Seamless correction of the sickle cell disease mutation of theHBBgene in human induced pluripotent stem cells using TALENs

2013 ◽  
Vol 111 (5) ◽  
pp. 1048-1053 ◽  
Author(s):  
Ning Sun ◽  
Huimin Zhao
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Induced Pluripotent Stem Cells ◽  
Sickle Cell ◽  
Pluripotent Stem Cells ◽  
Cell Disease ◽  
Disease Mutation ◽  
Induced Pluripotent

scholarly journals A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells

2017 ◽  
Vol 8 (4) ◽  
pp. 1076-1085 ◽  
Author(s):  
Seonmi Park ◽  
Andreia Gianotti-Sommer ◽  
Francisco Javier Molina-Estevez ◽  
Kim Vanuytsel ◽  
Nick Skvir ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Induced Pluripotent Stem Cells ◽  
Sickle Cell ◽  
Pluripotent Stem Cells ◽  
Ethnically Diverse ◽  
Cell Disease ◽  
Induced Pluripotent ◽  
Disease Specific

Induced Pluripotent Stem Cell Modeling of Sickle Cell Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3233-3233
Author(s):  
Sarah S Rozelle ◽  
Brenden W Smith ◽  
Efthymia Melista ◽  
Ehimen Aneni ◽  
Paola Sebastiani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Pluripotent Stem Cells ◽  
Cell Disease ◽  
Large Numbers ◽  
Induced Pluripotent ◽  
Disease Specific

Abstract Abstract 3233 As they can be generated from the somatic cells of any individual, induced pluripotent stem cells (iPSC) represent renewable, potentially unlimited cell sources that circumvent the possibility of inappropriate immune response and open the door to the advent of patient-specific, personalized medicine. Disease-specific iPSCs have the potential to elucidate disease mechanisms, revolutionize drug discovery, and improve patient care. We have built a large library of sickle cell disease-specific iPSCs containing more than 100 individual lines from both African American and Saudi Arab patients with different HbS gene haplotypes and HbF-modulating quantitative trait loci (QTL) genotypes. The differentiation of these lines into the erythroid lineage offers a novel opportunity to study erythroid development, the regulation of globin switching, small molecule drug development and the modeling of red blood cell linked diseases in vitro. Although several teams have published proof-of-principle examples for the derivation of hematopoietic cells from pluripotent stem cells, these protocols are technically demanding and result in the production of limited numbers of cells. Our conceptual approach has been to mimic the natural sequences of development in vitro in order to derive the range and number of cell types needed for the creation of a robust iPSC-based platform. We have developed a novel, chemically defined and feeder-free methodology for the production of large numbers of functionally mature red blood cells (RBCs) from both normal and disease-specific human iPSCs. This protocol utilizes a 2D/adherent approach and eliminates the need for embryoid body formation or xenogeneic agents resulting in a shorter production time (∼10 days). Large numbers of clinically relevant, high purity hematopoietic cells can be generated such that 15,000 cells yield 1 billion cells in two weeks. This protocol produces bipotential megakaryocyte-erythroid progenitors (MEPs) that co-express the surface markers CD235 (red cells) and CD41 (megakaryocytes) and demonstrate expression of accepted panels of both erythroid and megakaryocyte-specific genes. Use of an erythroid maturation media results in efficient maturation of MEPs to erythrocytes. Due to this novel approach and the robust nature of the methodology, we are able to generate large numbers of functionally mature RBCs that produce hemoglobin, respond to oxygen deprivation, and enucleate. Furthermore, these human iPSC-derived directly differentiated erythroid-lineage cells engraft robustly in Nod-SCID-Gamma (NSG) immunocompromised mice and demonstrate detectable chimerism in peripheral blood. Importantly, these cells respond to hydroxyurea (HU), the only FDA approved drug that increases HbF levels in sickle cell anemia. Our goals are to use these cells to further understand hemoglobin switching in carriers of varied HbS haplotypes and to harness our library of sickle cell disease-specific lines in combination with the developed differentiation protocol in order to create correlations between genetics and response to new and available HbF inducing agents, furthering the clinician's capability to personalize treatment plans for each patient. Disclosures: No relevant conflicts of interest to declare.

A Library of Sickle Cell Anemia Induced Pluripotent Stem Cells of Diverse Haplotypes and Ethnicities

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2354-2354
Author(s):  
Seonmi Park ◽  
Andreia Gianotti-Sommer ◽  
David H.K. Chui ◽  
Maria Stella Figueiredo ◽  
Abdulrahman Alsultan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Pluripotent Stem Cells ◽  
Globin Gene ◽  
Genetic Modifiers ◽  
Induced Pluripotent

Abstract The mutation causing sickle cell anemia (rs334, GAG-GTG, glu6val) had several independent origins in Africa, the Middle East and India and spread throughout parts of the world by wars, slave trading and population migrations. The genetic background upon which the HbS mutation occurred, or the β-globin gene (HBB) haplotype, is associated with differences in the phenotype of this disease and the ability of affected individuals to synthesize fetal hemoglobin (HbF). The main modifier of the disease phenotype is the level of HbF in the blood of affected individuals. HbF inhibits the polymerization of HbS, the proximate cause of disease pathophysiology. As part of the NHLBI NextGen consortium (U01HL107443) we established a library of induced pluripotent stem cells (iPSC) from patients with sickle cell anemia of diverse HBB haplotypes and HbF phenotypes. The purpose of establishing this library was to allow genetic studies of globin gene expression during the erythroid differentiation of iPSC of diverse genotypes. During these studies we have implemented an efficient and highly reproducible platform for the production of large numbers of sickle cell anemia-specific iPSC, derived and characterized a novel in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of normal and disease-specific iPSC and used this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes. We have recently finished whole genome DNA and RNA sequencing analysis in some of these lines aimed at identifying developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS as part of our search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment. Furthermore, our labs are also focusing on using a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers and explore globin switching. Cell lines established are shown in the table. Table 1. Number of subjects recruited to date 98 Number of subjects with iPSC lines established 56 Average number of iPSC lines per subject 3 (total of 158 lines generated) Quality control status of iPSC lines All lines are expanded and banked, mycoplasma free, express pluripotency markers Subjects with target cells differentiated (erythrocytes) 25 Samples have been collected on African American patients with sickle cell anemia with diverse HBB haplotypes, predominantly homozygotes and compound heterozygotes for the Benin and Bantu haplotypes, Saudi Arabian patients with the Arab-Indian haplotype and the Saudi Benin haplotype that is characterized by HbF levels about twice as high as in African Benin haplotype patients and from Brazilian patients who are predominantly homozygotes for the Bantu haplotype that typically is associated with the lowest HbF of all HBB haplotypes. This iPSC-based library and the data associated with it represents a valuable readily available resource for the sickle cell research community and all the generated lines will be available for distribution early in 2016 through WiCell. Disclosures No relevant conflicts of interest to declare.

scholarly journals 332. Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells

2016 ◽  
Vol 24 ◽  
pp. S132
Author(s):  
R. Geoffrey Sargent ◽  
Shingo Suzuki ◽  
Luke Gruenert ◽  
Fei Xie ◽  
Lin Ye ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent
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