As universal precursors for any cell type in the human body, human induced pluripotent stem cells (iPSCs) hold great promise for cell replacement therapies and regenerative medicine. Similar to embryonic stem (ES) cells, but avoiding ethical controversy and regulatory burdens, iPSCs can be expanded indefinitely and induced to differentiate into multiple cell types. With genetic manipulation to ensure immune compatibility, iPSCs can in principle serve as an unlimited source for “off-the-shelf” therapies (Lanza et al., 2019). Among the multitude of cell types that could be used to treat human disease, iPSC-derived blood cells are arguably the most amenable to clinical use, as human hematopoietic stem and progenitor cells (HSPCs), red blood cells, and platelets have been used in all corners of the globe to treat blood malignancies, genetic disorders, and cytopenias for decades with great success (Appelbaum, 2007). Bone marrow, red cell and platelet procurement entail a cumbersome and expensive blood donation system that critically depends on donor altruism and availability, and can be disrupted by unpredictability, supply chain inefficiency, and shortages and is subject to pathogen contamination (viral, parasitic, and bacterial). Large-scale manufacture of HSPCs, red blood cells, platelets, lymphoid and myeloid lineages from iPSCs provides an appealing alternative source for bone marrow transplantation and various hematopoietic therapies, and consequently has great therapeutic importance. Efficient in vitro differentiation of iPSCs into HSPCs and other blood lineages relies on lessons learned from developmental studies of embryonic hematopoiesis. In mammals, blood cells are produced in “waves” in temporally and anatomically distinct sites that support the emergence, maintenance, or proliferation of HSPCs. The first wave of hematopoiesis arises in the mammalian yolk sac, an extra-embryonic tissue that produces so-called “primitive” erythroid cells (Palis et al., 1999; Whitelaw et al., 1990), macrophages (Takahashi et al., 1989), and megakaryocytes (Tober et al., 2007). The multipotent erythromyeloid progenitors (EMPs) and HSCs capable of supporting lifelong production of all mature blood lineages emerge later during the “definitive” wave of hematopoiesis in the aorta-gonad-mesonephros (AGM) region of embryonic mesoderm (Medvinsky and Dzierzak, 1996). It has been shown in multiple vertebrate animal models, from zebrafish to human, that definitive hematopoiesis happens through a highly conserved trans-differentiation process known as endothelial-to-hematopoietic transition (EHT), during which a subset of endothelial cells with hemogenic (blood-forming) potential differentiate and egress from the ventral wall of the aorta to enter the circulation as multipotential EMPs as well as a cohort of bone fide HSCs (Jaffredo et al., 1998; Kissa and Herbomel, 2010; Zovein et al., 2008). The HSCs generated from EHT later colonize fetal liver to further mature and expand significantly before they seed the bone marrow to maintain life-long adult hematopoiesis (Houssaint, 1981; Kieusseian et al., 2012).
A detailed roadmap of the underlying mechanisms of embryonic blood ontogeny and the emergence of definitive hematopoiesis has been summarized in depth in several recent reviews (Dzierzak and Bigas, 2018; Liggett and Sankaran, 2020; Sugden and North, 2021). Employing principles of morphogen-induced mesodermal patterning and fate-sustaining cytokines, multiple groups have established protocols that direct the differentiation of human iPSCs into HSPCs in vitro that can give rise to a variety of more terminally differentiated blood lineages. In the first such study, stromal cell lines derived from mouse hematopoietic tissues were used to support the differentiation of human ES cells into progenitor cells that formed hematopoietic colonies (Kaufman et al., 2001). Subsequently, culture systems employing the spontaneous differentiation of ES cells into embryoid bodies (EBs) enabled stromal-free differentiation of ES cells into HSPCs (Chadwick et al., 2003; Zambidis et al., 2005). Most early in vitro differentiation protocols recapitulated the primitive wave of hematopoiesis, though subsequent methods enabled the generation of CD34+ cells with lymphoid potential (Kyba et al., 2002; Vodyanik et al., 2005; Wang et al., 2005). More recent studies have used T cell differentiation as an indication of definitive multilineage potential, and have established that activation of Wnt/β-catenin signaling combined with inhibition of Activin/Nodal signaling can pattern mesoderm towards definitive hematopoiesis (Kennedy et al., 2012; Sturgeon et al., 2014). Small molecule-based manipulation of these signaling pathways during the EB stage of differentiation enables the generation of definitive hemogenic endothelium (HE; defined as CD34+ KDR+ CD184- CD73-) from human pluripotent stem cells (PSCs). In the presence of a cocktail of cytokines and chemicals that promote hematopoiesis, these HE cells can go through a Notch-dependent EHT-like conversion to form CD34+ CD45+ HSPCs that are capable of giving rise to erythroid, myeloid, and lymphoid cells (Ditadi et al., 2015). Such a strategy has also been applied to human iPS cells (Kennedy et al., 2012). Chemical inhibition of ActivinA/TGFβ pathway and aryl hydrocarbon receptor (AHR) facilitates the generation of iPSC-derived HSPCs that produce erythroid cells expressing adult globin proteins (Leung et al., 2018). Despite considerable effort, to date no group has succeeded in deriving HSCs with long-term, self-renewing, multi-lineage hematopoietic reconstitution potential in lethally-irradiated murine hosts, considered the cardinal definition of the HSC. However, two groups including our own have leveraged transcription-factor driven conversion of embryoid-body derived hematopoietic progenitors into engrafting cells with durable multi-lineage differentiation potential, albeit with low efficiency (Sugimura et al., 2017; Tan et al., 2018). These studies produce iPSC-derived HSPCs that are capable of multilineage engraftment of primary and secondary mouse recipients, and establish that PSCs indeed hold promise as a source for HSPC transplantation.26 Nevertheless, such a system requires the expression of transgenes, which is not optimal for clinical translation. To date, in vitro protocols appear to lack key developmental and microenvironmental cues to promote proper differentiation of engraftable HSPCs. Key deficiencies are likely to include exposure to the proper dose and duration of retinoids, which are well known to pattern developmental processes (Luff et al., 2022), as well as biomechanical forces, as it is has been established that longitudinal shear and circumferential stress forces trigger the emergence of HSPCs from the HE of the aorta (Adamo et al., 2009; Diaz et al., 2015; Kim et al., 2015; Lundin et al., 2020; North et al., 2009).
Although the generation of bona fide HSC from iPSCs without genetic manipulation has not yet been achieved, considerably more success has been had by several groups in producing definitive HSPCs with the capacity to differentiate further into cells of the lymphoid lineage (Demirci et al., 2020; Kennedy et al., 2012; Park et al., 2018; Sturgeon et al., 2014). Given that chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy against several types of blood cancer (June et al., 2018), there is growing interest in the prospect that iPSC-derived lymphoid cells could become a more facile and readily available source for adoptive immunotherapies. Current CAR-T therapies utilize patient-derived T cells that are collected via apheresis, cultured in the lab and engineered to express CARs that can specifically recognize tumor antigens. After expansion, autologous CAR T cells can be infused back to the patient to target tumor cells, with a recent study showing that CAR T cells can persist and lead to decade-long leukemia remissions (Melenhorst et al., 2022). Despite success against CD19-positive lymphoid leukemia and lymphoma and BCMA-positive multiple myeloma, and much promise for targeting a wider array of solid tumors, adoptive immunotherapy is currently limited by its dependence on autologous T cells, which renders the manufacturing process cumbersome, time-consuming, and expensive (Fesnak et al., 2016). iPSC-derived T cells provide an appealing alternative source for the production of CAR T cells, which if “cloaked” to reduce immunogenicity may enable precisely manufactured off-the-shelf adoptive T cells therapies. Compared to other blood lineages, production of T cells from iPSCs has proven challenging because T cell maturation relies on signaling via the Notch receptor pathway normally provided by thymic epithelial cells (De Smedt et al., 2005). As a surrogate, engineered mouse stromal cells that express Notch ligands, such as OP9-DL1/DL4, have been included in co-culture with HSPCs to support in vitro T cell differentiation from various stem cell types (Schmitt and Zúñiga-Pflücker, 2002; Timmermans et al., 2009). Exploiting such an approach, iPSCs harboring tumor-specific T cell receptors (TCR) or expressing CARs have been used to generate tumor antigen-specific T cells or CAR T cells showing antitumor activities in vitro and in animal models (Themeli et al., 2013; Vizcardo et al., 2013). These studies provide proof-of-concept that iPSC-derived T cells have the potential for clinical applications such as cancer immunotherapy. However, in the first studies, molecular characterizations of the iPSC-derived CAR T cells revealed a transcriptional signature more akin to innate-like T cells that express γδ T Cell Receptors (TCR) rather than mature T Cells that express αβ TCR. Moreover, the first reported iPSC-CAR T cells predominantly expressed CD8αα homodimer, which does not engage major histocompatibility complex (MHC) as efficiently as the CD8 αβ co-receptors found on more mature αβ TCR-expressing T cells that circulate in our blood (Van Kaer et al., 2014). As a result of these innate-like features, iPSC-CAR T cells produced by these early methods were not as functionally robust as clinical grade CAR T cells derived from peripheral blood mononuclear cells (PBMC) (Themeli et al., 2013). More recent studies have explored new strategies to generate iPSC-T cells via three-dimensional organoid-like culture systems, and have shown enhanced efficiency in producing αβ TCR+ CD8+ T cells from human iPSCs (Montel-Hagen et al., 2019; Seet et al., 2017). Studies to date on these enhanced in vitro T cell culture systems have included only limited transcriptional analysis at the single cell level, thereby hindering comparison to PBMC-derived mature T cells. Moreover, the translational potential of some protocols remains limited by the usage of mouse stromal cells. To overcome these obstacles, the Daley lab has been using immobilized Notch ligands instead of stromal cells to support T cell differentiation from iPSCs. Similar stroma-free protocols have been used to generate T cells from iPSCs that are reprogrammed from T cells with pre-rearranged TCRs (Iriguchi et al., 2021; Shukla et al., 2017). We have further shown that in vitro stroma-free differentiation can recapitulate normal TCR rearrangement and yield T cells expressing a highly diverse TCR repertoire. Additionally, previous studies from the Daley lab have identified transcription factors and epigenetic modulators that act as important regulators for multilineage blood potential and lymphoid commitment (Doulatov et al., 2013; Vo et al., 2018). Leveraging this information, we have combined the stroma-free T cell differentiation protocol with epigenetic factor-mediated reprogramming to further facilitate T cell differentiation from iPSCs. As a result, we have generated iPSC-T cells that are more developmentally mature and functionally robust. These iPSC-derived T cells exhibit a molecular signature that resembles mature αβ TCR+ T cells from PBMC, and when engineered to express tumor-specific CARs, display enhanced efficacy against tumor cells in xenograft mouse models. Such a strategy is compatible with large-scale production of iPSC-derived T cells and is highly amendable to immune-cloaking techniques that would be needed to realize “off-the-shelf” adoptive T cell therapies.
In addition to T cells, immunotherapies using NK cells have likewise shown great promise in the treatment of both hematopoietic and solid tumors (Basar et al., 2020; Wrona et al., 2021). Unlike T cells, NK cell cytotoxicity is not restricted to specific MHC molecules but depends on a more versatile regulation of activating vs. inhibitory signals (Fauriat et al., 2010). As a result, NK cells have advantages in targeting MHC-downregulated tumor cells that can escape from T-mediated antitumor responses. Moreover, CAR NK cells are less likely to cause graft-versus-host disease (GVHD) and CAR T cell-associated toxicities such as cytokine release syndrome (Liu et al., 2020; Ruggeri et al., 2002). Multiple protocols have been developed to generate CD3-CD56+ NK cells from pluripotent stem cells for the production of CAR NK cells (Knorr et al., 2013; Woll et al., 2009; Zeng et al., 2017). Current studies on iPSC-derived NK cells are focusing on further engineering the NK cells to promote expansion capacity or enhance antitumor activities (Cichocki et al., 2020; Li et al., 2018; Zhu et al., 2020). We have shown that a set of epigenetic modulators acts during early lymphoid commitment to regulate the NK cell vs. T cell fate decision. Using various genetic editing tools, we are able to temporally manipulate the expression of these epigenetic regulators and fine-tune the in vitro differentiation of iPSCs to produce mature NK cells. Compared to control iPSC-derived NK cells, these epigenetically reprogrammed NK cells exhibit elevated surface expression of CD16 and other activation receptors that are essential for the NK cell-mediated killing response. Whether such developmentally mature NK cells show enhanced anti-tumor activity remains to be proven.
In summary (Figure 1), iPSCs represent a compelling source for the generation of hematopoietic cells for applications in research and clinical medicine. Advances in genetic editing of iPSCs holds additional value for producing cell products with defined antigenic and receptor profiles (e.g., universal donor O-Red Blood Cells, PLA1-negative platelets, and TCR-deficient T cells), while advances in bioprocess engineering are required to achieve efficient and cost-effective cell manufacture at clinical scale for truly off-the-shelf therapeutics. Clinical trials have already commenced for iPSC-derived platelets (Nakamura et al., 2021) and NK cells (Cichocki et al., 2020) with the expectation that T cells, red blood cells, and HSCs anticipated to follow, ushering in a future where cells become living medicines.
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