Efficient Erythroid Differentiation of a PGD-Derived Human Pluripotent Stem Cell Line Affected with Sickle Cell Hemoglobinopathy

The technology of preimplantation genetic diagnosis (PGD) screens IVF-derived cleavage-stage embryos or oocytes from parents with inherited disorders, and is routinely used to avoid births with severe genetic disorders. More than one hundred testable genetic conditions, including severe hematologic diseases such as beta thalassemia, sickle cell anemia, and Fanconi anemia can be PCR-screened from either a micro-manipulated blastomere, or a pre-fertilization ovarian polar body. Derivation of human embryonic stem cell (hESC) lines from diseased IVF blastocysts has recently been reported, and these PGD-hESC are untested yet potentially valuable tools for investigating cellular and molecular events of human embryogenesis in diseased states. For example, a great deal of interest has recently been generated in treating hematologic diseases with genetically-corrected hematopoietic stem cells (HSC) derived from patient-specific pluripotent stem cells. Generation of hematopoietic progenitors from PGD-hESC affected with genetic syndromes may thus provide novel opportunities for testing cell-based and gene therapeutic strategies. An important candidate for such cell-based therapy includes sickle cell disease (SSD) hemoglobinopathy, a classic inherited single gene disorder resulting from the substitution of glutamic acid by valine at position 6 of the beta chain of hemoglobin. Human pluripotent stem cells derived from PGD-selected blastocysts or induced pluripotency (iPS; e.g., using patient’s somatic cells), will serve as critical reagents for testing such therapeutic strategies. In these studies, we report the characterization and erythropoietic differentiation of a novel PGD-hESC line affected with SSD hemoglobinopathy. The sickle point mutation was confirmed in this PGD-hESC line with direct genomic sequencing of the beta globin locus. This hESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SSD-hESC can be efficiently differentiated to the hematopoietic lineage under serum-free conditions, and gave rise to robust primitive and definitive erythropoieses. The expression of embryonic, fetal and adult globin genes in SSD PGD-hESC derived erythroid cells was confirmed by qRT-PCR, intracytoplasmic FACS, and in situ immunostaining of PGD-hESC teratoma sections. Moreover, we defined culture conditions for massive, long-term liquid culture expansion of sickle affected erythroid progenitors that remained in an undifferentiated erythroblastic phenotype for at least two months. These sickle erythroblasts were continuously maintained as a primary cell line that could be frozen and thawed without loss of viability. In vitro-expanded sickle erythroblasts expressed CD71+CD36+ and CD71+CD235a+ phenotypes, and underwent developmentally appropriate embryonic, fetal, and adult hemoglobin switching over a period of several months. Moreover, hESC-derived erythroblasts were readily infected with Plasmodium falciparum malaria parasites, thus demonstrating their potential utility in studying the effects of this important pathogen in normal and diseased erythropoiesis. These data demonstrate the utility of using patient-specific, hemoglobinopathic hESC for generating significant numbers of erythroid progenitors for molecular, developmental, gene therapeutic, pharmacologic, and microbiological studies. We are currently conducting a comparative erythropoietic differentiation study using SSD PGD-hESC vs. SSD iPS that were generated from somatic fibroblasts using defined pluripotency factors.