These studies demonstrate the successful derivation of NK cells from iPSC-derived hematopoietic progenitors using a culture system previously demonstrated to be suitable to drive NK cell differentiation from hESC-derived hematopoietic progenitor cells and UCB CD34+
). To date, this provides the most functional and definitive evidence of human iPSC-derived blood cells. Indeed, we have now generated NK cells with similar activities from several other iPSC lines. The phenotypic profiles of hESC-NK and iPSC-NK cells are similar to those of their PB-NK cell counterparts. Also, both hESC-NK and iPSC-NK cells have a tumor-killing capacity comparable to that of PB-NK cells, indicating that both hESC-NK and iPSC-NK cells are fully functional (Fig. ). Interestingly, the UCB-derived NK cells are a mixture of phenotypically mature and immature populations. UCB-derived NK cells typically express lower levels of KIRs and CD94 than either pluripotent stem cell-derived NK cells or peripheral blood NK cells (50
). Both in vivo
and in vitro
models of NK cell development support a direct interaction with stromal cells, which are required for the acquisition of receptors, including KIRs (reviewed in reference 52
). Since the AFT024 stromal cells used in this study are murine derived and do not express human class I major histocompatibility complex (MHC) proteins, the low levels of KIRs on the in vitro
-derived NK cells fit a model where the interaction between KIR receptors and MHC class I proteins help regulate KIR acquisition during NK cell maturation. However, hESC- and iPSC-derived NK cells routinely have higher levels of KIR expression than do UCB-derived NK cells. This may be due to other signaling pathways activated in the hESCs/iPSCs during hematopoietic development under the initial M2-10B4 coculture conditions that were used to support the differentiation of hESCs and iPSCs into hematopoietic progenitor cells. For example, we have previously demonstrated that these conditions result in the upregulation of some lineage-specific transcription factors, including ID proteins that are known to promote NK cell development (33
). Therefore, these studies suggest that NK cell development from hESC- or iPSC-derived hematopoietic progenitors does not depend on stromal cell interactions in the same fashion as that of UCB progenitors.
NK cells have been associated with the control of HIV-1 infection both in vivo
and in vitro
). In this study, our results also clearly illustrate that hESC-NK and iPSC-NK cells exercise their anti-HIV-1 activity potentially through at least three distinct cellular mechanisms: direct lysis, ADCC, and the production of soluble mediators (β-chemokines and IFN-γ). Interestingly, we noticed that a higher percentage of hESC-NK and iPSC-NK cells showed degranulation (assessed by CD107a expression) than PB-NK and UCB-NK cells upon activation by HIV-1-infected CD4+
T cells, whereas PB-NK cells produced more CCL4 and IFN-γ than did hESC-NK and/or iPSC-NK cells. It is possible that these cellular mechanisms may be utilized differently by NK cells derived from human pluripotent stem cells (both hESCs and iPSCs) compared to NK cells isolated from peripheral blood. Several studies have shown that NK cell receptors, such as KIR3DS1, NKG2D, and NKp44, are associated with anti-HIV-1 activity (2
). Indeed, we demonstrate that all these receptors are expressed on hESC/iPSC-derived NK cells (Fig. ). Additionally, NK cells kill targets through the recognition of the receptors of FasL and TRAIL (4
). We find that both FasL and TRAIL are robustly expressed on the surface of hESC-NK and iPSC-NK cells (Fig. ), suggesting that these two (and possibly other) members of the TNFR (tumor necrosis factor receptor) gene superfamily of death receptors may also be involved in the NK cell-mediated killing of HIV-1-infected targets.
Overall, these results have at least two key implications. First, both hESCs and iPSCs provide an excellent model to study human lymphocyte development and function utilizing a homogeneous cell population that can be cultured and genetically modified under well-controlled conditions. Second, human pluripotent stem cells (hESCs and iPSCs) can serve as a novel “universal” source for a cellular immunotherapeutic approach for the treatment of HIV/AIDS and malignancies. Indeed, iPSC technology would potentially enable patient-specific anti-HIV-1 cellular treatment. It should also be possible to engineer hESCs and iPSCs to express anti-HIV-1 receptors, as was shown previously to be effective with anti-HIV cytotoxicity for CD8+
T cells isolated from peripheral blood or derived from hematopoietic stem cells (HSCs) (30
Notably, some recent studies have suggested that iPSCs are significantly more difficult to differentiate into specific cell lineages and therefore are potentially less useful for therapeutic applications than hESCs (16
). While our studies (and those of others) do find variation in the efficiency for hematopoietic differentiation between different iPSC lines (12
), we also demonstrate that once we obtain differentiated hematopoietic progenitor (CD34+
) cells from iPSCs, these populations do have similar potentials for NK cell development.
Clearly, we are entering into a new era of stem cell-based therapies to treat and cure a wider range of diseases (28
). Based on these studies, HIV/AIDS can be added to the list of conditions to be treated and potentially cured by hESC- and iPSC-derived cells. CCR5-deficient HSCs could also potentially be derived from hESCs/iPSCs (for example, creating iPSCs from a CCR5-deficient individual). However, to date, despite intense efforts by multiple groups, HSCs capable of long-term multilineage hematopoietic engraftment have not been efficiently derived from hESCs or iPSCs (31
). Therefore, at this time, a clinical scenario utilizing CCR5-deficient hESC/iPSC-derived HSCs as a starting cell population to transplant and potentially cure HIV/AIDS (24
) is not yet feasible. As described here, the use of hESC/iPSC-derived NK cells as a source of novel anti-HIV and anticancer (50
) immunotherapy currently provides a safer and more viable approach. This strategy of anti-HIV immunotherapy could be applied to successfully treat malignancies, as has been done previously (35
). Here, there are two possible scenarios. For patients who are refractory to standard highly active antiretroviral therapy (HAART), this anti-HIV immunotherapy could be used to better manage or possibly eliminate the viral burden. Alternatively, patients controlling viral load on HAART could receive adoptively transferred hESC/iPSC-derived NK cells to eliminate remaining disease reservoirs (22
). Additionally, as hESCs and iPSCs can be routinely genetically modified, it will also be possible to test the antiviral activities of hESC-NK/iPSC-NK cells modified to express receptors that specifically recognize HIV-1 targets, such as what has been defined for certain T-cell receptors (TCRs) (30
). In this manner, hESC/iPSC-derived NK cells can potentially serve as a universal cell source for targeted anti-HIV immunotherapies rather than having to modify immune effector cells on a patient-specific basis. While the translation of these approaches to clinical therapies still has significant hurdles to overcome (28
), clearly, the use of human pluripotent-derived NK cells opens up promising new approaches to treat lethal malignancies and infectious diseases.