Human natural killer (NK) cell-based adoptive anticancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if needed. These characteristics make hESC-/iPSC-derived NK cells an ideal cell population for developing standardized, "off-the-shelf" immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously has established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin (IL)-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single-cell adaptation of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESCs/hiPSCs are directly used to form embryoid body (EB) to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.

Adoptive immunotherapy using immune effector cells has revolutionized cancer treatments with approval of two autologous chimeric antigen receptor (CAR) T cell therapies by the US FDA. Clinical trials using natural killer (NK) cell-based adoptive immunotherapy have been shown to be safe and effective for treatment of multiple malignancies, especially acute myelogenous leukemia. However, most of these trails use primary NK cells isolated from peripheral or cord blood which can have donor-dependent variability and can be challenging to genetic engineer to improve antitumor functions, limiting the widespread use of this promising new therapy. NK cells can now be routinely produced from human pluripotent stem cells, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These pluripotent stem cells are homogenous, easy to genetically modify on a clonal level and can be used as unlimited source of NK cells, making them ideal population to develop standardized, off-the-shelf adoptive NK cell therapy products. In this review, we discuss recent advances of obtaining and expanding hESC and iPSC-derived NK cells and novel genetic engineering strategies that are being applied to improve their antitumor functions.

Purpose of review

We summarize current advances to define the role the aryl hydrocarbon receptor (AHR) plays in mammalian hematopoiesis. We emphasize approaches to modulate AHR throughout human hematopoietic development in vitro to support the production of clinically relevant blood products suitable for patient care.

Recent findings

Initial data demonstrate that both pharmacologic AHR inhibition and genetic deletion from human pluripotent stem cells provide useful strategies to enhance the yield of hematopoietic stem and progenitor cells. AHR hyperactivation following the induction of CD34 megakaryocyte-erythroid progenitors skews developed toward erythroid lineages, whereas AHR inhibition supports platelet production. At the level of lymphoid specification, AHR inhibition enhances the proliferation and differentiation of functional human natural killer cells, whereas hyperactivation leads to production of Group 3 innate lymphoid cells and provides a novel platform for studying human innate lymphoid cell development.

Summary

Modulation of AHR in human hematopoietic cells in vitro is a promising tool to mediate development of terminal hematopoietic cell populations with significant clinical implications to generate cells suitable for antitumor immunotherapy and bone marrow transplantation.

Abstract Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs). Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb. Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p <0.005 (Figure). Survival studies are still ongoing. Together, these studies demonstrate that iPSCs can be used to routinely and efficiently derive macrophages with potent anti-tumor activity. Additionally, CARs that are optimized for macrophage-mediated activity can be expressed to generate iPSC-CarMacs that effectively kill tumor cells in vitro and in vivo. These iPSC-CarMacs provide another approach to provide a standardized, targeted, off-the-shelf cell therapy product that can be used to treat both hematological malignancies as well as diverse solid tumors. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

Mouse and human pluripotent stem cells have been widely used to study the development of the hematopoietic and immune systems. Although not all cells can be derived with the same efficiency, immune cells such as natural killer (NK) cells and macrophages can be easily produced from PSCs to enable development of new cell-based therapies. NK cells and macrophages are part of the innate immune system, the first line of defense against malignancies and infectious disease. Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived NK cells can be produced at a clinical scale suitable for translation into clinical trials. Additionally, PSCs can be genetically modified to produce hESC/iPSC-derived human NK cells with enhanced antitumor activity. These engineered NK cells can express a stabilized version of the high-affinity Fc receptor CD16, chimeric antigen receptors, or other strategies to enable more potent and targeted cellular immunotherapies. Moreover, macrophages can also be routinely and efficiently produced from hESCs and iPSCs as a tool to expand our knowledge of the basic biology of these cells. hESC- and iPSC-derived macrophages can also be employed as a novel approach for cancer immunotherapy, as well as a strategy to repair or regenerate diseased and damaged tissues and organs.

Mouse and human pluripotent stem cells have been widely used to study the development of the hematopoietic and immune systems. Although not all cells can be derived with the same efficiency, immune cells such as natural killer (NK) cells and macrophages can be easily produced from PSCs to enable development of new cell-based therapies. NK cells and macrophages are part of the innate immune system, the first line of defense against malignancies and infectious disease. Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived NK cells can be produced at a clinical scale suitable for translation into clinical trials. Additionally, PSCs can be genetically modified to produce hESC/iPSC-derived human NK cells with enhanced antitumor activity. These engineered NK cells can express a stabilized version of the high-affinity Fc receptor CD16, chimeric antigen receptors, or other strategies to enable more potent and targeted cellular immunotherapies. Moreover, macrophages can also be routinely and efficiently produced from hESCs and iPSCs as a tool to expand our knowledge of the basic biology of these cells. hESC- and iPSC-derived macrophages can also be employed as a novel approach for cancer immunotherapy, as well as a strategy to repair or regenerate diseased and damaged tissues and organs.

Purpose of review

In this review, we summarize the current status of clinical trials using therapeutic cells produced from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We also discuss combined cell and gene therapy via correction of defined mutations in human pluripotent stem cells and provide commentary on key obstacles facing widescale clinical adoption of pluripotent stem cell-based therapy.

Recent findings

Initial data suggest that hESC/hiPSC-derived cell products used for retinal repair and spinal cord injury are safe for human use. Early-stage studies for treatment of cardiac injury and diabetes are also in progress. However, there remain key concerns regarding the safety and efficacy of these cells that need to be addressed in additional well designed clinical trials. Advances using the clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system offer an improved tool for more rapid and on-target gene correction of genetic diseases. Combined gene and cell therapy using human pluripotent stem cells may provide an additional curative approach for disabling or lethal genetic and degenerative diseases wherein there are currently limited therapeutic opportunities.

Summary

Human pluripotent stem cells are emerging as a promising tool to produce cells and tissues suitable for regenerative therapy for a variety of genetic and degenerative diseases.

Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains.

Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs), large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34, respectively, from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGFβ-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542 ⁺ hESC-ECs, SB431542^- hESC-ECs, and HUVECs showed similar permeability to 10,000 Da dextran, but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542 ⁺ hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542^- hESC-ECs and HUVECs responded differently to VEGF and bFGF, which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542^- hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 678-687, 2016.