In recent years, the progression of stem cell therapies has shown great promise in advancing the nascent field of regenerative medicine. Considering the non-regenerative nature of the mature central nervous system, the concept that "blank" cells could be reprogrammed and functionally integrated into host neural networks remained intriguing. Previous work has also demonstrated the ability of such cells to stimulate intrinsic growth programs in post-mitotic cells, such as neurons. While embryonic stem cells demonstrated great potential in treating central nervous system pathologies, ethical and technical concerns remained. These barriers, along with the clear necessity for this type of treatment, ultimately prompted the advent of induced pluripotent stem cells. The advantage of pluripotent cells in central nervous system regeneration is multifaceted, permitting differentiation into neural stem cells, neural progenitor cells, glia, and various neuronal subpopulations. The precise spatiotemporal application of extrinsic growth factors in vitro, in addition to microenvironmental signaling in vivo, influences the efficiency of this directed differentiation. While the pluri- or multipotency of these cells is appealing, it also poses the risk of unregulated differentiation and teratoma formation. Cells of the neuroectodermal lineage, such as neuronal subpopulations and glia, have been explored with varying degrees of success. Although the risk of cancer or teratoma formation is greatly reduced, each subpopulation varies in effectiveness and is influenced by a myriad of factors, such as the timing of the transplant, pathology type, and the ratio of accompanying progenitor cells. Furthermore, successful transplantation requires innovative approaches to develop delivery vectors that can mitigate cell death and support integration. Lastly, host immune responses to allogeneic grafts must be thoroughly characterized and further developed to reduce the need for immunosuppression. Translation to a clinical setting will involve careful consideration when assessing both physiologic and functional outcomes. This review will highlight both successes and challenges faced when using human induced pluripotent stem cell-derived cell transplantation therapies to promote endogenous regeneration.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and hPSC-derived cardiac progenitors (hPSC-CPs) represents a promising strategy for regenerating hearts damaged by myocardial infarction (MI). After nearly two decades of experience testing these cell populations in various small- and large-animal MI models, multiple clinical trials have recently been initiated. In this review, we consider the principal lessons learned from preclinical experience with hPSC-CMs and -CPs, focusing on three conclusions that have been supported by the majority of reported transplantation studies. First, hPSC-CMs and -CPs stably engraft in injured hearts and partially remuscularize the infarct scar, but more progress is needed to improve graft cell retention and survival. Second, the transplantation of hPSC-CMs and -CPs has been found to improve contractile function in infarcted hearts, but the mechanistic basis for these effects remains incompletely elucidated. Third, the graft tissue formed by these cells can integrate and activate synchronously with host myocardium, but this capacity for electromechanical integration has been associated with an elevated risk of graft-related arrhythmias. Here, we summarize the preclinical evidence supporting these three observations, identify the relevant gaps and barriers to translation, and summarize ongoing efforts to improve the safety and efficacy of hPSC-CM- and -CP-based regenerative therapies.

Human pluripotent stem cell (hPSC)-derived therapies are a realistic possibility for numerous disorders, including Parkinson's disease. While generating replacement neurons is achievable, immunosuppressive drug challenges, to prevent rejection, remain. Here we adopted a hPSC line (termed H1-FS-8IM), engineered to overexpress 8 immunomodulatory transgenes, to enable transplant immune evasion. In co-cultures, H1-FS-8IM PSC-derived midbrain neurons evaded rejection by T lymphocytes, natural killer cells, macrophages, and dendritic cells. In humanized mice, allogeneic H1-FS-8IM neural grafts evaded rejection, while control hPSC-derived neural grafts evoked activation of human immune cells, elevated inflammatory cytokines in blood and cerebrospinal fluid, and caused spleen and lymph node enlargement. H1-FS-8IM neural grafts retained functionality, reversing motor deficits in Parkinsonian rats. Additional incorporation of a suicide gene into the H1-FS-8IM hPSC line enabled proliferative cell elimination within grafts. Findings demonstrate feasibility of generating a population-wide applicable, safe, off-the-shelf cell product, suitable for treating diseases for which cell-based therapies are a viable option.

The new era of cell therapeutics has started with autologous products to avoid immune rejection. However, therapeutics derived from allogeneic cells could be scaled and made available for a much larger patient population if immune rejection could reliably be overcome. In this review, we outline gene engineering concepts aimed at generating immune-evasive cells. First, we summarize the current state of allogeneic immune cell therapies, and second, we compile the still limited data for allogeneic cell replacement therapies. We emphasize the advances in this fast-developing field and provide an optimistic outlook for future allogeneic cell therapies.

Currently, most cell or tissue transplantations using induced pluripotent stem cells (iPSCs) are anticipated to involve allogeneic iPSCs. However, the immunological properties of iPSCs in an allogeneic setting are not well understood. We previously established a mouse transplantation model of MHC-compatible/minor antigen-mismatched combinations, assuming a hypoimmunogenic iPSC-setting. Here, we found that iPSCs subcutaneously inoculated into MHC-compatible allogeneic host mice resisted rejection and formed teratomas without immunosuppressant administration. Notably, when skin grafts were transplanted onto hosts more than 40 d after the initial iPSCs inoculation, only the skin of the same strain as the initial iPSCs was engrafted. Therefore, donor-specific immune tolerance was induced by a single iPSC inoculation. Diverse analyses, including single-cell RNA-sequencing after transplantation, revealed an increase in regulatory T cell (Treg) population, particularly CD25+ CD103+ effector Tregs within the teratoma and skin grafts. The removal of CD25+ or Foxp3+ cells suppressed the increase in effector Tregs and disrupted graft acceptance, indicating the importance of these cells in the establishment of immune tolerance. Within the teratoma, we observed an increase in TGF-β2 levels, suggesting an association with the increase in effector Tregs. Our results provide important insights for future applications of allogeneic iPSC-based cell or tissue transplantation.

Several countries have either developed or are developing national induced pluripotent stem cell (iPSC) banks of cell lines derived from donors with HLA homozygous genotypes (two identical haplotypes) prevalent in their local populations to provide immune matched tissues and cells to support regenerative medicine therapies. This 'haplobank' approach relies on knowledge of the HLA genotypes of the population to identify the most beneficial haplotypes for patient coverage, and ultimately identify donors or cord blood units carrying two copies of the target haplotype.

A potentially more efficient alternative to a national bank approach is to assess the haplotypes required to provide global patient coverage and to produce a single, global haplobank. Toward that end, we have developed an algorithm to prioritize HLA haplotypes that optimize coverage across the global population.

We analyzed data from eighteen countries participating in the Global Alliance for iPS Therapy (GAiT). A representative pool of HLA genotypes, reflecting the HLA of patients, was derived by sampling from each country's WMDA hematopoietic stem cell donor registry, or surrogate population. An algorithm was created based on HLA-A, -B and -DRB1 haplotype homozygous types with population HLA matching coverage defined by the absence of Host versus Graft (HvG) mismatches at these loci. HLA matching coverage was determined by iteratively selecting HLA haplotypes that provide the largest coverage against patient HLA genotypes sampled from the same population, excluding genotypes compatible with previous iterations.

The top 10 haplotypes for each of the 18 countries were identified with patient coverage ranging from 19.5% in Brazil to 63.8% in Japan, with a mean coverage of 33.3%. In a 'global' model, utilizing the 180 most frequent haplotypes across all 18 populations (equivalent to 10 lines per country), the patient coverage ranged from 54.6% in India to 81.7% in Sweden, with a mean of 68.4%. Our findings demonstrate that global collaboration could more than double the potential for patient HLA matching coverage.

Interrogation of unrelated hematopoietic stem cell donor registry and cord blood bank HLA data demonstrated that HLA-A, -B, and -DRB1 homozygous donors for the top 180 global haplotypes are widely available. These results show that a globally coordinated strategy for haplobanking would reduce redundancy and allow more patients to be treated with the same investment.

Organoids have revolutionized the whole field of biology with their ability to model complex three-dimensional human organs in vitro. Intestinal organoids were especially consequential as the first successful long-term culture of intestinal stem cells, which raised hopes for translational medical applications. Despite significant contributions to basic research, challenges remain to develop intestinal organoids into clinical tools for diagnosis, prognosis, and therapy. In this review, we outline the current state of translational research involving adult stem cell and pluripotent stem cell derived intestinal organoids, highlighting the advances and limitations in disease modeling, drug-screening, personalized medicine, and stem cell therapy. Preclinical studies have demonstrated a remarkable functional recapitulation of infectious and genetic diseases, and there is mounting evidence for the reliability of intestinal organoids as a patient-specific avatar. Breakthroughs now allow the generation of structurally and cellularly complex intestinal models to better capture a wider range of intestinal pathophysiology. As the field develops and evolves, there is a need for standardized frameworks for generation, culture, storage, and analysis of intestinal organoids to ensure reproducibility, comparability, and interpretability of these preclinical and clinical studies to ultimately enable clinical translation.

The clinical success of autologous adoptive cell therapy (ACT) is substantial but wide application is challenged by the quality and quantity of the patient's immune cells and the need for personalized manufacturing processes. Induced pluripotent stem cells (iPSCs) can be differentiated into immune effectors and thus provide an alternative, allogeneic cell source for ACT. Here, we compare iPSC-derived immune effectors to their PBMC-derived counterparts and review iPSC-derived ACT products currently under preclinical and clinical development.

iPSC-derived T cells, NK cells, macrophages, and neutrophils largely mimic their PBMC-derived counterparts in terms of cell-surface marker expression and cytotoxic effector functions. iPSC-derived immune effectors can be engineered with chimeric antigen receptors and other activating receptors to redirect their cytotoxic potential specifically to tumor-associated antigens (TAAs). However, several differences between iPSC- and PBMC-derived immune effectors remain and have inspired additional engineering strategies to enhance the antitumor capacity of iPSC-derived immune effectors.

iPSCs can be engineered to facilitate the generation of immune effectors with homogenous specificity for TAAs and enhanced effector functions. TAA-specific and functionally enhanced iPSC-derived T and NK cells are currently undergoing clinical evaluation in phase 1 trials. Engineered iPSC-derived macrophages and neutrophils are in preclinical development.

Potential trend of regenerative treatment for type I diabetes has been introduced for more than a decade. However, the technologies regarding insulin-producing cell (IPC) production and transplantation are still being developed. Here, we propose the potential IPC production protocol employing mouse gingival fibroblast-derived induced pluripotent stem cells (mGF-iPSCs) as a resource and the pre-clinical approved subcutaneous IPC transplantation platform for further clinical confirmation study. With a multi-step induction protocol, the functional and matured IPCs were generated by 13 days with a long-term survival capability. Further double encapsulation of mGF-iPSC-derived IPCs (mGF-iPSC-IPCs) could preserve the insulin secretion capacity and the transplantation potential of the generated IPCs. To address the potential on IPC transplantation, a 2-step subcutaneous transplantation procedure was established, comprising 1) vascularized subcutaneous pocket formation and 2) encapsulated IPC bead transplantation. The in vivo testing confirmed the safety and efficiency of the platform along with less inflammatory response which may help minimize tissue reaction and graft rejection. Further preliminary in vivo testing on subcutaneous IPC-bead transplantation in an induced type I diabetic mouse model showed beneficial trends on blood glucose control and survival rate sustainability of diabetic mice. Taken together, an established mGF-iPSC-IPC generation protocol in this study will be the potential backbone for developing the iPSC-derived IPC production employing human and animal cell resources. As well as the potential further development of IPC transplantation platform for diabetes treatment in human and veterinary practices using an established subcutaneous encapsulated IPC-bead transplantation platform presented in this study.

The remarkable ability of human pluripotent stem cells (hPSCs) to differentiate into specialized cells of the human body emphasizes their immense potential in treating various diseases. Advances in hPSC technology are paving the way for personalized and allogeneic cell-based therapies. The first-in-human studies showed improved treatment of diseases with no adverse effects, which encouraged the industrial production of this type of medicine. To ensure the quality, safety and efficacy of hPSC-based products throughout their life cycle, it is important to monitor and control their clinical translation through good practices (GxP) regulations. Understanding these rules in advance will help ensure that the industrial development of hPSC-derived products for widespread clinical implementation is feasible and progresses rapidly.

In this review, we discuss the key translational obstacles of hPSCs, outline the current hPSC-based clinical trials, and present a workflow for putative clinical hPSC-based products. Finally, we highlight some future therapeutic opportunities for hPSC-derivatives.

hPSC-based products continue to show promise for the treatment of a variety of diseases. While clinical trials support the relative safety and efficacy of hPSC-based products, further investigation is required to explore the clinical challenges and achieve exclusive regulations for hPSC-based cell therapies.

The promise of curative outcomes for life-limiting diseases using cell therapies is starting to become a reality, not only for patients with end-stage cancer, but also increasingly for regenerative therapies, including dentistry, ocular, neurodegenerative, and cardiac diseases. The introduction of often genetically modified cells into a patient can come with an extensive range of safety considerations. From an immune perspective, cell-based therapies carry inherent consequences and consideration of factors, such as the cell source (donor-derived autologous cells versus allogeneic cells), the intrinsic cellular nature of the therapy, and engineering/manufacturing methods, all of which influence the likelihood of inducing unwanted immune responses. Here, we provide an overview of the potential immune safety risks associated with cell therapies and explore possible mitigation approaches.

Guided tissue regeneration (GTR) has been widely used in the periodontal treatment of intrabony and furcation defects for nearly four decades. The treatment outcomes have shown effectiveness in reducing pocket depth, improving attachment gain and bone filling in periodontal tissue. Although applying GTR could reconstruct the periodontal tissue, the surgical indications are relatively narrow, and some complications and race ethic problems bring new challenges. Therefore, it is challenging to achieve a consensus concerning the clinical benefits of GTR. With the appearance of stem cell-based regenerative medicine, mesenchymal stem/stromal cells (MSCs) have been considered a promising cell resource for periodontal regeneration. In this review, we highlight preclinical and clinical periodontal regeneration using MSCs derived from distinct origins, including non-odontogenic and odontogenic tissues and induced pluripotent stem cells, and discuss the transplantation procedures, therapeutic mechanisms, and concerns to evaluate the effectiveness of MSCs.

Adoptive immunotherapy, notably involving chimeric antigen receptor (CAR)-T cells, has obtained Food and Drug Administration (FDA) approval as a treatment for various hematological malignancies, demonstrating promising preclinical efficacy against cancers. However, the intricate and resource-intensive autologous cell processing, encompassing collection, expansion, engineering, isolation, and administration, hamper the efficacy of this therapeutic modality. Furthermore, conventional CAR T therapy is presently confined to addressing solid tumors due to impediments posed by physical barriers, the potential for cytokine release syndrome, and cellular exhaustion induced by the immunosuppressive and heterogeneous tumor microenvironment. Consequently, a strategic integration of adoptive immunotherapy with synergistic multimodal treatments, such as chemotherapy, radiotherapy, and vaccine therapy etc., emerges as a pivotal approach to surmount these inherent challenges. This collaborative strategy holds the key to addressing the limitations delineated above, thereby facilitating the realization of more precise personalized therapies characterized by heightened therapeutic efficacy. Such synergistic strategy not only serves to mitigate the constraints associated with adoptive immunotherapy but also fosters enhanced clinical applicability, thereby advancing the frontiers of therapeutic precision and effectiveness.

Wilson disease (WD), also known as hepatolenticular degeneration, is an autosomal recessive disorder characterized by disorganized copper metabolism caused by mutations in the ATP7B gene. Currently, the main treatment options for WD involve medications such as d-penicillamine, trientine hydrochloride, zinc acetate, and liver transplantation. However, there are challenges that encompass issues of poor compliance, adverse effects, and limited availability of liver sources that persist. Stem cell therapy for WD is currently a promising area of research. Due to the advancement in stem cell directed differentiation technology in vitro and the availability of sufficient stem cell donors, it is expected to be a potential treatment option for the permanent correction of abnormal copper metabolism. This article discusses the research progress of stem cell therapy for WD from various sources, as well as the challenges and future prospects of the clinical application of stem cell therapy for WD.

Chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy has revolutionized the treatment of various diseases, including cancers and autoimmune disorders. However, all US Food and Drug Administration (FDA)-approved CAR-T cell therapies are autologous, and their widespread clinical application is limited by several challenges, such as complex individualized manufacturing, high costs, and the need for patient-specific selection. Allogeneic off-the-shelf CAR-engineered cell therapy offers promising potential due to its immediate availability, consistent quality, potency, and scalability in manufacturing. Nonetheless, significant challenges, including the risks of graft-versus-host disease (GvHD) and host-cell-mediated allorejection, must be addressed. Strategies such as knocking out endogenous T cell receptors (TCRs) or using alternative therapeutic cells with low GvHD risk have shown promise in clinical trials aimed at reducing GvHD. However, mitigating allorejection remains critical for ensuring the long-term sustainability and efficacy of off-the-shelf cell products. In this review, we discuss the immunological basis of allorejection in CAR-engineered therapies and explore various strategies to overcome this challenge. We also highlight key insights from recent clinical trials, particularly related to the sustainability and immunogenicity of allogeneic CAR-engineered cell products, and address manufacturing considerations aimed at minimizing allorejection and optimizing the efficacy of this emerging therapeutic approach.

Human induced pluripotent stem cells (hiPSCs), derived from reprogrammed adult somatic cells, hold significant promise for disease modelling, personalized medicine, drug discovery, and regenerative therapies. Public awareness and understanding of hiPSCs are crucial for advancing research in this field. However, limited data exists on the general population's knowledge and attitudes toward their use.

This study aimed to assess the awareness and perceptions of hiPSCs among Italian adults through a web-based survey conducted via the EUSurvey platform, using a snowball sampling approach. The survey included demographic information and mandatory questions on knowledge, awareness, and concerns regarding hiPSC technology, with responses collected on a 3-point scale. Statistical analysis was performed using chi-squared tests, with significance set at p ≤ 0.05.

Out of 1874 respondents, the majority were aged 18-35 years (40.5%), female (63.4%), and university-educated (67.2%). Among those familiar with hiPSCs (54.1%, n = 1,201), 95.3% expressed willingness to donate blood samples for hiPSC generation to treat individuals with incurable diseases. Concerns about current research and therapeutic applications were low (less than 20%), but nearly half of the respondents were hesitant or opposed to the use of hiPSCs in animal experiments and their commercialization by pharmaceutical companies. Increased skepticism was observed in older, less educated, religious individuals, and those who were not blood donors. Overall, the Italian public shows strong support for hiPSC-based therapies, though reservations exist around specific ethical and economic issues.

These findings underscore the importance of addressing public concerns through targeted educational campaigns, not only in Italy but globally, to foster a more informed and supportive environment for advancing stem cell research and its clinical applications worldwide. Similar studies have been conducted in Japan, the United States, and Sweden, but there remains a need for all countries to engage with their citizens to better understand how stem cell research is perceived locally. Such engagement is crucial for guiding international strategies in personalized medicine and regenerative therapies, ensuring that emerging technologies are met with both ethical integrity and public trust.

Human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NSCs) hold promise for treating neurodegenerative and demyelinating disorders. However, comprehensive studies on their identity and safety remain limited. In this study, we demonstrate that hiPSC-NSCs adopt a radial glia-associated signature, sharing key epigenetic and transcriptional characteristics with human fetal neural stem cells (hfNSCs) while exhibiting divergent profiles from glioblastoma stem cells. Long-term transplantation studies in mice showed robust and stable engraftment of hiPSC-NSCs, with predominant differentiation into glial cells and no evidence of tumor formation. Additionally, we identified the Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1) as a regulator of astroglial differentiation in hiPSC-NSCs. These findings provide valuable transcriptional and epigenetic reference datasets to prospectively define the maturation stage of NSCs derived from different hiPSC sources and demonstrate the long-term safety of hiPSC-NSCs, reinforcing their potential as a viable alternative to hfNSCs for clinical applications.

The use of allogeneic induced pluripotent stem cell (iPSC)-derived cell therapies for regenerative medicine offers an affordable and realistic alternative to producing individual iPSC lines for each patient in need. Human Leukocyte Antigens (HLA)-homozygous iPSCs matched in hemi-similarity could provide cell therapies with reduced immune rejection covering a wide range of the population with a few iPSC lines. Several banks of HLA-homozygous iPSCs (haplobanks) have been established worldwide or are underway, to provide clinical grade starting material for cell therapies covering the most frequent HLA haplotypes for certain populations. Harmonizing quality standards among haplobanks and creating a global registry could minimize the collective effort and provide a much wider access to HLA-compatible cell therapies for patients with less frequent haplotypes. In this review we present all the current haplobank initiatives and their potential benefits for the global population.

Cell-based therapies for drug-resistant epilepsy using induced pluripotent stem cell-derived inhibitory interneurons are now in early-phase clinical trials, building on findings from trials in Parkinson's disease (PD) and Huntington's disease (HD). Graft rejection and the need for immunosuppressive therapy post-transplantation pose potential barriers to more epilepsy patients becoming potential candidates for inhibitory interneurons transplantation surgery.

The present literature review weighs the evidence for and against human leukocyte antigen (HLA)-mediated graft rejection in PD and HD and examines the potential advantages and drawbacks to five broad approaches to cell-based therapies, including autologous cell culture and transplantation, in vivo reprogramming of glial cells using viral vectors, allogeneic transplantation using off-the-shelf cell lines, transplantation using inhibitory interneurons cultured from HLA-matched cell lines, and the use of hypoimmunogenic-induced pluripotent stem cell-derived inhibitory interneurons. The impact of surgical technique and associated needle trauma on graft rejection is also discussed.

Non-systematic literature review.

While cell-based therapies have enjoyed early successes in treating a host of central nervous system disorders, the immunologic reaction against surgical procedures and implanted materials has remained a major obstacle.

Adapting cell-based therapies using iPSC-derived inhibitory interneurons for epilepsy surgery will similarly require surmounting the challenge of immunogenicity.

Primary bone tumors (PBT), although rare, could pose significant mortality and morbidity risks due to their high incidence of lung metastasis. Survival rates of patients with PBTs may vary based on the tumor type, therapeutic interventions, and the time of diagnosis. Despite advances in the management of patients with these tumors over the past four decades, the survival rates seem not to have improved significantly, implicating the need for novel therapeutic interventions. Surgical resection with wide margins, radiotherapy, and systemic chemotherapy are the main lines of treatment for PBTs. Neoadjuvant and adjuvant chemotherapy, along with emerging immunotherapeutic approaches such as chimeric antigen receptor (CAR)-T cell therapy, have the potential to improve the treatment outcomes for patients with PBTs. CAR-T cell therapy has been introduced as an option in hematologic malignancies, with FDA approval for several CD19-targeting CAR-T cell products. This review aims to highlight the potential of immunotherapeutic strategies, specifically CAR T cell therapy, in managing PBTs.

The motor symptoms of Parkinson's disease (PD) are caused by the progressive loss of dopamine neurons from the substantia nigra. There are currently no treatments that can slow or reverse the neurodegeneration. To restore the lost neurons, international groups have initiated clinical trials using human embryonic or induced pluripotent stem cells (PSCs) to derive dopamine neuron precursors that are used as transplants to replace the lost neurons. Proof-of-principle experiments in the 1980s and 1990s showed that grafts of fetal ventral mesencephalon, which contains the precursors of the substantial nigra, could, under rare circumstances, reverse symptoms of the disease. Improvements in PSC technology and genomics have inspired researchers to design clinical trials using PSC-derived dopamine neuron precursors as cell replacement therapy for PD. We focus here on 4 such first-in-human clinical trials that have begun in the US, Europe, and Japan. We provide an overview of the sources of PSCs and the methods used to generate cells for transplantation. We discuss pros and cons of strategies for allogeneic, immune-matched, and autologous approaches and novel methods for overcoming rejection by the immune system. We consider challenges for safety and efficacy of the cells for durable engraftment, focusing on the genomics-based quality control methods to assure that the cells will not become cancerous. Finally, since clinical trials like these have never been undertaken before, we comment on the value of cooperation among rivals to contribute to advancements that will finally provide relief for the millions suffering from the symptoms of PD.

Although current stem cell therapies exhibit promising potential, the extended process of employing autologous cells and the necessity for donor-host matching to avert the rejection of transplanted cells significantly limit the widespread applicability of these treatments. It would be highly advantageous to generate a pluripotent universal donor stem cell line that is immune-evasive and, therefore, not restricted by the individual's immune system, enabling unlimited application within cell replacement therapies. Before such immune-evasive stem cells can be moved forward to clinical trials, in vivo testing via transplantation experiments in immune-competent animals would be a favorable approach preceding preclinical testing. By using human stem cells in immune competent animals, results will be more translatable to a clinical setting, as no parts of the immune system have been altered, although in a xenogeneic setting. In this way, immune evasiveness, cell survival, and unwanted proliferative effects can be assessed before clinical trials in humans. The current study presents the generation and characterization of three human embryonic stem cell lines (hESCs) for xenogeneic transplantation in immune-competent mice. The major histocompatibility complexes I- and II-encoding genes, B2M and CIITA, have been deleted from the hESCs using CRISPR-Cas9-targeted gene replacement strategies and knockout. B2M was knocked out by the insertion of murine CD47. Human-secreted embryonic alkaline phosphatase (hSEAP) was inserted in a safe harbor site to track cells in vivo. The edited hESCs maintained their pluripotency, karyotypic normality, and stable expression of murine CD47 and hSEAP in vitro. In vivo transplantation of hESCs into immune-competent BALB/c mice was successfully monitored by measuring hSEAP in blood samples. Nevertheless, transplantation of immune-evasive hESCs resulted in complete rejection within 11 days, with clear immune infiltration of T-cells on day 8. Our results reveal that knockout of B2M and CIITA together with species-specific expression of CD47 are insufficient to prevent rejection in an immune-competent and xenogeneic context.

T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety.

In recent years, immunotherapy has become a standard cancer therapy, joining surgery, chemotherapy, and radiation therapy. This therapeutic approach involves the use of patient-derived antigen-specific T cells or genetically modified T cells engineered with chimeric antigen receptors (CAR) or T cell receptors (TCR) that specifically target cancer antigens. However, T cells require ex vivo stimulation for proliferation when used in therapy, and the resulting "exhaustion," which is characterized by a diminished proliferation capacity and anti-tumor activity, poses a significant challenge. As a solution, we reported "rejuvenated" CD8 + T cells that possess high proliferation capacity from induced pluripotent stem cells (iPSCs) in 2013. This review discusses the status and future developments in immunotherapy using iPSC-derived T cells, drawing insights from our research to overcome the exhaustion associated with antigen-specific T cell therapy.

Human allogeneic pancreatic islet transplantation is a life-changing treatment for patients with severe Type 1 Diabetes (T1D) who suffer from hypoglycemia unawareness and high risk of severe hypoglycemia. However, intensive immunosuppression is required to prevent immune rejection of the graft, that may in turn lead to undesirable side effects such as toxicity to the islet cells, kidney toxicity, occurrence of opportunistic infections, and malignancies. The shortage of cadaveric human islet donors further limits islet transplantation as a treatment option for widespread adoption. Alternatively, porcine islets have been considered as another source of insulin-secreting cells for transplantation in T1D patients, though xeno-transplants raise concerns over the risk of endogenous retrovirus transmission and immunological incompatibility. As a result, technological advancements have been made to protect transplanted islets from immune rejection and inflammation, ideally in the absence of chronic immunosuppression, to improve the outcomes and accessibility of allogeneic islet cell replacement therapies. These include the use of microencapsulation or macroencapsulation devices designed to provide an immunoprotective environment using a cell-impermeable layer, preventing immune cell attack of the transplanted cells. Other up and coming advancements are based on the use of stem cells as the starting source material for generating islet cells 'on-demand'. These starting stem cell sources include human induced pluripotent stem cells (hiPSCs) that have been genetically engineered to avoid the host immune response, curated HLA-selected donor hiPSCs that can be matched with recipients within a given population, and multipotent stem cells with natural immune privilege properties. These strategies are developed to provide an immune-evasive cell resource for allogeneic cell therapy. This review will summarize the immunological challenges facing islet transplantation and highlight recent bio-engineering and cell-based approaches aimed at avoiding immune rejection, to improve the accessibility of islet cell therapy and enhance treatment outcomes. Better understanding of the different approaches and their limitations can guide future research endeavors towards developing more comprehensive and targeted strategies for creating a more tolerogenic microenvironment, and improve the effectiveness and sustainability of islet transplantation to benefit more patients.

Acute liver failure (ALF) results from severe liver damage or end-stage liver disease. It is extremely fatal and causes serious health and economic burdens worldwide. Once ALF occurs, liver transplantation (LT) is the only definitive and recommended treatment; however, LT is limited by the scarcity of liver grafts. Consequently, the clinical use of bioartificial liver (BAL) has been proposed as a treatment strategy for ALF. Human primary hepatocytes are an ideal cell source for these methods. However, their high demand and superior viability prevent their widespread use. Hence, finding alternatives that meet the seed cell quality and quantity requirements is imperative. Stem cells with self-renewing, immunogenic, and differentiative capacities are potential cell sources. MSCs and its secretomes encompass a spectrum of beneficial properties, such as anti-inflammatory, immunomodulatory, anti-ROS (reactive oxygen species), anti-apoptotic, pro-metabolomic, anti-fibrogenesis, and pro-regenerative attributes. This review focused on the recent status and future directions of stem cell-based strategies in BAL for ALF. Additionally, we discussed the opportunities and challenges associated with promoting such strategies for clinical applications.

The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.

Stem cell therapy is an emerging treatment paradigm for stroke patients with remaining neurological deficits. While allogeneic cell transplants overcome the manufacturing constraints of autologous grafts, they can be rejected by the recipient's immune system, which identifies foreign cells through the human leukocyte antigen (HLA) system. The heterogeneity of HLA molecules in the human population would require a very high number of cell lines, which may still be inadequate for patients with rare genetic HLAs. Here, we outline key progress in genetic HLA engineering in pluripotent stem and derived cells to evade the host's immune system, reducing the number of allogeneic cell lines required, and examine safety measures explored in both preclinical studies and upcoming clinical trials.

With the aging population, the incidence of degenerative diseases such as dementia and arthritis is on the rise. To combat these diseases, cell therapies using induced pluripotent stem cells (iPSCs) are being developed worldwide. However, challenges such as high development costs and immune compatibility persist. Thus, methods such as generating patient-specific iPSCs or genetically edited iPSCs with deleted immune-related genes are being researched. Applying these approaches is limited due to high cost and safety concerns of gene editing. Therefore, we focused on an alternative method using human leukocyte antigen (HLA)-homozygous cell lines, which could overcome immune rejection issues economically. We investigated diseases that could potentially be treated with cell therapy and identified which HLA-homozygous cell lines could be most effectively used for the efficient production of therapeutic cell lines. The results of the study showed that cell therapy could be applied to a wide range of diseases, and expanding the population that can benefit from HLA-homozygous iPSC lines could help popularize these treatment methods. We highlight the necessity of a global HLA-homozygous iPSC bank.

Regenerative medicine is an emerging and rapidly evolving field of research and therapeutics aimed to restore, maintain, and improve body functions. In the adult mammalian brain, very few neurons, if any, are generated after disease onset or an injury, and its ability to self-repair is therefore limited. Replacing neurons that are lost during neurodegenerative diseases or due to injury therefore represents one of the major challenges to modern medicine. In this introductory chapter, we describe the basic biology of stem cells and outline how stem cells and cell reprogramming can be utilized to create new neurons for therapeutic purposes that are discussed in detail in other chapters in this handbook.

Human iPSCs' derivation and use in clinical studies are transforming medicine. Yet, there is a high cost and long waiting time associated with autologous iPS-based cellular therapy, and the genetic engineering of hypo-immunogenic iPS cell lines is hampered with numerous hurdles. Therefore, it is increasingly interesting to create cell stocks based on HLA haplotype distribution in a given population. This study aimed to assess the potential of HLA-based iPS banking for the Saudi population.

In this study, we interrogated the HLA database of the Saudi Stem Cell Donor Registry (SSCDR), containing high-resolution HLA genotype data from 64,315 registered Saudi donors at the time of analysis. This database was considered to be a representative sample of the Saudi population. The most frequent HLA haplotypes in the Saudi population were determined, and an in-house developed iterative algorithm was used to identify their HLA matching percentages in the SSCDR database and cumulative coverage. Subsequently, to develop a clinically relevant protocol for iPSCs generation, and to illustrate the applicability of the concept of HLA-based banking for cell therapy purposes, the first HLA-based iPS cell line in Saudi Arabia was generated. Clinically relevant methods were employed to generate the two iPS clones from a homozygous donor for the most prevalent HLA haplotype in the Saudi population. The generated lines were then assessed for pluripotency markers, and their ability to differentiate into all three germ layers, beating cardiomyocytes, and neural progenitors was examined. Additionally, the genetic stability of the HLA-iPS cell lines was verified by comparing the mutational burden in the clones and the original blood sample, using whole-genome sequencing. The standards set by the American College of Medical Genetics and Genomics (ACMG) were used to determine the clinical significance of identified variants.

The analysis revealed that the establishment of only 13 iPSC lines would match 30% of the Saudi population, 39 lines would attain 50% coverage, and 596 lines would be necessary for over 90% coverage. The proof-of-concept HLA-iPSCs, which cover 6.1% of the Saudi population, successfully demonstrated pluripotency and the ability to differentiate into various cell types including beating cardiomyocytes and neuronal progenitors. The comprehensive genetic analysis corroborated that all identified variants in the derived iPSCs were inherently present in the original donor sample and were classified as benign according to the standards set by the ACMG.

Our study sets a road map for introducing iPS-based cell therapy in the Kingdom of Saudi Arabia. It underscores the pragmatic approach of HLA-based iPSC banking which circumvents the limitations of autologous iPS-based cellular therapies. The successful generation and validation of iPSC lines based on the most prevalent HLA haplotype in the Saudi population signify a promising step toward broadening the accessibility and applicability of stem cell therapies and regenerative medicine in Saudi Arabia.

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing β-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to β-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a "Trojan horse".

Mesenchymal stem cells (MSCs) are effective in treating autoimmune diseases and managing various conditions, such as engraftment of allogeneic islets. Additionally, autologous and HLA-matched allogeneic MSCs can aid in the engraftment of human allogeneic kidneys with or without low doses of tacrolimus, respectively. However, HLA alloantigens are problematic because cell therapy uses more HLA-mismatched allogeneic cells than autologous for convenience and standardization. In particular, HLA-mismatched MSCs showed increased Ag-specific T/B cells and reduced viability faster than HLA-matched MSCs. In CRISPR/Cas9-based cell therapy, Cas9 induce T cell activation in the recipient's immune system. Interestingly, despite their immunogenicity being limited to the cells with foreign Ags, the accumulation of HLA alloantigen-sensitized T/B cells may lead to allograft rejection, suggesting that alloantigens may have a greater scope of adverse effects than foreign Ags. To avoid alloantigen recognition, the β2-microglobulin knockout (B2MKO) system, eliminating class-I MHC, was able to avoid rejection by alloreactive CD8 T cells compared to controls. Moreover, universal donor cells in which both B2M and Class II MHC transactivator (CIITA) were knocked out was more effective in avoiding immune rejection than single KO. However, B2MKO and CIITA KO system remain to be controlled and validated for adverse effects such as the development of tumorigenicity due to deficient Ag recognition by CD8 T and CD4 T cells, respectively. Overall, better HLA-matching or depletion of HLA alloantigens prior to cell therapy can reduce repetitive transplantation through the long-term survival of allogeneic cell therapy, which may be especially important for patients seeking allogeneic transplantation.

Adoptive cell therapy (ACT) has transformed the treatment landscape for cancer and infectious disease through the investigational use of chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and viral-specific T-cells (VSTs). Whilst these represent breakthrough treatments, there are subsets of patients who fail to respond to autologous ACT products. This is frequently due to impaired patient T-cell function or "fitness" as a consequence of prior treatments and age, and can be exacerbated by complex manufacturing protocols. Further, the manufacture of autologous, patient-specific products is time-consuming, expensive and non-standardised. Induced pluripotent stem cells (iPSCs) as an allogeneic alternative to patient-specific products can potentially overcome the issues outlined above. iPSC technology provides an unlimited source of rejuvenated iPSC-derived T-cells (T-iPSCs) or natural killer (NK) cells (NK-iPSCs), and in the context of the growing field of allogeneic ACT, iPSCs have enormous potential as a platform for generating off-the-shelf, standardised, "fit" therapeutics for patients. In this review, we evaluate current and future applications of iPSC technology in the CAR-T/NK, TIL and VST space. We discuss current and next-generation iPSC manufacturing protocols, and report on current iPSC-based adoptive therapy clinical trials to elucidate the potential of this technology as the future of ACT.

Identifying human leukocyte antigen (HLA) haplotype-homozygous donors for the generation of induced pluripotent stem (iPS) cell lines permits the construction of biobanks immunologically compatible with significant numbers of individuals for use in therapy. However, two questions must be addressed to create such a bank: how many cell lines are necessary to match most of the recipient population and how many people should be tested to find these donors? In Japan and the UK, 50 and 100 distinct HLA-A, -B, and -DRB1 triple-homozygous haplotypes would cover 90% of those populations, respectively. Using data from the Brazilian National Registry of Bone Marrow Donors (REDOME), encompassing 4,017,239 individuals, we identified 1,906 distinct triple-homozygous HLA haplotypes. In Brazil, 559 triple-homozygous cell lines cover 95% of the population, and 3.8 million people would have to be screened. Finally, we show the contribution of the 30 most frequent triple-homozygous HLA haplotypes in Brazil to populations of different countries.

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

Stem cell therapies can potentially treat various retinal degenerative diseases, including age-related macular degeneration (AMD) and inherited retinal diseases like retinitis pigmentosa. For these diseases, transplanted cells may include stem cell-derived retinal pigmented epithelial (RPE) cells, photoreceptors, or a combination of both. Although stem cell-derived RPE cells have progressed to human clinical trials, therapies using photoreceptors and other retinal cell types are lagging. In this review, we discuss the potential use of human pluripotent stem cell (hPSC)-derived photoreceptors for the treatment of retinal degeneration and highlight the progress and challenges for their efficient production and clinical application in regenerative medicine.

In transplantation using allogeneic induced pluripotent stem cells (iPSCs), strategies focused on major histocompatibility complexes were adopted to avoid immune rejection. We showed that minor antigen mismatches are a risk factor for graft rejection, indicating that immune regulation remains one of the most important issues. In organ transplantation, it has been known that mixed chimerism using donor-derived hematopoietic stem/progenitor cells (HSPCs) can induce donor-specific tolerance. However, it is unclear whether iPSC-derived HSPCs (iHSPCs) can induce allograft tolerance. We showed that 2 hematopoietic transcription factors, Hoxb4 and Lhx2, can efficiently expand iHSPCs with a c-Kit+Sca-1+Lineage- phenotype, which possesses long-term hematopoietic repopulating potential. We also demonstrated that these iHSPCs can form hematopoietic chimeras in allogeneic recipients and induce allograft tolerance in murine skin and iPSC transplantation. With mechanistic analyses, both central and peripheral mechanisms were suggested. We demonstrated the basic concept of tolerance induction using iHSPCs in allogeneic iPSC-based transplantation.

Corneal endothelial dysfunction is one of the leading causes of corneal blindness, and the current conventional treatment option is corneal transplantation using a cadaveric donor cornea. However, there is a global shortage of suitable donor graft material, necessitating the exploration of novel therapeutic approaches. A stem cell-based regenerative medicine approach using induced pluripotent stem cells (iPSCs) offers a promising solution, as they possess self-renewal capabilities, can be derived from adult somatic cells, and can be differentiated into all cell types including corneal endothelial cells (CECs). This review discusses the progress and challenges in developing protocols to induce iPSCs into CECs, focusing on the different media formulations used to differentiate iPSCs to neural crest cells (NCCs) and subsequently to CECs, as well as the characterization methods and markers that define iPSC-derived CECs. The hurdles and solutions for the clinical application of iPSC-derived cell therapy are also addressed, including the establishment of protocols that adhere to good manufacturing practice (GMP) guidelines. The potential risks of genetic mutations in iPSC-derived CECs associated with long-term in vitro culture and the danger of potential tumorigenicity following transplantation are evaluated. In all, this review provides insights into the advancement and obstacles of using iPSC in the treatment of corneal endothelial dysfunction.

While cord blood (CB) is primarily utilized in allogeneic hematopoietic cell transplantation (HCT), the development of novel cell therapy products from CB is a growing and developing field. Compared to adult blood, CB is characterized by a higher percentage of hematopoietic stem cells (HSCs) and progenitor cells, less mature immune cells that retain a high capacity of proliferation, and stronger immune tolerance that requires less stringent HLA-matching when used in the allogenic setting. Given that CB is an FDA regulated product and along with its unique cellular composition, CB lends itself as a readily available and safe starting material for the development of off-the-shelf cell therapies. Moreover, non-hematologic cells such as mesenchymal stem cell (MSCs) residing in CB or CB tissue also have potential in regenerative medicine and inflammatory and autoimmune conditions. In this review, we will focus on recent clinical development on CB-derived cellular therapies in the field of oncology, including T-cell therapies such as chimeric antigen receptor (CAR) T-cells, regulatory T-cells, and virus-specific T-cells; NK-cell therapies, such as NK cell engagers and CAR NK-cells; CB-HCT and various modifications; as well as applications of MSCs in HCT.

Pluripotent stem cell (PSC)-derived cardiomyocytes are a promising source of cells in myocardial regeneration therapy for end-stage heart failure. Because most previous reports have focussed on xenotransplantation models using immunocompromised animals, studies on immune rejection in allogeneic transplantation models are needed for preclinical and clinical applications. Human leukocyte antigen (HLA) plays an important role in allogeneic transplantation, and cell bank projects are currently underway worldwide to stock induced pluripotent stem cells (iPSCs) generated from healthy individuals with homozygous HLA haplotypes. However, it is difficult to stock iPSCs that match the entire population in these cell banks; thus, several groups have produced hypoimmunogenic PSCs by knocking out HLA. These HLA-knockout PSCs were able to avoid rejection by T cells but still suffered rejection by natural killer (NK) cells caused by 'missing self-recognition'. Recent studies have attempted to generate hypoimmunogenic PSCs with gene editing to inhibit NK cell activation. Regenerative medicine using autologous iPSCs can be an ideal transplantation therapy, but, currently, there are major hurdles to its practical application. Hopefully, further research will resolve these issues. This review provides an overview of the current understanding and progress in this field.

In recent years, great strides have been made toward the development of immune cell-based therapies in the treatment of refractory malignancies. Primary T cells and NK cells armed with chimeric antigen receptors have achieved tremendous clinical success especially in patients with leukaemia and lymphoma. However, the autologous origin of these effector cells means that a single batch of laboriously engineered cells treats only a certain patient, leading to high cost, ununiform product quality, and risk of delay in treatment, and therefore results in restricted accessibility of these therapies to the overwhelming majority of the patients. Addressing these tricky obstacles calls for the development of universal immune cell products that can be provided 'off the shelf' in a large amount. Pluripotent stem cells (PSCs), owing to their unique capacity of self-renewal and the potential of multi-lineage differentiation, offer an unlimited cell source to generate uniform and scalable engineered immune cells. This review discusses the major advances in the development of PSC-derived immune cell differentiation approaches and their therapeutic potential in treating both hematologic malignancies and solid tumours. We also consider the potency of PSC-derived immune cells as an alternative therapeutic strategy for other diseases, such as autoimmune diseases, fibrosis, infections, et al.

The development of methods to derive induced pluripotent stem cells (iPSCs) has propelled stem cell research, and has the potential to revolutionize many areas of medicine, including cancer immunotherapy. These cells can be propagated limitlessly and can differentiate into nearly any specialized cell type. The ability to perform precise multigene engineering at the iPSC stage, generate master cell lines after clonal selection, and faithfully promote differentiation along natural killer (NK) cells and T-cell lineages is now leading to new opportunities for the administration of off-the-shelf cytotoxic lymphocytes with direct antigen targeting to treat patients with relapsed/refractory cancer. In this review, we highlight the recent progress in iPSC editing and guided differentiation in the development of NK- and T-cell products for immunotherapy. We also discuss some of the potential barriers that remain in unleashing the full potential of iPSC-derived cytotoxic effector cells in the adoptive transfer setting, and how some of these limitations may be overcome through gene editing.