol-glycan class A), which is required for the first step of GPI synthesis. GPI-AP-deficient hES cells were capable of forming embryoid bodies and initiating cell differentiation into the three embryonic germ layers. However, GPI-AP-deficient hES cells failed to form trophoblasts after differentiation induction by embryoid body formation or by adding exogenous BMP4. The defect in trophoblast formation was due to the lack of GPI-anchored BMP coreceptors, resulting in the impairment of full BMP4 signaling activation in the GPI-AP-deficient hES cells. These data reveal that GPI-AP-enhanced full activation of BMP signaling is required for human trophoblast formation.

An AAVS1-Targeted Minigene Platform for Correction of iPSCs From All Five Types of Chronic Granulomatous Disease

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Generation of Glycosylphosphatidylinositol Anchor Protein-Deficient Blood Cells From Human Induced Pluripotent Stem Cells

PIG‐A is an X‐linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG‐A mutant cells have a deficiency or absence of all GPI‐anchored proteins (GPI‐APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG‐A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG‐A gene targeting and an inducible PIG‐A expression system, we have established, for the first time, a conditional PIG‐A knockout model in human iPSCs that allows for the production of GPI‐AP‐deficient blood cells. PIG‐A‐null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG‐A‐null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG‐A transgene expression allowed for the generation of GPI‐AP‐deficient blood cells. This conditional PIG‐A knockout model should be a valuable tool for studying the importance of GPI‐APs in hematopoiesis and human development.

57. Seamless Targeted Correction of CYBB Exon 5 Mutations Restores Granulocyte Function in X-Linked Chronic Granulomatous Disease iPSCs

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted “safe-harbor” gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with “excision-only” piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.