Characterization of Hematopoietic Progenitor Cells Derived from Human Embryonic Stem Cells That Differentiate into Natural Killer Cells Capable of In Vivo Anti-Tumor Activity.

Derivation of multiple blood cell lineages from human embryonic stem cells (hESCs) clearly establishes these cells as an important model system to better characterize human hematopoietic ontogeny. Previously we have demonstrated development of myeloid, erythroid and lymphoid cells, as well as endothelial cells, from hESCs. However, the progenitor cells that give rise to these more mature cells remain poorly characterized. Here we use combined phenotypic and genetic analysis to discriminate between distinct progenitor cell populations. We also demonstrate that natural killer (NK) cells derived from these hESC-derived progenitors are capable of killing diverse tumors both in vitro and in vivo. By detailed flow cytometric analysis of differentiated hESCs over a defined time course, we identify development of two waves of CD34⁺ cells. The first wave consists of CD34^brightCD31⁺Flk1⁺ cells, whereas the second wave consists of CD34^dimCD45⁺ cells. Novel combined morphologic and phenotypic analysis by image scanning flow cytometry demonstrates these phenotypically different cell populations also are morphologically distinct. CD34^brightCD31⁺ cells derived from hESCs appear to have a heterogeneous cell morphology, with an irregular cell shape, higher mean cell size and complex cytoplasmic organization. In contrast, CD34^dimCD45⁺ cells are a more homogeneous cell population with a uniform spherical morphology and smaller cell size. Purified CD34^brightCD31⁺Flk1⁺ cells express transcription factors associated with both the hematopoietic and endothelial lineages and can differentiate into both these lineages in vitro. In contrast, CD34^dimCD45⁺ cells display a transcription profile suggestive of hematopoietic commitment and are significantly enriched for hematopoietic progenitors. Taken together, these results suggest that the CD34^brightCD31⁺Flk1⁺ and CD34^dimCD45⁺ cell populations are distinct cell lineages that represent early hemato-endothelial and hematopoietic precursors, respectively. Next, we better characterized the lymphoid developmental potential of the CD34^dimCD45⁺ cells by the ability of these cells to differentiate into NK cells. Using a two-step differentiation process we find that hESC-derived NK cells express the wide repertoire of activating and inhibitory receptors similar to NK cells derived from other sources. We now demonstrate that these hESC-derived NK cells acquire cytolytic activity against breast cancer and glioma cell lines, as well as leukemia and lymphoma cells, in vitro. Finally, we have begun to test the in vivo efficacy of hESC-derived NK cells against established tumors. Here, luciferase (luc)-expressing K562 cells are inoculated into NOD/SCID mice, and several days later 2x10⁶ NK cells derived from hESCs or from umbilical cord blood (UCB) cells are injected iv. The luc⁺ K562 cells allows serial bioluminescent imaging to follow growth of the tumor cells non-invasively over a prolonged time course. These studies demonstrate a beneficial effect of hESC-derived NK cells on tumor growth and metastasis, comparable to the effect of UCB-derived NK cells. Taken together, these studies characterize the earliest hematopoietic progenitors as they develop from hESCs during in vitro differentiation, providing a starting point to evaluate the effect of endogenous and exogenous factors on differentiation of distinct human hematopoietic lineages. This will translate into even more efficient derivation of NK cells from hESCs with a potential use for cancer immunotherapy.