Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor T Cells: The Intersection of Stem Cells and Immunotherapy

Abstract

Chimeric antigen receptor (CAR) T cell therapy is a promising cell-based immunotherapy applicable to various cancers. High cost of production, immune rejection, heterogeneity of cell product, limited cell source, limited expandability, and relatively long production time have created the need to achieve a universal allogeneic CAR-T cell product for “off-the-shelf” application. Since the innovation of induced pluripotent stem cells (iPSCs) by Yamanaka et al., extensive efforts have been made to prepare an unlimited cell source for regenerative medicine, that is, immunotherapy. In the autologous grafting approach, iPSCs prepare the desired cell source for generating autologous CAR-T cells through more accessible and available sources. In addition, generating iPSC-derived CAR-T cells is a promising approach to achieving a suitable source for producing an allogeneic CAR-T cell product. In brief, the first step is reprogramming somatic cells (accessible from peripheral blood, skin, etc.) to iPSCs. In the next step, CAR expression and T cell lineage differentiation should be applied in different arrangements. In addition, in an allogeneic manner, human leukocyte antigen/T cell receptor (TCR) deficiency should be applied in iPSC colonies. The allogeneic iPSC-derived CAR-T cell experiments showed that simultaneous performance of HLA/TCR deficiency, CAR expression, and T cell lineage differentiation could bring the production to the highest efficacy in generating allogeneic iPSC-derived CAR-T cells.

Introduction

The first adoptive immune cell therapy was referred to in 1980 when autologous and allogeneic lymphocytes were infused in patients with metastatic melanoma and relapsed leukemia (Rosenberg et al., 1988). With the advent of gene transfer techniques, using immune cells for therapeutic targets was developed, and the first chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) were introduced (Kuwana et al., 1987). Over the past decade, CAR-T cell therapy has been considered an advanced therapeutic approach in the field of cancer, particularly hematological malignancies. CAR cells can recognize the tumor cells and hammer the immune tolerance to self-tumor antigens (Azad et al., 2015; Jamnani et al., 2012; Sharifzadeh et al., 2013; Yu et al., 2023), so they can eradicate the tumor cells in an independent manner of the major histocompatibility complex (MHC) (Sadelain et al., 2013).

In the recent decade, CAR-T cell products have achieved remarkable clinical success in some hematological malignancies, and six CAR-based drugs are approved by the Food Drug Administration, including tisagenlecleucel (Kymriah^®), lisocabtagene maraleucel (Breyanzi^®), axicabtagene ciloleucel (Yescarta^®), idecabtagene vicleucel (ABECMA^®), brexucabtagene autoleucel (Tecartus^®), and ciltacabtagene autoleucel (CARVYKTI^®), which are currently available in the market (Denlinger et al., 2022; Mazinani and Rahbarizadeh, 2022; Sterner and Sterner, 2021). Despite all advances regarding a clinical opportunity to struggle with cancer, there are some challenges, that is, high cost of production, achieving a homogeneous functional T cell population to enhance the efficiency, autologous grafting, hard-to-expanding the final cell product, hard gene manipulation, the limited count of origin cells to the generation of CAR-T cells due to the leukopenia in cancer patients, and others (Hajari Taheri et al., 2019; Zhang et al., 2022b).

To overcome these obstacles, the attention of many scientists has been attracted to other immune cells, including natural killer cells, macrophages, gamma delta (γδ) T, invariant natural killer (NK) T (iNKT), and mucosal-associated invariant T (MAIT), that can be used for generating CARs with better efficacy and minimal side effects. Stem cells are another candidate for CAR generation due to the golden ability of self-renewal and differentiation into multiple cell types (Hojjatipour et al., 2023b). Recent advances in stem cell engineering display promising stem cell-based therapies with higher efficacy and lower side effects to change the treatment landscape for many clinical conditions, importantly for cancers. However, the widespread clinical use of traditional stem cells such as umbilical cord blood stem cells and bone marrow stem cell reserves has faced some complications due to the lack of reliable sources and the difficulties of their in vivo reproduction.

Regarding the innovation by Yamanaka et al., which led to the generation of induced pluripotent stem cells (iPSCs), an unlimited source of origin cells aimed at regenerative medicine and cell therapy strategies was achieved (Aboul-Soud et al., 2021; Takahashi and Yamanaka, 2006). The embryonic state of iPSC allows scientists to regenerate all human/nonhuman cell lineage, such as T cells and natural killer cells. This attainable approach can provide a wide range of manipulated immune cells to tackle cancer. Using iPSC to regenerate Ag-specific T cells was first reported in 2013 (Nishimura et al., 2013; Themeli et al., 2013; Vizcardo et al., 2013). In this method, T cells are reprogrammed into the pluripotency stage (iPSCs) and then differentiated back into T cells with desired phenotypes.

Moreover, due to the longer life span and less density of chromatin of iPSC, the gene manipulation aimed at disruption of human leukocyte antigen and TCR is easier compared with mature T cells, the conventional source of CAR-T cells (Kaneko, 2022; Zhang and Zhang, 2021). Therefore, iPSCs provide a desired source to generate “off-the-shelf” CAR-T cells (Table 1). In this review, we discuss the potential of iPSCs-based immunotherapies as a therapeutic approach. The first part explains CAR-T cells' application and sources as well as its limitations; in the second part, iPSCs were overviewed, and in the following, iPSC-derived CAR-T cells and related subjects were surveyed.

Table 1.

Comparison of Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-T Cells and Conventional Chimeric Antigen Receptor-T Cells

	Conventional CAR-T cells	iPSC-derived CAR-T cells
Origin cells	Peripheral T cells/limited in leukopenia condition	Any cells/no limitations
Expandability	Limited	High rate
Gene reception status	Hard to transfection/transduction	Highly efficient transfection/transduction
HLA/TCR manipulation	Limited	Less restriction
Cost (in autologous grafts)	High	High
Cost (in allogeneic grafts)	High	Cost–benefit
Heterogeneity of resulted T cells	Often heterogeneous	Often homogeneous
Time to generation	≈2 Weeks	A few months (depending on the origin)
“Off-the-shelf” producibility	Low	High

CAR, chimeric antigen receptor; iPSC, induced pluripotent stem cell; TCR, T cell receptor.

CAR-T Cell Therapy: Structure, Generations, Cell Sources, and Limitations

CAR-T cells are engineered synthetic receptors with remarkable effects, which have revolutionized immunotherapy-based cancer treatment (June et al., 2018; Maali et al., 2023). Their Ag-specific receptors can recognize target Ags that are expressed on the malignant cell, independent of MHC expression, contrary to natural T cells. These binding triggers antitumor responses in high intensity to eliminate malignant cells. Another point is in in vivo conditions, tumor Ags are considered as self-Ags, and natural immune cells tolerate them; consequently, the immune system fails to restrict enigmatic tumors. Fortunately, this problem was debugged in synthetic CAR-T cells due to their unique design.

Each CAR consists of the following four main components (Sterner and Sterner, 2021): (1) an extracellular domain, (2) a hinge region, (3) a transmembrane domain, and (4) one or more intracellular domains. As their names suggest, the extracellular domain responsible for the Ag-recognizing function consists of an antibody's single-chain variable fragment (scFv), which brings proper affinity and specificity to CAR (Dwivedi et al., 2018; Rahbarizadeh et al., 2011). Hinge regions contrive between transmembrane and extracellular domains and confer more flexibility to Ag-binding domains to participate effectively in immunological synapse formation (Srivastava and Riddell, 2015).

The transmembrane domain acts as an anchor to join CAR to the T cell membrane, which through this can affect CAR-T cell function. Natural proteins derived from CD3ζ, CD4, CD8α, or CD28 take part in transmembrane (TM) domain formation. According to studies, CD8α or CD28 transmembrane domains can enhance the stability of CAR expression. The CD28 TM domain triggers TNF and IFNγ secretion, whereas the CD3ζ TM domain facilitates CAR dimerization (Bridgeman et al., 2010; Fujiwara et al., 2020; Muller et al., 2021).

The intracellular domain or signaling domain, which has taken up a large part of the CAR engineering research, commonly consists of two components: (1) proteins derived from immunoreceptors CD3ζ chain as a necessary component for signaling, T cell proliferation, and cytokine secretion, and (2) one or more co-stimulatory domains including CD28, CD137, CD134, ICOS34, CD27, MYD88 and CD40, and others, as a helper for activity, efficacy, and persistence of CAR-T cells (Weinkove et al., 2019). Notably, the main stimulatory domains have immunoreceptor tyrosine-based activation motifs (ITAMs), which are derived from TCRs and responsible for signal transduction (Guedan et al., 2018). Today, much research is being conducted on the effects of CAR co-stimulation to generate optimal CAR signaling domains.

According to this structure, five generations of CAR have been launched (Zhang et al., 2017); the simple first generation contains an scFv and an intracellular CD3ζ domain, without any co-stimulatory agents, in vivo proliferation, and cytokine response. In the second generation, one co-stimulatory domain such as CD137 or CD28 was added to CD3ζ, and unsurprisingly, the efficiency and stability of CARs were promoted in some clinical trials (van der Stegen et al., 2015). This process continued, and with the addition of multiple co-stimulatory domains, the third-generation CARs were produced that have more substantial cytokine production, antitumor ability, and T cell proliferation (Smith et al., 2016; Tomasik et al., 2022).

The fourth-generation CARs, which are also named T cell redirected for universal cytokine-mediated killing (TRUCK) or armored CAR, were modified to express cytokines such as IL-7, IL-12, IL-15, and IL-21, and suicide genes such as iCaspase-9. These changes result in running various functions related to each cytokine and producing more resistant CARs in the immunosuppressive tumor microenvironment (TME); for instance, an IL-12-secreted CAR-T cell was generated, which has multifunctional activity including enhancement of T cell viability, activation of immune cells, and elevation of safety (Chmielewski and Abken, 2015; Chmielewski et al., 2014; Tian et al., 2020). Interestingly, in the fifth generation, an extra intracellular domain was added to another intracellular domain. This doubling results in enhancing CAR signaling and stimulates the immune system (Fu et al., 2020; Tokarew et al., 2019). Next-generation CARs with higher efficacy and safety include tandem, combinatorial, ON-switch, inhibitory, universal, and T cells reprogrammed as global cytokine killing (TRUCK) CARs based on their function (Khawar and Sun, 2021).

To date, conventional Τ cells derived directly from cancer patients (autologous CARs) are the most common source for CAR production, and they have also shown promising results in clinical trials (Graham et al., 2018); but their high cost, labor-intensive manufacturing process, long times, poor quality, low efficacy, and accessibility hamper their popularization in widespread use (Sterner and Sterner, 2021); therefore, a scientist looks for other cell sources or other ways to diminish such issues (Mazinani and Rahbarizadeh, 2023). Many efforts have been made to utilize high-quality T cells from healthy donors to generate allogeneic CAR-T products named universal CAR-T cell (UCAR-T), which can be prescreened for the desired attributes to provide readily administrable “off-the-shelf” cell therapies (Lin et al., 2021).

Compared with autologous CARs from leukemic patients under heavy chemotherapies, allogeneic CAR-T products have better quality and efficacy and will be cost-effective, but they are not completely safe; transplant rejection, graft-versus-host disease (GVHD), and consequent acute toxicities, such as CRS (cytokine release syndrome) and immune effector cell-associated neurotoxicity syndrome (ICANS), remain their main problem (Siegler and Kenderian, 2020; Sterner and Sterner, 2021). However, studies showed that using memory T cells or knockout MHC-related genes (e.g., β2-microglobulin [β2M] and class II major histocompatibility complex transactivator [CIITA]), TCR (e.g., T cell receptor alpha constant [TRAC] and T cell receptor beta constant [TRBC]) and immune checkpoint gene (e.g., PDCD1, LAG3, CTLA4, and DGKa) by gene editing tools, and lymphodepletion chemotherapy before infusion can reduce GVHD and alloimmunization risk and improves immune cell antitumor efficacy (Dimitri et al., 2022; Ghaderi et al., 2022; Li et al., 2023).

Apart from auto- or allo-CARs, exclusive use of T cells associated with the development of exhaustion, tumor escaping by shedding desired Ags, limited persistency, and restricted penetrance into solid tumors microenvironment are other issues that hamper T cell's effective activities. As designing pens for astronauts, focusing on improving CAR-T cells may be the best solution; exploring alternative cellular sources for CAR generation may overcome some deficiencies of CAR-T cells. Fighting cancers in the body is performed by some other cells, including γδ T, iNKT, MAIT, macrophages, and NK cells (Bagheri et al., 2020; Hojjatipour et al., 2023b). Studies suggest that they can be considered a potential candidate for generating CARs with better efficacy and minimal side effects, but there are still some drawbacks (Mazinani and Rahbarizadeh, 2023).

NK cells offer several advantages over T cells, including quick response due to their innate immunity without former exposure, various strong defense mechanisms such as perforin/granzyme secretion or antibody-dependent cellular cytotoxicity, their ability to recognize self-missing HLA-I tumor cells, less toxicity, efficacy safety, persistency, and tolerability and low-cost manufacturing as well as easy transduction (Hojjatipour et al., 2023a; Pan et al., 2022; Zhang et al., 2022a). Although some evidence suggests that NK cell immunotherapy incorporates limited capacity in proliferation and activation, less durability, and a low proportion of NK cells in the blood, its gene transfection efficacy is not convincing (Daher et al., 2021; Karadimitris, 2020). Similar to NK cells, iNKT cells' characteristics, including their direct cytotoxicity, naturally traffic to the tumor site, and modulation of immune-suppressive cells with no risk of GVHD due to lack of MHC engagement, make them a favorable target for allogeneic immunotherapy; but low frequency and persistency hinder the application of these cells (Liu et al., 2022).

γδ T cells' characteristics include expanding in high numbers, having the ability to recognize a broad spectrum of tumor Ags, reducing GVHD and tumor escaping, strong antitumor responses, releasing effective cytokines, and having cytolytic and MHC-independent activity. Even their regulatory role in immune responses makes them an attractive source for adoptive cell immunotherapy, particularly in allogeneic settings. Exhaustion in some subtypes of γδ T cells is its drawback in related studies (Ferry and Anderson, 2022; Fisher et al., 2019; Rozenbaum et al., 2020).

MAIT cells are another unconventional source for CAR-based immunotherapy. Their advantages consist of abundancy in tissues and blood, migration into tissues, rapid response, cytotoxic activity, having intrinsic effector memory phenotype (CD45RA⁻CD45RO⁺CD62L^lowCD161⁺), and no risk of GVHD due to their MHC-unrestricted nature. CAR-MAIT hurdles include displaying exhausted phenotypes and limited ex vivo expansion (Bohineust et al., 2021; Lukasik et al., 2020). In addition, regulatory T cells (T-regs), which have a central role in maintaining immunological homeostasis, can also be used as a new source for generating CARs. Studies have shown that despite Treg's rarity in circulation, they can be largely isolated, manipulated, and expanded. Also, using specific CAR-Tregs accompanied by decreased GVHD reduced dependency on immunosuppressive drugs and persistence in the target tissue, as we expected. Although systemic immunosuppression induces CRS and ICANS, exhaustion and their high cost have limited their widespread use in clinics (Arjomandnejad et al., 2022; Ferreira et al., 2019; Zhang et al., 2018).

Moreover, CAR-edited macrophages have displayed promising potency in cancer therapy by overcoming some conventional CAR-T cells; for instance, macrophages can penetrate the TME and perform phagocytosis, releasing proinflammatory cytokines. In addition, they can act as antigen presenters and trigger adaptive responses. Such advantages make macrophages attractive platforms for CARs (Pan et al., 2022; Su et al., 2022). However, their limited cell number, lack of explanation, and gene transfer troubles are major challenges en route to their clinical application. Furthermore, their cytotoxicity is like a double-edged sword, and their dosage must be balanced (Chen et al., 2021). In addition, we must consider the anti-immune M2 macrophages in TME, which promote tumor growth and metastasis (Boutilier and Elsawa, 2021). Overall, TME is highly intricate, and further studies need to improve current understanding.

Using stem cell resources, including hematopoietic stem cells (HSCs) and pluripotent stem cells (PSCs), is another way to provide a sustained supply and to overcome mentioned problems related to auto- and allo-CARs (Gschweng et al., 2014). PSCs are more applicable than HSCs due to providing unlimited cell sources to generate desired immune cells. The noteworthy point here is that restoring somatic cells such as periphery blood mononuclear cells (PBMCs) into pluripotency and reprogramming into differentiated desired cells such as rejuvenated immune cells has come true. These immune cells-iPSCs can undergo further engineering with CARs for elevated antitumor efficiency (Deng et al., 2023; van der Stegen and Rivière, 2023).

An Overview of iPSCs

The first experiments on generating murine embryonic stem cells (ESC)-mimicking cells results by Yamanaka and colleagues in 2006 (Takahashi and Yamanaka, 2006). In their study, the murine fibroblasts were reprogrammed to ESC-like cells by retroviral transduction of four embryonic-associated transcription factors, including octamer-binding transcription factor 4 (Oct4), Kruppel-like zinc-finger domain transcription factor 4 (Klf4), SRY-related HMG-box2 (Sox2), and c-Myc. The generated iPSC colonies showed the pluripotency state and were able to form all three germ lines in in vitro conditions. Next year, in 2007, they generated human iPSCs (Takahashi et al., 2007). Till now, various reprogramming factors and agents have been used to reprogram different sources of cells to iPSC. OCT4, SOX2, KLF4, c-MYC, NANOG, estrogen-related receptor beta (ESRRB), and LIN28 are the most common reprogramming factors used in reprogramming studies (Maali et al., 2021).

In brief, OCT4 is enrolled in stem cell development, survival, and re-establishment to pluripotency. SOX2 acts through the maintenance of pluripotency behaviors of iPSC and the formation of trophoblast formation in ESC. KLF4 is enrolled in the expansion and maintenance of iPSC. c-MYC prepares flatter morphology, stemness maintenance, and chromatin remodeling. NANOG is a reprogramming facilitator, and ESSRB increases the pluripotency-associated genes' expression. Finally, LIN28, as a microRNA-processing protein, acts for embryogenesis and development of iPSC through splicing of stemness-associated microRNAs, that is, miR-let7. Also, it should be noted that the pluripotency network regulates the pluripotency of iPSC (Karagiannis et al., 2019; Maali et al., 2021).

Three major steps are needed to reprogram a somatic cell to iPSC. First, the cell source, for example, fibroblast and lymphocytes, should be obtained from a donor (Wang et al., 2021b). It should be noted that it is better to choose a cell source from the same cell lineage due to the partial similarity of the epigenetic pattern (Li and Darabi, 2022). For example, if iPSC-derived T cell is required for generation, it is better to use T cell progenitor/precursor, HSCs, or mesodermal stem cells. After selecting a suitable source, reprogramming is the next step. For this aim, reprogramming factors should be delivered to obtain somatic cells in ex vivo conditions. Reprogramming factors are developed in DNA-, RNA-, and protein-based platforms. RNA platform leads to transient expression of the desired gene (Ilieva and Uchida, 2022; Wang et al., 2021b). After the coronavirus infectious disease 2019 (COVID-19) pandemic, the platforms of RNA vaccines faced a progressive development, which leads to the development of RNA delivery for the expression of the desired protein (Eygeris et al., 2022).

In addition, the protein platform for reprogramming is based on the delivery of reprogramming factor proteins. It is the safest platform to manipulate the proteome because it does not contain any nucleic acid residues, which threaten the off-target/on-target integration of mentioned genes (Liu et al., 2019b). RNA- and protein-based platforms do not contain a suitable efficiency; therefore, DNA-based reprogramming is the most suitable method used for the expression of reprogramming factors. Various studies used viral and nonviral methods to deliver plasmids containing reprogramming factor genes (Maali et al., 2021; Maali et al., 2018). Retroviral, adenoviral, adeno-associated viral, Sendai viral, and simple vectors are the most commonly used DNA-based platforms for reprogramming. The third step is characterization, purification, and differentiation. As mentioned, iPSCs are potent to differentiate to all human cell lineages, that is, ectoderm, endoderm, and mesoderm; therefore, iPSCs are a suitable source for the generation of T cells through mesodermal differentiation (Deng et al., 2021).

iPSC-Derived CAR-T Cells: Generation and Advances

Due to the pluripotency of iPSC, a step-by-step differentiation is required to avoid unwanted differentiation (Liu et al., 2019a). Various studies used different sources for the generation of iPSC-derived T cells (Netsrithong and Wattanapanitch, 2021). However, it is better to use hematopoietic lineage as a source for T cell differentiation from iPSC. It should be noted that the generation of iPSC-derived CAR-T cells can be achieved by reprogramming somatic cells to iPSCs, then delivering CAR structure to iPSC, and entering differentiation process to T lineage or induction of CAR expression to expand iPSC-derived T cells (Harada et al., 2022; Jafari et al., 2022; Ueda et al., 2023; Wang et al., 2021a).

After selecting proper sources, iPSCs must be differentiated into the mesodermal lineage. Different mesodermal-inductive media are used for mesodermal induction (Ebrahimi et al., 2020). In addition, bone morphogenic proteins (BMPs), vascular endothelial growth factors (VEGFs), and fibroblast growth factors (FGFs) could trigger mesodermal differentiation. After achievement on induced mesodermal progenitor (characterized by CD56⁺ immunophenotype), hematopoietic induction should be started to achieve HSC (Inoue-Yokoo et al., 2013; Kitagawa and Era, 2010). The next step is for HSCs to differentiate into T cells. After the achievement of iPSC-derived T cells, it should be expanded by various supplementations to achieve a suitable cell count for immunotherapeutic purposes. IL-2, IL7, anti-CD3, and anti-CD28 are the most common cytokines and antibodies used for T cell expansion and activation (Chapman et al., 2020; Hajari Taheri et al., 2019; Hassani et al., 2020; Neurauter et al., 2007; Pietrobon et al., 2021).

For the first time, Themeli et al. (2013) generated CD19-targeting iPSC-derived CAR-T cells in which they reprogrammed peripheral blood T lymphocytes by KLF4, SOX2, OCT-4, and c-MYC reprogramming factors via lentiviral transduction. After the achievement of iPSC colonies, lentiviral transduction was applied to express CD19-CD28-CD3ζ (1928z-T-iPSC-T cells) CAR construct on the surface of iPSCs (Themeli et al., 2013). However, they reported that in vitro T-iPSC-derived cells may be intrinsically skewed toward embryonic characteristics; T-iPSC-derived expanded T cells have CD3⁺, CD7⁺, CD5^low, CD8α⁺CD8β⁻, and CD161⁺ phenotype, which are shared between adult γδ T cells and innate-like T cells generated in fetal development.

Wang et al. used primary CD62L⁺-naive and memory T cells from a healthy donor to generate iPSCs using KLF4, SOX2, OCT-4, c-MYC, LIN28, and short hairpin RNA (shRNA) delivered by electroporation of episomal plasmid. After the achievement of iPSC colonies, a CAR construction containing antiCD19, CD28, CD3ζ, and truncated EGFR (EGFRt) was transduced by a lentiviral vector to iPSCs (Wang et al., 2021a). In other studies regarding the generation of T cells from iPSC, Staerk et al. (2010) developed iPSC-derived T cells from PBMCs. They used OCT3/4, KLF4, SOX2, and c-MYC reprogramming factors in polycistronic lentiviral vector to generate iPSC-derived T cells using granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-6 (Staerk et al., 2010).

Seki et al. (2010) developed iPSC-derived T cells from peripheral blood mononuclear cells using OCT3/4, KLF4, SOX2, and c-MYC reprogramming factors delivered by temperature-sensitive mutated Sendai viral vector. Loh et al. (2010) performed a PBMC-derived iPSC experiment using two lentiviral vectors. Melanoma tumor-infiltrating lymphocytes (TILs) were used by Saito et al. (2016) for the generation of iPSC-derived TILs. OCT3/4, KLF4, SOX2, and c-MYC reprogramming factors were delivered by a Sendai viral vector (Saito et al., 2016). Also, Minagawa et al. (2018) generated iPSC-derived CD8αβ T cells using OCT3/4, KLF4, SOX2, and c-MYC reprogramming factors delivery to CD8⁺ cytotoxic T lymphocytes (CTLs) by Sendai viral vector.

Before moving on, there are two points that we must take notice of: TCR stimulation is a fundamental step in T cell development. The strength and length of stimulation can determine the T cell fate. For instance, when naive CD4⁺ T cells are exposed to strong TCR stimulation, T cell differentiation tends toward Th1 than Th2 cells. In contrast, weak TCR signals trigger Th2 cell differentiation or memory CD8⁺ T cells (Gomez-Rodriguez et al., 2009). Of note, the strength of TCR signaling also affects Treg cell differentiation. Regarding the TCR deficiency in iPSC-derived CAR-T cells, FT819 is currently being used in a Phase I study (NCT04629729) as monotherapy and in combination with IL-2 in subjects with B cell malignancies (Mehta et al., 2022). FT819 is a novel and optimized anti-CD19 engineered off-the-shelf CAR-T cell with a downregulated TRAC locus.

TCR-less iPSC-derived CAR-T cells showed significant antitumor activity as well as reduced GVHD rate and provided the large-scale development of potent allogeneic CD8αβ⁺ T cells for a broad range of immunotherapies (van der Stegen et al., 2022). Of note, cells that lack an αβ TCR, including NK cells, γδ T cells, or invariant NKT cells, may be used as an alternative source for universal CAR generation (Mazza and Maher, 2021). The second point is that knockout HLA genes can solve consequence problems. Universal iPSCs are HLA/TCR-deficient ones for broad clinic usage (Shamshirgaran et al., 2022; van der Stegen et al., 2022). Clarke et al. (2018) displayed some preclinical data for an iPSC-derived Universal CD19 CAR-T cell product with TCR knockout and the expression of a high affinity to address tumor antigen escape and control B cell malignancy progression effectively and safely in vitro and in vivo in a mouse model, without alloreactivity.

Differentiating iPSC into any cell lineage requires various modified media and supplementations, for generating T lineage from PSCs, three methods can be utilized: feeder-based, feeder-free, and artificial thymic organoid. In the feeder-based method, serum or undefined cellular extracts are added to immortalized stromal cell lines to support T cell differentiation (Galic et al., 2006); however, this is accompanied by undesirable interactions. For instance, Wang et al. used endothelial cell growth medium 2 (EGM-2) supplemented with SB-431542 (inhibitor of the transforming growth factor-β [TGF-β] type I receptor/ALK5), stem cell factor (SCF; a cytokine that binds to the c-KIT receptor), human Flt3 ligand (inductor of early hematopoiesis), and thrombopoietin; this step (differentiation of induced mesodermal progenitor to HSC) took 2 weeks.

They also used the MS5-hDLL1 stromal cell line as a feeder layer (Wang et al., 2022). Furthermore, Sugimura et al. (2017) reported that seven transcription factors, including ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1, are sufficient to convert hemogenic endothelium into HSCs and myeloid, B and T progenitor cells in primary and secondary mouse recipients.

In the second method, a serum- and feeder-free system is used to differentiate human PSCs into hematopoietic progenitors and T cells. Recently, Iriguchi et al. (2021) developed a feeder-free protocol for differentiating iPSCs into CD8⁺, CD4⁻, CD3⁺, and TCRαβ⁺ T cells; however, iPSCs without a prearranged TCR failed to develop into TCRαβ⁺ T cells. In the same year, Trotman-Grant et al. (2021) generated CD4⁺, CD8⁺, CD3⁺, and TCRαβ⁺ T cell progenitors from iPSCs in a feeder-free manner, but the rate of efficiency was low. In another study, Michaels et al. (2022) reported that utilizing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition can provide better control at this stage and enhance downstream progenitor T cell output.

In the third method, the three-dimensional (3D) scaffold of primary thymic stromal cells has been shown to have a positive effect on promoting positive selection and TCR rearrangement of human T cells (Lee et al., 2017; Yasui et al., 2021). Wang et al.'s (2022) study supports the development of “off-the-shelf” producing strategies by establishing the possible methodologies in the presence of a 3D iPSC-based artificial thymic organoid (PSC-ATO) system to generate highly functional CAR-T cells from iPSCs. They generated CD19-targeting iPSC-derived CD8⁺CAR-T cells with higher cytolytic effect on CD19-expressing tumor cells and enhanced cytokine secretion profile (GM-CSF, INF-γ, and TNF-α). Also, their in vivo trial on xenograft models showed that iPSC-derived CAR-T cells have more tumor inhibition potency in combination with irradiated IL15-secreting nurse cells (NS0-hIL15) than conventional CAR-T cells with the same combination regimen.

Some studies showed that co-transduction of SV40 large T antigen has been necessary to reach high efficiency in reprogramming original CTL single-cell clones (Vizcardo et al., 2013). SV40 LT antigen is one of the most commonly used immortalizing genes through the inactivation of P53, causing genomic instabilities and tumorigenesis (Park et al., 2008; Yu et al., 2023). Nishimura et al. (2013) found a synergistic relation between the coexpression of SV40 LT antigen and reprogramming factors to enhance the efficacy of T cells' reprogramming.

In terms of signaling pathways, studies showed that many of them are involved in TCR signaling, including the protein kinase C (PKC)θ-IĸB kinase (IKK)-nuclear factor (NF)-κB pathway, the Ras-extracellular signal-related kinase (Staerk et al., 2010)-activator protein (AP)-1 pathway, the tuberous sclerosis complex (TSC)1/2-mammalian target of rapamycin (mTOR) pathway, and the inositol triphosphate (IP3)-Ca⁺² nuclear factor of activated T cells (NFAT) pathway (Gaud et al., 2018). PTEN is a negative regulator of PIP3, and the lack of PTEN results in inducing the differentiation of double positive (DP) thymocytes. Therefore, early T cell development requires a balance between PI3K and PTEN (Fayard et al., 2010).

Moreover, Zap70, a member of the Syk family kinases, plays a crucial role in TCR signal transduction, which is regulated by Lck during a signaling cascade in αβ T lineage cells (Wang et al., 2010). Moreover, studies suggest that CD4⁺ and CD8⁺ lineage commitment requires anachronistic TCR signaling and the induced expression of Zbtb7b (Thpok) and Runx3 as the critical transcription factors in T cell differentiation. Of note, Runx3d restricts the Thpok expression, which is required for CD4⁺ T cell differentiation (Taniuchi, 2018). Promoting Runx3d expression by intrathymic cytokines, which are released from MHC-I-restricted thymocytes, culminates in decreased expression of the CD4 and induction of CD8 re-expression (Park et al., 2010).

Besides, the Notch signaling pathway plays a regulatory role in T cell development as well as Ikaros and GATA3 transcription factors (Brandstadter and Maillard, 2019; Hwang et al., 2020). Studies showed that in the presence of Notch1 expression, the differentiation of stem cells into B cells was completely blocked in studies mice while commitment to T cell was increased (Pui et al., 1999). The results of reverse studies, which were knocked-out Notch1, confirmed the critical role of the Notch signaling pathway in T cell lineage development (Izon et al., 2002; Koch et al., 2001; Wilson et al., 2001). Also, the Notch signaling pathway is involved in T cell differentiation into different subtypes and serves as a therapeutic target (Tindemans et al., 2017).

van der Stegen et al.'s study illustrated that premature CAR expression at the double negative (Arjomandnejad et al., 2022) stage interferes with Notch signaling as a critical role at the αβ- and γδ-lineage commitment; therefore, skews differentiation away from DP differentiation (innate-like phenotype). Their results showed that inducing DP cells during T lineage development from precursor cells required Notch ligand DLL1, while T(αβ)-iPSCs need DLL4 to progress to the DP stage. However, in constitutive CAR expression, NOTCH1s expression decreased; thus, some downstream genes, including DTX1, TCF7, and PTCRA, deregulated and skewed differentiation away from DP commitment in αβTCR T cells and navigated them toward innate/γδTCR-like fate. In addition, they suggested that DP T cell development and CD8αβ iT cell expansion could be produced by delayed expression and calibrated CAR signaling in the absence of a TCR (van der Stegen et al., 2022).

Furthermore, studies showed that NOTCH signaling provokes T cell-mediated antitumor immunity and cytokine release (Kelliher and Roderick, 2018); for instance, activation of NOTCH1/2 in CD8 T cells increases IFNγ production and consequently improves antitumor response (Sierra et al., 2014). Generating CAR-T cells with synthetic NOTCH receptors (synNOTCH) provides specific cytotoxic responses, which result in enhanced CAR efficacy (Morsut et al., 2016; Roybal et al., 2016).

Ikaros is another factor that plays a critical role in the differentiation of HSCs into T cell development. Ikaros-null mutant mice lack fetal T and B cells, adult γδ T, B, NK, and thymic Ag-presenting cells, while CD4⁺ T cells increase postnatally in them (Georgopoulos et al., 1994; Wang et al., 1996). Of note, GATA3 also has a crucial role in T cell development, especially commitment to the CD4 SP lineage (Wang et al., 2008; Ting et al., 1996).

In addition to signaling pathways, the presence or abundance of some specific cytokines greatly influences CAR-T cell manufacturing (Zhang et al., 2020a). T cells are cultured in specific mediums supplemented with determinant cytokines such as IL-2, IL-7, IL15, and IL-21 (Stock et al., 2019). IL-2 promotes mature effector T cells with low CD62L, CCR7, CD27, and CD28 expression and incorporates reduced blood persistence. Also, IL-2 regulates effector and memory CD8⁺ T cell (CTL) generation (Kalia and Sarkar, 2018; Pipkin et al., 2010). Conversely, IL-7 elevates proliferation and survival of T cells; interestingly, co-expression of IL-7 has shown increased cell expansion and suppressed cell apoptosis and exhaustion in NKG2D-based CAR-T cell therapy (He et al., 2020). Similarly, expression of IL-15 and IL-21 in CAR-T cells manifested increased expansion, persistence, and antitumor activity with elevated memory cells (Batra et al., 2020). IL-12 is another cytokine implicated with CAR-T cell's cytotoxic function, which can reduce Treg responses (Koneru et al., 2015).

Moreover, epigenetic regulation is a key factor in gene-manipulating-free reprogramming and directed differentiation cells (Henning et al., 2018; Maali et al., 2021; Simpson et al., 2021). In this regard, Jing et al. (2022) showed a significant directed differentiation of iPSCs to mature CAR-T cells via EZH1 (required for HSC maintenance) repression. They used an established human iPSC cell line; the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated EZH1 knocking down was applied by lentiviral transduction to iPSCs. After the achievement of the T cell population, the CAR construct was delivered by lentiviral transduction. The resulting iPSC-derived CAR-T cells showed significant tumor Ag-specific cytolysis compared with conventional CAR-T cells. However, the cytokine secretion profile was higher in conventional CAR-T cells compared with iPSC-derived CAR-T cells. They significantly achieved the functional αβ T cells derived from iPSCs (Jing et al., 2022) (Table 2; Fig. 1).

FIG. 1.

Overview of iPSC-derived CAR-T cells. (a) Conventional method for generation of CAR-T cells. (b) Generation of iPSC-derived CAR-T cells from T cell lineage differentiation of CAR iPSC. (c) Generation of iPSC-derived CAR-T cells by CAR expression of iPSC-derived T cell. (d) Generation of HLA-deficient iPSC-derived CAR-T cells for the “off-the-shelf” approach. CAR, chimeric antigen receptor; iPSC, induced pluripotent stem cell.

Table 2.

Overview of Induced Pluripotent Stem Cell-Derived T/Chimeric Antigen Receptor-T Cell Studies

The phenotype of T cell	Antigen-specific targeting construct	Gene delivery method for CAR expression	Origin	Reprogramming factors	Gene delivery method for reprogramming	Year	Results	References
Innate γδ T cells	aCD19-CD28-CD3ζ (19-28z CAR)	Lentiviral vector	Peripheral blood heterogeneous T lymphocytes	KLF4, SOX2, OCT-4, and C-MYC	Retroviral transduction	2013	First report of generation of iPSC-derived CAR-T cells, which led to generation innate γδ T phenotype.	Themeli et al. (2013)
CTLs	CD8-targeting rearranged TCR	NA	Nef138-8(WT)-specific CTL line	OCT3/4, SOX2, KLF4, c-MYC, NANOG, and LIN28A	Retroviral transduction+Sendai viral transduction	2013	HIV-1-infected patient-derived CTLs were reprogrammed to iPSCs and then differentiated into CD8⁺ T cells aimed at rejuvenation through elongated telomere.	Nishimura et al. (2013)
INFγ-producing CTLs	MART-1-targeting rearranged TCR	NA	MART-1-specific CD8⁺ T cell line JKF6	KLF4, SOX2, OCT4, and c-MYC+SV40 large T antigen	Sendai viral transduction	2013	MART-1-specific iPSC-derived CTLs were significantly regenerated from CD8⁺ cells.	Vizcardo et al. (2013)
CD8ab T cells	LMP2-targeting TCR	NA	LMP2-specific CTLs	KLF4, SOX2, OCT4, and c-MYC+SV40 Large T Ag	Sendai viral transduction	2016	OP9 and OP9/DLL1 stromal cell coculture was used for redifferentiation.	Maeda et al. (2016)
RAG2^−/− T-iPSC-derived CTL	GPC3-targeting TCR	NA	GPC3 peptide-specific CTL	OCT3/4, SOX2, KLF4, c-MYC, NANOG, and LIN28A	Sendai viral transduction	2018	CRISPR-mediated RAG2 knockout significantly avoided TCR rearrangement in iPSC-derived CTLs.	Minagawa et al. (2018)
iPSC-derived thymic emigrants (iTE)	NA	NA	T-iPSC	NA	NA	2019	Differentiation to T lineage using 3D FTOC system.	Vizcardo et al. (2019)
Pseudo-homozygous iPSCs HLA-C-retained iPSCs	NA	NA	Cellosaurus cell line 604B1 and F f-XT28s05 cell line	NA	NA	2019	HLA disruption by CRISPR-Cas9 significantly led to generating allogeneic iPSC-derived T cells.	Xu et al. (2019)
Polarized CD4 single positive Th cells	NA	NA	HLA II-restricted Th cells	NA	TBX21 ^KO/KO iPSCs and IL4 ^KO/KO iPSCs	2019	iPSC-derived CD4 single positive T helper cells were subjected to TBX21 and IL4 knocking out using CRISPR, which led to selective secretion of IL4 or IFN-γ. Differentiation to T lineage was applied using 3D ATO.	Yano et al. (2019)
FT819	CD19CAR-CD28-CD3ζ (19-28z CAR)	NA	αβ T cells	NA	CRISPR-cas9 integration into the TRAC locus	2019	Clinical trial on FT819 (iPSC-derived CAR-T cells) was the first report of application of “off-the-shelf” iPSC-derived CAR-T cells.	Chang et al. (2019)
TCR-deficient CD3ζ-nonfunctionalized αβ T cell (FT819)	CD19CAR-CD28-CD3ζ (19-28z CAR)	Retroviral transduction	αβ T cells	KLF4, SOX2, OCT-4, and C-MYC	CRISPR-cas9 integration into the TRAC locus	2020	NOTCH stimulation facilitated CD4⁺CD8αβ⁺ T cell formation from iPSCs.	van der Stegen et al. (2020)
β2M^KOMHC-II^Δ-CD155^KOiPSC-derived CTLs	CD20-targeting TCR	Retroviral transduction	Human iPSC line GPC3-16-1	NA	NA	2021	Hypoimmunogenic iPSC-derived T cells were generated from β2M^KOMHC-II^Δ-CD155^KOiPSCs.	Wang et al. (2021a)
CD8αβ iPSC-CTLs	NA	NA	CD3⁺ T cells HIV Nef and Gag Ag-specific CD8⁺ T cell	OCT3/4, KLF-4, SOX-2, and C-MYC	Retroviral transduction Sendai viral vectors	2021	Rejuvenated iPSC-derived CTLs showed early memory phenotype (CD8αβ⁺CD5⁺CCR7⁺CD45RA⁺CD56⁻).	Kawai et al. (2021)
iCD8αβ T cells iCAR-T cells	GPC3-specific TCR CD19 scFv-4- 1BB-CD3ζ	NA Retroviral transduction	Peripheral blood T cells (CTLs)	KLF4, SOX2, OCT-4, and C-MYC	Sendai viral transduction	2021	Application of DF1α and p38 inhibitors increased the T cell commitment in TCR Ag-specific T cell and CAR-T cells.	Iriguchi et al. (2021)
Dual antigen receptor rejuvenated CTLs (DRrejTs)	LMP1CAR-CD28TM-CD28/OX40-CD3ζ	Lentiviral transduction	EBV Ag-directed LMP2-CTL	OCT3/4, SOX2, KLF4, and c-MYC and SV40 large T Ag	Sendai viral transduction	2022	LMP2-specific CTLs derived from EBV-associated lymphoma patients were reprogrammed into iPSCs, and then, LMP1-CAR iPSCs were generated and differentiated into CTLs.	Harada et al. (2022)
αβ T cells	CD19CAR-CD8-4-1BB-CD3ζ	Lentiviral transduction	Human 1157.2 iPSCs	NA	NA	2022	EZH1 repression through epigenetic knocking down increased the T cell lineage differentiation from iPSCs.	Jing et al. (2022)
CD8⁺ T cell	CD19CAR-CD28-CD3ζ -EGFRt	Lentiviral transduction	CD62L⁺-naive and memory T cells	KLF4, SOX2, OCT-4, C-MYC, LIN28, and shRNA for TP53	Episomal plasmid electroporation	2022	CD62L⁺-naive and memory T cells subjected to reprogramming showed T cell differentiation into CD8αβ⁺ phenotype in a 3D organoid construct.	Wang et al. (2022)
TCR-CAR+CD8αβ iT	CD19(19-28z CAR)	NA	CD8αβ T cells from healthy donors	KLF4, SOX2, OCT-4, and C-MYC	Retroviral transduction	2022	TCR loci were disrupted in iPSC-derived CD8αβ⁺ CAR-T cells to avoid GVHD.	van der Stegen et al. (2022)
CD8αβ iPS-T cells	CD19(19-28z CAR)	Lentiviral transduction	αβT-iPSC	NA	NA	2023	Persistency of CAR-T cells will be increased through the diacylglycerol kinase knocking out and IL-15/IL-15R transduction.	Ueda et al. (2023)
Rag2^−/−-OT1-iT cells	OT1-specific TCR	NA	Rag2^−/−-OT1-iR9-PSCs	NA	NA	2023	iPSCs were differentiated into hematopoietic progenitor cells in vitro conditions, followed by in vivo maturation into TCR-specific T cells. Also, the Rag-2 locus was disrupted to avoid TCR rearrangement.	Wu et al. (2023)

β2M, β2-microglobulin; γδ, gamma delta; 3D, three-dimensional; ATO, artificial thymic organoid; CRISPR, clustered regularly interspaced short palindromic repeats; CTL, cytotoxic T lymphocyte; EGFRt, truncated EGFR; FTOC, fetal thymus organ culture; GVHD, graft-versus-host disease; KLF4, Kruppel-like zinc-finger domain transcription factor 4; LMP1, latent membrane protein 1; MHC, major histocompatibility complex; NA, not applicable/not available; OCT4, octamer-binding transcription factor 4; PSC, pluripotent stem cell; scFv, single-chain variable fragment; shRNA, short hairpin RNA; SOX2, SRY-related HMG-box2; TRAC, T cell receptor alpha constant.

iPSC-Derived CAR-T Cell Limitations and Overcoming

As mentioned earlier, the generation of CAR-T cells from an iPSC source could overcome some limitations, that is, lack of access to a sufficient number of origin cells, lack of expanded cells, heterogeneity of final CAR-T cell population, and high off-target effects. The brute fact is that side effects related to iPSC-derived CAR-T cells have not yet been reported; however, several manufacturing and regulatory hurdles need to be overcome. For example, potential mutagenesis, transgene reactivation, chromosomal alterations, and copy number variations are feasible following the reprogramming methods. Therefore, checking obtained cells by whole-exome or genome sequencing and SNP array seems essential (Wattanapanitch, 2019).

Another problem is to complete suppression of HLA expression, both HLA-I and HLA-II must be deleted; however, reaching this goal has some troubles with current gene editing tools due to the large region of the HLAs gene. Studies showed that there are some resolutions to cover this weakness: knockout the β2M gene as a subunit forming a heterodimer or knockout the CIITA gene as an essential transcription factor for HLA-II expression (Wang et al., 2015; Zha et al., 2020). Han et al. (2019) used the CRISPR/Cas9 system to delete the major HLA class I and HLA class II molecules by knockout of the CIITA gene. Also, they knocked in the PD-L1, HLA-G, and CD47 (immune checkpoints that reduce NK cell and macrophage activity) to regulate the T cells, NK cells, and macrophage-mediated immune responses. Their results showed T cell response suppression as well as a significant decrease in NK and macrophage activities (Han et al., 2019). Also, the disruption of CD155 (a ligand for DNAM-1 [NK cell-activating receptor]) showed longer survival and more resistant NK cell-mediated cytolysis in the generated hypoimmunogenic β2M^KO-iPSC-derived CAR-T cells (Wang et al., 2021a).

In addition, engineering iPSC-CARs by the CRISPR/Cas9 system can bring off-target effects, and consistent quality control, as mentioned above, is necessary. Besides, we can use autologous iPSCs, but in reality, high costs and longer preparation times diminish their benefits (Maroufi et al., 2020). Another approach is generating iPSCs from common HLA haplotypes (Taylor et al., 2012) and establishing a haplobank of iPSCs that contain HLA haplotype homozygous donors to provide HLA-matched iPSC products with lower cost to significant numbers of patients (Stacey, 2023; Yoshida et al., 2023).

Tumor escaping is another issue that CAR-T cells face; the generation of dual-targeting iPSC-derived CAR-T cells is a promising tool to eliminate tumor escaping. Harada et al. (2022) targeted latent membrane protein 1 (LMP1) and LMP2 using dual-targeting iPSC-derived CAR-T cells. In this experiment, LMP2-specific CTLs were reprogrammed to iPSCs. Then, LMP1-CAR (anti-LMP1-CD28TM-CD28/OX40-CD3ζ) was expressed in iPSCs, and the resulting cells were differentiated to rejuvenated LMP1/LMP2-targeting CTLs (Harada et al., 2022). Also, iPSC-derived LMP2-targeting CAR-T cells were generated as control. iPSC-derived rejuvenated LMP1/LMP2-targeting CTLs showed a significant targeting and elimination of tumor cells in vitro and in vivo (xenograft model). Moreover, significant survival has occurred in tumor models that received iPSC-derived rejuvenated LMP1/LMP2-targeting CTLs compared with iPSC-derived LMP2-targeting CAR-T cells.

Furthermore, there is an important concern that can reverse all efforts: tumor formation risk after transplantation due to residual pluripotent cells. All-round laboratory and immunodeficient mice checking are essential (Doi et al., 2020; Takahashi, 2020). Another solution is using a suicide system, which by a suicide gene such as inducible Caspase 9 (iCas9) induce into the cells, and in the presence of the inducer, apoptosis starts (Ando et al., 2015). Also, this system can be effective in other adverse situations that occur because of CAR transplantation, such as CRS, GVHD, and off-tumor toxicities (Ando et al., 2015). Generating controllable CAR cells in a different way is another solution; Eyquem et al. (2017) introduced a CAR into the TCR locus, which could control its expression under physiological conditions of the endogenous TCR promoter.

In terms of solid tumors, CAR-T cells cannot penetrate and survive in the TME. Overcoming this hurdle comes true by using other immune cells such as macrophages that can penetrate the TME, conduct antigen presentation, and phagocytosis. Zhang et al. (2020b) generated iPSC-derived CAR macrophages (CAR-iMac), and their results showed that CAR-iMAC were polarized to the M1 subtype, and tumor cells were phagocyted by them in an antigen-dependent manner.

Despite these limitations, iPSC-derived CAR cells offer great promise toward generating off-the-shelf cell products for immunotherapy, and advances in iPSC and engineering technologies open new perspectives to overcome such limitations.

Conclusions

With the advent of CAR-T cell engineering, the field of immunotherapy revolutionized. CAR-T cell therapy has had great success in hematologic malignancies and seems promising for other solid tumors, but some major limitations traditionally hamper its wide clinical use. So scientists turned to investigate the potential of other immune cells such as CAR-NK cells and CAR-macrophages due to their intrinsic killing capacity and infiltration into the TME or stem cells due to their HLA-lack nature and high rate self-renewal to generate proper CAR cells and overcome CAR-based therapies hurdles. iPSC-derived CAR-T cells promise therapeutic cell-based products applicable in immunotherapy. Various studies report different methods for the generation of these cells. Autologous and homozygous HLA haplotype iCAR-T cells showed no desirable results and incorporated some obstacles.

The engineered HLA and TCR-deficient iPSC-derived CAR-T cells prepare a suitable source for generating allogeneic iPSC-derived CAR-T cells to avoid immune rejection and GVHD. Feasibility, safety, and efficacy of such editing strategies by gene editing tools such as CRISPR have been confirmed in studies, and they are undergoing clinical trials for the actual in vivo results. Although due to complex production processes and unknown points about cells, these new strategies may be accompanied by new problems and side effects such as adverse oncogenic mutations related to gene editions. The advanced bioengineering in CAR-T cells, such as dual/multispecific targeting CAR, universal Ags targeting CAR, immune checkpoint-blocking CAR, tyrosine-depended cleaving CAR, and so on, can provide a controllable switch for activation of autologous/allogeneic iPSC-derived CAR-T cells, and the progression of knowledge, technology, and equipment in the near future will bind personalized medicine with regenerative medicine and surmount these barriers.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Footnotes

Authors' Contributions

M.R.L. collected the data and drafted this article. F.M. revised scientifically. A.M. participated in the scientific supervising of the article.

Author Disclosure Statement

The authors declare they have no conflicting financial interests.

Funding Information

No funding was received for this study.

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