One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
Methichit Wattanapanitch1,2,
Nattaya Damkham1,
Ponthip Potirat1,
Kongtana Trakarnsanga3,
Montira Janan1,
Yaowalak U-pratya1,4,
Pakpoom Kheolamai5,
Nuttha Klincumhom6 &
Surapol Issaragrisil1,4
Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia.
We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression.
The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein.
Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.
HbE/β-thalassemia, a double heterozygosity of hemoglobin E (HbE) and β-thalassemia, is the most common thalassemic syndrome found in adults in Southeast Asia. The clinical manifestations are heterogeneous; at one end the mutation may be very mild, whereas at the other end it is very severe similar to homozygous β-thalassemia or thalassemia major. In HbE/β-thalassemia, one allele (β0) produces no β-globin chain and the other allele (βE) produces a HbE globin chain resulting from nucleotide substitution at codon 26 (GAG → AAG, glutamic acid to lysine) [1]. Previous reports have shown the success of genetic correction of a β-globin gene in β-thalassemia-specific induced pluripotent stem cells (iPSCs) using a lentiviral vector [2, 3] or homologous recombination [4]. However, the lentiviral gene therapy results in random integration of the functional gene into the genome, leading to undesired mutations. It is therefore necessary to screen for the clones that integrate the transgene to genomic safe harbor sites. On the other hand, the classic homologous recombination is very inefficient, especially in human pluripotent stem cells [5]. The more efficient genome editing technologies using custom-engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been used to correct β-thalassemia iPSCs [6, 7].
The RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been used recently to correct HBB mutation in iPSCs derived from β-thalassemia [8,9,10,11] and sickle cell disease patients [12]. However, these studies relied on a donor plasmid containing a wild-type HBB gene and an antibiotic selection cassette for enrichment, thereby requiring subsequent excision and clonal selection steps. To overcome these limitations, a single-stranded DNA oligonucleotide (ssODN) donor template can be used to provide seamless correction [13, 14]. In this study, we used the CRISPR/Cas9 system and the ssODN donor template to efficiently correct the HbE mutation in iPSCs derived from a patient with HbE/β-thalassemia, resulting in the corrected iPSCs, which is a β-thalassemia heterozygote. The corrected iPSCs are capable of differentiating into hematopoietic stem cells, which can be used for autologous transplantation to the patient in the future. In addition, our study further demonstrates that these cells can differentiate in vitro to reticulocytes, which can be developed for therapeutic use.
Sample collection and generation of induced pluripotent stem cells
The study was approved by the Siriraj Institutional Review Board (no. Si248/2011), in accordance with the Helsinki Declaration of 1975. All patients were provided with an explanation and with a participant information sheet and signed the informed consent. Skin biopsies were collected from HbE/β-thalassemia patients for further mutation analysis and isolation of fibroblasts. Briefly, the skin specimens were washed with sterile phosphate buffered saline (PBS) containing 25 U/ml penicillin, 25 μg/ml streptomycin, chopped into small pieces of 1 mm3 and transferred into a T-25 tissue culture flask containing DMEM supplemented with 10% fetal bovine serum (FBS) (Lonza, Switzerland), 2 mM GlutaMAX™ and 25 U/ml penicillin, 25 μg/ml streptomycin. Fibroblasts were subcultured once every 5 days or whenever they reached 80% confluency by incubation with 0.25% Trypsin for 2 min. Generation and characterization of Eβ-iPSCs from a HbE/β-thalassemic patient's HDFs were performed as described previously [15]. iPSCs were maintained in mTeSR™1 medium (StemCell Technologies, Canada) on Matrigel™-coated (BD Bioscience, USA) plates and subcultured using 1 mg/ml Dispase (StemCell Technologies) according to the manufacturer's instructions.
Gene expression analysis
Total RNA was obtained using TRIzol® reagent (Invitrogen). cDNA was prepared using 2 μg of RNA and reverse-transcribed using the SuperScript III First-Strand Synthesis System and Oligo (dT) primers (Invitrogen). PCR analysis of pluripotent genes was performed on a T100™ Thermal Cycler (Bio-Rad, USA) using Platinum Taq DNA polymerase (Invitrogen). Primer sequences are presented in Additional file 1: Table S1. For quantitative RT-PCR (qRT-PCR) analysis, primers and probes were designed using the Universal Probe Library Assay Design Center (Roche Diagnostics). The qRT-PCR analysis was carried out on the CFX96™ Real-Time PCR detection system (Bio-Rad). Data were normalized with a housekeeping gene, GAPDH, and expressions were plotted against the undifferentiated normal iPSCs. Primer sequences are presented in Additional file 1: Table S2.
Multiplex PCR analysis for hemoglobin E
Genomic DNA was isolated using the Gentra® Puregene® Cell Kit (Qiagen) according to the manufacturer's instructions. HbE mutation was detected by multiplex PCR. The PCR reaction consisted of 1.5 U of DNA polymerase (Platinum Taq; Invitrogen), 1× PCR buffer, 1.5 mM MgCl2, 0.1 mM dNTPs, 0.4 μM HbE-Fc primer, 0.4 μM HbE-Rc primer, 0.5 μM HbE-Rn primer, 0.5 μM HbE-Fm primer and 2 μl of DNA sample in a total volume of 30 μl. The PCR was performed after an initial denaturation at 95 °C for 15 min followed by 30 cycles of denaturation (94 °C for 45 s), annealing (68 °C for 45 s) and extension (72 °C for 1 min), and a final extension step (72 °C for 7 min). Primer sequences are presented in Additional file 1: Table S3.
Generation of CRISPR/Cas plasmid, single-guide RNA and single-stranded DNA oligonucleotide template
CRISPR/Cas plasmids pSpCas9(BB)-2A-GFP (PX458) and pSpCas9(BB)-2A-Puro (PX459) were obtained from Addgene (Cambridge, USA). Seven gRNAs targeting the HbE mutation were designed and their potential off-target sites were identified using the Crispr Design Tool (http://crispr.mit.edu/). Top and bottom strands of each gRNA were annealed, phosphorylated and cloned into the BbsI site of the PX459 plasmid according to a protocol published previously [16]. The ssODN donor template for HbE correction was designed to have homology arms of 90 nucleotides on either side of the point mutation (a total of 181 nucleotides). The gRNA sequences and the ssODN template are presented in Additional file 1: Tables S4 and S5.
Gene targeting of the HbE mutation in Eβ-iPSCs
Transfection of Eβ-iPSCs was performed using Amaxa 4D-nucleofector as described previously [17]. Briefly, 5 × 105 Eβ-iPSCs were resuspended in nucleofection mixture containing 20 μl of P3 Primary Cell Solution (Lonza) and 2 μg of DNA. The mixture was transferred to Nucleocuvette strips and nucleofection was performed using the CB-150 program according to the manufacturer's instructions. The PX459 plasmid was used for gRNA construction and the PX458 was used as a positive control. The transfected iPSCs were plated onto Matrigel™-coated 24-well plates and cultured in mTESR1 medium supplemented with 10 μM Y-27632. At 24 hours post nucleofection, the PX458 transfected cells were subjected to flow cytometry analysis and the percentage of GFP+ cells was analyzed by FACSCalibur™ (BD Biosciences). To determine the gRNA efficiency, the genomic DNA of PX459-gRNA transfected cells was extracted after being cultured for 5 days. The PCR product of 306 bp around the target site was amplified and digested with T7 endonuclease I (T7EI) enzyme (New England Biolabs) according to the manufacturer's instructions.
To target the HbE mutation in Eβ-iPSCs, 200 pmol of the ssODN template was cotransfected with 2 μg of the PX459-gRNA with the highest specificity to the HbE site, as determined by T7EI assay. At 3 days post nucleofection, we performed a clonal isolation by limiting dilution. Briefly, transfected iPSCs were pretreated with SMC4 (Corning) prior to dissociation with accutase (Merck) and plated into 96-well plates at a density of 20 cells/96-well plate in mTESR1 medium supplemented with SMC4 for 8 days. Single colonies were picked and screened for the HbE mutation. The DNA sequence of HbE negative clones was further confirmed by direct sequencing. For off-target analysis, five potential off-target sites were amplified by PCR. In brief, genomic DNA was extracted using QuickExtract DNA Extraction Solution (Epicentric) according to the manufacturer's instructions. The PCR reaction consisted of 0.2 U of Q5 High-Fidelity DNA polymerase (New England Biolabs), 1× Q5 PCR buffer with MgCl2, 0.2 mM dNTPs, 0.5 μM of each primer and 2 μl of DNA sample in a total volume of 50 μl. The PCR was performed with an initial denaturation at 98 °C for 30 s followed by 35 cycles of denaturation (98 °C for 10 s), annealing (68 °C for 30 s) and extension (72 °C for 30 s), followed by a final extension step (72 °C for 2 min). Primer sequences are presented in Additional file 1: Table S6. DNA sequences were validated by Sanger sequencing.
Hematopoietic differentiation of iPSCs to myeloid and erythroid lineages
Hematopoietic differentiation was performed according to the previous study [18]. Briefly, iPSCs were subcultured on Matrigel™-coated six-well plates at 20–30% confluency in mTESR1 medium. After 24 hours of culture (day 0), the medium was replaced with differentiation medium: day 0–1 medium, RPMI (Gibco) supplemented with 5 ng/ml of human bone morphogenetic protein 4 (hBMP4) (Peprotech), 50 ng/ml of human vascular endothelial growth factor (hVEGF) (Peprotech), 25 ng/ml of hWnt3a (R&D) and 5% Knockout serum replacement (KOSR) (Gibco); day 2 medium, RPMI supplemented with 5 ng/ml of hBMP4, 50 ng/ml of hVEGF, 20 ng/ml basic fibroblast growth factor (bFGF) (Peprotech) and 5% KOSR; day 3 medium, StemPro-34 (Invitrogen), 5 ng/ml of hBMP4, 50 ng/ml of hVEGF and 20 ng/ml bFGF; day 4–5 medium, StemPro-34, 15 ng/ml of hVEGF and 5 ng/ml bFGF; day 6 medium, 74% Iscove modified Dulbecco medium (IMDM) (Gibco), 24% Ham's F12 (Gibco), 1% B27 supplement (Gibco), 0.5% N2 supplement (Gibco), 0.5% bovine serum albumin (BSA), 50 ng/ml of hVEGF, 100 ng/ml of bFGF, 100 ng/ml of human stem cell factor (SCF) (R&D) and 25 ng/ml of hFlt3 ligand (R&D); and day 7 medium, 74% IMDM, 24% Ham's F12, 1% B27 supplement, 0.5% N2 supplement, 0.5% BSA, 50 ng/ml of hVEGF, 100 ng/ml of bFGF, 100 ng/ml of hSCF, 25 ng/ml of hFlt3 ligand, 50 ng/ml of human thrombopoietin (TPO) (R&D), 10 ng/ml of IL-6 (Peprotech), 0.5 U/ml of hEPO (Eprex) and 0.2 μM of 6-formylindolo[3,2-b]carbazole (FICZ) (Abcam). After day 7, 0.5 ml of day 7 medium was added to the culture daily without removing the medium. All basal media mixes included 2 mM of GlutaMAX™, 0.4 mM of monothioglycerol (MTG) (Sigma-Aldrich), 100 μg/ml of Primocin (Invivogen) and 50 μg/ml of ascorbic acid (Sigma-Aldrich). Differentiated cells on days 10–14 were collected for gene expression and flow cytometry analysis. Adherent cells were dissociated with accutase at 37 °C for 15 min, washed twice with PBS/EDTA + 2% FBS and stained with antibodies specific to hematopoietic markers (CD34-PE, CD43-FITC, CD235a-PE and CD71-FITC; all from BioLegend) at room temperature for 15 min. The stained cells were washed twice with PBS/EDTA+ 2% FBS before being fixed with 300 μl of 1% paraformaldehyde and analyzed by FACSCalibur and CELLQuest software (BD Biosciences, USA). The floating cells on day 12 were also collected for colony-forming unit (CFU) assay by culturing in the methylcellulose-based medium, MethoCult™ H4435 Enriched (StemCell Technologies). The CFU number was analyzed after 14 days of culture.
Alternatively, iPSCs were also differentiated on OP9 mouse stromal cells. Briefly, small clumps of iPSCs were seeded onto overgrown OP9 cells in differentiation medium containing α-MEM (Invitrogen), 10% defined FBS (Hyclone), 100 mM MTG and 25 U/ml penicillin, 25 μg/ml streptomycin for 5 days. A half medium change was performed on day 3. iPSCs were harvested on day 6 using type IV collagenase (Invitrogen) for 20 min, followed by 0.25% trypsin for 15 min. CD34+ cells were isolated from differentiated cells using EasySep (StemCell Technologies) according to the manufacturer's instructions. To promote erythroid maturation, the purified CD34+ cells were cultured in a three-stage culture system according to Griffiths et al. [19]. Briefly, cells were seeded in stage 1 medium containing basic medium (IMDM (Biochrom), 3% AB serum, 2% defined FBS, 10 μg/ml insulin, 3 U/ml heparin, 3 U/ml EPO, 200 μg/ml transferrin and 100 U/ml penicillin/streptomycin) supplemented with 10 ng/ml SCF and 1 ng/ml IL-3 for 8 days, stage 2 medium containing basic medium supplemented with 10 ng/ml SCF for 3 days and stage 3 medium containing basic medium supplemented with extra transferrin to a final concentration of 500 μg/ml for 13 days. Morphological analysis was performed by Wright's staining at the indicated time points. The differentiated cells (1.5 × 104–3 × 104) were spun onto glass slides at 1000 rpm for 5 min using a Cytospin™ 4 Cytocentrifuge (Thermo Scientific) and stained with Wright-Giemsa (Merck). Images were taken with a light microscope (Carl Zeiss, Axio Star Plus).
Western blotting analysis
Differentiated iPSCs at day 24 of erythroid liquid culture were harvested, washed with PBS and lysed in lysis buffer (Cell Signaling) for 1 hour on ice. The protein concentration was measured by the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Samples were loaded onto 18% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 10% skimmed milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBS-T) for 1 hour at room temperature and probed with the primary antibodies, anti-hemoglobin alpha and anti-hemoglobin beta (Santa Cruz) overnight at 4 °C. Subsequently, the membrane was incubated with the HRP-conjugated secondary antibody for 1 hour at room temperature. After washing with TBS-T, the membrane was incubated with ECL substrate (Thermo Scientific) and the results were visualized by an ImageQuant LAS 4010 Biomolecular imager (GE Healthcare).
Generation and characterization of HbE/β-thalassemia patient-specific iPSCs
We obtained human dermal fibroblasts (HDFs) from a patient with HbE/β-thalassemia who had a 4-bp deletion (–TCTT) at codon 41/42 in one allele, and a point mutation (G → A) at codon 26 resulting in abnormal hemoglobin E (HbE) production in the other, and generated iPSCs from these HbE/β-thalassemia patient's HDFs. Several iPSC lines, designated Eβ-iPSCs, were obtained. Of these, two iPSC lines (Eβ-iPSC1 and Eβ-iPSC2) were expanded and characterized. To confirm the HbE mutation, multiplex PCR analysis was performed with the genomic DNA of the Eβ-iPSC1 and the Eβ-iPSC2 cells. Both Eβ-iPSCs had a product size of 529 bp, which represents the HbE point mutation, similar to that of fibroblasts derived from the patient's skin (Eβ-HDFs) but not in the wild-type HDFs or iPSCs (Fig. 1a). Further characterization showed that both Eβ-iPSCs expressed pluripotent markers and were able to differentiate both in vitro and in vivo into cells/tissues of three embryonic germ layers. The Eβ-iPSCs exhibited a normal karyotype of 46, XY at passage 19 (Additional file 2: Figure S1). We then selected the Eβ-iPSC2 cells for further analyses.
Strategy to target HbE mutation in Eβ-iPSCs using CRISPR/Cas9 to induce a double-stranded break at the HBB locus and a single-stranded oligo donor (ssODN) template to repair the mutation. a Multiplex PCR analysis for HbE mutation. wt-HDFs and wt-iPSCs indicate wild-type human dermal fibroblasts and iPSCs from a healthy individual. Eβ-HDFs, Eβ-iPSC1 and Eβ-iPSC2 are human dermal fibroblasts and two iPSC lines derived from a patient with HbE/β-thalassemia. b Schematic of targeted region of the HBB locus of the patient with HbE/β-thalassemia. The patient has a 4-bp deletion (–TCTT) in one allele and a point mutation at codon 26 (G → A) resulting in a structural variant hemoglobin E (HbE) in the other. Seamless and efficient correction of HbE is achieved using a gRNA targeting the point mutation (A) and the ssODN template carrying the correct nucleotide (G) with the left and right homology arms of 90 bp. Orange boxes indicate exons; red lines indicate introns. Red arrowheads show mutation sites; black arrowhead shows cleavage site of Cas9 by gRNA1; purple and red arrows indicate primer pairs for T7E1 assay and multiplex PCR for HbE detection, respectively. c Sequence of seven gRNAs designed to target the HbE mutation. Red "A" indicates point mutation in the Eβ-iPSC2 cells and gRNAs at HBB locus. HBB beta hemoglobin
Correction of HbE mutation in Eβ-iPSC2 cells using the CRISPR/Cas9 system
To correct the mutation in the Eβ-iPSC2 cells, guide RNAs (gRNAs) were designed to target the HbE mutation at codon 26 where nucleotide substitution occurs (G → A) (Fig. 1b). We first designed seven 20-nucleotide (nt) or truncated (18-nt or 19-nt) gRNAs targeting regions close to the point mutation (Fig. 1c and Additional file 1: Table S4). Each gRNA was constructed into a PX459 plasmid, which contains the Cas9 nuclease and puromycin selection cassettes. We used PX458, which has the same backbone as PX459 except for GFP expression, instead of the puromycin selection cassette, to determine the transfection efficiency. At 24 hours post nucleofection, the PX458 transfected cells were harvested for flow cytometry analysis. We obtained approximately 30% transfection efficiency in the Eβ-iPSC2 cells (Additional file 3: Figure S2). We then used the same conditions for transfecting PX459-gRNA into the Eβ-iPSC2 cells. We harvested the genomic DNA of the PX459-gRNA transfected pool at 5 days post nucleofection and examined for gRNA-Cas9 cleavage efficiency and specificity using T7EI assay. Of these seven gRNAs, gRNA1, which targeted DSB at two nucleotides upstream of the HbE point mutation, gave the highest efficiency and was then selected for further experiments (Fig. 2a).
Genetic correction of HbE mutation of the HBB gene. a T7E1 assay of gRNA target sites. At 5 days post transfection, genomic DNA of PX459-gRNA transfected cells was extracted and the region spanning the gRNA target sites was PCR amplified using on-target primer pairs (arrows), giving PCR products of 306 bp (uncut) and 150 and 156 bp (cut). M indicates marker. b Multiplex PCR screening for HbE mutation of isolated clones after genetic correction by CRISPR/Cas9 and the ssODN template. HbE-negative clone (clone 297) indicated in red. c Representative DNA sequences of PCR products of the region spanning the gRNA target site in HbE-negative clones with HDR in the corrected clones and indels in others. DSB indicates the double-stranded break site generated by gRNA1. d Chromatogram of the parental Eβ-iPSC2 cells and the corrected C46 cells at the mutation site. Red box indicates HbE mutation (G → A) at codon 26. Note the overlapped peaks in the Eβ-iPSC2 cells, which occurred as a result of G in one allele (normal) and A in another allele (HbE). After genetic correction, both alleles contained the right nucleotide "G". e Representative karyotype of the corrected C46 cells, which exhibited a normal karyotype (46, XY). f Potential off-target sites for gRNA1 as identified by BLAST search. Mismatch nucleotides are indicated in red. Eβ-iPSC2 iPSC lines derived from a patient with HbE/β-thalassemia, HbE hemoglobin E, HBD delta hemoglobin
For HbE correction, we cotransfected PX459-gRNA1 and the ssODN template. At 3 days post nucleofection, the transfected pool was harvested and seeded at a density of 20 cells/well of a 96-well plate. Eight to 10 days after plating, one to five colonies were observed in each well. Single colonies were individually picked for multiplex PCR analysis for HbE mutation. From a total of 312 individual clones screened, we estimated that 93 clones were transfected (according to 30% transfection efficiency with the GFP plasmid control). We obtained 23 HbE-negative clones from PCR screening (7.4% DSB efficiency) (Fig. 2b). Of these, 14 clones showed indels (4.5% nonhomologous end joining (NHEJ)) and nine clones showed successful seamless correction of HbE mutation (2.9% homology-directed repair (HDR)) as confirmed by direct sequencing (Fig. 2c, d). We selected five clones (C22, C46, C134, C137 and C258) for karyotyping analysis. These corrected clones exhibited a normal karyotype (Fig. 2e and Additional file 4: Figure S3) and three clones (C22, C46 and C137) were randomly expanded for off-target analysis.
Off-target analysis
We selected five potential off-target sites of gRNA1 including the HBD gene and other genes that have similar homology with the gRNA1-PAM sequence using NCBI BLAST. PCR amplification of these regions was performed and DNA sequences of the PCR products were validated by direct sequencing as compared to that of the Eβ-iPSC2 cells. We detected no off-target cleavage in the corrected C46 cells, whereas a point mutation in the HBD gene was observed in both of the corrected C22 and C137 cells (Fig. 2f).
Generation of hematopoietic cells from the corrected iPSCs
To evaluate whether the genetic correction in Eβ-iPSCs could restore the HBB expression, hematopoietic differentiation of the wild-type iPSCs (HDF-iPSCs), the Eβ-iPSC2 cells and the corrected clones (C22, C46, C134, C137 and C258) was performed in a feeder-free condition (Fig. 3a) at the following stages of hematopoietic development: mesoderm progenitor, hematovascular specification, endothelial hematopoietic transition and hematopoietic progenitor cells. At days 5–6 of culture, the differentiated cells appeared to be a monolayer of endothelial-like cells, which later formed three-dimensional structures, observed from day 8 onward. The nonadherent cells started to emerge from both monolayer and three-dimensional structures on days 8–12 (Fig. 3b). During hematopoietic differentiation, the Eβ-iPSC2 cells showed impaired hematopoietic differentiation as indicated by a lower number of cells expressing hematopoietic progenitor and erythroid markers, CD43 and CD71, when compared to the HDF-iPSCs in the adherent cell population. In contrast to the HDF-iPSCs, which could give rise to the nonadherent cell population that highly expressed CD43, CD71 and CD235, the Eβ-iPSC2 cells produced a very low number of nonadherent cells, which were mainly nonviable. After genetic correction, all of the five corrected clones were able to differentiate into hematopoietic progenitor cells which expressed CD34 and CD43, and erythroid markers CD71 and CD235a at comparable levels to those of the HDF-iPSCs in both adherent and nonadherent cell population (Fig. 3c, d). We also examined the gene expression profile of these corrected cells during hematopoietic specification by quantitative real-time PCR. All corrected clones expressed SOX17 and RUNX1, which play an important role in blood formation from hemogenic endothelium, and GATA1 and KLF1, which are erythroid-specific markers (Additional file 5: Figure S4a). We harvested the differentiated floating cells from the HDF-iPSCs and the corrected clones on day 12 and seeded them onto methylcellulose plates. After 2 weeks, the HDF-iPSCs and three of the corrected clones (C22, C46 and C137) gave rise to all types of colonies, mainly CFU-E and CFU-GM, confirming the functional characteristic of hematopoietic progenitor cells. However, two of the corrected clones (C134 and C258) could only give rise to CFU-GM and a small number of CFU-E (Fig. 3e, f). In contrast to the corrected cells, the Eβ-iPSC2 cells could not produce any CFU colonies. The BFU-E obtained from the corrected clones expressed high levels of fetal gamma hemoglobin (HBG) and low levels of adult beta hemoglobin (HBB) transcripts, when examined by qRT-PCR (Additional file 5: Figure S4b).
Hematopoietic differentiation of iPSCs using the feeder-free system. a Schematic of the feeder-free hematopoietic differentiation protocol used in this study. b Morphological changes of the corrected C46 cells during hematopoietic differentiation. c, d Numbers of CD34, CD43, CD235a and CD71-expressing cells in adherent and nonadherent systems at day 12 of differentiation. e Representative images of CFU from HDF-iPSCs. Differentiated cells at day 12 were harvested and seeded in MethoCult media. f Numbers of CFU colonies counted on day 14 of culture in MethoCult media. Data obtained from two independent experiments. Scale bars = 200 μm. IMDM Iscove modified Dulbecco medium, BMP-4 bone morphogenetic protein 4, VEGF vascular endothelial growth factor, KOSR knockout serum replacement, bFGF basic fibroblast growth factor, BSA bovine serum albumin, SCF stem cell factor, TPO thrombopoietin, IL interleukin, FICZ 6-formylindolo[3,2-b]carbazole, HDF human dermal fibroblasts, iPSC induced pluripotent stem cell, Eβ-iPSC2 iPSC lines derived from a patient with HbE/β-thalassemia, CFU-E colony-forming unit erythroid, EPO erythropoietin, BFU-E burst-forming unit erythroid, GM granulocyte, macrophage, GEMM granulocyte, erythrocyte, macrophage, megakaryocyte
Since the Eβ-iPSC2 cells seemed to be refractory to the hematopoietic differentiation protocol under the feeder-free condition, we turned to the OP9 coculture system for hematopoietic differentiation followed by an erythroid liquid culture (Fig. 4a) [20]. The supportive OP9 stromal cells have been shown to efficiently induce hematopoietic differentiation [21]. We selected the corrected C46 cells, which differentiated well under the feeder-free condition and contained no off-target mutation, for comparison with the Eβ-iPSC2 cells. Small clumps of iPSCs were seeded onto overgrown OP9 cells and cultured for 6 days. In contrast to the feeder-free hematopoietic differentiation system, both the Eβ-iPSC2 cells and the corrected C46 cells were able to differentiate into sac-like structures (Fig. 4b). We isolated CD34+ cells from the differentiated cells on day 6 of the OP9 coculture system and further expanded erythroid cells using the three-stage culture system [19]. Upon erythroid culture, both the Eβ-iPSC2 cells and the corrected C46 cells gradually changed their morphology from that representing proerythroblasts/basophilic erythroblasts on day 13 of culture to that representing polychromatic/orthochromatic erythroblasts on day 23 and finally became orthochromatic erythroblasts/reticulocytes on day 29 of culture. Analysis of gene expression during the erythroid liquid culture demonstrated that both differentiated Eβ-iPSC2 and the corrected C46 cells at day 19 of differentiation (when the morphological stages are equivalent to those of day 13 erythroid cells derived from peripheral blood progenitors) expressed lower levels of erythroid-associated transcription factors KLF1 and BCL11A as compared to the cultured erythroblasts from peripheral blood CD34+ cells (Fig. 4c). We harvested the differentiated cells at day 30 and analyzed hemoglobin protein expression. Both differentiated Eβ-iPSC2 and the corrected C46 cells expressed similar levels of beta hemoglobin and alpha hemoglobin proteins, indicating successful hematopoietic differentiation under the OP9 coculture system (Fig. 4d).
Hematopoietic differentiation of iPSCs using the OP9 coculture system and erythroid liquid culture. a Schematic of hematopoietic differentiation protocol used in this study. b Morphological changes of the Eβ-iPSC2 cells and the corrected C46 cells during hematopoietic differentiation on day 6 of OP9 coculture (scale bar = 500 μm), and Wright's staining on days 13, 23 and 29 of differentiation. c Quantitative RT-PCR analysis of erythroid-associated transcription factors at day 19 of differentiation (equivalent to day 13 of erythroid liquid culture) of the Eβ-iPSC2 cells and the corrected C46 cells as compared to peripheral blood CD34+ cell-derived erythroblasts at day 13 (PB). Data presented as mean ± SD of triplicate samples from a representative experiment. d Western blot analysis of alpha and beta hemoglobin expression of the Eβ-iPSC2 cells and the corrected C46 cells at day 30 of differentiation as compared to peripheral blood CD34+ cell-derived erythroblasts at day 24 of erythroid liquid culture. MEM minimal essential medium, IMDM Iscove modified Dulbecco medium, FBS fetal bovine serum, SCF stem cell factor, IL interleukin, EPO erythropoietin, Eβ-iPSC2 iPSC lines derived from a patient with HbE/β-thalassemia, Hb hemoglobin
HbE is the hallmark of Southeast Asia. Double heterozygosity of HbE and β-thalassemia, namely HbE/β-thalassemia, can cause a severe clinical syndrome similar to homozygous β-thalassemia [22]. These severe patients manifest with anemia requiring red cell transfusion during the first year of life. The only curative therapy at present is hematopoietic stem cell transplantation. However, unavailability of HLA-matched donors and risk of transplant-related morbidity, mortality as well as immunologic complications, especially graft failure and graft versus host disease (GVHD), have limited the use of allogeneic transplantation [23]. Recently, in vitro genetic correction of hematopoietic stem cells (HSCs) has been successfully reported; however, culturing and maintaining HSCs remain difficult [24].
Advances in iPSC technology offer promise for autologous cell-based therapy as it provides a stem cell source, which can be continuously expanded in vitro and is amendable to genetic manipulation before differentiation into functional HSCs. We performed an efficient one-step seamless genetic correction of iPSCs from a HbE/β-thalassemic patient using the CRISPR/Cas9 system and the ssODN template. The genetic correction of the HbE mutation in one allele is easier than correcting the β-globin gene mutation in the other allele in which the mutations are heterogeneous [25]. Our approach does not require an antibiotic selection cassette that may interfere with expression of the corrected gene [6, 9, 12].
We designed gRNAs to recognize only HbE mutation and used the ssODN template possessing the correct nucleotide at the center of the oligo, which provides the highest targeting efficiency especially when the mutation site is less than 10 bp away from the cutting sites [26]. The ssODN template is easy to design and synthesize. It is footprint-free after genetic correction and has a high targeting efficiency. Our results show that transfection of PX459-gRNA1 with ssODN template resulted in a gene disruption frequency (DSB) of 7.4% and homologous gene targeting frequency (HDR) of 2.9%. Our transfection efficiency varied among iPSC lines, ranging from 30 to 60%. The number of screened clones could be further reduced if the transfection efficiency increased. To improve HDR efficiency, small molecule inhibitors may be used to suppress the nonhomologous end joining (NHEJ) pathway, thus facilitating the downstream screening process [27, 28].
Off-target mutagenesis is a major safety concern since genetic modifications are permanent and will have devastating consequences if the mutations are to be at important sites [29]. We identified five potential off-target sites of gRNA1 including the HBD gene, which has similar homology to HBB gene, and confirmed them by Sanger sequencing. Of the three corrected clones screened, one had no indel mutation whereas two harbored a point mutation. Interestingly, the presence of the point mutation in the delta hemoglobin (HBD) gene did not affect the hematopoietic differentiation potential when using the feeder-free system. A previous report on the genetic correction of a beta hemoglobin gene to treat sickle cell disease in human hematopoietic stem/progenitor cells using ZFNs showed off-target cleavage in the highly homologous, but functionally dispensable, HBD gene [30]. Measures to improve targeting specificity of Cas9 include the use of truncated gRNA with shorter regions of target complementarity [31], or modifying the Cas9 components so that two gRNA/Cas9 complexes are required to cleave DNA. The latter can be achieved via nickases that induce single-strand breaks [32, 33] or single-guide RNA (sgRNA)-guided catalytically inactive Cas9 (dCas9) fused to the FokI nuclease [34,35,36]. Recently, an alternative CRISPR system to Cas9 nuclease called Cpf1 nuclease was used for genome editing with simpler crRNA synthesis and more effective delivery and targeting results [37]. Nevertheless, whole genome sequencing is still required before using these corrected clones in clinical applications.
Future therapeutic use of ex vivo genetically corrected HSCs will require efficient conversion of these stem cells to differentiated hematopoietic precursors and safety testing. We used two in vitro differentiation schemes to assess the potential of our cells for conversion to hematopoietic cells making normal hemoglobin products. We first induced hematopoietic differentiation using a chemically defined, serum and feeder cell-free protocol based on aryl hydrocarbon receptor (AhR) activation [18]. This protocol has been shown to facilitate an expansion of both erythroid and megakaryocyte progenitor cells. We compared the number of hematopoietic progenitor and erythroid markers of the wild-type iPSCs (HDF-iPSCs), the Eβ-iPSC2 cells and the five corrected clones. The number of adherent and nonadherent cells expressing those markers was comparable in the wild-type iPSCs and the five corrected clones; however, we noticed varying numbers of multilineage CFUs among these cell types due to clonal variability. In contrast, the Eβ-iPSC2 cells could differentiate to some extent, as demonstrated by the presence of CD34+ and CD71+ cells in adherent cells, although these cells could not give rise to CFUs. Hematopoietic differentiation based on the OP9 coculture system seemed to be more supportive for the Eβ-iPSC2 cells. Morphological changes of the Eβ-iPSC2 cells on the OP9 feeder layer were similar to those of the corrected C46 cells. Both cells produced significant numbers of erythroblasts with occasional enucleated mature erythrocytes using the three-stage erythroid liquid culture system. The erythroblasts from both the Eβ-iPSC2 cells and the corrected C46 cells expressed KLF1 and BCL11A transcripts at lower levels compared to those of the peripheral blood-derived erythroblasts. Similarly, analysis of beta and alpha hemoglobin protein expressions showed that both the Eβ-iPSC2 cells and the corrected C46 cells expressed the proteins at similar levels; however, the beta hemoglobin levels were much lower than those of the peripheral blood-derived erythroblasts. A previous study demonstrated that the transcription factors KLF1 and BCL11A are required for the induction of beta hemoglobin levels [38]; therefore, low levels of KLF1 and BCL11A transcripts in the corrected C46 cell-derived erythroid cells could result in lower beta hemoglobin expression. In addition, the number of erythroid cells derived from iPSCs is still limited; further optimization of the differentiation protocol to increase the number of erythroid cells should be performed to further facilitate an in vitro functional assay. In this study, the anti-human beta hemoglobin antibody could not distinguish the one amino acid difference from abnormal hemoglobin E in the Eβ-iPSC2 cells. Therefore, validation with other techniques such as HPLC should be performed to confirm the result at protein levels. In addition, further development of long-term repopulating hematopoietic stem/progenitor cells is required for successful engraftment in immunodeficient mouse models, where the clinical efficacy of corrected human hematopoietic stem cells can be more properly assessed.
Our study provides a successful strategy to correct HbE mutation in one step and could be employed as a universal approach in the future correction of the HBB gene in iPSCs derived from other HbE/β0-thalassemia or β+-thalassemia patients. The results indicate that genetic correction of HbE mutation in one allele is sufficient to restore HBB protein expression upon hematopoietic differentiation by the OP9 coculture system followed by an erythroid liquid culture. Similarly, previous studies demonstrated that genetic correction of mutation in one allele—to a heterozygous state—in iPSCs derived from homozygous β-thalassemia or sickle cell disease was achieved using CRISPR/Cas9 or TALENs [6, 8, 9, 12]. Occasionally, homozygous corrections of homozygous β-thalassemia iPSCs were observed [10, 13]. The knowledge and protocols obtained from this study will facilitate and be applicable to genetic correction of patient-specific iPSCs with other genetic disorders.
AhR:
BMP4:
Bone morphogenetic protein 4
CFU:
Colony-forming unit
CRISPR/Cas9:
Clustered regularly interspaced short palindromic repeat/Cas9
FICZ:
6-Formylindolo[3,2-b]carbazole
gRNA:
Guide RNA
GVHD:
Graft versus host disease
HBB:
Beta hemoglobin
HDF:
Human dermal fibroblasts
Homology-directed repair
HSC:
Hematopoietic stem cell
HSCT:
Hematopoietic stem cell transplantation
IMDM:
Iscove modified Dulbecco medium
iPSC:
Induced pluripotent stem cell
KOSR:
Knockout serum replacement
MTG:
Monothioglycerol
NHEJ:
Nonhomologous end joining
SCF:
ssODN:
Single-stranded DNA oligonucleotide
Transcription activator-like effector nuclease
TBS:
Tris-buffered saline
TBST:
Tris-buffered saline with 0.1% Tween-20
TPO:
Thrombopoietin
VEGF:
ZFN:
Zinc-finger nuclease
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The authors thank Kamthorn Pruksananonda from the Department of Obstetrics and Gynaecology, Faculty of Medicine, Chulalongkorn University, for providing the Chula2.hES line.
This study was supported by grants from the Thailand Research Fund (grant no. RTA 488–0007 to SI and grant no. TRG5880200 to MW) and the Commission on Higher Education (grant no. CHE-RES-RG-49 to SI). SI is a Senior Research Scholar of Thailand Research Fund. MW and KT are supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.
All data generated or analyzed during this study are included in this published article (and its supplementary information files).
Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Montira Janan, Yaowalak U-pratya & Surapol Issaragrisil
Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Methichit Wattanapanitch
Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Kongtana Trakarnsanga
Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Yaowalak U-pratya & Surapol Issaragrisil
Division of Cell Biology, Department of Pre-clinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand
Pakpoom Kheolamai
Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Nuttha Klincumhom
Nattaya Damkham
Ponthip Potirat
Montira Janan
Yaowalak U-pratya
Surapol Issaragrisil
MW designed and performed the experiments, supervised the study and wrote the manuscript. ND, PP, KT and MJ performed the experiments, analyzed data and cowrote the paper. YU and PK analyzed data. NK performed animal study. SI designed the experiments, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
Correspondence to Surapol Issaragrisil.
The study was approved by the Siriraj Institutional Review Board (no. Si248/2011), in accordance with the Helsinki Declaration of 1975. All patients were provided with an explanation and with a participant information sheet and signed the informed consent.
Additional file 1: Tables S1–S6.
Presenting primer and oligo sequences. (DOCX 20 kb)
Showing characterization of iPSCs derived from skin fibroblasts of a patient with hemoglobin E/beta-thalassemia. (a) Pluripotent gene expression of wild-type human dermal fibroblasts (wt-HDFs), parental human dermal fibroblasts (Eβ-HDFs) and Eβ-iPSCs compared with hESC line Chula2.hES, analyzed by RT-PCR. (b) Immunofluorescent staining shows expression of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81 in the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars = 100 μm. (c) Immunofluorescent staining shows expression of lineage markers NESTIN (ectoderm), AFP (endoderm) and SMA (mesoderm) of differentiated embryoid bodies generated from the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars: for NESTIN and SMA = 100 μm; for AFP = 50 μm. (d) Hematoxylin and eosin (H&E) staining of teratomas derived from the Eβ-iPSC2 cells at 8 weeks post implantation into nude mice. Teratomas contained tissues derived from three embryonic germ layers, sebaceous tissue (ectoderm), cartilage (mesoderm) and gut-like epithelium (endoderm). Scale bars = 100 μm. (e) Representative karyotypic analysis of the Eβ-iPSC2 cells at passage 19 shows normal karyotype (46, XY) (TIFF 9760 kb)
Showing transfection efficiency of PX458 in the Eβ-iPSC2 cells. (a) Phase contrast and fluorescent images of the Eβ-iPSC2 cells 1 day post transfection with PX458. (b) Flow cytometry analysis of GFP-expressing cells in the untransfected cells (negative control) and the PX458 transfected cells (TIFF 4645 kb)
Showing representative karyotypes of the corrected C22, C134, C137 and C258 cells, which exhibited normal karyotypes (46, XY) (TIFF 3596 kb)
Showing gene expression profile of the differentiated cells. (a) qRT-PCR analysis of hematopoietic and erythroid-specific markers: RUNX1, SOX17, GATA1 and KLF1. Data presented as relative expression to day 0 of each sample, N = 2. (b) qRT-PCR analysis of fetal (HBG) and adult (HBB) globin gene expressions of day 0 normal iPSCs (D0 iPSC) and BFU-E and CFU-E colonies of the corrected C22 and C46 cells on day 14 of culture in MethoCult media. Data presented as relative expression to GAPDH, N = 2 (TIFF 1576 kb)
Wattanapanitch, M., Damkham, N., Potirat, P. et al. One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system. Stem Cell Res Ther 9, 46 (2018). https://doi.org/10.1186/s13287-018-0779-3
Genetic correction
Pluripotent Stem Cells: Research Review


1125 Fashion Drive
P.F. Chang's Clarkstown Catering
9pm-Close, Monday-Friday
Nanuet Mall
1125 Fashion Drive Nanuet, NY 10954 US
W Rte 59 and Middletown Rd
Kelly Blau
About P.F. Chang's Clarkstown Catering
With roots in Chinese cooking, the culinary landscape at P.F. Chang's stretches across Asia with diverse, culturally inspired recipes. Your P.F. Chang's Clarkstown serves a variety of Asian-inspired dishes, including hand-rolled sushi, Vietnamese-style soups and bowls, traditional Chinese dim sum, wok-fired chicken and beef entrées, and family-style noodles and rice. All made from scratch every day. P.F. Chang's honors the 2,000-year-old Asian tradition of wok cooking and believes in making food from scratch every day in every restaurant. With a Farm to Wok® food philosophy at its core, P.F. Chang's chefs hand-roll dim sum, hand chop and slice all vegetables and meats, handcraft every sauce and wok-cook each recipe, every day in every restaurant using wholesome, fresh ingredients. P.F. Chang's is known for its Chang's Lettuce Wraps, a secret Chiang family recipe that hasn't changed since the very first menu. Other signature classics include Mongolian Beef, Chang's Spicy Chicken, Crispy Honey Shrimp, and Cecilia's Dumplings. Visit P.F. Chang's Clarkstown. Order catering for pick up or delivery at catering.pfchangs.com.
P.F. Chang's Clarkstown Services
P.F. Chang's White Plains
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125 Westchester Ave. Spc D315 White Plains, NY 10601 US


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Chartwell's mission to provide students the skills, strategies, tools, and social-emotional mindset to grow and succeed extends to our Athletics program. Coaches and staff focus on giving students opportunities to learn and participate in any sport we offer, regardless of their experience or ability. Sportsmanship, teamwork, commitment to a goal, sacrifice of one's personal gain for that of a team, self-discipline, managing success, and coping with adversity are core lessons we instill in our athletes. Chartwell student-athletes gain confidence, learn accountability and build relationships with coaches and teammates that will help them to be successful now and in life beyond school.
Calendars in progress. Please check back soon for complete schedule.
Track & Field (2023)
CHS Athlete Brings Home First Victories for the School
High school wrestler Enzo O. makes Chartwell history by bringing home the first victory for the school.
MS Soccer Team Develops as Season Progresses
The Chartwell middle school soccer team began the season with many of the players new to the sport. Under the leadership of Coach Chad Miller, players started with conditioning and skill development and have worked their way up to scrimmages and position development.
Middle School Volleyball and Cross Country in Full Swing
This fall, Chartwell athletes in our middle school sports program are engaging in volleyball matches and cross country meets.
Chartwell's Inaugural High School Athletics Season
With the growth of the high school and an increased interest in sports, Chartwell High School is launching its first competitive athletics teams this year. We will start the year with basketball and wrestling, and offer golf in the spring. Garrett Benjamin joined Chartwell this year and will be the athletic director for the high school. Mr. Benjamin joins us from Saint Francis School in Mountain View where he coached golf, wrestling, football, and softball, in addition to his teaching and administrative responsibilities.
Electives & Clubs Introduce New Opportunities to Learn (opens in new window/tab)
Electives and clubs offer students engaging and different ways to learn and develop skills - many of which they might have never been exposed to. This year several new offerings have students challenging themselves and growing socially and emotionally - including judo.
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The Impact of the Eton Union Sanitary Authority
Bell Farm at the edge of the Parish of Eton
Nineteenth-century reform took another turn locally in 1849 with the formation of the Eton Urban Sanitary Authority, or Local Health Board as it was also known. Eton Wick was not included and thus the village was not subject to the Authority's new by-laws or supervision by its new officials and committees the Inspector of Nuisances, the Medical Officer, Street Committee and others. Perhaps the villagers were relieved that they were outside this control, but of course they did not benefit from the steady improvements in sanitary conditions which were the aim of the Board - an increase in privies for cottages, the removal of manure heaps and pigsties from close proximity to houses and laying of new drains. For another sixty years or more the village had to manage with bucket toilets and cesspits while the Eton Sewage Farm lay at their backdoor.
Bell Farm was bought from William Goddard in 1870 for the sewage farm, and the land freed from lammas rights in the early months of the following year. Compensation for this loss of rights was negotiated by a committee appointed at a meeting of persons entitled to commonable rights and included George Lillywhite of Manor Farm. However, it was to be six years before Mr Tough took up his appointment as manager. Perhaps this was the remaining length of the lease of Mr Aldridge, farmer of Cippenham Court and tenant of Bell Farm. Not all the land was needed for the disposal of sewage, and year after year in the Minute Books of the Authority an inventory of produce, livestock and implements is given.
In earlier years after the initial purchase, some of the land was sold, but under the management of Mr Tough the farm prospered and more land was leased. The farm continued to give employment to workers from the village and indeed treated them well, as judged by a decision of 1881 to pay a man who broke his ankle at work the then princely sum of 8s 6d a week while he was off sick.
Perhaps the achievement of the Board which must have given rise to the most bitter feelings in the village was the building of the Cottage Hospital. The story began with a young man of Meadow Lane who had the misfortune to catch smallpox, but who was determined to not be considered a pauper and so be sent to the Workhouse Infirmary at Slough. There was nowhere in the parish where he could be isolated and treated. Medical help could be obtained from the Windsor Dispensary, thus putting at risk other patients. The disease did spread, not in Windsor, but in Eton itself. It was not yet possible to prevent such outbreaks, but it was now understood how they could be contained by isolating the patients. The need for an Infectious Diseases Hospital was now obvious to members of the Board.
Plans were drawn up, sites inspected and central authorities consulted; so much is clear from the Minute Books, but underneath the meagre statements is the hint of conflict between the Board and the village. Plans for converting the Bell Farm Cottages into a room for the nurses and living quarters for a caretaker were well ahead and negotiations were progressing towards the purchase of the adjoining land, when suddenly there was a change of mind and suggestions of the inadvisability of building a hospital so near the village. An alternative site was found on the Board's own land between Bell Farm and Saddocks, quite isolated from other houses. By 1883 the building was completed and its first matron, a Mrs Sarah Hopkins, was engaged at £40 per annum. A brougham was bought to do duty as ambulance and the latest disinfecting apparatus in-stalled. By May, 1884 the first patients were accepted, a mother and her three children, all suffering from smallpox.- Did they recover ? We do not know.
This is an extract from The Story of a Village: Eton Wick 1217 to 1977 by Judith Hunter.
Labels: 19th century, Eton, farms, Judith Hunter, Local history, School, The story of a village
Location: Bell Farm House, Bell Ln, Eton Wick, Windsor SL4 6LH, UK
The First Memorial Committee Meeting - January 191...
The Mystery of the rise of the Lillywhites
Our Village December 2011 - The Way Things Were


Pyongyang To Deport American Who Entered North Korea Illegally
North Korea says it will deport a U.S. citizen who entered the country illegally from neighboring China last month — a move seen as a conciliatory gesture aimed at maintaining ties with Washington.
North Korea's official KCNA news agency identified the American as Bruce Byron Lowrance and said that he had told his captors that he was controlled by the CIA.
According to Reuters, "An American man of the same name was deported from South Korea in November 2017 after being found wandering near the heavily fortified border with North Korea, but there was no immediate confirmation of the identity of the man held by North Korea."
As The Associated Press notes, the announcement of a quick release, "suggests that North Korea still wants to maintain the mood for dialogue with the United States despite stalled nuclear diplomacy."
The deportation announced Friday stands in sharp contrast to North Korea's treatment of American citizens detained by authorities there in the past.
American college student Otto Warmbier, arrested in North Korea in 2016, was tried on charges of attempted theft, allegedly for trying to steal a propaganda poster from his hotel room. He was sentenced to 15 years hard labor, but was later released in a state of a coma and died days later.
In May, the Trump administration secured the release of three Americans, Kim Dong Chul, Kim Hak Song and Tony Kim, who had been held on espionage charges. The release of the three was seen as a good will gesture by North Korean leader Kim Jong Un ahead of a summit in Singapore with President Trump the following month.


Home Science Research: Researchers plan tiny wasp counterattack —
Research: Researchers plan tiny wasp counterattack —
UC Riverside is testing whether a sesame seed-sized wasp can control a pest that could seriously damage California crops including wine, walnuts, and avocados.
The pest, a sap-sucking spotted lantern fly, is originally from China and was first detected five years ago in Pennsylvania. Since then, large populations have spread rapidly to grape vines, apple trees, and other plants in New York, Delaware, New Jersey, Maryland, and Virginia.
Experts believe the lantern fly is likely to make its way to California soon.
Mark Hoddle, director of the Center for Invasive Species Research at UC Riverside, explains why the lantern fly is so harmful.
"It secretes copious amounts of "honeydew," a waste product that encourages black, sooty mold and damages a plant's ability to grow," he said. The honeydew also attracts undesirable insects such as ants and hornets.
The impacts could extend well beyond California. According to industry reports, the state is the world's fourth-largest wine producer, selling an estimated $35 billion domestically and exporting $1.5 billion annually.
READ Research: Gains of only 22 percent over the next six decades represent best-case scenario --
Around 44% of nonnative insects arriving in California were first established elsewhere in the U.S. Given the speed with which the spotted lantern fly has spread, Hoddle realized the state needed a proactive approach to this predictable problem.
"Normally, when a bug shows up, we try to contain and eradicate it," Hoddle said. "But by the time the population is found, it tends to already be widespread and hard to handle."
The state Department of Food and Agriculture recently granted Hoddle $544,000 to test whether a tiny parasitic wasp, also originally from China, could be the solution to the looming problem. Hoddle explained that the wasp has a needle-like appendage it uses to lay its own eggs inside the lantern fly's eggs. While developing, the wasp larvae eat and kill their hosts, and then emerge after chewing escape holes through the lantern fly eggs.
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These wasps pose no threat to plants or people, but before they can be used to control the lantern fly, Hoddle must prove they won't cause unnecessary harm to other native insects. "We can't just release a Chinese parasite into the wild in California," Hoddle said. "Chances are low it will harm the wrong targets, but we have to be sure."
Safety testing will be conducted in a highly secure quarantine facility at UC Riverside. Native lantern flies, the subjects of safety testing, will be collected from natural areas in California and southern Arizona this summer.
Though the wasp is now being evaluated as a biological control on the East Coast, populations of lantern fly there have already grown large enough to cause significant concern for the grape industry, Hoddle said.
A spotted lantern fly's wingspan is about 1.5 inches, and at most they can fly a few hundred feet at a time if they're assisted by the wind. The lantern fly has spread so fast in part because the females lay eggs on nonbiological materials, such as train cars, motor homes, wooden pallets, and trucks that inadvertently move them into new territories.
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"Anyone on the East Coast driving to California should be especially vigilant about checking their vehicle for egg masses before they make the journey," Hoddle warned. "Failing to notice them could have serious consequences."
Hoddle's testing will take roughly three years, and he estimates that this may be around the time when the wasps will be needed in California. "We hope to be ready to release these wasps immediately when the spotted lantern fly shows up, giving us a really strong head start on the invasion," he said.
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Jazz Guitar Lessons • Jazz Chord Substitution Part Two • Altered Chords, Theory, Charts, Videos.
Lorne Hemmerling
To purchase a PDF copy, please follow this link:
Learning Blues Guitar | Lorne K. Hemmerling - Sellfy.com
Learning Blues Guitar
I have been teaching guitar professionally since 1992, when Don't Fret Guitar Instruction was established. Over the years, I have taught countless students (beginners to advanced) how to play or improve their chops. Past students include four members of PROTEST THE HERO.
With this book, my goal is to relate the scales with chords and rhythms as opposed to just learning solos or licks and having no idea how to apply them. Good rhythm playing and knowledge is crucial to good soloing and vice versa. This comes through understanding the relationship between chords and scales. This book provides that important foundation.
The book is unique in the fact that each chapter is based around a different key signature and an open (contains unfretted notes), pattern of the pentatonic scale. There are five chapters covering the key signatures of E, A, D, G and C, and the five open 'box patterns' (scale patterns) of the pentatonic scale. Eventually all the box patterns are covered, from the open strings to the fifteenth fret.
There is no endless scale practice or useless licks to learn. Instead, each chapter begins with a chord progression, moves into various rhythm patterns derived from the chord progression, and then culminates with solos based on the scale and key covered. These solos tie in with the chord progression and rhythm patterns to form a complete lesson for each chapter.
The book is progressive. Upon completion, the student will have a solid foundation in blues guitar, and will understand the rhythm, lead connection.
The book is best studied from beginning to end, without slighting any material. All theory is explained in the simplest terms. There are fretboard diagrams for the scales, chord grids, and photos of hand positions as well as videos posted on YouTube to aid in the learning process.
It is best, but not necessary, to have a knowledge of barre and open chord shapes before beginning this course. All the chords have fretboard grids associated with them.
Good luck and have fun. Music is a celebration. Enjoy!
Lorne K. Hemmerling
Jazz and blues are all about substitution. This is especially true in blues music where most tunes consist of the famous one four five, twelve bar progression. Chords, riffs, lick, etc are substituted to break up the repetition. Of course, there is nothing saying that you must do this, but with the same progression repeating over and over again, it can get quite boring for the musicians and the audience.
What I have done here is the same as in Part One. I have re-harmonized the basic blues progression with more intricate chords to give it a jazzy overhaul. I have transcribed all the variations in the key of G, the same as Part One. It is good practice to transpose the progressions to other keys. Most jazz is performed in flat keys. I suggest learning all the variations in the written key, then make the first transposition to G♭(one fret lower). Continue to A ♭, B♭, D♭, etc.
Part Two introduces altered chords. These are tricky to improvise over. If in doubt about what scales to use (I will address this in future lessons), stick with chord tones.
Progression One
This is a standard blues progression in the key of G. This is the famous 'One, Four, Five' progression. This version contains 'the quick change', that is, the movement in the second measure is to the 'Four' chord (C7). C7 and G7 are not actually in the key of G Major, but blues players would take liberties here and call this out as the key of G.
I have transcribed this with a mixture of Root 5 and Root 6 barre chords.
Many blues and jazz players work with barres as opposed to open chords for a number of reasons. They are easier to transpose by simply moving the shapes to another fret, they are easier to mute by releasing the fret hand pressure, and quite a few of the altered chords have no open shapes.
Jazz Guitar Lessons • Jazz Chord Substitution Part Two | Source
Progression Two
The G Major in measure one from Progression One has been replaced by G Major Seven (GMaj7). The Major Seven chord is formed by adding a fourth note to the G Major triad. The triad is built on thirds. The root is G, a third above G is B and a third above B is D. All the notes are three Major scale steps away from each other.
Climbing up three more scale steps in the key of G Major yields F♯. This the the note that forms the Major Seventh from the Major chord. GMaj7 is a common substitution for G Major in blues and especially jazz. The Ionian mode (in this case the G Major Scale played from G to G), fits perfectly across this chord, because of the Major Seventh degree.
Root G
Third B
Fifth G
G Major Seventh
Seventh F♯
http://lornehemmerling.hubpages.com/hub/Jazz-Guitar-Lessons-Jazz-Chord-Substitution-Part-Two-Charts-Altered-Chords-Videos
In measures two, five, six and ten Gm7 is substituted for C7. Once again, this is a normal substitution for a dominant seventh chord.
I have been asked in a previous post to explain this. Look at the structure of the minor seventh chord. It is very close to a dominate C9. In fact, the Gm7 chord could be renamed C9sus4/G. Lets look at the chord spellings:
Root C
Third E
Seventh B♭
Ninth D
C9sus4/G (Gm7)
Suspended Fourth F
The only note that forces a change on the chord (and the related G Major scale) is the F, the suspended fourth. Also, there is no Root C in C9sus4/G.
Progression Three
This version substitutes Major and minor sixth chords for the Major and minor seventh chords in Progression Two. The minor sixth chord is actually the dominant ninth chord with the fifth in the root. There is no suspended fourth in this chord. Lets compare the dominant ninth and minor sixth in terms of chord construction:
C9/G (Gm6)
All the notes from Gm6 are the same as C9, which can replace the standard dominant seventh from the original progression (in this case, C7), anytime.
Progression Four
I have notated the last progressions with two measures per staff to allow for better spacing of the chords.
Measure one combines the Major seventh chord with the Major sixth. These chords substitute for the normal G Major chord. This is a very pleasant, consonant sound. When moving between the chords, hold the fourth finger. This will make the transition much easier.
Measure two moves from the minor seventh to the dominant seventh chord. Try employing different voicings for the C7. A Root 5 barre would place the root C on the fifth string, the same as it is with the open shape. In fact, you may want to try playing the notated open shape for the third beat, then moving to the Root 5 chord on the fourth beat.
In measure four, I have substituted the minor seventh for the first two beats. The Dm7 forms a G9sus4/D, the same as explained above for the Gm7-C9sus4/G. To find this substitution, play the minor seventh or minor sixth a fifth higher from the root of the dominant seventh chord. The G7 chord in beats two and three places the minor seventh degree (F) in two places in the chord, not the fourth string and an octave higher on the second string. This gives the chord more 'bite'.
In the turnaround, measures eleven and twelve, I have notated a very common chromatic movement. The diminished chords add that flavour with the ascending bass note from GMaj6 to G♯dim7, then the descending intervals on the fourth and second strings form Am7 to Adim7. This is a very cool sound!
Progression Five
This version is similar to Progression Four.
Most of the dominant chords have been substituted by the minor seventh to minor sixth change. The only exceptions are the G13 movement to G7 in measures four and eight. This voicing for the thirteenth chord is difficult to get to (with that fourth finger barre), but well worth the effort. If you play this fingerstyle by 'grabbing' the chords, you can leave the barre out. I have notated that shape in the next Progression Six.
The turnaround is a very common sound in jazz. In measure eleven, the minor seventh chord has been substituted for the Major seventh. This is another common use for the minor seventh chord, replacing the root chord (in this case, GMaj7), a third higher. The Bm7 can be respelled as a GMaj9/B. Lets take a look:
Major Seventh F♯
Fifth D
GMaj9/B (Bm7)
Major Ninth A
Once again, there is no root in the GMaj9/B.
Progression Six
I play this version fingerstyle. I have notated the chords exactly as played.
This is the first progression with an altered chord, the G7♯5. If you are jamming this with another player such as a keyboardist or another guitar player, they would have to know that this change is coming up. Obviously, you cannot have another player voicing a G7, G9 or G13 on top of this chord. The clash of the sharp fifth with the natural fifth would sound wrong.
The descending chromatic change on the second string from the E in G13 to the D♯ in G7♯5, to the D natural in Gm7 (occurring in measures four and five) is an excellent example of voice leading within chords.
In measure eight, the G13 to G7♯5 has been substituted with the movement from Bm7 to B♭m7. This is a complete change and sounds great moving into the Am7 in measure nine. As far as I know, there is no real theoretical explanation for this substitution, unless you treat this measure as one bar of G Major instead of trying o associate these chords (Bm7 and B♭m7) with the substituted dominant chords (G13 and G7♯5). Even at that, it is a stretch. As stated above, the Bm7 can be respelled as GMaj9/B, but the B♭m7 bears little resemblance to a G Major chord.
In the measure before this change, measure seven, I have notated different voicings for the Major seventh and Major sixth chords. These song great leading into measure eight.
Measures nine and ten would stay on the fifth (D7) in the original progression, instead of the normal movement from D7 to C7. There is much chromatic movement in jazz in both chord and melody work. The G♯7 leading into the turnaround is an excellent sound. Once again, the other players would have to know that this is coming up.
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5 years ago from Oshawa
Thanks so very much for the kind words, my friend!
Walter Holokai
5 years ago from Youngstown, Ohio
Man! Thank you so much! It took some time to put that together and it came out beautiful! I am very impressed. You deserve your high rating. It is perhaps kismet that I would have seen this since I am about to embark on a guitar journey in the very near future where these exercises may become very helpful to me. Aloha!


Why Democrats Lost
Kenneth Whitley, WPA poster, 1939
I voted for Hillary Clinton. Barack Obama was absolutely right when he said that there was no candidate better qualified to be president than Hillary Clinton. So, why did Democrats lose?
When asked about raising the minimum wage–while Bernie Sanders was calling for a $15.00 an hour minimum wage–Hillary took the politically safe route and said we should pursue "incremental changes." Holy crap. My head exploded. Talk about poor optics. Talk about being completely out of touch with your base of support. Let's do another fundraiser with Wall Street bankers in the freaking Hamptons. Can you imagine how that sounded to a single mother trying to feed her kids on a $9.00 an hour job? Incremental change? We don't need incremental change. We need someone to call 911. We're dying here.
Hillary Clinton lost one million Democratic and independent women voters in 2016. Imagine that.
So here's the not-so-secret answer to why Democrats lost in 2016: Bernie Sanders supporters aren't a radical fringe element. They are the Democratic Party. They are the ideological heirs of FDR, John F. Kennedy and LBJ. They are the same social democrats who created social security, medicaid, passed the civil rights acts, and protested the unnecessary wars and the military-industrial complex. They didn't abandon the Democratic Party. They were abandoned by the Democratic Party. The Democratic Party has strived to become more conservative and politically safe. And in doing so has become politically irrelevant.
Author konigludwigPosted on May 21, 2017 June 13, 2017 Categories Opinion/Editorial, Political commentary, PoliticsTags 2016 presidential race, Bernie Sanders, Democratic Party, Hillary Clinton, income inequality2 Comments on Why Democrats Lost
Do Trump's Murky Financial Ties to Russia Connect to Money Laundering?
Investigators have only begun to follow the money.
aluxum/Getty
By Bill Buzenberg
Federal investigators continue to dig into Russia's cyberattack on the US election and the Trump administration's possible involvement—but as bad as that intrusion and collusion may be, Trump's opaque financial dealings could prove even more perilous for the president.
Trump has blazed a decades-long trail of questionable financial dealings with Russian sources that could provide investigators with the grist they need for legal action. A wide array of Russian oligarchs with links to Vladimir Putin have invested tens of millions of hard-to-explain dollars in Trump properties. And Trump professes never to know who these people are or where they got the big bucks for their mostly cash deals.
Money laundering is the process of taking proceeds from criminal activity (dirty money) and making them appear legal (clean). Although the news didn't make much of a splash during the 2016 campaign, Trump paid a $10 million fine to the U.S. Treasury in 2015 for his bankrupt Taj Mahal casino in Atlantic City because it failed to meet anti-money-laundering requirements. According to the Wall Street Journal, "Regulators said the casino failed to establish and implement an effective anti-money-laundering program, failed to implement an adequate system of internal controls, and failed to properly file currency transaction reports or keep other required records."
It is already a matter of public record that several Trump-affiliated businesses and associates are connected to alleged Russian money-laundering operations.
Many of Trump's business dealings involve Deutsche Bank or the Bank of Cyprus—both known for their connections with Russian oligarchs. Deutsche Bank is also Trump's biggest lender; he owes the bank some $300 million, as first reported by Mother Jones' Russ Choma and David Corn. The Guardian reported that Deutsche Bank paid $630 million in fines for failing to prevent $10 billion in Russian money laundering between 2012 and 2015.
The Bank of Cyprus is also well known as a money-laundering haven for Russian oligarchs. Trump's Commerce Secretary Wilbur Ross has invested heavily in the Bank of Cyprus (some $424 million in 2014, giving him an 18% stake) and he was once vice-chairman of the bank, according to the Guardian. Ross presided over deals that raise questions about his tenure at the bank and his ties to politically connected Russian oligarchs.
Ross shared his vice-chairman post at the bank with a deposit holder-turned-shareholder, Vladimir Strzhalkovsky, referred to in Russian media as a former KGB official and Putin ally.
Author konigludwigPosted on May 13, 2017 April 20, 2018 Categories Exposé, HistoryTags Bank of Cyprus, Commerce Secretary Wilbur Ross, Deutches Bank, Donald Trump, money laundering, Paul Manafort, Russian mafia, Vladimir Putin3 Comments on Do Trump's Murky Financial Ties to Russia Connect to Money Laundering?


Neurosurgery Practice
Mazda Turel | STEM
Dr. Mazda Turel is a general practice neurosurgeon who specializes in the treatment of diseases of the brain and spine and advocates an approach to neurosurgery that is both balanced and aggressive. Born in 1981, Mazda was ordained a priest in the Zoroastrian faith in 1992. He completed his degree in medicine (MBBS) from Grant Medical College and Sir JJ groups of Hospitals in Mumbai, and went on to pursue a MCh degree in Neurosurgery from the prestigious Christian Medical College in Vellore, India.
He was awarded the Jacob Chandy Gold Medal at his graduation; a distinction that has only been conferred previously to four other recipients since its institution 35 years ago.
His prolific clinical research has garnered several academic accolades both nationally and internationally and has resulted in numerous publications in esteemed scientific journals.
After completing his residency he visited several departments of neurosurgery across the world and earned a Diploma in Minimally Invasive Neurosurgery from Beijing, China. He then went onto complete a fellowship in skull base surgery and neuro-oncology at the illustrious Toronto Western Hospital and another fellowship in minimally invasive and complex spine surgery at the Rush University in Chicago after which he crossed continents to travel to Shanghai to specialize in cerebro-vascular surgery, with an emphasis on arterial bypasses in the brain.
Latika Nath | Environmentalist, Wildlife Photographer


Julia Arriola Biography
Click here to view available works by Julia Arriola
Julia Arriola, an artist with eclectic ideas, draws on history for conceptual renderings that address past, present and future with an indigenous flair. Trained as a sculptor, she likes to look at all sides of an image whether 2d or 3d. Currently her works are drawings using colored pencils and ink on ledger paper that dates from 1859.
Born in Tucson, Arizona, and daughter of a career master sergeant, she has lived in other states but always returned to her beloved city. One of the favorite subjects in early schooling was art period and history in high school. "As a child she was always drawing and trying to make things," her mother once said of her. During the teenage and early adult years the thoughts of art went away and did not fully return until the early 80s. After jobs in factories, a three year tour in the Navy, and more factory jobs she decided to go back to school, making her a late comer into the world of higher education and art. After graduating with a Masters in Fine Art the race was on to 'make it' in the art world, or at least try to make a living on ones art. Easier said than done, and definitely not the 9 to 5 job that the factories offered, she was recommended for a curatorial position at the Arizona Historical Society, a museum where she had been hired as a work study student during undergrad and graduate school.
Always grateful to the Arizona Historical Society for not only the position but throughout the career there were many creative outlets that the museum provided to bring art into the interpretation of history exhibits. After recent retirement, Arriola is now able to spend 6 to 7 hours a day in her studio once again becoming a full time artist.
Although art came back at a later time in life, Arriola would not change a thing, as all experiences led her through a rich life with amazing outcomes that now are manifested through ledger drawings, sculpture.
Julia Arriola "Steampunk Bear" Colored pencil and ink on c. 1859 Ledger paper
Julia says about her art and inspiration:
"The ledger drawings began a little over a year ago starting with my love for Steampunk. Love the design, fashion, and technology that came from that great but horrible time period, especially for NA, the 19th century. These drawings suggest how objects/concepts from the 19th century were not only forced on Native Americans, but was also embraced. Different tribes, traditions, and ceremonies are included because all were impacted by the overwhelming force of Manifest Destiny. Steampunk fashion is a subgenre of the steampunk movement in science fiction. It is a mixture of the Victorian era's romantic view of science in literature and elements from the Industrial Revolution in Europe during the 1800s."