Induced Pluripotent Stem Cell Modeling of Sickle Cell Anemia

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.