Stem cells-derived natural killer cells for cancer immunotherapy: current protocols, feasibility, and benefits of ex vivo generated natural killer cells in treatment of advanced solid tumors

Abstract

Nowadays, natural killer (NK) cell-based immunotherapy provides a practical therapeutic strategy for patients with advanced solid tumors (STs). This approach is adaptively conducted by the autologous and identical NK cells after in vitro expansion and overnight activation. However, the NK cell-based cancer immunotherapy has been faced with some fundamental and technical limitations. Moreover, the desirable outcomes of the NK cell therapy may not be achieved due to the complex tumor microenvironment by inhibition of intra-tumoral polarization and cytotoxicity of implanted NK cells. Currently, stem cells (SCs) technology provides a powerful opportunity to generate more effective and universal sources of the NK cells. Till now, several strategies have been developed to differentiate types of the pluripotent and adult SCs into the mature NK cells, with both feeder layer-dependent and/or feeder laye-free strategies. Higher cytokine production and intra-tumoral polarization capabilities as well as stronger anti-tumor properties are the main features of these SCs-derived NK cells. The present review article focuses on the principal barriers through the conventional NK cell immunotherapies for patients with advanced STs. It also provides a comprehensive resource of protocols regarding the generation of SCs-derived NK cells in an ex vivo condition.

Keywords: Advanced solid tumors; Cancer; Immunotherapy; NK cell therapy; Stem cells technology.

Fig. 1

A schematic representation of the NK cells development and division in human BM and thymus. Briefly, in the BM stroma, human CD34⁺/CD135⁺ HSCs differentiate into CD34⁺/CD44⁺ CLPCs under the stimulation of SCF, IL-7, and IL-17. Inside the BM, CLPCs directly generate CD34⁺/IL2/15Rβ⁺/15Rα⁺ NK cells precursors. CD19⁺ B cells are also generated indirectly from the CLPCs in the BM. Unlikely, inside the thymus, the CLPCs transition to CD34⁺/IL2/15Rβ⁺/15Rα⁺ NK cells precursors is indirectly occurred through differentiation of the CD34⁺/CD44⁺ CLPCs to CD2⁺/CD25⁺ pro-T cells. Besides the generation of NK cells precursors, all of the CD3⁺ T cell lineages are produced within the differentiation of CD2⁺/CD25⁺ pro-T cells in the thymus. Finally, under the stimulation with IL-2 and IL-17, the mature CD16⁺/CD56⁺/CD3⁻ NK cells are generated through CD34⁺/IL2/15Rβ⁺/15Rα⁺ NK cells precursors differentiation in both BM stroma and thymus. Below each cell, lineage-specific markers are shown. For more details, please refer to the text

Fig. 2

A schematic representation of the standard protocols for the NK cells ex vivo generation. Human PSCs including ESCs and iPSCs as well as HSCs are presented as the main SCs sources for NK cells production. As the first step, blastocyst-isolated ESCs and also somatic cells-generated iPSCs are differentiated into CD34⁺ HSCs through three different methods: feeder-free, feeder dependent, and also spin EB. In a feeder-free method, HSCs are generated only through stimulation of the expanded PSCs with SCF, VEGF, and BMP-4 after 11 days. Without the cytokines application, HSCs are differentiated by the co-culture of the PSCs with murine-irradiated M210-B4, O19, S17, and AFT024 stroma cell lines in a 17–20 period of days. In spin EB method, through the application of low-density mouse embryonic fibroblasts (MEFs) and under the SCF, VEGF, and BMP-4 stimulation, human ESCs- and iPSCs-derived spin EBs are differentiated into the HSCs after 10–20 passages. In the next step, the functional NK cells are generated from both the PSCs-derived HSCs and PB-, BM-, and UCB-isolated HSCs through one of the feeder-free and/or feeder-dependent conditions. In a feeder-free system, under the stimulation with various cytokines and growth factors including SCF, IL-3, IL-6, IL-12, IL-15, IL-21, Flt-3L-GM-CSF, TPO, and HC, the human HSCs are differentiated into the NK cells during a 21–30 period of days. In addition to the cytokines and growth factors, mature NK cells are generated through co-culture of human HSCs with murine-irradiated M210-B4, Sro-11, and AFT024 stroma cell lines in 11–35 days. The generated NK cells can be isolated under the exposure of CD3, CD16, and CD56 antibodies