Publications by authors named "Zhaohui Ye"

70 Publications

Convergence of human pluripotent stem cell, organoid, and genome editing technologies.

Exp Biol Med (Maywood) 2021 Apr 19;246(7):861-875. Epub 2021 Jan 19.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, John Hopkins University, Baltimore, MD 21218, USA.

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.
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http://dx.doi.org/10.1177/1535370220985808DOI Listing
April 2021

MiR-140-3p Ameliorates the Progression of Osteoarthritis via Targeting CXCR4.

Biol Pharm Bull 2020 May 26;43(5):810-816. Epub 2020 Feb 26.

Department of Orthopaedics, The First Affiliated Hospital of Soochow University.

Osteoarthritis is a common disease character with progressive destruction of cartilage. MicroRNA (miR)-140-3p was validated as a biomarker for osteoarthritis. However, the mechanism by which miRNA-140-3p regulates osteoarthritis remains unclear. Thus, this study aims to evaluate the potential function of miRNA-140-3p during the pathogenesis of osteoarthritis. MiRNA-140-3p expression in tissue and CHON-001 chondrocyte cells was determined with quantitative real time (qRT)-PCR. In vitro osteoarthritis model was established by treatment of the chondrocyte cells CHON-001 with interleukin (IL)-1β. Cell proliferation and apoptosis were measured with cell counting kit-8 (CCK8) and Annexin V/propidium iodide (PI) apoptosis assay, respectively. Protein expressions were evaluated using Western blot. The target gene of miR-140-3p was predicted using Targetscan and miRDB. MiR-140-3p was downregulated in knee tissue from patients with osteoarthritis. IL-1β inhibited the proliferation of CHON-001 cells via inducing apoptosis. In addition, IL-1β significantly inhibited the expressions of collagen II and aggrecan and increased the level of matrix metalloproteinase (MMP)13. However, the effects of IL-1β could be ameliorated by the addition of miR-140-3p mimics. Moreover, luciferase reporter assay demonstrated CXCR4 as a target gene of miR-140-3p. IL-1β-induced upregulation of CXCR4 could be blocked by miR-140-3p mimics. Our study indicated that miR-140-3p could suppress the progression of osteoarthritis by directly targeting CXCR4. Therefore, miR-140-3p might serve as a potential therapeutic target for the treatment of osteoarthritis.
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http://dx.doi.org/10.1248/bpb.b19-00959DOI Listing
May 2020

Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing.

Nat Commun 2019 11 25;10(1):5353. Epub 2019 Nov 25.

Division of Cellular and Gene Therapies, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.

DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.
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http://dx.doi.org/10.1038/s41467-019-13342-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877639PMC
November 2019

Human-relevant preclinical in vitro models for studying hepatobiliary development and liver diseases using induced pluripotent stem cells.

Exp Biol Med (Maywood) 2019 05 26;244(8):702-708. Epub 2019 Feb 26.

1 Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21205, USA.

Impact Statement: In this review, we address the potential of human-induced pluripotent stem cell-based hepatobiliary differentiation technology as a means to study human liver development and cell fate determination, and to model liver diseases in an effort to develop a new human-relevant preclinical platform for drug development.
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http://dx.doi.org/10.1177/1535370219834895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552401PMC
May 2019

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Stem Cells 2019 03 17;37(3):306-317. Epub 2018 Dec 17.

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.
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http://dx.doi.org/10.1002/stem.2950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435276PMC
March 2019

Biliary Atresia Relevant Human Induced Pluripotent Stem Cells Recapitulate Key Disease Features in a Dish.

J Pediatr Gastroenterol Nutr 2019 01;68(1):56-63

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center.

Biliary atresia (BA) is the most common cause of pediatric end-stage liver disease and the etiology is poorly understood. There is no effective therapy for BA partly due to lack of human BA models. Towards developing in vitro human models of BA, disease-specific induced pluripotent stem cells (iPSCs) from 6 BA patients were generated using non-integrating episomal plasmids. In addition, to determine the functional significance of BA-susceptibility genes identified by genome-wide association studies (GWAS) in biliary development, a genome-editing approach was used to create iPSCs with defined mutations in these GWAS BA loci. Using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, isogenic iPSCs deficient in BA-associated genes (GPC1 and ADD3) were created from healthy iPSCs. Both the BA patient-iPSCs and the knock out (KO) iPSCs were studied for their in vitro biliary differentiation potential. These BA-specific iPSCs demonstrated significantly decreased formation of ductal structures, decreased expression of biliary markers including CK7, EpCAM, SOX9, CK19, AE2, and CFTR and increased fibrosis markers such as alpha smooth muscle actin, Loxl2, and Collagen1 compared to controls. Both the patient- and the KO-iPSCs also showed increased yes-associated protein (YAP, a marker of bile duct proliferation/fibrosis). Collagen and YAP were reduced by treatment with the anti-fibrogenic drug pentoxifylline. In summary, these BA-specific human iPSCs showed deficiency in biliary differentiation along with increased fibrosis, the 2 key disease features of BA. These iPSCs can provide new human BA models for understanding the molecular basis of abnormal biliary development and opportunities to identify drugs that have therapeutic effects on BA.
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http://dx.doi.org/10.1097/MPG.0000000000002187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314509PMC
January 2019

A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease.

Stem Cells Transl Med 2018 01 21;7(1):87-97. Epub 2017 Nov 21.

Division of Hematology, Department of Medicine, Baltimore, Maryland, USA.

Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with β-thalassemia (β-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site-specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, targeting various HBB mutations of β-thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two β-thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in β-thal and SCD. Stem Cells Translational Medicine 2018;7:87-97.
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http://dx.doi.org/10.1002/sctm.17-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746148PMC
January 2018

Derivation of a disease-specific human induced pluripotent stem cell line from a biliary atresia patient.

Stem Cell Res 2017 10 8;24:25-28. Epub 2017 Aug 8.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, USA; Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, USA; Institute for Cell Engineering, Johns Hopkins University School of Medicine, USA. Electronic address:

Biliary atresia (BA) is a common cause of pediatric end-stage liver disease. While its etiology is not yet clear, evidence has suggested that BA results from interactions between genetic susceptibility and environmental factors. Disease relevant human cellular models of BA will facilitate identification of both genetic and environmental factors that are important for disease prevention and treatment. Here we report the generation of a human induced pluripotent stem cell line from a BA patient using episomal vectors. Patient-specific BA iPSC lines provide valuable tools for disease mechanism study and drug development.
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http://dx.doi.org/10.1016/j.scr.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881114PMC
October 2017

A hypomorphic PIGA gene mutation causes severe defects in neuron development and susceptibility to complement-mediated toxicity in a human iPSC model.

PLoS One 2017 25;12(4):e0174074. Epub 2017 Apr 25.

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

Mutations in genes involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis underlie a group of congenital syndromes characterized by severe neurodevelopmental defects. GPI anchored proteins have diverse roles in cell adhesion, signaling, metabolism and complement regulation. Over 30 enzymes are required for GPI anchor biosynthesis and PIGA is involved in the first step of this process. A hypomorphic mutation in the X-linked PIGA gene (c.1234C>T) causes multiple congenital anomalies hypotonia seizure syndrome 2 (MCAHS2), indicating that even partial reduction of GPI anchored proteins dramatically impairs central nervous system development, but the mechanism is unclear. Here, we established a human induced pluripotent stem cell (hiPSC) model containing the PIGAc.1234C>T mutation to study the effects of a hypomorphic allele of PIGA on neuronal development. Neuronal differentiation from neural progenitor cells generated by EB formation in PIGAc.1234C>T is significantly impaired with decreased proliferation, aberrant synapse formation and abnormal membrane depolarization. The results provide direct evidence for a critical role of GPI anchor proteins in early neurodevelopment. Furthermore, neural progenitors derived from PIGAc.1234C>T hiPSCs demonstrate increased susceptibility to complement-mediated cytotoxicity, suggesting that defective complement regulation may contribute to neurodevelopmental disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174074PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404867PMC
September 2017

Generation of human iPSCs from an essential thrombocythemia patient carrying a V501L mutation in the MPL gene.

Stem Cell Res 2017 01 11;18:57-59. Epub 2016 Dec 11.

Division of Hematology, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Activating point mutations in the MPL gene encoding the thrombopoietin receptor are found in 3%-10% of essential thrombocythemia (ET) and myelofibrosis patients. Here, we report the derivation of induced pluripotent stem cells (iPSCs) from an ET patient with a heterozygous MPL V501L mutation. Peripheral blood CD34 progenitor cells were reprogrammed by transient plasmid expression of OCT4, SOX2, KLF4, c-MYC plus BCL2L1 (BCL-xL) genes. The derived line M494 carries a MPL V501L mutation, displays typical iPSC morphology and characteristics, are pluripotent and karyotypically normal. Upon differentiation, the iPSCs are able to differentiate into cells derived from three germ layers.
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http://dx.doi.org/10.1016/j.scr.2016.12.012DOI Listing
January 2017

Gene correction in patient-specific iPSCs for therapy development and disease modeling.

Hum Genet 2016 09 2;135(9):1041-58. Epub 2016 Jun 2.

Department of Medicine, Johns Hopkins University School of Medicine, Ross Research Building, Room 1032, 720 Rutland Ave., Baltimore, 21205, MD, USA.

The discovery that mature cells can be reprogrammed to become pluripotent and the development of engineered endonucleases for enhancing genome editing are two of the most exciting and impactful technology advances in modern medicine and science. Human pluripotent stem cells have the potential to establish new model systems for studying human developmental biology and disease mechanisms. Gene correction in patient-specific iPSCs can also provide a novel source for autologous cell therapy. Although historically challenging, precise genome editing in human iPSCs is becoming more feasible with the development of new genome-editing tools, including ZFNs, TALENs, and CRISPR. iPSCs derived from patients of a variety of diseases have been edited to correct disease-associated mutations and to generate isogenic cell lines. After directed differentiation, many of the corrected iPSCs showed restored functionality and demonstrated their potential in cell replacement therapy. Genome-wide analyses of gene-corrected iPSCs have collectively demonstrated a high fidelity of the engineered endonucleases. Remaining challenges in clinical translation of these technologies include maintaining genome integrity of the iPSC clones and the differentiated cells. Given the rapid advances in genome-editing technologies, gene correction is no longer the bottleneck in developing iPSC-based gene and cell therapies; generating functional and transplantable cell types from iPSCs remains the biggest challenge needing to be addressed by the research field.
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http://dx.doi.org/10.1007/s00439-016-1691-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881117PMC
September 2016

Efficient and Controlled Generation of 2D and 3D Bile Duct Tissue from Human Pluripotent Stem Cell-Derived Spheroids.

Stem Cell Rev Rep 2016 Aug;12(4):500-8

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

While in vitro liver tissue engineering has been increasingly studied during the last several years, presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver, but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly, generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions, and has been inefficient so far. Towards generating a fully functional liver containing biliary system, we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver, EpCAM, is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can, not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes), in a 2D differentiation condition, but also form functional ductal structures in a 3D condition. Importantly, this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition, we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues, which may facilitate engineering of complete and functional liver tissue in the future.
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http://dx.doi.org/10.1007/s12015-016-9657-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186008PMC
August 2016

Genome Editing in Human Pluripotent Stem Cells.

Cold Spring Harb Protoc 2016 Apr 1;2016(4):pdb.top086819. Epub 2016 Apr 1.

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities.
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http://dx.doi.org/10.1101/pdb.top086819DOI Listing
April 2016

A Method for Genome Editing in Human Pluripotent Stem Cells.

Cold Spring Harb Protoc 2016 Apr 1;2016(4):pdb.prot090217. Epub 2016 Apr 1.

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

Human pluripotent stem cells (PSCs) hold great potential for regenerative medicine and currently are being used as a research tool for basic discovery and disease modeling. To evaluate the role of a single genetic variant, a system of genome editing is needed to precisely mutate any desired DNA sequence in isolation and measure its effect on phenotype when compared to the isogenic parental PSC from which it was derived. This protocol describes the general targeting schemes used by researchers to edit PSCs to knock out, knock-in, or precisely alter a single nucleotide, covering conditions for electroporation, clonal isolation, and screening of edited PSCs for the targeted mutation. These recent advances simplify the procedure for genome editing, allowing individual researchers to induce nearly any desired mutation to further study its function or to reverse a disease-causing variant for future applications in regenerative medicine.
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http://dx.doi.org/10.1101/pdb.prot090217DOI Listing
April 2016

Genome editing systems in novel therapies.

Discov Med 2016 Jan;21(113):57-64

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.
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January 2016

Modified Ham test for atypical hemolytic uremic syndrome.

Blood 2015 Jun 10;125(23):3637-46. Epub 2015 Apr 10.

Division of Hematology, Department of Medicine.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.
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http://dx.doi.org/10.1182/blood-2015-02-629683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784297PMC
June 2015

A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.

Stem Cells Transl Med 2015 Apr 5;4(4):320-32. Epub 2015 Mar 5.

Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications.
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http://dx.doi.org/10.5966/sctm.2014-0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4367508PMC
April 2015

Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.

Stem Cells 2015 May;33(5):1470-9

Division of Hematology, Department of Medicine; Institute for Cell Engineering.

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.
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http://dx.doi.org/10.1002/stem.1969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628786PMC
May 2015

Covalent modification of a cysteine residue in the XPB subunit of the general transcription factor TFIIH through single epoxide cleavage of the transcription inhibitor triptolide.

Angew Chem Int Ed Engl 2015 Feb 12;54(6):1859-63. Epub 2014 Dec 12.

Department of Pharmacology, Johns Hopkins School of Medicine, 725 North Wolfe Street, Hunterian Building, Room 516, Baltimore, MD 21205 (USA).

Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.
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http://dx.doi.org/10.1002/anie.201408817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314353PMC
February 2015

Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders.

Sci Transl Med 2014 Dec;6(265):265ra168

Center for Pediatric Genomic Medicine, Children's Mercy-Kansas City, Kansas City, MO 64108, USA. Department of Pediatrics, Children's Mercy-Kansas City, Kansas City, MO 64108, USA. School of Medicine, University of Missouri-Kansas City, Kansas City, MO 64108, USA. Department of Pathology, Children's Mercy-Kansas City, Kansas City, MO 64108, USA.

Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.
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http://dx.doi.org/10.1126/scitranslmed.3010076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286868PMC
December 2014

Efficient and allele-specific genome editing of disease loci in human iPSCs.

Mol Ther 2015 Mar 24;23(3):570-7. Epub 2014 Nov 24.

1] Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA [2] Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.
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http://dx.doi.org/10.1038/mt.2014.226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351458PMC
March 2015

Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs.

Cell Stem Cell 2014 Jul;15(1):12-3

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:

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http://dx.doi.org/10.1016/j.stem.2014.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338993PMC
July 2014

Early frameshift mutation in PIGA identified in a large XLID family without neonatal lethality.

Hum Mutat 2014 Mar 13;35(3):350-5. Epub 2014 Jan 13.

Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium; Human Genome Laboratory, Department of Human Genetics, KU Leuven, Leuven, Belgium.

The phosphatidylinositol glycan class A (PIGA) protein is a member of the glycosylphosphatidylinositol anchor pathway. Germline mutations in PIGA located at Xp22.2 are thought to be lethal in males. However, a nonsense mutation in the last coding exon was recently described in two brothers with multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) who survived through birth likely because of the hypomorphic nature of the truncated protein, but died in their first weeks of life. Here, we report on a frameshift mutation early in the PIGA cDNA (c.76dupT; p.Y26Lfs*3) that cosegregates with the disease in a large family diagnosed with a severe syndromic form of X-linked intellectual disability. Unexpectedly, CD59 surface expression suggested the production of a shorter PIGA protein with residual functionality. We provide evidence that the second methionine at position 37 may be used for the translation of a 36 amino acids shorter PIGA. Complementation assays confirmed that this shorter PIGA cDNA was able to partially rescue the surface expression of CD59 in a PIGA-null cell line. Taken together, our data strongly suggest that the early frameshift mutation in PIGA produces a truncated hypomorph, which is sufficient to rescue the lethality in males but not the MCAHS2-like phenotype.
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http://dx.doi.org/10.1002/humu.22498DOI Listing
March 2014

Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.

Stem Cells Transl Med 2013 Nov 10;2(11):819-29. Epub 2013 Oct 10.

Division of Hematology, Department of Medicine, School of Medicine, and.

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.
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http://dx.doi.org/10.5966/sctm.2013-0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808197PMC
November 2013

Differential sensitivity to JAK inhibitory drugs by isogenic human erythroblasts and hematopoietic progenitors generated from patient-specific induced pluripotent stem cells.

Stem Cells 2014 Jan;32(1):269-78

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD34+ hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.
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http://dx.doi.org/10.1002/stem.1545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096297PMC
January 2014

Extensive ex vivo expansion of functional human erythroid precursors established from umbilical cord blood cells by defined factors.

Mol Ther 2014 Feb 3;22(2):451-463. Epub 2013 Sep 3.

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Electronic address:

There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10(6)-10(7)-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~10(68)-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine.
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http://dx.doi.org/10.1038/mt.2013.201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3916033PMC
February 2014

Generation and homing of iPSC-derived hematopoietic cells in vivo.

Mol Ther 2013 Jul;21(7):1292-3

Division of Hematology, Department of Medicine and Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

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http://dx.doi.org/10.1038/mt.2013.129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702102PMC
July 2013

RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells.

Blood 2013 Apr 31;121(15):2882-90. Epub 2013 Jan 31.

Moores University of California at San Diego Cancer Center, University of California at San Diego, La Jolla, CA, USA.

Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.
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http://dx.doi.org/10.1182/blood-2012-08-451641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624936PMC
April 2013

Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells.

Hepatology 2013 Jun;57(6):2458-68

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Unlabelled: Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug or gene therapy available, we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay, based on our hepatic differentiation protocol, was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease technology, we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications.

Conclusions: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs, both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases.
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http://dx.doi.org/10.1002/hep.26237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633649PMC
June 2013