Derivation and Characterization of Hematopoietic Cells From Human Embryonic Stem Cells

Lisheng Wang, Chantal Cerdan, Pablo Menendez, and Mickie Bhatia

Summary

In vitro, the aggregation of pluripotent human embryonic stem cells (hESC) into cell clusters termed embryoid bodies (EB) allows for the spontaneous differentiation of hESC into progeny representing endoderm, mesoderm, and ectoderm lineages. During human EB (hEB) differentiation, stochastic emergence of hematopoietic cells can be enhanced by a combination of hematopoietic cytokines and the ventral mesoderm inducer bone morpho-genetic protein (BMP)-4. Dependent on the presence of hematopoietic cytokines and BMP-4, vascular endothelial growth factor (VEGF-A165) selectively promotes erythropoietic development toward the primitive lineage. The effects of VEGF-A165 can be augmented by erythropoietin (EPO). Hematopoietic cells are derived from a rare subpopulation of hemogenic precursors during hEB development. These hemogenic precursors lack CD45, but express PECAM-1, Flk-1, and VE-cadherin (hereinafter CD45negPFV) and are solely responsible for hematopoietic cell fate. Human ESC-derived hematopoietic cells have similar colony and cellular morphologies to those derived from committed adult hematopoietic tissues, and also show repopulating capacity in immune deficient mice after intrabone marrow transplantation. In this chapter, we describe methods that have been successfully applied in our laboratory, including (1) generation of hematopoietic cells by EB formation; (2) augmentation of hematopoiesis by use of hematopoietic cytokines and BMP-4; (3) promotion of erythropoietic development by addition of VEGF-A165 and EPO; (4) isolation of CD45negPFV hemogenic precursors and generation of hematopoietic cells from these precursors; and (5) characterization of hESC-derived hematopoietic cells in vitro and in vivo.

Key Words: hESC; hematopoiesis; BMP-4; VEGF; precursor; transplantation; NOD/ SCID mice.

1. Introduction

Human embryonic stem cells (hESC) are pluripotent cells and can grow infinitively in feeder-free media (1). In vitro, the aggregation of hESC into cell

From: Methods in Molecular Biology, vol. 331: Human Embryonic Stem Cell Protocols Edited by: K. Turksen © Humana Press Inc., Totowa, NJ

clusters termed embryoid bodies (EB) allows for the spontaneous differentiation of hESC into progeny representing endoderm, mesoderm, and ectoderm lineages (2,3). During human EB (hEB) differentiation, stochastic emergence of hematopoietic cells can be enhanced by a combination of hematopoietic cytokines and the ventral mesoderm inducer bone morphogenetic protein (BMP)-4 (4,5). Functional CD45+ hematopoietic cells capable of in vitro colony forming activity emerge after 10 d of hEB development and the role of hematopoietic cytokines is restricted during these first 10 d (5). Under treatment with hematopoietic cytokines and BMP-4, up to 90% of cells dissociated from 22 d EB express pan-leukocyte marker CD45 (see Fig. 1) (5). In addition, dependent on the presence of hematopoietic cytokines and BMP-4, vascular endothelial growth factor (VEGF-A165) selectively promotes erythropoietic development toward the primitive lineage. The effects of VEGF-A165 can be augmented by erythropoietin (EPO) (6), suggesting that hematopoietic differentiation from hESC can be influenced.

Recently, we have demonstrated that hematopoietic cells are derived from a rare subpopulation of hemogenic precursors during hEB development (7). These hemogenic precursors lack CD45, but express PECAM-1, Flk-1, and VE-cadherin (hereinafter CD45negPFV) and are solely responsible for hematopoietic cell fate (7). CD45negPFV precursors can be fractioned from d 10 hEB development by a FACSVantage (see Fig. 2). Up to 98.5% of CD45+ cells are generated from CD45negPFV precursors, but not the remaining d 10 hEB cells after culture in a hematopoietic conducive condition for 7 d, with approx 8% of these CD45neg cells coexpressing CD34 (see Fig. 3) (7).

hESC-derived hematopoietic cells show similar colony and cellular morphologies to those derived from committed adult hematopoietic tissues (5-7). They possess characteristic progenitors of all myeloid lineages, including macrophage, granulocyte, erythroid, and multipotent hematopoietic progenitors containing granulocytic, erythroid, macrophage, and megakaryocytic lineages. Further, similar to adult peripheral blood, cord blood, and bone marrow, hESC-derived hematopoietic progenitor capacity is enriched in the CD34+ subfraction, suggesting that, in addition to producing mature hematopoietic cells, these precursors possess the ability to sustain production of primitive blood cells with appropriate function and phenotype (7).

Despite these in vitro phenotypic and functional assays, clinical promise of hESC-derived hematopoietic cells can only be functionally defined by sustained multilineage in vivo reconstitution on transplantation. Experimentally, the non-obese diabetic-severe combined immunodeficient (NOD/SCID) xenotransplant assay has provided a powerful tool to functionally define candidate human hematopoietic stem cells, defined as SCID-repopulating cells (8). However, recent evidence from our laboratory indicates that intra venous transplantation of hESC-derived hematopoietic cells causes mortality in recipient immune deficient mice because of emboli formed from rapid cellular aggregation in response to mouse serum (9). We suggest that intrabone marrow transplantation (IBMT) that bypasses the recipient mouse circulation may be necessary to observe hematopoietic engraftment from hESC-derived hematopoietic cells (9).

In this chapter, we will describe methods that have been successfully applied in our laboratory, including: (1) generation of hematopoietic cells by EB formation; (2) augmentation of hematopoiesis by use of hematopoietic cytokines and BMP-4; (3) promotion of erythropoietic development by addition of VEGF-A165; (4) isolation of CD45negPFV hemogenic precursors and generation of hematopoietic cells from these precursors; and (5) characterization of hESC-derived hematopoietic cells in vitro and in vivo.

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