Characterization of Developmental Pathway of Natural Killer Cells from Embryonic Stem Cells In Vitro

Abstract

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34+ EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34+ EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34+ EB cells, NK and T cell potentials were detected in a CD45− subset, whereas CD45+ EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34+CD45− EB cells showed that CD45+Mac-1−Ter119− ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45−CD34+ EB cells have lymphoid potential, and they differentiate into more mature CD45+Lin− hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122− and they rapidly acquire CD122 as they differentiate along the NK lineage.

Generation of “Off-the-Shelf” Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells

“…This cell-sorting step not only complicates the protocol, but also reduces yield of NK cells because it excludes many other hematopoietic progenitors that are also competent for differentiation into NK cells. Another approach is to form EBs ( Tabatabaei-Zavareh et al., 2007 ) and recently spin EBs ( Knorr et al., 2013 ). Spin EB formation requires a prior time-consuming adaptation of hPSCs to TrypLE digestion for at least ten passages on mouse embryonic fibroblasts, which may not be easy for many hPSC lines.…”

A Dual Role for Talin in NK Cell Cytotoxicity: Activation of LFA-1-Mediated Cell Adhesion and Polarization of NK Cells

“…Since homozygous Tln1 KO in mice results in embryonic lethality (36), we generated NK cells from Tln1 Ϫ/Ϫ ES cells (clone A28) and the corresponding WT ES cells (clone HM1) by in vitro differentiation cultures as previously described (32,33). As reported previously (28), the two ES cell lines exhibited different morphologies.…”

Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells

“…Although lymphoid progenitors have not been identified in this early phase of ESC-derived hematopoiesis, they have been detected in later-stage stromal-based cultures, suggesting that a P-Sp-like program is established under these conditions (Cho et al, 2001;Cho et al, 1999;Nakano et al, 1994;Schmitt and Zuniga-Pflucker, 2002;Tabatabaei-Zavareh et al, 2007;Yamane et al, 2009). The notion that P-Sp hematopoiesis can be generated from ESCs is further supported by observations that cells with properties of HSCs can be generated in ESC differentiation cultures following the enforced expression of either Hoxb4 or the combination of Cdx4 and Hoxb4 (Kyba et al, 2002;Wang et al, 2005b).…”