Abstract
Human embryonic stem cells derived after nuclear transfer (NT-hESCs) may be used to study the earliest events in human hematopoietic stem cell development. They may also be potential candidates for cell therapy in degenerative and genetic diseases because of immunological compatibility, important for eventual transplantation. We developed an efficient, step-wise system for differentiating hESC to CD34+ hematopoietic stem cells from embryoid bodies (EB) in liquid cultures and, subsequently, further differentiating these into myeloid and lymphoid precursors using different combinations of cytokines and stromal cells. The H1 and NT-hESC-1 lines were maintained on mouse embryonic fibroblasts and NT-hESC-2 was cultured on autologous human fibroblasts with basic fibroblast growth factor in serum-replacement containing medium. In all three lines initiation of hematopoiesis begins during the first week of EB differentiation in the presence of different combinations of hematopoietic growth factors and in the absence of stromal support. Gene expression analysis, using RT-PCR methods, revealed that the first wave of embryonic hematopoiesis at 4–8 days of EB development was associated with expression of SCL/TAL1, flt3, KDR and GATA2. In FACS analysis, CD34+, KDR+, CD31+ cells were firstly detected in 6–10 days and CD34+, KDR+, CD45+, CD14+ (monocyte/macrophage marker), CD33+ (early myeloid cell marker), CD56+ (natural killer cell marker), Glycoporin A+ (erythroid cell marker), CD86 + (dendritic cell marker) cells were observed up to 15–20 days of EB development. CD34+ cell derived from 10–12 day EB’s from all three hESC lines, formed multiple lineage colonies in methylcellulose culture. These CD34+ cells can be maintained on OP9- stroma transduced with an adenovector expressing thrombopoietin (Tpo). We developed selective culture conditions for differentiation of CD34+ cells into different lineages. Isolated CD34+ cells from 10 to 12 Day EB’s differentiated in CD86+ dendritic cells in the presence of SCF, Tpo, Flt3 ligand, G-CSF, and TNF-alpha. Mature multinucleated CD41a+ megakaryocytes developed in cultures with SCF, Flt3L, and Tpo. Glycophorin A+ erythroid differentiation through to enucleated red cell stage was obtained with SCF, Epo, and IGF-1. B cell precursors (CD19+) developed in MS-5 stromal co-culture with SCF and G-SCF while CD44+CD25+ (early T progenitor marker) cells were detected in co-culture with OP9 transduced with the Notch ligand delta like-1 and IL-7. We have developed GFP and luciferase-expressing H1 and NT-hES-2 lines by lentiviral-vector-mediated gene transfer. These lines will allow us to monitor ES-derived cell localization and quantitation in immunodeficient mice using whole animal luciferase imaging systems. We are examining the potential for GFP-luciferase-transduced NT-hES-derived hematopoietic cells to engraft in NOD/SCID beta2−/ − mice. Our data demonstrate that NT-hESC lines as well as H1 lines are capable of differentiation to various lymphoid and hematopoietic lineages.
Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells
