Multi-Lineage Differentiation of Human Pluripotent Stem Cells

#### **Chapter 3**

## Human Pluripotent Stem Cell-Derived Mesenchymal Stem Cells for Oncotherapy

*Hao Yu, Xiaonan Yang, Shuang Chen, Xianghong Xu, Zhihai Han, Hui Cai, Zheng Guan and Leisheng Zhang*

#### **Abstract**

Mesenchymal stem/stromal cells (MSCs) with hematopoietic-supporting and immunoregulatory properties have aroused great expectations in the field of regenerative medicine and the concomitant pathogenesis. However, many obstacles still remain before the large-scale preparation of homogeneous and standardized MSCs with high cellular vitality for clinical purposes ascribe to elusive nature and biofunction of MSCs derived from various adult and fetal sources. Current progress in human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), have highlighted the feasibility of MSC development and disease remodeling, together with robust MSC generation dispense from the inherent disadvantages of the aforementioned MSCs including ethical and pathogenic risks, donor heterogeneity and invasiveness. Herein, we review the state-of-the-art updates of advances for MSC preparation from hPSCs and multiple tissues (perinatal tissue, adult tissue) as well as tumor intervention with biomaterials, and thus propose a framework for MSCs-based oncotherapy in regenerative medicine. Collectively, we describe the landscape of *in vitro* generation and functional hierarchical organization of hPSC-MSCs, which will supply overwhelming new references for further dissecting MSC-based tissue engineering and disease remodeling.

**Keywords:** hPSCs, MSCs, drug delivery, oncotherapy, biomaterials

#### **1. Introduction**

Human pluripotent stem cells (hPSCs), including human induced PSCs (hiPSCs) and human embryonic stem cells (hESCs), are cell population with unique selfrenewal and multi-lineage differentiation potential [1–3]. Attribute to the aforementioned properties, hPSCs have been considered as splendid alternatives for tissue engineering and disease remodeling [3, 4]. For instance, we and other investigators have been devoted to verifying the feasibility of high-efficient generation of MSCs from hPSCs (hPSCS-MSCs) for diverse disease treatment, including osteoarthritis, colitis, liver fibrosis [4–6]. Therewith, hPSCs have served as advantageous alternative sources for MSC preparation for regenerative medicine [7].

MSCs with unique immunoregulatory properties and tissue-repair capacity have been considered as advantageous cytotherapy for various refractory and recurrent disorders. For instance, preclinical studies and clinical practice have suggested the safety and efficiency of MSCs against hematological diseases, articular diseases, neurological diseases, digestive diseases, immune diseases and vascular diseases [8–11]. Meanwhile, the unique characteristic of MSCs with a lower immunogenicity as recommended by the International Society for Cellular Therapy (ISCT), which is appropriate for cell-based cancer immunotherapy [4, 12].

Currently, a certain number of studies have been reported that the capability of MSCs can migrate directionally to tumor sites and contribute to tumor microenvironment formation. Moreover, MSCs exert therapeutic function through an immune evasive mechanism, which will protect MSCs from immune detection and prolong their persistence *in vivo* [13, 14]. Numerous preclinical studies have indicated MSCs as gene transfer systems and ideal drug carries for targeted tumor therapy by releasing cytokines or suppressing tumor cells [15]. For examples, MSCs can load with anti-tumor drugs (as PTX or GBA), enzyme prodrug (as 5-FC/CD, GCV/HSV-TK or CPT-11) or oncolytic viruses, which thus provide antitumor effects with improved safety profiles. In addition, MSCs genetically modified to express interleukin (e.g., IL-2, IL-10, IL-12, IL-15, IL-18, IL-21) and interferon (e.g., IFN-a, IFN-β) could elicit antitumor immunity *in vivo* and inhibit tumor growth in vitro. Although, a large number of pre-clinical studies have been conducted to investigate engineering MSCs and revealed that the effects of it on tumor progress, only a small number of registered and completed clinical trials of engineering MSCs for tumors treatments. In this review, we briefly review the pre-clinical and clinical trials of engineered MSCs as gene transfer systems or drug delivery vehicles for the treatment of solid tumors, as well as summarize the therapeutic mechanism of cancers with engineered MSCs and future prospects.

#### **2. Cell sources for MSC preparation**

#### **2.1 Adult tissue-derived MSCs**

Since the 1960s, MSCs have been isolated from various sources, including adult tissues (e.g., bone marrow, adipose, dental pulp), perinatal tissues (e.g., umbilical cord, amniotic membrane, placenta) and even derived from human pluripotent stem cells (e.g., hESCs and hiPSCs) [16, 17]. Of them, MSCs were firstly isolated from bone marrow in clinical practice, followed by relative tissues such as adipose tissue, dental pulp and apical root papilla [18]. Bone marrow-derived MSCs (BM-MSCs) have been considered with the widest range of clinical applications, whereas adipose tissuederive MSCs (AD-MSCs) have been recognized with superiority in adipogenesis over the relative tissue-derived MSCs [4, 19, 20].

#### **2.2 Perinatal tissue-derived MSCs**

To date, diverse perinatal tissues have been applied for MSC preparation, including umbilical cord, umbilical cord blood, amniotic membrane, amniotic fluid and placenta. For instance, Zhao *et al.* reported the generation of MSCs from umbilical cord (UC-MSCs) as well as the variations in biological and molecular properties at series passages [12]. Instead, Wei *et al.* and Du *et al.* took advantage of the cytokine

cocktail-based strategies for the high-efficient generation of VCAM-1+ UC-MSCs with preferable immunoregulatory and proangiogenic properties [21, 22]. Of note, we and other investigators in the field verified the superiority of UC-MSCs over relative counterpart in immunoregulatory properties [8, 23]. As to placenta tissue-derived MSCs (P-MSCs), Hou *et al.* reported the spatio-temporal metabolokinetics as well as the efficacy upon mice with refractory Crohn's-like enterocutaneous fistula as well [24].

#### **2.3 Human PSCs-derived MSCs**

State-of-the-art literatures have reported the generation of MSCs from both hESCs and hiPSCs. Generally, there are four typical procedures for high-efficient hPSC-MSC preparation, including the monolayer model, the coculture model, the embryonic body (EB) model, and the cell programming strategy. For instance, we took advantage of a transcription factor, MSX2, for the initiation of MSC differentiation within 2 weeks [4]. Furthermore, we turned to small molecular cocktail-based strategies for high-efficient hPSC-MSC generation [5]. Notably, the hPSC-MSCs revealed considerable efficacy for the management of colitis, critical limb ischemia (CLI) and osteoarthritis [4–6]. Meanwhile, Li *et al.* and Yan *et al.* reported the therapeutic effect of hESC-MSCs for the treatment of autoimmune and inflammatory diseases under serum-containing or serum-free condition, respectively [25, 26]. Additionally, Wang and the colleagues generated hESC-MSCs with immune modulatory property via a trophoblast-like intermediate stage, which would also help understand the early mesengenesis in vitro [27].

#### **3. Current strategies for MSC engineering**

#### **3.1 Nano-engineered mesenchymal stem cells**

The therapeutic index of chemotherapeutic drugs can be improved by site-designed administration by reducing the exposure of drugs in non-target tissues. Current methods of targeted drug delivery mainly rely on nano-drug carriers, which can be accumulated in solid tumors. However, this passive accumulation is very inefficient, resulting in less than 5% of the dosage is delivered to the tumor, and the distribution of nanodrug carriers within the tumor is unevenly. More interestingly, MSCs can load with anti-tumor drugs as chemotherapeutic drug paclitaxel (PTX), galbanic acid (GBA) and doxorubicin (DOX), which can uniformly infiltrate into tumor tissue, and improve the distribution of therapeutic drugs within the tumor as shown in **Table 1**. For examples, Pessina *et al.* have demonstrated that MSCs-PTX could produce dramatic antitumor effects in MOLT-4 cells *in vitro* through negatively regulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression on microvascular endothelium in 2013 [48]. Moreover, PTX-loaded BM-MSCs and AT-MSCs were respectively co-cultured with NCI-H28 cells and CG5 cells *in vitro*, it showed that both of them could intensely suppress these cancer cell line proliferation and significant improvement in these cell apoptosis [20, 49]. Dual drug loading modalities including cell surface conjugation or endocytosis have been investigated in order to overcome the limited drug loading of MSCs. MSCs have been engineered with various types of organic or inorganic nanoparticles with aimed to improve their drug loading and therapeutic efficacy [35, 50]. For examples, to investigate the efficacy of adipose tissue-derived from MSCs as drug carriers for delivery of galbanic acid



#### **Table 1.**

*Pre-clinical experiments of nano-engineered MSCs for cancer therapy within 5 years (2018–2023).*

(GBA)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (nano-engineered MSCs) against tumor cells, the results have performed the nano-engineered MSCs could effectively induce cell death in C26 cells, which is considered to be as a valuable platform for drug delivery in cancer therapy [35]. Remarkably, exosomes derived from MSCs can delivery chemotherapeutic agents (DOX) in the treatment of various cancer. For instance, Liu Y *et al*. have indicated doxorubicin-loaded MSCs encapsulated into superparamagnetic iron oxide (SPIO) nanoparticles could mainly enhance anti-tumor effects and reduce the immune system response in the treatment of colon cancer [45].

#### **3.2 Genetically modified MSCs via non-viral and viral vector systems**

During previous years, cytokine-mediated cancer therapy has the potential to enhance immunotherapeutic approaches through the endowing of the immune



#### **Table 2.**

*Pre-clinical experiments of genetically modified MSCs for cancer therapy within 5 years (2018–2023).*

system by providing improved anti-cancer immunity. Nevertheless, the influence of interleukins originated therapeutics is still restricted by short half-life, systemic dose-limiting toxicities, and side-effects. In order to overcome these defects, as gene delivery platform, MSCs have been genetically modified by using viral and non-viral vectors result in the secretion of proinflammatory cytokines to enhance the host immune response to cancer cells, as well as to directly mediate tumor cell death, which have already been reported in several preclinical and clinical trials [51]. Hererin, we have summarized several cytokines engineered MSCs as drug vehicles in the treatment of cancers as seen in **Table 2**.

IFN-β is known to exhibit the classic antitumor effect, which has been certified to inhibit the proliferation of tumor cells and induce apoptosis *in vitro*, however, IFN-β could not generate and maintain therapeutic dose in the tumor sites due to its short half-life; Meanwhile, it leads to the toxicity of organ with serious side effects [52]. To overcome this problem, mesenchymal stem cells (MSCs) have been utilized as drug carriers for IFN-β gene delivery. This IFN-β expressing MSCs as therapeutic agents via systemic administration have been demonstrated effective in attenuation of cancers as melanoma [69], breast cancer [70], pancreatic cancer [71], lung cancer [52], squamous cell carcinoma [53].

IFN-γ can not only enhance the antigen presentation of dendritic cells, up-regulate co-stimulatory molecules, and promote lymphocyte differentiation, and effectively stimulate the activation of effector cells in immune system. Although IFN-γ has many advantages, the ability to induce apoptosis and inhibit angiogenesis will also influence on the normal tissues of body, resulting in side effects. In clinical trials, large doses of IFN-γ have been found to cause the side effects of nervous, blood and liver system. However, using MSCs as a drug carrier with chemotropism and precisely delivery characters, which can not only improve the concentration of IFN-γ in tumor tissues and achieve better therapeutic effectiveness, but also significantly reduce the side effects of IFN-γ on normal tissues.

IL-2 as an immunomodulatory agent was firstly approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma and carcinoma, which is required by both effector T lymphocyte and regulatory T cell. However, the short half-life and high-dose toxicity caused by IL-2 limit the clinical application [72, 73]. For instance, Joonbeom Bae and the colleagues reported that exogenous IL-2 gene modified mesenchymal stem cells elicited antitumor immunity and rejuvenate CD8+ tumor-infiltrating lymphocytes (TILs) [74].

IL-10 is produced by innate and adaptive immune cells, and mainly functions as an immune suppressor that inhibits the cancer immunity cycle. However, the half-life of IL-10 in the body is very short. For example, Zhao *et al.* verified that IL-10 modified MSCs could inhibit the growth of the transplanted tumor *in vivo* and prolong survival of bearing animals [61].

IL-12 is mainly produced by antigen-presenting cells (APCs) that regulate the immune response and serves as an effective inducer for T lymphocytes and NK cells to produce interferon-γ (IFN-γ), which is a promising therapeutic agent for the treatment of cancers. However, a short half-life and dose-limited toxicity of IL-12 limits its clinical application [75]. Numerous studies have reported that IL-12 gene modified MSCs could exhibit strengthen the anti-tumor effect in various cancer. For examples, Wu *et al.* have demonstrated that IL-12 derived from lentivirus-mediated IL-12-modified BM-MSCs combined with Fuzheng Yiliu decoction shows a strong inhibitory effect against tumor growth of glioma-nude mice, which have shown promise as an excellent drug delivery vehicle for antitumor-targeted therapy [62]. In another research, Ryu *et al.* took advantage of a delivery system based on IL-12-expressing human umbilical cord blood-derived MSCs (UC-MSCs) significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice, which thus induced long-term antitumor immunity against intracranial gliomas [76].

IL-15 is mainly secreted by activated myeloid cells that are structurally and functionally similar to IL-2. IL-15 supports the persistence of CD8<sup>+</sup> memory T cells, while inhibits IL-2-induced T cell death that better maintains long-term anti-tumor immunity [77]. For instance, Wei *et al.* have demonstrated that umbilical cord blood derived MSCs (UCB-MSCs)-transduced with lentivirus vector coding IL-15 could significantly inhibit tumor growth and prolong the survival of Pan02 pancreatic tumor mice, which were associated with tumor cell apoptosis, natural killer (NK) cell—and T-cell accumulation [78].

IL-18 as an interferon (IFN)-γ-inducing factor, which has been reported to be involved in Th1- and Th2- mediated immune responses, as well as in the activation of NK cells and macrophages. IL-18 plays a pivotal role in linking inflammatory immune responses, tumor progression and macrophage activation [79, 80]. For instance, Liu *et al.* indicated that UC-MSCs genetically modified with IL-18 could inhibit the proliferation and metastasis of breast cancer cells *in vivo* by activating immunocytes and immune cytokines, and inhibiting tumor angiogenesis [68].

IL-21 has been reported to induce a cell mediated immune responses, including NK cells and T cells. Moreover, IL-21 as an immunotherapeutic agent has been extensively applied for tumor administration. For examples, Kim *et al.* found that IL-21 expressing MSCs could inhibit the development of disseminated B-cell lymphoma and prolonged survival, which were associated with the infusion of IL-21/MSCs led to induction of effector T and NK cells [81].

#### **4. Programmed MSCs for cytotherapy**

#### **4.1 Gene-directed enzyme prodrug therapy**

Gene-directed enzyme prodrug therapy (GDEPT) is a novel approach to cancer treatment. Genetically engineered MSCs expressing suicide genes (cytosine deaminase, thymidine kinase, and carboxylesterase) have been indicated to have significant anti-tumor responses as shown in **Table 3**. To date, there are three common pro-drug activating enzymes to modify MSCs (including herpes simplex virus-hymidine kinase (HSV-TK), cytosine deaminase (CD), and rCE) to combine with ganciclovir (GCV), 5-fluorocytosine (5-FC), or Irinotecan hydrochloride (CPT-11), which can effectively inhibit DNA synthesis of tumor, as well as decrease systemic toxicity [86]. As to CD/5-FC, a certain number of researchers have reported MSCs with CD suicide gene expression have been conformed to suppress the development of breast cancer, glioma, melanoma, osteosarcoma and lung carcinoma via converting non-toxic prodrug 5-FC into cytotoxic chemotherapeutic drug 5-FU [92–96]. For instance, Daniela Klimova *et al.* have demonstrated that intravenous injection of adiposetissue and BM-MSCs-CD/5FC inhibited the progression of tumor in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model [97]. The authors have proposed that MSC/CD combined with 5-FC and TMZ could increase cell cycle arrest and DNA breakage, which could be used in patients with glioblastoma multiforme (GBM) during the immediate postoperative period to sensitize tumors to subsequent adjuvant chemo- and radiotherapy [98]. Moreover, it has been suggested that extracellular vesicles derived from MSCs with CD gene delivery as cargo have an inhibitory effect on the growth of tumor cell lines *in vitro*, as Daniela Klimova engineered the MSCs-EV were cultivated with gemcitabine (GCB), which significantly inhibited the cell growth of pancreatic carcinoma cell lines *in vitro* via converting non-toxic prodrug 5-fluorocytosine (5-FC) to highly cytotoxic prodrug 5-fluorouracil (5-FU), and thereby provide a therapeutic option for tumors [82]. In addition, the transduced iPSC-MSCs both limited growth of preformed tumors and decreased lung metastases after administration of the prodrug (5-FC) [99]. As HSV-TK/GCV, the thymidine kinase (TK)/ganciclovir (GCV) system is a gene-directed enzyme prodrug therapy. Therefore, the herpes simplex virus 1 thymidine kinase (HSV-TK) gene as a suicide gene is introduced into cells phosphorylates a prodrug GCV, which inhibits DNA synthesis and causes cell apoptosis. Although the group of HSV-TK/GCV as suicide gene therapy method is safe and effective in pre-clinical experiments, yet it is not effective in clinical trials due to the lower transfection rate of target cells [100]. In this regard, using engineered MSCs as drug carriers to induce tumor regression in human tumors mainly based on the strong migration ability to especially invasive tumors. For examples, Wei *et al.* have reported that HSV-TK-expressing UC-MSCs combined with prodrug GCV exerted a better effect in the treatment of subcutaneous tumor models and brain intracranial tumor models [88]. Azra Kenarkoohi *et al.* further investigated the anti-tumor activity of MSCs transduced with the HSV/TK in a mouse cervical cancer model via intratumoral injection, which performed significant reduction in tumor size and improvement of NK and CTL activity [85]. As rCE/CPT-11, carboxylesterases (CEs) are enzymes that can convert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, which has significant cytotoxicity to tumor cells [101]. For example, Seung Ah Choi *et al.* reported that adipose tissue-derived from MSCs


*DP-MSCs: dental pulp MSCs; AD: Adipose tissue; BM: bone marrow; DP: dental pulp; UC: umbilical cord; BP: blood platelets; P-MSCs: placenta MSCs; and PEI-PLL: polylysine-modified polyethylenimine copolymer.*

#### **Table 3.**

*Pre-clinical experiments of MSCs-based enzyme prodrug for cancer therapy within 5 years (2018–2023).*

expressing rCE as cellular vehicles could convert CPT-11 to SN-38, which revealed cytotoxic effect on F98 cell *in vitro* and effectively inhibited the progression of tumor in a rat brainstem glioma model. Therewith, the genetically modified MSCs-rCE as drug delivery have showed therapeutic potential against brainstem gliomas [102].

#### **4.2 Trail prodrug therapy**

The death ligand tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), a member of the TNF cytokine superfamily, has long been recognized for its

potential as a cancer therapeutic due to its capacity to induce apoptosis in many types of cancer cells via the receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2/KILLER), and Fas ligand (FasL) binding to the Fas receptor [103, 104]. Based on the previous research, TRAIL-MSCs as delivery vehicles could induce strength cytotoxicity against cancer cells, which furtherly inhibited tumor growth and prolonged survival in cancer


*B-All: B-cell acute lymphocytic leukemia; AML: acute myeloid leukemia; VPA: valproic acid; AAV: adeno-associated virus; PEI: polyethylenimine; and VPA: valproic acid.*

#### **Table 4.**

*Pre-clinical experiments of TRAIL-MSCs for cancer therapy within 5 years (2018–2023).*

models as shown in **Tables 2** and **4**. For instance, Young Un Choi *et al.* constructed the genetically engineered AD-MSCs with TRAIL expression and verified the suppressive effects upon tumor growth in an H460 xenograft model [108]. Chen *et al.* found that TRAIL-MSCs could significantly inhibit the proliferation and promote the apoptosis of B-cell acute lymphocytic leukemia (B-ALL) cells *in vitro* and *in vivo* [106]. Moreover, iPSC-MSCs overexpressing TRAIL are also considered an effective option for the treatment of cancer. For example, Wang and the colleagues have reported that genetically modified iPSCs-MSCs with TRAIL could significantly induce apoptosis in various tumor cell lines *in vitro*, as well as inhibit tumor growth in tumor-bearing mice models via the activation of apoptosis-associated signaling pathways [116].

#### **5. Clinical application of engineering MSCs in tumor**

Although numerous preclinical trials have been published, only a small number of clinical trials were registered and completed for the treatment of solid tumors with engineering MSCs. For example, Hanno Niess *et al.* conducted a single-arm phase I/II study for the treatment of gastrointestinal tumors by genetically modified autologous BM-MSCs. According to another clinical trial in the stage of phase I, the safety of the investigational medicinal product (IMP) is evaluated in six patients by 3 times injection of MSCs at diverse concentrations followed by administration of the prodrug Ganciclovir. In the stage of phase II, 16 patients will be enrolled receiving IMP treatment [117]. One completed clinical trial is an investigational study for INF-β modified MSCs in the treatment of ovarian cancer with the aim to evaluated the safety of MSCs/INF-β in the stage of Phase I (without published results). For the treatment of lung cancer, TRAIL engineered allogeneic MSCs as therapeutic agent to treat the metastatic non-small cell lung cancer (NSCLC) patients in a Phase I/II clinical trial. Furthermore, an exploratory trial reported four children with metastatic neuroblastoma to received autologous MSCs infected with ICOVIR-5, and the results exhibited a well-tolerance and safety of MSCs delivered with oncolytic adenoviruses in the treatment of metastatic neuroblastoma [118].

In summary, according to preclinical investigations and clinical trials, we suppose that engineered MSCs as drug delivery is a multifaceted player in oncotherapy development and the clinical transformation of MSCs is urgently needed to accelerate tumor therapy.

#### **6. Prospective and challenges**

Longitudinal studies have indicated hPSCs as advantageous cell sources for functional cell generation and the concomitant therapeutic strategy for regenerative medicine and oncotherapy. As mentioned above, the unique property, including self-renewal and multipotent differentiation, have endowed hPSCs with first-rate potential for disease remodeling and alternative cell source preparation. Even though, the significant disadvantages such as teratoma formation and the low differentiation efficiency should not be neglected [3]. Distinguish from the other counterparts, hPSC-MSCs revealed more robust cellular viability and considerable therapeutic effect upon diverse diseases, which thus hold promising prospects for serving as alternative sources of adult tissue- or perinatal tissue-derived MSCs [4].

Notably, considering the rapid progress in gene-editing and MSC-based cytotherapy, it would be of great interesting to further explore the feasibility of generating hESC-MSCs or hiPSC-MSCs with specific targets for the next-generation of oncotherapy in preclinical and clinical practice.

#### **Acknowledgements**

The authors would like to thank the members in Gansu Provincial Hospital and Chinese Academy of Sciences for their technical support. This study was supported by grants from the project Youth Fund funded by Shandong Provincial Natural Science Foundation (ZR2020QC097), Project funded by China Postdoctoral Science Foundation (2023 M730723), Postdoctoral Program of Natural Science Foundation of Gansu Province (23JRRA1319), Science and technology projects of Guizhou Province (QKH-J-ZK[2021]-107, QKH-J-ZK[2022]-436), the Joint Funds of Yunnan Provincial Science and Technology Department and Kunming Medical University (202301AY070001-221), National Natural Science Foundation of China (82260031), the Natural Science Foundation of Gansu Province (21JR7RA594), Gansu Provincial Hospital Intra-Hospital Research Fund Project (22GSSYB-6), The 2022 Master/ Doctor/Postdoctoral program of NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor (NHCDP2022004, NHCDP2022008), Jiangxi Provincial Novel Research & Development Institutions of Shangrao City (2020AB002, 2021F013, 2022A001, 2022AB003), Jiangxi Provincial Key New Product Incubation Program from Technical Innovation Guidance Program of Shangrao city (2020G002, 2020 K003), Spring City Plan of the High-level Talent Promotion and Training Project of Kunming (2022SCP002), and Major Science and Technology Project of Science and Technology Department of Yunnan Province (202302AA310018), Jiangxi Provincial Natural Science Foundation (20224BAB206077, 20212BAB216073), Jiangxi Provincial Leading Talent of "Double Thousand Plan" (jxsq2023102017).

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

Not applicable.

#### **Appendices and nomenclature**


#### *Advances in Pluripotent Stem Cells*


### **Author details**

Hao Yu1†, Xiaonan Yang2†, Shuang Chen3,4†, Xianghong Xu5 , Zhihai Han3 , Hui Cai<sup>5</sup> \*, Zheng Guan6 \* and Leisheng Zhang3,4,5,7,8,9\*

1 School of Medicine, Nankai University, Tianjin, China

2 Department of Plastic and Reconstructive Surgery and Department of Hemangioma and Vescular Malformation, Plastic Surgery Hospital Affiliated to Chinese Academy of Medical Sciences and Peiking Union Medical College, Beijing, China

3 Jiangxi Research Center of Stem Cell Engineering, Jiangxi Health-Biotech Stem Cell Technology Co., Ltd., Shangrao, China

4 Institute of Stem Cells, Health-Biotech (Tianjin) Stem Cell Research Institute Co., Ltd., Tianjin, China

5 Key Laboratory of Molecular Diagnostics and Precision Medicine for Surgical Oncology in Gansu Province and NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor, Gansu Provincial Hospital, Lanzhou, China

6 Biomedical Research Center, Affiliated Calmette Hospital of Kunming Medical University (the First Hospital of Kunming), Kunming, China

7 Center for Cellular Therapies, The First Affiliated Hospital of Shandong First Medical University, Jinan, China

8 Key Laboratory of Radiation Technology and Biophysics, Hefei Institute of Physical Science, Chinese Academy of Sciences, Hefei, China

9 Center Laboratory, The Fourth People's Hospital of Jian and The Teaching Hospital of Shandong First Medical University, Jinan, China

\*Address all correspondence to: caialon@163.com; jasmin\_067@163.com and leisheng\_zhang@163.com

† These authors contributed equally.

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 4**

## Hematopoietic Development of Human Pluripotent Stem Cells

*Igor M. Samokhvalov and Anna Liakhovitskaia*

#### **Abstract**

Blood development proceeds through several waves of hematopoietic progenitors with unclear lineage relationships, which convolute the understanding of the process. Thinking of the hematopoietic precursors as the "blood germ layer" can integrate these waves into a unified hematopoietic lineage that originates in the yolk sac, the earliest site of blood development. Hematopoietic differentiation of pluripotent stem cells (PSCs) reflects to a certain extent the complexities of the yolk sac hematopoiesis. In the unified version of blood issue development, the PSC-derived hematopoiesis can also generate post-yolk sac hematopoietic progenitors. To do this, the differentiation has to be arranged for the reproduction of the intraembryonic hematopoiesis. Inflammatory signaling was recently shown to be actively engaged in blood ontogenesis. In addition, a highly recapitulative differentiation of human PSCs was found to spontaneously ignite intense sterile inflammation that has both instructive and destructive roles in the hPSC-hematopoiesis. Inflammatory induction of blood progenitors during hPSC-derived hematopoietic development has to be properly contained. A possible explanation of problems associated with *in vitro* blood development is the failure of inflammation containment and resolution.

**Keywords:** pluripotent stem cells, hematopoietic differentiation, inflammation, hematopoietic stem cells, tissue macrophages

#### **1. Introduction**

Hematopoietic differentiation initiated by human pluripotent stem cells (hPSCs) is a surrogate model for studying early human hematopoietic development. It is generally accepted that the transition of PSCs into blood cells essentially recapitulates the yolk sac stage of hematopoiesis [1, 2], which turns out to be significantly more complicated than previously thought. Most of the information on blood origin came from mouse studies despite the fact that mouse development is highly specialized and adapted to specific living conditions of the species. The mouse hematopoietic system emerges in the early ontogenesis to support embryo development after the initiation of the heartbeat. The first blood cells arise directly from extraembryonic mesoderm at murine embryonic day 7.0 (E7.0) and consist of primitive erythrocytes or erythroblasts, primitive megakaryocytes, and macrophages that perform the tissue oxygenation, preventing embryonic blood loss, and clearing of apoptotic cells, respectively [3]. The role of progenitors for these cell types is taken by mesodermal precursors of the yolk sac.

A second wave of erythromyeloid progenitors (EMPs) initiates in the yolk sac at E8.0–8.5, and progenitors with lymphoid or lymphomyeloid potential emerge in the yolk sac and embryo proper at around E9.5 [3–7]. These data picture an obvious trend of yolk sac hematopoiesis from primitive monopotential and bipotential progenitors [8–10] through EMPs toward the lymphoid-primed multipotent progenitors (LMPPs). This fast evolution of hematopoietic potentials does not culminate in the self-renewing multipotential progenitors, although it does not mean that precursors of these progenitors do not emerge in the yolk sac. Hematopoietic stem cells (HSCs) that can self-renew and repopulate the hematopoietic system of a conditioned recipient are first observed at E10.5, emerging in the aorta–gonad–mesonephros (AGM) region [11]. At this stage, very few HSCs arise alongside numerous HSC-independent hematopoietic progenitors from the endothelium of the dorsal aorta in an extremely peculiar process designated as endothelial to hematopoietic transition (EHT) [12, 13]. EHT is thought to be also involved in the formation of EMPs and possibly LMPPs in the yolk sac [3, 14]. Circulating HSCs and their precursors colonize the extravascular territories of the fetal liver (FL) at E12.0–12.5, where they undergo massive expansion before migrating to incipient bone marrow at E17.5, the main site of hematopoiesis during the adulthood [15, 16]. Of note, the FL colonization looks very similar to leukocyte extravasation during inflammation [17], and the FL niche harbors and cultivates both HSC-dependent as well as HSC-independent progenitors during development.

Human hematopoiesis development is far less amenable for systemic molecular and cellular studies but, based on several lines of evidence, follows a similar logic. Primordial angioblastic cords appear in the human yolk sac around Day 16 of gestation [18] whereas the formation of blood islands, observed starting from Day 19 [19], leads to the production of primitive erythrocytes and macrophages [20], as well as primitive megakaryocytes [21]. Clonogenic multilineage hematopoietic progenitors, the analogs of murine EMPs, are functionally identified in the human yolk sac at four to five weeks of gestation [22, 23] when systemic circulation already started. Under the influence of the discovery that the AGM region is a niche of the first mouse HSCs, early human studies had also presumed the intraembryonic origin of adult hematopoiesis and HSCs [24], although the autonomous generation of HSCs in the human AGM region was not confirmed [25].

Human PSC-hematopoiesis struggles to generate authentic HSCs [26] perhaps due to the yolk sac mode of the differentiation. The yolk sac-like concept of hPSC-hematopoiesis, however, does not overrule the possibility of HSC generation *in vitro*. There are two main reasons for the continuation of the efforts: (1) no solid data prove that the yolk sac does not produce precursors of HSCs, while the opposite may be correct [27]; (2) only primary hPSC differentiation is similar to the yolk sac hematopoiesis, and additional steps with selected cell populations may improve the HSC chances.

This chapter will discuss the role of archetypal cell interactions of sterile inflammation in hPSC-hematopoiesis. A recapitulative hPSC differentiation reproduces the inflammatory mechanisms that participate in the *in vivo* hematopoietic development. The spontaneous sterile inflammation and inefficient resolution can be important factors contributing to the difficulties in the derivation of HSCs from hPSCs.

#### **2. Induction of hematopoiesis in the human conceptus**

Early human development is drastically and conceptually different from the development of the mouse embryo, the standard developmental model. In mice, a

#### *Hematopoietic Development of Human Pluripotent Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.112554*

number of fate mapping and molecular studies have established that during the blastocyst formation ICM cells lose their potential for the trophectoderm specification. In contrast, more ontogenically advanced epiblast cells in human preimplantation embryos still showed outstanding developmental plasticity. Pre-gastrulation human epiblast was demonstrated to give rise not only to all three germ layers but also to the trophectoderm [28]. These findings provided compelling experimental evidence of the regulative embryonic development in humans and showed that the regulative development, inherent to all mammals and birds, is more prominently manifested in humans compared to mice. The increased developmental plasticity may be an adaptation to the long and relatively slow gestation so that a majority of developmental deviations or failures can be effectively fixed by other embryonic cells. Alternatively, stricter lineage bifurcation in the mouse embryo suits better to fast and short-term ontogenesis. The outstanding human plasticity may not be limited to epiblast cells. Later conceptal locations, including extraembryonic mesoderm with the emerging hematopoietic system, are likely to possess the increased regulative potential, although experimental proof of the postimplantation plasticity is difficult to obtain.

Due to the limited access to the tissues of the early human conceptus, the initiation and structure of the human hematopoietic development is less well-known compared to that of the mouse. In mice, the founding members of the blood germ layer are located in the pre-gastrulation epiblast giving rise to the primitive blood and hemogenic endothelial cells (HECs) within the yolk sac vascular endothelium [29]. A body of evidence [18, 30–32] indicates that human hypoblast or primitive endoderm rather than epiblast gives rise to the extraembryonic mesoderm (EXM) of the primary yolk sac. The anatomical origin of EXM had been debated and other anatomical regions of the pre-gastrulation embryo were thought to contribute to its emergence [18, 33]. Nevertheless, the most compelling cell tracing evidence points to the hypoblast origin [32]. Close alignment of EXM and epiblast transcriptomes in monkey pre-streak embryo [34] is an indication of the early commitment of epiblast cells to mesodermal specification before gastrulation. It is also possible that the secondary mesoderm arising from the primitive streak during gastrulation also contributes to expanding EXM [35], although the extent of this contribution is unknown. The major role of EXM is the generation of first blood cells and vasculature in primordial structures traditionally called blood islands. These tissues sustain the developing human embryo for a period, but this is not the only function of the EXM derivatives. Mouse studies demonstrate the long-term contribution of the yolk sac hematopoiesis into various cohorts of adult cells [36–38], including adult HSCs [27, 39].

The hypoblast origin of the early hematopoiesis implies that the human blood tissue develops in parallel with primitive streak-derived secondary mesoderm. The origin of the yolk sac blood islands is polyclonal [40], otherwise, there would be not enough cells to sustain embryonic development by providing erythroblast-based tissue oxygenation and timely clearing of cell debris by primitive macrophages. This polyclonal, definitive mesoderm-independent development means that blood induction is a morphogenic event analogous to the formation of ectoderm, trophectoderm, and gastrulation. Then, EXM and its progeny—blood can be considered a cryptic or atavistic germ layer containing all elements required for its development and function.

The concept of the blood germ layer helps to alleviate an obvious problem of the strange mode of hematopoietic ontogenesis with two seemingly separate developmental lineages, the primitive and the definitive hematopoiesis. In the blood germ layer hypothesis, EXM is a founder conceptus location for the whole of the hematopoietic lineage developing as a special germ layer. As in the classical germ layers, blood has

transitory primordial tissue, the primitive blood, within primordial organs, blood islands, that contain precursors of definitive tissue. These precursors represented by HECs use the vascular niche for their survival and maintenance of the undifferentiated state to serve as an origin of definitive tissues and organs. During ontogenesis, the derivatives of the definitive HECs migrate throughout the vascular system probing the potential niches for settling down and being sometimes or somewhere retained on vascular beds due to the affinity of their surface molecules to cognate receptors expressed on endothelial cells. Later on, the blood germ layer, in collaboration with the derivatives of other germ layers, develops into the immune system with its multiple organs and hematopoietic system settled in the bone marrow. In mice, under strong selective pressure to minimize the gestation period, the regulative development canceled the primary mesoderm and fused the blood layer to the secondary mesoderm.

Supporting the blood germ layer concept, recent studies show that the yolk sac hematopoiesis is more complicated than was previously thought. Early evidence of the key role of the yolk sac in the initiation of fetal and adult hematopoiesis [41–43] was dismissed based on experimental data supporting the intraembryonic origin of definitive hematopoiesis, which was located in the pre-circulation para-aortic splanchnopleura and its derivative—the AGM region. The presence of definitive EMPs and LMPPs in the yolk sac was rediscovered by the new post-AGM generation of researchers using modern approaches and techniques. The lineage tracing experiments in mice demonstrated that populations of tissue-resident macrophages in the adult, including Kupffer cells, alveolar macrophages, and microglia develop from the progenitors originating in the yolk sac [36–38, 44]. The role of the yolk sac in the establishment of adult cell populations became even more apparent after it was found that subpopulations of mast and T cells descend from hematopoietic programs localized in the yolk sac [45–47]. The unifying feature of the abovementioned adult, yolk sac-derived cell populations is their capacity to maintain themselves within the tissues by lifelong self-renewal. Taken together, these data indicate that at least some yolk sac progenitors can acquire self-renewal potential during development. In other words, the ability to self-renew is not something impossible for yolk sac cells to gain on their way toward adulthood. It is then not too untrustworthy that cell tracing and gene reactivation studies indicate the yolk sac origin of the pre-HSCs [27, 39].

The signaling events participating in the induction of human blood are still poorly understood. In the mESC differentiation model, Wnt/β-catenin signaling promotes, whereas Notch signaling suppresses primitive erythroblast development [48]. It was also found that the transient expression of Numb in mesodermal precursors led to the inhibition of the Notch signaling. BMP4 promotes the generation of VEGFR2+ cells within the mESC-derived lateral mesoderm and VEGF supports the subsequent specification and expansion of hematopoietic and endothelial cells [49–51]. Short-term exposure to BMP4 was also instrumental in driving the mesodermal specification of human ESCs [52]. It is highly likely that BMP4 is among the human conceptal factors that induce EXM and help to initiate the hematopoiesis transition. Recent hPSC differentiation data showed that FGF2, BMP4, VEGF, and possibly signals evoked by the collagen IV adherence are sufficient to induce efficient primitive and definitive human hematopoiesis [53].

#### **3. Hematopoietic differentiation of hPSCs**

Since their derivation in 1998, human embryonic stem cells (hESCs) have become a useful model for studying human development and molecular mechanisms of

lineage specification *in vitro*. Reprogramming human somatic cells into embryoniclike stem cells [54], named human induced pluripotent stem cells (hiPSCs), opened realistic perspectives for generating various therapeutic cell populations, disease modeling, and drug discovery [55]. The hope is boosted by experiments, in which mouse iPSC-derived primitive macrophages differentiated into tissue-resident macrophages and microglia upon injection into recipient animals [56]. Nevertheless, common usage of instructive hematopoietic cytokines shifts the ontogenic program of PSC-derived mesodermal precursors into the development of cells with a limited functional diversity [57].

Conventional, not naïve, hPSCs that possess so-called primed pluripotency are considered to represent postimplantation epiblast. In order to begin their way to hematopoiesis these cells have to transit from epiblast-like though hypoblast-like to mesodermal epigenetic state under the influence of internal and external effector molecules. This transition is facilitated by a standard variety of factors, and over the decades, there were no significant changes in basic approaches to the hematopoietic differentiation of hPSCs. The traditional methods include coculture with supportive stromal cells, most prominently the M-CSF-negative OP9 cell line [58, 59]; various modifications of the planar differentiation [60, 61]; 3D cultures through the formation of embryoid bodies (EBs) [62, 63], or a combination of 2D and 3D differentiation [53, 64]. In the well-substantiated trend, the protocols are modified in order to remove the fetal calf serum from the culture medium. The *in vitro* analog of the primary mesoderm is generally induced through the BMP signaling [52, 65, 66], followed by hemogenic endothelium induction by VEGF, SCF, FLT3L, IL3, and FGF2 [67], or similar recipes of exogenous growth factors and hematopoietic cytokines. The shift toward hematopoiesis is first manifested by the expression of CD235a, CD43, and CD34, a marker of hematopoietic progenitors and vascular endothelial cells. The endothelium is always present in the hematopoietic differentiation of PSCs since both lineages have common ancestor cells: the hemangioblasts—a mouse primitive streak mesendodermal entity [68], and hemogenic endothelial cells, VEGFR2-positive mesodermal precursors expressing CD34 and the endothelial marker VE-cadherin [69, 70]. CD34 is expressed at a substantially higher level on endothelial cells compared to emerging CD43<sup>+</sup> hematopoietic progenitors. The objective of the first step of differentiation is usually to obtain CD34<sup>+</sup> CD45<sup>+</sup> cells that can be used as hematopoietic progenitors in downstream cultures or applications. Omitting the hematopoietic cytokines and minimizing the use of the mesodermal/endothelial growth factors can strongly improve the recovery of the clonogenic hematopoietic progenitors, including the multilineage ones [53, 64]. The hPSC-derived progenitors can be used to generate clinically relevant blood and immune cell populations [71, 72]. Nonetheless, a practical biotechnological design for getting large numbers of safe functional cells is missing. There is also a serious concern about the genomic stability of ethically acceptable induced pluripotent stem cells, which threaten to prevent the efficient use of hPSC differentiation for therapeutical purposes [73].

The search for a powerful inducer of hematopoiesis led to the conclusion that BMP4 signaling plays a key role in the induction of blood-competent mesoderm upon hPSC differentiation. In mice, there is compelling evidence that Bmp4 signaling is critically required for mesodermal induction [50, 74–79]. In the recently developed model of the early human peri-implantation development, BMP4 was shown to participate in the maintenance of EXM [80]. Induction of EXM was achieved by inhibition of Nodal signaling and GSK3β. This suppression only indirectly reflects the induction events in the developing human embryo and actual signals leading to the suppression are unknown. We, therefore, can only guess which factors induce EXM in the peri-implantation embryo. It seems that the BMP4 signaling does not only participate in the maintenance of EXM *in vitro* but also may induce the emergence of the tissue. Indeed, previous studies showed that Nodal inhibition results in enhanced BMP4 signaling in the pluripotent stem cell context [81]. Activin/Nodal/TGF-β pathway was not active in the naïve hPSC-derived EXM cells, and no data were available on the role of the FGF2 signaling.

In addition to BMP4, hematopoietic induction in hPSC-derived extraembryonic mesoderm is strongly dependent on VEGF, a growth factor of outstanding pleiotropy. In human development, VEGF treatment enhances blastocyst outgrowth and stimulates embryo implantation [82]. In postnatal development, accumulating evidence indicates the crucial role of VEGF in body growth and organ development [83]. Other findings demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection, and glial growth independently of the angiogenic function of the growth factor [84]. The major developmental function of VEGF is organizing and stimulating embryonic vasculogenesis and angiogenesis [85, 86]. In inflammation, VEGF and one of its receptors, VEGFR1, previously identified as a decoy receptor, were found to play a role in the recruitment and activation of monocytes and macrophages [87, 88]. In hPSC biology, VEGF signaling participates in the mesendodermal induction of hESCs [89] and selectively promotes erythropoietic development from hESCs, which have been strongly augmented by BMP4 [90]. Together with FGF2, VEGF is crucial for the progression of mesoderm to hemogenic endothelium [91], which was identified as CD31+ CD34+ VE-CADHERIN+ KDR+ cell population [92]. These hPSC-HECs can initiate both primitive and broadly defined definitive hematopoiesis.

Strictly defined definitive hematopoiesis arises only from fetal or adult-type hematopoietic stem cells (HSCs). Generation of lymphoid cells from PSCs was previously considered proof of the definitive lineage potential, although most likely it recapitulates the emergence of pre-HSC hematopoiesis of the yolk sac type. The derivation of HSCs of adult or fetal type remains elusive despite many efforts, including the most recent one [93]. One possible reason for the HSC failure is a low recapitulative quality of hPSC hematopoietic differentiation *in vitro*. HSC precursors change several locations within developing conceptus until they land in the bone marrow [94]. During their development, the HSC lineage cells seem to focus on segregation from progenitor-driven embryonic hematopoiesis and settling in a safe haven of fetal liver, spleen, and bone marrow sinusoids. Therefore, a good recapitulation of HSC development should include a second leg of differentiation so that pre-HSCs are isolated from active hPSC-hematopoiesis. The secondary differentiation most likely should include a stromal culture supported by selected cytokines and small molecules such as stable derivatives of ascorbic acid.

Excessive use of growth factors and cytokines at a nonphysiological concentration to induce the emergence of EXMCs and HECs is another reason for the poor generation of multipotent hematopoietic progenitors and HSCs by differentiating hPSCs. We do not know the exact makeup of the signaling factors participating in the hematopoietic induction within the developing human conceptus. Moreover, inductive events in early mammalian embryos are performed by short-range signaling proteins and growth factors [65]. Therefore, any use of exogenous hematopoietic cytokines to initiate hPSC-derived hematopoiesis may *a priori* disturb the developmental pathway of HSC precursors due to the strong instructive influence of these cytokines [95]. The published protocols use complex compositions and excessive concentrations of

#### *Hematopoietic Development of Human Pluripotent Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.112554*

cytokines, growth factors, and signaling molecules to ensure efficient conversion of hPSCs into hematopoietic cells at the cost of a proper recapitulation of hematopoietic development in the conceptus. Such an approach decreases the value of the hPSC differentiation as a model of early hematopoietic ontogenesis. Furthermore, the use of external cytokines accelerates terminal differentiation of hematopoietic progenitors that negatively influences the length of the proliferative stage and the yield of clinically relevant cellular material.

In the structural aspect of hPSC-hematopoiesis, planar, 3D, or stromal differentiations do not reproduce to any acceptable extent the anatomy of the peri-implantation human embryo. In the early postimplantation human embryo, EXM cells spread from the 3D embryo proper part over the distal trophoblast in an essentially planar fashion (**Figure 1A**). Therefore, the optimal recapitulation of mesodermal induction

#### **Figure 1.**

*Forced attachment of hPSC-EBs reproduces the early EXM development. (A) Day 8–9 human peri-implantation embryo. The induction of EXM. (B) Early stages of hPSC differentiation. Shortly after attachment to the collagen surface, EB undergoes epithelialization (left panel). Next day, the EB-derived EXM starts to spread over the surface.*

in peri-implantation embryos should include the spreading of differentiating hPSCs from a central 3D cell mass in a planar circumferential fashion. Stromal support from adult sources, the fetal liver, and the AGM region creates artificial differentiation conditions that are not encountered at the start of hematopoietic development.

In the recently published protocol for recapitulative differentiation of hPSCs, no external cytokines were used [53]. The onset of EXM development was reproduced by forced attachment of hPSC-EBs to collagen-covered surfaces in the presence of BMP4, VEGF, and mTeSR1-derived FGF2. The attachment initiated active planar spreading of mesenchymal cells (**Figure 1B**) followed by spontaneous formation of blood island-like aggregates (**Figure 2**) in which hematopoietic induction occurs. Differentiating cultures produced a variety of endogenous cytokines that supported hematopoietic development and the production of large numbers of progenitors.

#### **Figure 2.**

*After attachment to collagen-coated surface, hPSC-derived embryonic bodies form angioblastic cords (upper panel) that transform later on into blood island-like structures (lower panel).*

Among others, M-CSF, a pro-inflammatory cytokine [96], was secreted at an exceptionally high level. After 1 week of the differentiation, emerging myeloid cells activate genetic programs that induce and control spontaneous sterile inflammation.

#### **4. Inflammation**

Inflammation is a complex, local and systemic, defensive, and adaptive response of the immune system triggered by a variety of agents that disturb homeostasis on an organismal or cellular level. The inflammatory factors include pathogens, damaged cells, toxic compounds, irradiation, and irritation. The variety of the factors boils down to the intrusion of external organisms and the noninfectious damage to tissue cells. Cell damage and infectious agents activate inflammatory cells and trigger inflammatory signaling pathways. In most cases, these are the NF-κB, MAPK, and JAK-STAT signaling [97]. The cellular challenges are sensed by special sentinel receptor molecules that activate the immune system.

Damage-associated molecular patterns (DAMPs) such as oxidized lipoproteins, HMGB1, S100 calcium-binding proteins, heat-shock proteins, or pathogen-associated molecular patterns (PAMPs) such as uncapped viral RNA, lipopeptides, flagellin, and lipopolysaccharides are identified by pattern recognition receptors (PRRs) on the cell surface or endosomes in the cytoplasm. These PRRs are represented by a vast variety of cell surface and cytoplasmic molecules, including the TLRs (Toll-like receptors), the CLRs (C-type lectin receptors), NLRs (NOD-like receptors), the RLRs (RIG1 like receptors), and the RAGE (receptor for advanced glycosylation endproducts). PRRs are preferentially expressed on sentinel immune cells, including mast cells, macrophages, dendritic cells, innate lymphoid cells, and basophils. In addition, many tissues have nonimmune-tissue sentinel cells [98, 99]. Binding the DAMPs/ PAMPs ligands to cognate PRRs activates the NF-κB transcriptional effector complex, which then induces the expression of inflammatory cytokines. These cytokines then initiate the cellular phase of the inflammatory reaction. The key step is upregulating cell adhesion molecules on endothelial cells. VCAM-1, ICAM-1, and E-selectin, all inducible by inflammatory cytokines, promote, in cooperation with chemokines and other endothelial adhesion molecules, the adherence and extravasation, or diapedesis, of neutrophils and monocytes into damaged tissue. In sterile inflammation, the major post-diapedesis role belongs to monocytes, which differentiate into tissue macrophages that clean up cellular and extracellular matrix debris.

To preserve tissue homeostasis, acute inflammation has to be suppressed to avoid persistent, chronic inflammation that leads to additional and broader tissue damage. Inflammatory neutrophils are major culprits in collateral tissue and cell breakdown [100]. Stable chemokine gradients may attract an excess of neutrophils even when the microbial infection is already contained. These granulocytes discharge their immense cytotoxic arsenal into the extracellular space of surrounding tissues even if they fail to encounter a microbial agent for a short period of time. Similar cell damage occurs upon neutrophil activation and degranulation in sterile inflammation conditions [101]. Neutrophils release their own set of proteases, activate proteases that are expressed in a latent form by cells resident in the tissues, and inactivate anti-proteases by oxidation [102, 103] using reactive oxygen species (ROS). In addition to neutrophils, activated macrophages initiate nitric oxide-dependent killing of resident cells [104].

Inflammation resolution is an active, well-coordinated process that normally initiates shortly after the start of an inflammatory reaction. It involves the spatially- and

temporally-controlled production of specialized pro-resolving mediators (SPMs) [105], which coincide with a gradual dilution of chemokine gradients across an inflamed tissue. In consequence, neutrophil recruitment becomes attenuated and eventually stops, and then programmed death by apoptosis is engaged. Apoptotic neutrophils are cleared by macrophage phagocytosis followed by the release of anti-inflammatory and reparative cytokines such as IL-10 and TGFβ1, which can suppress pro-inflammatory signaling from Toll-like receptors [106, 107]. The inflammation resolution sequence ends with the conversion of macrophages into the M2 reparative type and/or the departure of macrophages through the lymphatic vessels. Phagocytosis of apoptotic cells inhibits activated macrophage killing of resident tissue cells and triggers the secretion of VEGF, which participates in the repair of endothelial and epithelial injury.

#### **5. Inflammatory hematopoietic development**

Most of the research on the role of inflammatory signaling in hematopoiesis is concentrated on the emergence and regulation of HSCs. Adult HSCs are capable not just to respond to inflammatory signals but also to secrete pro-inflammatory/ anti-inflammatory cytokines and chemokines [108, 109], including IFN-α [110, 111], IFN-γ [112], TNF-α [113], TGF-β [114], IL-1, and IL-6 [115]. All these cytokines are pleiotropic, and their expression in the blood stem cells may not translate into classical inflammatory cell interactions. However, the expression of PRRs and their co-receptors in HSCs and hematopoietic progenitors [116] directly indicates the involvement of the progenitor domain in sensing stress situations in the hematopoietic system. Hematopoietic progenitors respond to the ligation of TLRs by entering cell cycling, proliferation, and differentiation, and, therefore, can be considered as a part of the innate immune system. In the developmental aspect, hematopoietic progenitors, including HSCs, may arise as highly specialized innate immune cells possessing substantial proliferation and differentiation potential. Indirectly, this notion is supported by another mouse study, which has shown that HSCs in the AGM region exhibit lower levels of IFN-α expression compared to fetal liver HSCs, and this trait can contribute to the lower engraftment potential of the AGM HSCs. Similar to innate immune cells, these HSCs strongly reacted to IFN-α treatment by improvement of their proliferation capacity upon transplantation [117].

Homeostatic expression of another interferon, IFN-γ, a powerful anti-viral cytokine, also activates the proliferation of HSCs *in vivo* during normal hematopoiesis [112]. However, in the hPSC differentiation model, exogenous IFN-γ failed to stimulate the emergence of CD34+ HECs and CD34<sup>+</sup> CD43+ CD45+ CD235a− definitive hematopoietic cells [118]. The iconic pro-inflammatory cytokine IL-1β affects murine adult hematopoiesis by accelerating HSC proliferation and myeloid differentiation through activation of the PU.1 gene program [119]. TNF-α has been implied to play an important role in hematopoietic development based on its abundant expression in the murine yolk sac and fetal liver [120]. Zebrafish studies strongly indicate TNF-α importance for HSC emergence and specification [121]. The data is especially interesting because it implies the involvement of primitive neutrophils in the maintenance and emergence of HSCs in the zebrafish AGM region. Nevertheless, IL-1β and TNF-α signaling did not improve the hematopoietic differentiation of hPSCs [118]. The most straightforward explanation of IFN-γ, IL-1β, and TNF-α failure to influence hPSCderived hematopoiesis is a non-recapitulative differentiation protocol, although, in

#### *Hematopoietic Development of Human Pluripotent Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.112554*

the more positive attitude, it is possible that strong and instructive inflammatory signaling was spontaneously activated during the hematopoietic transition of hPSCs and additional external stimulation could not have any visible effect.

The notion of spontaneous sterile inflammation is supported by recent bioinformatics studies of highly recapitulative hematopoietic differentiation of hPSCs [53]. In the study, efficient hematopoiesis was induced and supported by hematopoietic cytokines produced endogenously in differentiated cultures. Many of the secreted cytokines, such as IL-8, IL-11, IL-16, and M-CSF, were pro-inflammatory, some of them were detected before the emergence of hematopoietic cells. These cytokines programmed the early hematopoietic cells to create an inflammatory milieu that included neutrophil activation, T cell activation, response to bacterial molecular patterns, activation and regulation of innate immune response, phagocytosis, viral response, and others. These observations strongly suggest the instructive role of inflammation in the recapitulative hPSC-hematopoiesis. Such hematopoiesis, however, cannot be sustained long-term in the primary hPSC differentiation probably due to the failure of inflammation resolution, so poorly controlled neutrophil activation leads to the gradual destruction of generated hematopoietic progenitors and blood cells. The optimal solution is to recapitulate the post-yolk sac phase of embryonic hematopoiesis by transferring early hPSC-derived hematopoietic progenitors into secondary differentiating cultures supported by stromal cells. The transfer allows the progenitors to escape from the inflammatory environment before the neutrophil activation. In the secondary differentiation, the escapees undergo extensive proliferation and can develop into lymphoid cells and HSCs in proper culture conditions.

The induction of sterile inflammatory programs in the hPSC differentiation is mediated by DAMPs released from dead or dying hPSCs. Many hPSC cannot easily enter the commitment sequence and have to undergo apoptosis. The major effector of the sterile inflammation is possibly BMP4, the growth factor that is required for the initiation of hematopoietic development. Corroborating evidence [122–124] shows that BMP4 signaling is linked to the induction of inflammatory nuclear factor-κB (NF-κB), nicotinamide adenine dinucleotide phosphate oxidase-1 (NOX1), and intracellular adhesions molecule-1 (ICAM-1), which are the key factors of inflammation initiation. This inflammatory role of BMP4 was described in the adult context, but it demonstrated that there exist molecular mechanisms involving BMP4 in the inflammatory response. It is unknown whether BMP4 induces inflammation at early stages of embryo development, but it is safer to minimize both the concentration and duration of the BMP4 treatment to avoid its participation in the ignition of a potent inflammatory reaction, including neutrophil activation and degranulation.

#### **6. Conclusions**

Accumulating evidence suggests that hematopoiesis develops as a single germ layer. Emerging hematopoietic progenitor cells adapt to the constantly changing conceptal environment by modulating their proliferative and self-renewal potential. They have to use inflammatory mechanisms to penetrate endothelial barriers and settle into transitory or permanent niches. Human PSC-derived hematopoiesis, despite evident problems, has the capacity to develop the self-renewal potential but has to reproduce the *in vivo* development as close as possible.

Inflammatory signaling can play an instructive role in hPSC-derived hematopoiesis. It is not just an artifact of the culture and may reflect some aspects of the hematopoietic development in the conceptus. Inflammation can also negatively influence the expansion of the hematopoietic populations due to the failure of regulated resolution. For successful cell engineering, the early hematopoietic progenitors have to be removed from the primary differentiation culture and used in the secondary differentiation with the immunosuppressive environment.

#### **Acknowledgement**

This work was supported by the Russian Science Foundation, grant #22-15-20063.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Igor M. Samokhvalov1 \* and Anna Liakhovitskaia<sup>2</sup>

1 Engineering Center of Genetic and Cellular Biotechnology, V.I. Vernadsky Crimean Federal University, Simferopol, Crimea

2 Faculty of Health Sciences, Bristol Medical School, Bristol, UK

\*Address all correspondence to: igormikhail@netscape.net

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Hematopoietic Development of Human Pluripotent Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.112554*

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#### **Chapter 5**

## Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status and Challenges

*Yu-Yun Xiong and Yun-Wen Zheng*

#### **Abstract**

The immune system plays a crucial role in recognizing and eliminating foreign antigens, working in conjunction with other bodily systems to maintain the stability and physiological balance of the internal environment. Cell-based immunotherapy has revolutionized the treatment of various diseases, including cancers and infections. However, utilizing autologous immune cells for such therapies is costly, time-consuming, and heavily reliant on the availability and quality of immune cells, which are limited in patients. Induced pluripotent stem cell (iPSC)-derived immune cells, such as T cells, natural killer (NK) cells, macrophages, and dendritic cells (DCs), offer promising opportunities in disease modeling, cancer therapy, and regenerative medicine. This chapter provides an overview of different culture methods for generating iPSC-derived T cells, NK cells, macrophages, and DCs, highlighting their applications in cell therapies. Furthermore, we discuss the existing challenges and future prospects in this field, envisioning the potential applications of iPSC-based immune therapy.

**Keywords:** NK cells, macrophage, iPSC-derived cells, cellular therapy, T cells

#### **1. Introduction**

Immunotherapy has emerged as a highly promising therapeutic approach, particularly in the field of anticancer treatment, showcasing remarkable clinical efficacy. It encompasses various strategies, such as adoptive cell transfer (ACT) and immune checkpoint inhibitors (ICIs). Immune cells, being central players in the pathogenesis and progression of numerous diseases, serve as the fundamental components underlying the effectiveness of immunotherapy [1, 2]. Currently, a Phase I/IIa clinical trial (NCT03666000) is underway, investigating the efficacy of allogeneic CD19 chimeric antigen receptor (CAR)-T cell therapy in the treatment of relapsed/refractory B-non-Hodgkin lymphoma (NHL) and B-cell acute lymphoblastic leukemia (ALL). Furthermore, a Phase I clinical trial (NCT04220684) is evaluating the potential of allogeneic natural killer (NK) cell therapy for acute myeloid leukemia (AML). However, the use of autologous immune cells in such immunotherapy approaches is associated with significant drawbacks, including

high costs, time-intensive procedures, and a heavy reliance on the availability and quality of immune cells, which are limited in patients. Consequently, there is an urgent need to develop an "off the shelf" strategy in an allogeneic setting, allowing for the unlimited proliferation of immune cells to address these challenges and facilitate clinical advancements.

In 2006, the Takahashi and Yamanaka research group achieved a groundbreaking milestone by generating induced pluripotent stem cells (iPSCs) from fibroblasts through the transfection of key factors known as the "Yamanaka factors." This pioneering approach involved the introduction of OCT3/4, SOX2, KLF4, MYC, NANOG, and LIN28 into the cells, resulting in their reprogramming into a pluripotent state [3, 4]. These iPSCs possess similar properties to those of embryonic stem cells (ESCs) in terms of morphology, growth characteristics, and developmental potential [5, 6]. iPSCs offer distinct advantages in the production of immunotherapeutic cells, primarily due to their ability to undergo unlimited reproduction *in vitro* and their ease of genetic modification. These characteristics make iPSCs highly valuable in the generation of immunotherapeutic cells for various applications [7, 8]. Furthermore, unlike ESCs, the clinical use of iPSCs does not raise ethical concerns. As a result, iPSC technology holds great potential for the development of allogeneic "off-the-shelf" cellular therapeutics that can benefit a larger number of patients. This approach is anticipated to be the most effective method for treating various types of malignancies. The advent of iPSC-derived immune cells signifies the beginning of a new era in immunotherapy, paving the way for innovative advancements in the field [9]. In this chapter, we will provide an overview of the key methods utilized for generating immune cells from iPSCs, along with their potential clinical applications and inherent limitations.

#### **2. iPSCs-T cells**

T lymphocytes are derived from hematopoietic stem cells (HSCs) located within the bone marrow. Following their production in the bone marrow, hematopoietic progenitors migrate to the thymus, where they undergo maturation into functional T cells under the influence of thymic hormones. Matured T cells are then distributed throughout the body via the bloodstream, reaching thymus-dependent regions of peripheral immune organs. They can also circulate through lymphatic vessels, peripheral blood, and tissue fluid, performing essential functions in cellular immunity and immune regulation [10]. Adoptive T-cell immunotherapy has emerged as a promising therapeutic strategy for treating various cancers and viral infections [11–14]. However, the current processes involved in generating T-cell lines from donors or genetically modifying autologous T cells for each patient are timeconsuming and expensive. These limitations hinder the widespread and convenient utilization of T cells with antigen specificity. Moreover, the exhaustion of antigenspecific T cells remains a significant challenge in this approach. There is an urgent need for an unlimited supply of T lymphocytes with antigen-specific characteristics to enhance the effectiveness of T-cell therapies. In this regard, potential sources for such T cells include peripheral blood T cells from healthy donors and T cells generated from iPSCs. iPSC technology, as an "off-the-shelf " source of T cells, holds the potential to generate antigen-specific T cells that not only fulfill the requirements for large-scale clinical applications but also ensure the expression of identical T-cell receptor (TCR) genes [15, 16].

*Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

#### **2.1 Generation**

In 2002, Hochedlinger and Jaenisch conducted a groundbreaking experiment, demonstrating the successful transfer of mature lymphocyte nuclei into oocytes. This pioneering approach enabled them to establish ESCs from cloned blastocysts. To advance their research, they further injected these ESCs into tetraploid blastocysts, leading to the generation of monoclonal mice. These significant findings provided compelling evidence that a fully differentiated cell possesses the capacity for reprogramming and can give rise to an adult cloned animal [17]. In nearly all protocols for iPSC-T cell differentiation, the initial step involves reprogramming T-iPSCs from sorted CD4<sup>+</sup> helper T cells and CD8<sup>+</sup> cytolytic T cells obtained from a healthy donor. This reprogramming process is typically achieved through the transfection of Sendai virus vectors or episomal plasmid vectors carrying the four Yamanaka factors [15, 18–21]. During the process of reprogramming, it is possible to retain the antigenic specificity of T cells. This is because T-iPSCs inherit the same rearranged TCR genes at the T-cell receptor loci as the original T cells, which allows for the generation of functional T cells with the desired antigenic specificity [15, 22].

To differentiate T-iPSCs into iPSC-T cells, three different methods have been employed. These methods include the two-dimensional (2D) Delta-like ligand (DLL)1/DLL4-expressing stroma system [22–26], 2D stroma-free system [27, 28], and three-dimensional (3D) artificial thymus organoid (pluripotent stem cell-artificial thymus organoid (PSC-ATO)) system [29, 30]. In 2013, three separate research groups pursued a similar approach by employing feeder cells to differentiate iPSC-T cells. Typically, T-iPSCs were initially cultured on mouse embryonic feeder (MEF) cells to expand pluripotent stem cells *ex vivo*. Subsequently, they were redifferentiated into hematopoietic progenitors using OP9 or C3H10T1/2 feeder cells. It is worth noting that the Notch pathway plays a critical role in the generation of HSCs during this process [31–33]. In mammals, the Notch signaling pathway comprises four Notch receptors (Notch 1–4) and five Notch ligands, including Delta-like ligands (Dll) 1, 3, and 4, and Jagged 1 and 2 (JAG 1 and 2). This pathway is highly conserved and plays a crucial role in various developmental processes, particularly in hematopoiesis [34, 35]. Dar Heinze et al. conducted a study where they discovered that early stimulation of the Notch pathway, achieved through the use of OP9-hDLL4 feeder cells or hDLL4 coated plates, directed hematopoietic progenitors toward differentiation into NK cells and T cells. This finding highlights the critical role of Notch signaling in guiding the fate determination of hematopoietic progenitors toward these specific immune cell lineages [36]. The final step in the differentiation of iPSC-T cells involved seeding the cells onto OP9-DL1 feeder cells and utilizing a combination of cytokines to promote the production of functional T cells [23, 24, 26]. This standardized approach proved successful in generating a significant number of CD8+ T cells, with over 90% of these cells originating from the same T-iPSC source. The results demonstrated that iPSCs are a potent tool for generating and developing T-cell lineages *in vitro*. This advancement holds great potential in the field of regenerative medicine, particularly for the progress of allogeneic therapies [37].

Feeder cells were employed in the generation of iPSC-derived T cells at various stages of the process. Nevertheless, the use of murine-derived stroma feeder layers raises concerns about potential cross-species contamination [27, 38]. Moreover, the utilization of different feeder cells necessitates distinct combinations of serum and basal media for maintenance culture. This complexity in culturing conditions can

increase the risks of uncontrolled differentiation and pose challenges for ensuring quality control of feeder cells and serum. To address this issue, Iriguchi et al. devised a feeder-free and serum-free culture system for differentiating iPSC-T cells [27]. First, they induced hematopoietic progenitors from T-iPSC cell lines. Embryoid bodies (EBs) were generated from single cells without the need for feeder cells and serum. Next, hematopoietic cells were induced in the presence of CHIR99021, bone morphogenetic protein 4 (BMP-4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), and they underwent proliferation in the presence of hematopoietic cytokines, specifically SB431542. Second, to enhance the production of T cells, a combination of CXCL12-SDF1α and a p38 inhibitor, SB203580, was utilized to generate CD4+ CD8αβ<sup>+</sup> double-positive (DP) cells. This innovative approach demonstrates remarkable efficiency and scalability in generating fully functional CD8αβ T cells from iPSCs. More recently, a 3D organoid culture system was reported to successfully generate CAR T cells for "off-the-shelf" manufacturing strategies [29]. Montel-Hagen et al. introduced a 3D artificial thymus organoid (ATO) culture system for the *in vitro* differentiation of human hematopoietic stem and progenitor cells (HSPCs) into functional, mature T cells. They achieved this by utilizing a standardized stromal cell line expressing Notch ligands in a serum-free environment [39]. This innovative continuous culture system facilitated both the specification of hematopoietic cells and their subsequent terminal differentiation into naïve CD3+ CD8αβ + and CD3<sup>+</sup> CD4+ conventional T cells.

While the redifferentiation strategy allows for the realization of "off-the-shelf" T cells [8, 40, 41], there is a risk of rejection by the patient's immune system when using allogeneic T cells. Additionally, a significant concern arises from the production of heterogeneous T cells, which can lead to the generation of potentially harmful alloreactive T cells at varying frequencies. In order to circumvent immune rejection and the production of polyclonal T cells, T-iPSCs can also be derived from patients themselves. Daopeng Yang conducted a study where T-iPSCs were generated from cytotoxic T lymphocytes infiltrating hepatocellular carcinoma (HCC). This was achieved using an integrative Sendai virus vector. The resulting pluripotent cell line exhibited a normal karyotype and could be redifferentiated into rejuvenated CTLs specifically targeting HCC [42]. Munenari Itoh conducted a study where T-iPSCs were generated from monocytes of a melanoma patient. CD8+ T cells were sorted after stimulation with tumor antigens, and then reprogrammed into iPSCs through the exogenous expression of reprogramming factors, utilizing the Sendai virus vector [43].

The generation of T lymphocytes from induced pluripotent stem cells (iPSCs) *in vitro* holds promise for adoptive T-cell therapy. However, the yield and efficiency of lymphoid cells have been limited, and their properties are still only partially understood [7, 44]. Studying T cells derived from ESCs and iPSCs faces challenges due to a limited understanding of their antigen specificity and human leukocyte antigen (HLA) restriction. T cells generated in the laboratory from ESCs or iPSCs exhibit unpredictable T-cell receptor (TCR) repertoires due to random rearrangements of TCR genes, and the mechanisms involved in their selection during *in vitro* differentiation are not yet well understood. Nevertheless, this limitation can be overcome by utilizing iPSCs that possess an endogenous TCR with known antigen specificity [23, 24]. However, this approach requires a time-consuming procedure of cloning T cells specific to the antigen, and it is limited to antigens that can be identified using unique T cells from each patient. Additionally, the clinical use of T cells that recognize antigens through their inherent TCR is constrained by the need to match their specificity with the HLA molecules of the recipient patient.

*Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

#### **2.2 Translation**

One of the notable advantages of iPSCs is their versatility in genetic modification. iPSC-derived T cells have emerged as a prominent tool in cancer immunotherapy. By introducing a chimeric antigen receptor (CAR) or transgenic T-cell receptor (TCR) gene, iPSCs can be transformed into antigen-specific T cells capable of effectively targeting and eliminating cancer cells *in vitro* and *in vivo* [23, 24, 26]. The incorporation of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology further enhances the efficacy and safety of this approach. Through CRISPR-Cas9-mediated insertion of CAR genes into the TRAC locus of the endogenous TCRα constant (TRAC) gene, the risk of host-related alloreactions can be minimized, thus improving immune compatibility [45]. Genetic editing of iPSCs has proven to be a valuable strategy in broadening immune compatibility, supporting the potential for developing "off-the-shelf" cell products. In the context of T-iPSCs (T cell-derived iPSCs), reprogrammed iPSC clones can inherit the original TCR and subsequently be redifferentiated into iPSC-T cells (T cells redifferentiated from iPSCs). However, this approach necessitates time-consuming cloning of antigen-specific T cells and is limited to antigens that can be identified from patientspecific T cells. Furthermore, the therapeutic application of iPSC-T cells is restricted by the need for HLA compatibility between the donors and recipients, significantly limiting the universality of potential "off-the-shelf" applications [16, 25].

Engineering strategies for iPSC-derived T cells involve the introduction of TCR and CAR constructs. TCR-mediated therapies using iPSC-T cells have been carried out by either inheriting the endogenous TCR genes from antigen-specific T cells [22–24] or genetically incorporating exogenous TCRs [25, 29]. Clinical trials have shown the effectiveness of TCR-engineered primary T cells targeting melanoma antigen MART1 [46] and germline antigen New York esophageal squamous cell carcinoma 1 (NY-ESO1) [47]. In these studies, TCR-engineered iPSC-T cells have demonstrated promising results in clinical settings, validating their potential as therapeutic agents for cancer treatment.

Successful outcomes have been observed when introducing a CAR into iPSCs or directly into iPSC-derived T cells to generate antigen-specific T cells [26–28, 30, 48]. CAR engineering effectively redirects T-cell specificity in an HLA-independent manner, eliminating the need for HLA restriction and enhancing the antitumor properties. In a study conducted by Themeli et al. in 2013, T-iPSC clones were generated by reprogramming peripheral T cells from a healthy donor. Subsequently, a secondgeneration CAR specific for CD19 was transduced into the selected T-iPSC clone. The resulting CAR-expressing iPSC-T cells exhibited remarkable antitumor efficacy in a xenograft model, although they exhibited phenotypic similarities to innate γδT cells [26]. This method effectively generated an innate type of T cell expressing a CD8αα homodimer, which influenced the antigen-specific cytotoxic capacity of the redifferentiated T cells in a manner akin to MART-1-specific T cells. To improve upon the conventional approach, Maeda et al. modified the method by purifying differential pressure (DP) cells, which were subsequently stimulated with monoclonal anti-CD3 antibodies to generate CD8αβ T cells. These modified T cells displayed comparable antigen-specific cytotoxicity to the original cytotoxic T lymphocytes (CTLs) [15].

In recent years, iPSC-T cells have emerged as a promising approach in the treatment of various diseases. One notable example is their application in highly aggressive lymphoma, specifically extranodal NK/T-cell lymphoma of the nasal type (ENKL). iPSC-derived cytotoxic T lymphocytes, specifically designed to target the EBV

antigen, have exhibited remarkable results. These cytotoxic T lymphocytes have significantly prolonged patient survival, demonstrated potent tumor-suppressive effects, and persisted as central memory T cells *in vivo* for a minimum of 6 months [49–55]. Rejuvenated cytotoxic T lymphocytes have also exhibited promising outcomes in the treatment of various solid tumors, including cervical cancer associated with human papillomavirus (HPV) infection [56] and renal cell carcinoma [57]. For instance, in cervical cancer cases, T-iPSCs derived from human papillomavirus type 16 (HPV16) E6- or E7-specific cytotoxic T lymphocytes efficiently differentiated into rejuvenated CTLs that specifically targeted HPV16. These rejuvenated CTLs not only displayed prolonged survival compared to the original CTLs but also induced increased tumor shrinkage and significantly prolonged survival in an *in vivo* mouse model [56]. Similarly, in renal cell carcinomas, T-iPSCs established from Wilms tumor 1-specific CTLs effectively suppressed tumor growth in an *in vivo* mouse model [57]. These findings underscore the potential of iPSC-T cells as a valuable therapeutic strategy for targeting and treating various types of cancers, opening new avenues for therapeutic interventions.

#### **3. iPSCs-NK cells**

Natural killer cells are a type of lymphocyte that originates from common lymphoid progenitors (CLPs) during hematopoiesis [58]. They are predominantly found in the bone marrow, peripheral blood, liver, spleen, lung, and lymph nodes. Unlike T and B cells, NK cells possess the unique ability to eliminate tumor cells and virusinfected cells without prior sensitization. NK cells play a crucial role in immune responses, including antitumor activity, defense against viral infections, immune regulation, and even involvement in hypersensitivity and autoimmune diseases in certain cases. They possess the ability to recognize target cells and mediate their killing. When compared to T cells, allogeneic NK cell transfers have a lower risk of graft-versus-host disease (GVHD) and may even decrease the overall risk [59, 60]. However, current strategies dependent on donor cells can only provide a limited supply of custom-made therapeutic NK cells for a restricted number of patients. To overcome this limitation and offer a more accessible treatment option, induced pluripotent stem cells (iPSCs) have been employed in immunotherapy to enable the mass production of NK cells from iPSCs. This approach holds the potential to provide an unlimited supply of "off-the-shelf" NK cells, benefiting a larger number of recipients.

#### **3.1 Generation**

In contrast to the redifferentiation process of iPSCs into iPSC-T cells, NK cells can be reprogrammed directly from iPSC cell lines [61–63] or generate from peripheral blood cells [64] and human fibroblasts [65]. The iPSC cell lines used in these approaches were established from various sources, including umbilical cord blood CD34+ cells and newborn human foreskin fibroblasts [66, 67].

Two methods for the production of iPSC-derived NK cells can be categorized based on the use of feeder cells. In the standard protocol, iPSC cell lines were cultured on MEFs and differentiated into hematopoietic progenitors using M210-B4 cells. To generate spin EBs suitable for aggregation, iPSCs were passaged in TrypLE Select on a low-density MEF layer. Subsequently, the spin EBs were seeded onto plates either with or without EL08-1D2 (a murine embryonic liver cell line) for NK cell differentiation.

#### *Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

This differentiation process was carried out in the presence of specific NK cell initiating cytokines, including interleukin (IL)-3, IL-7, IL-15, stem cell factor (SCF), and fms-like tyrosine kinase receptor-3 ligand (FLT3L) [62, 63, 68].

A method developed by Frank Cichocki et al. eliminates the need for spin EB generation. In this approach, iPSCs were cultured in a combination of small molecules and cytokines to generate CD34<sup>+</sup> hematopoietic progenitor cells. These CD34+ cells were then cocultured with stromal cells that were transduced with Notch ligand and supplemented with cytokines that support the proliferation and differentiation of hematopoietic progenitor cells toward the NK cell lineage. Subsequently, the cells were cocultured with modified K562 cells to further expand the differentiation of iPSC-derived NK cells. This method offers a streamlined process for the efficient production and expansion of iPSC-NK cells [65].

In 2021, Kyle B Lupo et al. developed a serum- and feeder-free system for differentiating iPSCs into NK cells [69]. The differentiation process involved several key steps. First, iPSCs were differentiated into hematopoietic cells using a hematopoietic differentiation medium comprising STEMdiff APEL 2, SCF, bone morphogenetic protein 4, vascular endothelial growth factor, and a rho-associated protein kinase (Rock) inhibitor. To facilitate the formation of EBs, the cells were subjected to a spinning step. After 11 days, hematopoietic progenitor cells were collected from the EBs and seeded in a specialized NK cell differentiation medium containing STEMdiff APEL 2, SCF, IL-7, IL-15, and FLT3L to initiate the differentiation into NK cells. This novel system offers a serum- and feeder-free approach for efficient and controlled differentiation of iPSCs into functional NK cells.

#### **3.2 Translation**

Natural killer cells derived from iPSCs offer the advantage of not being HLA restricted, making iPSC-NK cells an excellent candidate for allogeneic "off-the-shelf" immunotherapy [66]. These iPSC-NK cells serve as a readily available source of cells for immunotherapy, capable of targeting tumors and activating the adaptive immune system to transform a "cold" tumor into a "hot" one by facilitating the recruitment of activated T cells, thus enhancing the efficacy of checkpoint inhibitor therapies [65]. The ability to produce iPSC-NK cells under defined conditions and their demonstrated functional responses indicate their potential as effective therapeutic agents in adoptive transfer settings for treating solid tumors. They offer a renewable source of donor-independent NK cells for immunotherapy, holding great promise in clinical applications [69].

Moreover, iPSC-NK cells, being derived from iPSCs, possess the characteristic feature of being amenable to genetic editing. One strategy for genetic modification of iPSC-NK cells is the incorporation of CARs to enhance their antitumor cytotoxicity. Reports have shown that CARs effectively reprogram NK cell specificity [70]. Notably, Laurent Boissel et al. observed that CAR-NK cells exhibited enhanced elimination of primary chronic lymphocytic leukemia (CLL) cells through antibodydependent cell-mediated cytotoxicity (ADCC) mediated by anti-CD20 monoclonal antibodies [71]. iPSC-NK cells engineered with CARs offer several advantages: (1) they have fewer complications such as cytokine release syndrome (CRS), neurotoxicity, or GVHD; (2) they are not restricted by HLA; and (3) they can activate cytotoxic effects independently of the CAR itself [72–77]. In a study by Dan Kaufman's group, a first-generation CAR incorporating CD4/CD3ζ was introduced into iPSC-NK cells, demonstrating their ability to suppress human immunodeficiency virus (HIV)

replication in CD4<sup>+</sup> T cells [68]. Furthermore, Li et al. tested a series of specialized CARs incorporating costimulatory molecule intracellular domains and found that iPSC-derived NK cells expressing CAR (NK-CAR-iPSC-NK cells) exhibited a typical NK cell phenotype and demonstrated superior antitumor activity compared to iPSC-derived NK cells expressing T-cell CARs (T-CAR-iPSC-NK cells) or non-CARexpressing cells, both *in vitro* and *in vivo* [78].

The persistence and enhanced functional activity of NK cells rely on their interaction with various immune cells that release different cytokines. Among these cytokines, IL-15 plays a crucial role in the differentiation of NK cells [79]. However, during *in vitro* culturing, the frequent addition of IL-15 is necessary due to its short half-life [80]. To overcome this limitation, innovative approaches have been explored, including the use of IL-15 constructs such as secreted IL-15 or an IL-15/IL-15-receptor fusion construct (IL-15RF). Woan et al. developed triple-gene-edited iPSC-NK cells with a high-affinity, noncleavable version of the Fc receptor CD16a, a membranebound interleukin (IL)-15/IL-15R fusion protein, and a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+ . They discovered that these engineered iPSC-NK cells exhibited enhanced anticancer effects in leukemia and multiple myeloma [81]. Another important negative regulator of IL-15 signaling in NK cells is cytokineinducible SH2-containing protein (CIS), encoded by the CISH gene. Huang Zhu et al. found that knockout of CISH in iPSC-NK cells improved the expansion capacity of NK cells and increased their cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations [82]. These modified IL-15 forms provide sustained proliferation signals, thereby augmenting the antitumor efficacy of NK cells both in laboratory settings and in living organisms [83, 84].

Antibody-dependent cell-mediated cytotoxicity is a mechanism by which NK cells exert cytotoxicity through the Fc receptor CD16a. However, CD16a has a low affinity for tumor-bound IgG antibodies and is susceptible to cleavage by a disintegrin and metalloprotease 17 (ADAM17) upon NK cell activation. To address these limitations, Kristin M Snyder et al. enhanced the binding ability of NK cells to antitumor monoclonal antibodies (mAbs) by constructing a fusion protein comprising CD64, the highest-affinity Fc-gamma receptor (FcγR) expressed by leukocytes, and CD16A. This CD64/16A fusion protein lacked the ADAM17 cleavage region in CD16A, preventing downregulation of expression following NK cell activation during ADCC. The CD64/16A iPSC-NK cells exhibited enhanced conjugation to antibody-treated tumor cells, improved ADCC, cytokine production, and ultimately mediated effective tumor cell killing [85]. Another strategy involves mutating CD16a to produce a high-affinity noncleavable variant known as hnCD16. When hnCD16 was incorporated into iPSC-NK cells, the resulting hnCD16-iPSC-NK cells exhibited functional maturity and demonstrated enhanced ADCC against multiple tumor targets. In *in vivo* xenograft studies using a human B-cell lymphoma model, the combination of hnCD16-iPSC-NK cells and anti-CD20 monoclonal antibodies significantly improved regression of B-cell lymphoma and increased overall survival [86]. Additionally, Fanyi Meng fused the ectodomain of hnCD16 with NK cell-specific activating domains in the cytoplasm. This fusion protein showed improved ADCC and cytotoxicity *in vitro* and *in vivo*, as observed in coculture experiments with tumor cell lines and in a xenograft mouse model bearing human B-cell lymphoma [87].

As mentioned above, iPSC-NK cell technology has been utilized for the treatment of hematologic malignancies. However, its applications extend beyond that and encompass the field of solid tumors and viral infections as well. Studies have demonstrated the efficacy of iPSC-derived NK cells in various contexts. For instance, *Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

Hermanson DL found that iPSC-derived NK cells enhanced the antitumor effect and prolonged survival in ovarian cancer [66]. Furthermore, iPSC-derived NK cells have shown promise as an improved approach for treating HIV infection [61, 68] and COVID-19 [88]. These findings highlight the broad potential of iPSC-NK cells in combating a range of diseases, including both cancers and viral infections.

#### **4. iPSCs-macrophages**

Macrophages are a type of white blood cells that reside within tissues and are derived from monocytes, which themselves originate from precursor cells in the bone marrow. Macrophages, along with monocytes, function as phagocytes involved in both nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates. Their primary role is to engulf and digest cell fragments and pathogens, whether in the form of stationary or free cells, and to activate lymphocytes or other immune cells to mount a response against pathogens. Macrophages are immune cells with diverse functions, making them crucial subjects for the study of cellular immunity and molecular immunology. These nonreproductive cells can survive for 2–3 weeks under favorable conditions. While primary cultures of macrophages are often used, they are challenging to maintain for extended periods. Immortalized macrophage cell lines are not suitable for clinical applications, and engineering bone marrow or peripheral blood mononuclear cell (PBMC)-derived primary macrophages is not efficient. Therefore, iPSC-derived macrophages represent a valuable source for myeloid cell-based immunotherapy, offering great potential in the field of immunotherapy [89].

#### **4.1 Generation**

Similar to the production of iPSC-NK cells, iPSCs utilized for differentiating into macrophages originate from iPSC cell lines derived through the reprogramming of fibroblasts using iPSC reprogramming vectors such as OCT4, SOX2, KLF4, and c-MYC [90–92], CD34+ bone marrow cells [93] or peripheral blood monocytes [94, 95]. The methods employed to generate iPSC-macrophages can also be categorized based on the use of feeder cells.

In standard protocols, iPSC cells are initially cocultured with feeder cells such as OP9 mouse stromal cells, in the presence of bone morphogenetic protein 4. This culture condition leads to the differentiation of iPSCs into either 37.8% CD133 HSCs or 9–17% CD43+ hematopoietic progenitors. To generate macrophages, myelomonocytic colonies are cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). The resulting iPSCderived macrophages exhibit functionality and, upon stimulation, secrete substantial amounts of IL-6, IL-10, and tumor necrosis factor alpha (TNF-α) compared to nonstimulated macrophages [90]. IL-3 plays a crucial role in promoting the proliferation of various types of hematopoietic cells during early primitive hematopoiesis and definitive hematopoietic specification. Lachmann, N. et al. combined the use of IL-3 with M-CSF or G-CSF to achieve prolonged and large-scale production of functional granulocytes as well as monocytes/macrophages through EB-based hematopoietic *in vitro* differentiation [96]. They initiated EB formation in ESC medium supplemented with basic fibroblast growth factor (bFGF) and a Rock inhibitor. Subsequently, an intermediate myeloid-cell-forming complex (MCFC) was generated by culturing the EBs in albumin polyvinylalcohol essential lipid (APEL) medium supplemented

with human IL-3, human M-CSF, human G-CSF, or human GM-CSF for a period of 7 days. From day 10 to day 15 onward, monocytes/macrophages or granulocytes were generated. To further promote maturation, the generated monocytes/macrophages or granulocytes were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal serum, L-glutamine, human M-CSF, human G-CSF, or human GM-CSF for 7–10 days.

The use of feeder cells or serum in the culture system adds additional biological and regulatory complexities, which may limit the clinical utility of iPSC-derived monocytes and macrophages. To overcome these challenges, a fully chemically defined, serum- and feeder-free protocol has been developed, significantly improving reproducibility [97, 98]. In this protocol, iPSC cells are used to generate spin embryoid bodies (EBs) in a culture medium supplemented with BMP4, VEGF, and SCF. Subsequently, the EBs are collected and passed through a 40-μm strainer before being transferred to a "factory" medium. This medium, known as X-VIVO 15, is supplemented with Glutamax, 1% penicillin/streptomycin, mercaptoethanol, M-CSF, and IL-3. Utilizing this serum-free protocol, differentiation cultures are established, which continue to produce harvestable and uniform monocytes for extended periods, often lasting up to 1 year [98]. López-Yrigoyen, M. also successfully generated macrophages from an iPSC line using this method [99].

During the differentiation process, the generation of EBs typically involves reseeding and size control steps. However, alternative protocols modified by Cao et al. have been developed to eliminate the need for EB generation by employing serum-free culture conditions. Despite this improvement, the yield obtained from these protocols is relatively low, thereby limiting the scalability of the studies [100]. To address this limitation, Cui, D. et al. present a fully optimized differentiation protocol that incorporates precise timing of steps and the addition of specific cytokines, chemokines, or chemicals. This optimized protocol enables large-scale production of macrophages under serum- and feeder-free conditions without the need for the EB generation step [101]. The development of this fully optimized differentiation protocol represents a significant advancement in the field, providing a reliable and scalable method for generating macrophages from iPSCs. By incorporating precise timing and additional factors, this protocol enhances the efficiency and yield of macrophage production, enabling largescale studies and expanding the potential applications of iPSC-derived macrophages.

#### **4.2 Translation**

Induced pluripotent stem cell (iPSC)-derived macrophages can be engineered with CARs through gene modification. Klichinsky et al. engineered human primary macrophages with an anti-CD19 CAR containing a CD3ζ intracellular domain. These CAR-macrophages exhibited M1-like pro-inflammatory phenotypes and were resistant to the immunosuppressive effects of the tumor microenvironment (TME) through stimulation by the adenovirus vector [102]. Zhang et al. established a platform to engineer iPSCs with a CAR and differentiate them into macrophages, referred to as CAR-iPSCs-macrophages [89]. CAR expression conferred antigen-dependent macrophage functions, including cytokine expression and secretion, polarization toward a pro-inflammatory/antitumor state, enhanced phagocytosis of tumor cells, and demonstrated *in vivo* anticancer activity [89]. Fusing a CD20 single-chain variable fragment (scFv) to FcγR1 in iPSC-macrophages enhanced their ability to engulf and eliminate B-cell leukemic cells both *in vitro* and *in vivo* [103]. Zhang et al. successfully developed iPSC-derived CAR macrophages expressing either a CD19-specific

#### *Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

or a mesothelin-specific fusion receptor, utilizing two distinct endodomain configurations [89]. These iPSC-derived CAR macrophages exhibited antigen-dependent anticancer functions, including cytokine expression and secretion, polarization toward a pro-inflammatory/antitumor state, enhanced phagocytosis of tumor cells, and demonstrated anticancer activity *in vivo* [89].

Macrophages are terminally differentiated cells with limited capacity for expansion. To overcome this limitation and enable the large-scale clinical use of iPSCderived macrophages, Azusa Miyashita et al. employed gene transduction techniques to introduce genes involved in cell growth or senescence suppression, such as c-MYC, in combination with BMI1, murine double minute (MDM2), or enhancer of zeste homolog 2 (EZH2). This approach resulted in the production of human iPSC-derived macrophages that could be propagated for extended periods, functioning as primary macrophages [104]. The engineered cell lines demonstrated a low risk of tumorigenicity, as they exhibited cytokine-dependent proliferation *in vitro*. Importantly, the cytokine-rich conditions required for their growth could not be replicated in the physiological environment *in vivo*.

Patient-derived iPSCs provide valuable cellular models for studying disease pathogenesis and evaluating potential treatments. iPSC-derived macrophages, in particular, hold great promise for investigating various diseases, including cancer. Both macrophages derived from patients' monocytes and iPSCs derived from patients' fibroblasts have been utilized as models in diseases such as Gaucher disease [93]. In Gaucher disease, iPSC-derived macrophages generated from fibroblast lines obtained from patients with type 1 or type 2 Gaucher disease displayed similar characteristics. These macrophages exhibited reduced glucocerebrosidase activity and increased accumulation of glucocerebrosidase and glucosylsphingosine in lysosomes, mirroring the observations in patient monocytes. Furthermore, all the macrophages demonstrated effective phagocytosis of bacteria but exhibited reduced production of intracellular reactive oxygen species (ROS) and impaired chemotaxis [91]. Another example involves iPSCs derived from a patient with hereditary pulmonary alveolar proteinosis. When differentiated into macrophages, these cells exhibited defects in GM-CSF-dependent functions, characteristic of the disease phenotype [93].

#### **5. iPSCs-DCs**

Dendritic cells (DCs) are derived from myeloid pluripotent hematopoietic stem cells and undergo differentiation through two main pathways. Myeloid dendritic cells (MDCs) are generated by stimulation with GM-CSF and differentiate from common precursor cells shared with monocytes and granulocytes. On the other hand, lymphoid dendritic cells (LDCs) or plasmacytoid dendritic cells (pDCs) arise from lymphoid stem cells and share precursor cells with T cells and NK cells. These LDCs are also known as DC2 cells. DCs exhibit widespread distribution in various tissues, including the skin, airways, and lymphatic organs, with notable heterogeneity. Consequently, different tissues have distinct names for DCs. For instance, DCs present in the basal layer of the skin epidermis and spinous cells are referred to as Langerhans cells. DCs are considered the most potent professional antigen-presenting cells (APCs) in the body. They efficiently capture, process, and present antigens to other immune cells. Mature DCs play a crucial role in the initiation, regulation, and maintenance of immune responses by effectively activating naïve T cells, which are central to the immune response.

#### **5.1 Generation**

Dendritic cells derived from iPSCs exhibit characteristics similar to those of other immune cells. Kitadani et al. utilized dermal fibroblasts transfected with Sendai virus vectors to generate iPSCs. These iPSCs were then differentiated into hematopoietic progenitors using a combination of recombinant human bone morphogenetic protein 4 (rhBMP4), recombinant human vascular endothelial growth factor (rhVEGF), growth factor (GF), and recombinant human stem cell factor (rhSCF). Following the addition of a cytokine mixture and CD14 cell sorting, monocytic cell cultures were established and further differentiated into DC cells [105]. In contrast to Kitadani et al.'s method, a feederdependent system was employed for the production of iPSC-derived DCs (iPS-DCs). In this approach, iPSCs were seeded onto OP9 cell layers and cultured in the presence of GM-CSF. Once the cells differentiated into hematopoietic progenitors, the floating cells were collected and transferred to Petri dishes without feeder cells. After 5–7 days, the majority of the floating cells had differentiated into iPS-DCs. To promote their maturation, the cells were transferred and cultured in RPMI-1640/10% fetal calf serum (FCS) supplemented with GM-CSF, IL-4, TNF-α, and anti-CD40 monoclonal antibody [103, 106, 107]. These two different methods demonstrate distinct approaches for generating iPSC-derived DCs. Both techniques have proven effective in producing functional DCs from iPSCs, providing valuable tools for studying the biology of DCs and their potential applications in immunotherapy and disease modeling.

#### **5.2 Translation**

Dentritic cell vaccines have been considered as a promising option for immune cell therapy against cancer. As APCs with robust T-cell stimulating activity, DCs play a pivotal role in orchestrating the immune response. Despite their potential, clinical trials utilizing DC vaccines have encountered challenges, and the outcomes have been largely disappointing [108]. One possible reason for the limited success of DC vaccines in clinical trials is the presence of immune exhaustion in cancer patients, which compromises the ability to generate a sufficient T-cell response [109].

Dentritic cells play a crucial role in immune responses, particularly in stimulating cytotoxic T lymphocytes against viral and tumor-associated antigens through the process of cross-presentation in an MHC class I-restricted manner. Under steadystate conditions, CD141 DCs, residing in interstitial tissues, are primarily involved in maintaining immune homeostasis and inducing tolerance to local antigens. However, iPSC-derived DCs have emerged as a promising avenue for antitumor immunotherapy. In a study by Junya Kitadani et al., iPSCs derived from three healthy donors were differentiated into DCs using feeder-free culturing protocol. Carcinoembryonic antigen (CEA) complementary DNA (cDNA) was then introduced into the iPSCderived DCs through transduction. The researchers demonstrated that these genetically modified iPSC-derived DCs were capable of inducing CEA-specific cytotoxic T lymphocytes in a human model and exhibited significant antitumor effects in a CEA transgenic mouse model [105]. Building upon their previous work, the same research group, in 2023, designed iPSC-derived DCs targeting mesothelin (MSLN) and focused on enhancing the antigen-presenting ability of these cells through the ubiquitin-proteasome system. By simultaneously expressing ubiquitin and MSLN, genetically modified iPSC-derived DCs exhibited potent cytotoxicity against tumors that naturally express MSLN, thereby overcoming immune tolerance and eliciting robust antitumor immune responses [110]. These findings highlight the potential of

*Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

iPSC-derived DCs in antitumor immunotherapy. The ability to genetically modify iPSC-derived DCs to express specific antigens opens up opportunities for personalized and targeted therapies. Further research and development of iPSC-derived DC-based immunotherapies hold promise for enhancing the efficacy of cancer treatments and improving patient outcomes.

#### **6. Limitations and challenges**

Although iPSC-derived cells hold great potential for immunotherapy in clinical applications, several limitations currently hinder their widespread use. One major challenge is the low efficiency of pluripotent reprogramming across various cell types. Reprogramming adult human fibroblasts, for instance, yields a conversion rate of only 0.02–0.05% [111]. Similarly, differentiation of CD34+ mobilized human peripheral blood cells results in a conversion rate of just 0.01–0.02% [112]. In order to achieve successful cell transplantation in patients, a high yield of immune cells is required. The low reprogramming efficiency not only limits the final cell yield but also poses challenges for scaling up the process for clinical applications. Another limitation is the time-consuming nature of the differentiation process. As mentioned earlier, the differentiation of iPSCs into functional immune cells often takes 1–2 months for the development of mature properties. For iPSC-derived T cells, it typically requires 3–7 weeks to expand and reach maturity [27, 29, 38, 44]. iPSC-derived NK cell production takes at least 4 weeks [113], while iPSC-derived macrophages require around 3–7 weeks [114, 115]. The lengthy duration of expansion and differentiation not only increases costs but also prolongs the overall treatment time in clinical therapy. Furthermore, the use of murine-derived feeder cells and serum in current


**Table 1.** *Advantages and limitation of induced pluripotent stem cell (iPSC)-immune cells.* culture approaches introduces the risk of cross-species contamination and variations in the final cell products. Moreover, iPSCs derived from fibroblasts pose challenges in terms of product heterogeneity. Although researchers have developed serum-free and feeder-free culture systems that are more suitable for industrial applications [69, 116], achieving standardization and consistency of the final immune cell products remains a challenge for large-scale industrial production and their use in real-patient applications and clinical trials. The safety of iPSCs is also a concern before large-scale clinical application. Recent studies have reported the tumorigenic potential of undifferentiated iPSCs and the potential for malignant transformation in differentiated iPSCs [117, 118]. Additionally, investigations into TCR gene usage in T cells derived from T-iPSCs and TCR-iPSCs have revealed a small portion of rearranged TCRs, raising further safety considerations [15].

Despite the limitations discussed above, iPSC-derived immune cells offer several advantages (see **Table 1**). To address the challenges associated with iPSC-based immunotherapy, it is crucial to develop standardized protocols and identify novel targets for cell therapy. Additionally, the influence of the immune microenvironment should be carefully considered and investigated to optimize the efficacy of iPSCderived immune cells. By overcoming these limitations and leveraging the strengths of iPSC-based approaches, we can unlock the full potential of iPSC-immune cells in the field of immunotherapy.

#### **7. Conclusion and future perspectives**

The utilization of iPSC technology in immunotherapy has revolutionized traditional immune therapies, as it offers a virtually limitless supply of genetically engineered immune cells that can be readily available for patients' therapeutic needs. This approach eliminates the dependence on scarce cell sources from individual patients, which may not be sufficient for therapeutic purposes. By establishing iPSC banks, standardized protocols for immune cell differentiation can be implemented to ensure scalability and quality control of the generated cell products before administration. Currently, there are approximately 10 iPSC banks that have been established, with a focus on stem cell research and disease-specific cell lines, catering to the needs of both academic and industrial research endeavors [119].

The application of iPSC technology in cell therapy carries certain risks, including tumorigenicity and immune suppression. To address these concerns, novel strategies have been developed to enhance iPSC differentiation and modification. Various approaches can be employed to mitigate the tumorigenic risks associated with iPSCs. For instance, undifferentiated cells can be selectively sorted out using antibodies that target surface biomarkers [120] or eliminated through the use of cytotoxic antibodies [121]. Additionally, chemical inhibitors can be utilized to eradicate any remaining undifferentiated pluripotent cells [122, 123]. While these strategies have shown promise in reducing the risk, it is important to note that long-term culture for reprogramming and redifferentiation may still give rise to unexpected events that contribute to tumorigenicity. Consequently, caution must be exercised during the first-in-human clinical studies to anticipate and address potential issues. To enhance safety in iPSCbased cell therapies, suicide systems can be implemented as a precautionary measure. These systems are designed to induce apoptosis in transduced cells, thereby potentiating therapy without increasing toxicity or evoking cross-resistance to conventional agents. One example is the HSV-TK (herpes simplex virus thymidine kinase) gene,

#### *Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

**Figure 1.** *Application of iPSC immune cells.*

which can be combined with the administration of ganciclovir (GCV) as a safety switch in adoptive T-cell therapy or cancer treatment. However, it is important to note that certain suicide gene systems have shown limitations and may not be as clinically effective as desired [124, 125]. To safeguard against potential risks during clinical and translational investigations of iPSC-based cell therapy, Miki Ando et al. utilized the inducible caspase 9 (iC9) system as a safety mechanism. This system, consisting of an inducible caspase 9 (iC9) gene, can be activated to induce apoptosis in iPSCs if any unexpected issues arise [126].

In addition to their applications in cancer therapy, iPSC immune cells also play significant roles in establishing human disease models [91, 127, 128], drug screening, toxicity assessment [91, 97, 129], and clinical cell banking for "off-the-shelf " therapy (**Figure 1**). The versatility of iPSC immune cells enables their potential use in treating various pathological conditions beyond cancer through genetic modifications [94, 130–133]. This opens up exciting possibilities for utilizing genetically engineered iPSC immune cells as regenerative medical products in clinical practice. With further advancements and research, these cells could offer new avenues for personalized and targeted therapies in the future.

#### **Funding statement**

This research was funded partly by the National Natural Science Foundation of China (82,070,638 and 82,270,697), Jiangsu Provincial Medical Key Discipline Cultivation Unit (JSDW202229), the Science and Technology Planning Project of Guangdong Province of China (2021B1212040016), Japan Society for the Promotion of Science (JSPS), KAKENHI (JP18H02866), and the Grant for International Joint Research Project of the Institute of Medical Science, the University of Tokyo.

*Advances in Pluripotent Stem Cells*

### **Author details**

Yu-Yun Xiong1 and Yun-Wen Zheng1,2,3,4\*

1 Institute of Regenerative Medicine, Department of Dermatology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, China

2 Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, China

3 Department of Medicinal and Life Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Japan

4 Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

\*Address all correspondence to: zheng.yunwen.ld@alumni.tsukuba.ac.jp

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Immune Cell Generation from Human-Induced Pluripotent Stem Cells: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.112657*

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Section 3
