scholarly journals Efficient CRISPR/Cas9-based gene correction in induced pluripotent stem cells established from fibroblasts of patients with sickle cell disease

2016 ◽  
Vol 3 ◽  
pp. 78-78 ◽  
Author(s):  
Masahiro Sato ◽  
Issei Saitoh ◽  
Emi Inada
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Induced Pluripotent Stem Cells ◽  
Sickle Cell ◽  
Pluripotent Stem Cells ◽  
Cell Disease ◽  
Gene Correction ◽  
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.

Modeling Congenital Amegakaryocytic Thrombocytopenia Using Patient-Specific Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 703-703
Author(s):  
Naoya Takayama ◽  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Sou Nakamura ◽  
Sho-ichi Hirose ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Receptor Gene ◽  
Patient Specific ◽  
Gene Correction ◽  
Donor Skin ◽  
Induced Pluripotent

Abstract Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts

2015 ◽  
Vol 5 (4) ◽  
pp. 558-568 ◽  
Author(s):  
Tui Neri ◽  
Sharon Muggeo ◽  
Marianna Paulis ◽  
Maria Elena Caldana ◽  
Laura Crisafulli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Gene Correction ◽  
Induced Pluripotent
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