Acquired genetic changes in human pluripotent stem cells: origins and consequences.

In the 20 years since human embryonic stem cells, and subsequently induced pluripotent stem cells, were first described, it has become apparent that during long-term culture these cells (collectively referred to as 'pluripotent stem cells' (PSCs)) can acquire genetic changes, which commonly include gains or losses of particular chromosomal regions, or mutations in certain cancer-associated genes, especially TP53. Such changes raise concerns for the safety of PSC-derived cellular therapies for regenerative medicine. Although acquired genetic changes may not be present in a cell line at the start of a research programme, the low sensitivity of current detection methods means that mutations may be difficult to detect if they arise but are present in only a small proportion of the cells. In this Review, we discuss the types of mutations acquired by human PSCs and the mechanisms that lead to their accumulation. Recent work suggests that the underlying mutation rate in PSCs is low, although they also seem to be particularly susceptible to genomic damage. This apparent contradiction can be reconciled by the observations that, in contrast to somatic cells, PSCs are programmed to die in response to genomic damage, which may reflect the requirements of early embryogenesis. Thus, the common genetic variants that are observed are probably rare events that give the cells with a selective growth advantage.

CAR immune cells: design principles, resistance and the next generation.

The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years.

Gene and cell therapy of human genetic diseases: Recent advances and future directions.

Disruptions in normal development and the emergence of health conditions often result from the malfunction of vital genes in the human body. Decades of scientific research have focused on techniques to modify or substitute defective genes with healthy alternatives, marking a new era in disease treatment, prevention and cure. Recent strides in science and technology have reshaped our understanding of disorders, medication development and treatment recommendations, with human gene and cell therapy at the forefront of this transformative shift. Its primary objective is the modification of genes or adjustment of cell behaviour for therapeutic purposes. In this review, we focus on the latest advances in gene and cell therapy for treating human genetic diseases, with a particular emphasis on FDA and EMA-approved therapies and the evolving landscape of genome editing. We examine the current state of innovative gene editing technologies, particularly the CRISPR-Cas systems. As we explore the progress, ethical considerations and prospects of these innovations, we gain insight into their potential to revolutionize the treatment of genetic diseases, along with a discussion of the challenges associated with their regulatory pathways. This review traces the origins and evolution of these therapies, from conceptual ideas to practical clinical applications, marking a significant milestone in the field of medical science.

Upskilling the cell therapy manufacturing workforce: design, implementation, and evaluation of a massive open online course.

Cell therapies have gained prominence as a promising therapeutic modality for treating a range of diseases. Despite the recent clinical successes of cell therapy products, very few formal training programs exist for cell therapy manufacturing. To meet the demand for a well-trained workforce, we assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing. The course covers the basic cell and systems physiology underlying cell therapy products, in addition to explaining end-to-end manufacturing from cell acquisition through to patient treatment, industrialization, and regulatory processes. As of September 2023, >10,000 learners have enrolled in the course, and >90% of respondents to the course exit survey indicated that they were "very likely" or "likely" to recommend the course to a peer. In this article, we discuss our experience in the collaborative design and implementation of the online course as well as lessons learned from quantitative and qualitative student feedback. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.NEW & NOTEWORTHY We assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.

Potential applications of deep learning in single-cell RNA sequencing analysis for cell therapy and regenerative medicine.

When used in cell therapy and regenerative medicine strategies, stem cells have potential to treat many previously incurable diseases. However, current application methods using stem cells are underdeveloped, as these cells are used directly regardless of their culture medium and subgroup. For example, when using mesenchymal stem cells (MSCs) in cell therapy, researchers do not consider their source and culture method nor their application angle and function (soft tissue regeneration, hard tissue regeneration, suppression of immune function, or promotion of immune function). By combining machine learning methods (such as deep learning) with data sets obtained through single-cell RNA sequencing (scRNA-seq) technology, we can discover the hidden structure of these cells, predict their effects more accurately, and effectively use subpopulations with differentiation potential for stem cell therapy. scRNA-seq technology has changed the study of transcription, because it can express single-cell genes with single-cell anatomical resolution. However, this powerful technology is sensitive to biological and technical noise. The subsequent data analysis can be computationally difficult for a variety of reasons, such as denoising single cell data, reducing dimensionality, imputing missing values, and accounting for the zero-inflated nature. In this review, we discussed how deep learning methods combined with scRNA-seq data for research, how to interpret scRNA-seq data in more depth, improve the follow-up analysis of stem cells, identify potential subgroups, and promote the implementation of cell therapy and regenerative medicine measures.

Cell therapy in Huntington's disease: Taking stock of past studies to move the field forward.

Huntington's disease (HD) is a rare inherited neurodegenerative disease that manifests mostly in adulthood with progressive cognitive, behavioral, and motor dysfunction. Neuronal loss occurs predominantly in the striatum but also extends to other brain regions, notably the cortex. Most patients die around 20 years after motor onset, although there is variability in the rate of progression and some phenotypic heterogeneity. The most advanced experimental therapies currently are huntingtin-lowering strategies, some of which are in stage 3 clinical trials. However, even if these approaches are successful, it is unlikely that they will be applicable to all patients or will completely halt continued loss of neural cells in all cases. On the other hand, cellular therapies have the potential to restore atrophied tissues and may therefore provide an important complementary therapeutic avenue. Pilot studies of fetal cell grafts in the 2000s reported the most dramatic clinical improvements yet achieved for this disease, but subsequent studies have so far failed to identify methodology to reliably reproduce these results. Moving forward, a major challenge will be to generate suitable donor cells from (nonfetal) cell sources, but in parallel there are a host of procedural and trial design issues that will be important for improving reliability of transplants and so urgently need attention. Here, we consider findings that have emerged from clinical transplant studies in HD to date, in particular new findings emerging from the recent multicenter intracerebral transplant HD study, and consider how these data may be used to inform future cell therapy trials.

Using chimeric antigen receptor T-cell therapy to fight glioblastoma multiforme: past, present and future developments.

INTRODUCTION: Glioblastoma multiforme (GBM) constitutes one of the deadliest tumors to afflict humans, although it is still considered an orphan disease. Despite testing multiple new and innovative therapies in ongoing clinical trials, the median survival for this type of malignancy is less than two years after initial diagnosis, regardless of therapy. One class of promising new therapies are chimeric antigen receptor T cells or CAR-T which have been shown to be very effective at treating refractory liquid tumors such as B-cell malignancies. However, CAR-T effectivity against solid tumors such as GBM has been limited thus far. METHODS: A Pubmed, Google Scholar, Directory of Open Access Journals, and Web of Science literature search using the terms chimeric antigen receptor or CAR-T, GBM, solid tumor immunotherapy, immunotherapy, and CAR-T combination was performed for publication dates between January 1987 and November 2021. RESULTS: In the current review, we present a comprehensive list of CAR-T cells developed to treat GBM, we describe new possible T-cell engineering strategies against GBM while presenting a short introductory history to the reader regarding the origin(s) of this cutting-edge therapy. We have also compiled a unique list of anti-GBM CAR-Ts with their specific protein sequences and their functions as well as an inventory of clinical trials involving CAR-T and GBM. CONCLUSIONS: The aim of this review is to introduce the reader to the field of T-cell engineering using CAR-Ts to treat GBM and describe the obstacles that may need to be addressed in order to significantly delay the relentless growth of GBM.

Cell therapy for advanced liver diseases: Repair or rebuild.

Advanced liver disease presents a significant worldwide health and economic burden and accounts for 3.5% of global mortality. When liver disease progresses to organ failure the only effective treatment is liver transplantation, which necessitates lifelong immunosuppression and carries associated risks. Furthermore, the shortage of suitable donor organs means patients may die waiting for a suitable transplant organ. Cell therapies have made their way from animal studies to a small number of early clinical trials. Herein, we review the current state of cell therapies for liver disease and the mechanisms underpinning their actions (to repair liver tissue or rebuild functional parenchyma). We also discuss cellular therapies that are on the clinical horizon and challenges that must be overcome before routine clinical use is a possibility.

Cord blood beyond transplantation: can we use the experience to advance all cell therapies?

Unrelated cord blood (CB) units, already manufactured, fully tested and stored, are high-quality products for haematopoietic stem cell transplantation and cell therapies, as well as an optimal starting material for cell expansion, cell engineering or cell re-programming technologies. CB banks have been pioneers in the development and implementation of Current Good Manufacturing Practices for cell-therapy products. Sharing their technological and regulatory experience will help advance all cell therapies, CB-derived or not, particularly as they transition from autologous, individually manufactured products to stored, 'off-the shelf' treatments. Such strategies will allow broader patient access and wide product utilisation.

Revisiting cell and gene therapies in Huntington's disease.

Neurodegenerative movement disorders, such as Huntington's disease (HD), share a progressive and relentless course with increasing motor disability, linked with neuropsychiatric impairment. These diseases exhibit diverse pathophysiological processes and are a topic of intense experimental and clinical research due to the lack of therapeutic options. Restorative therapies are promising approaches with the potential to restore brain circuits. However, there were less compelling results in the few clinical trials. In this review, we discuss cell replacement therapies applied to animal models and HD patients. We thoroughly describe the initial trials using fetal neural tissue transplantation and recent approaches based on alternative cell sources tested in several animal models. Stem cells were shown to generate the desired neuron phenotype and/or provide growth factors to the degenerating host cells. Besides, genetic approaches such as RNA interference and the CRISPR/Cas9 system have been studied in animal models and human-derived cells. New genetic manipulations have revealed the capability to control or counteract the effect of human gene mutations as described by the use of antisense oligonucleotides in a clinical trial. In HD, innovative strategies are at forefront of human testing and thus other brain genetic diseases may follow similar therapeutic strategies.

Protection of retinal ganglion cells in glaucoma: Current status and future.

Glaucoma is a neuropathic disease that causes optic nerve damage, loss of retinal ganglion cells (RGCs), and visual field defects. Most glaucoma patients have no early signs or symptoms. Conventional pharmacological glaucoma medications and surgeries that focus on lowering intraocular pressure are not sufficient; RGCs continue to die, and the patient's vision continues to decline. Recent evidence has demonstrated that neuroprotective approaches could be a promising strategy for protecting against glaucoma. In the case of glaucoma, neuroprotection aims to prevent or slow down disease progression by mitigating RGCs death and optic nerve degeneration. Notably, new pharmacologic medications such as antiglaucomatous agents, antibiotics, dietary supplementation, novel neuroprotective molecules, neurotrophic factors, translational methods such as gene therapy and cell therapy, and electrical stimulation-based physiotherapy are emerging to attenuate the death of RGCs, or to make RGCs resilient to attacks. Understanding the roles of these interventions in RGC protection may offer benefits over traditional pharmacological medications and surgeries. In this review, we summarize the recent neuroprotective strategy for glaucoma, both in clinical trials and in laboratory research.

Induced Pluripotent Stem Cells (iPSCs) Provide a Potentially Unlimited T Cell Source for CAR-T Cell Development and Off-the-Shelf Products.

Chimeric antigen receptor T (CAR-T) cell therapy has been increasingly conducted for cancer patients in clinical settings. Progress in this therapeutic approach is hampered by the lack of a solid manufacturing process, T lymphocytes, and tumor-specific antigens. T cell source used in CAR-T cell therapy is derived predominantly from the patient's own T lymphocytes, which makes this approach impracticable to patients with progressive diseases and T leukemia. The generation of autologous CAR-T cells is time-consuming due to the lack of readily available T lymphocytes and is not applicable for third-party patients. Pluripotent stem cells, such as human induced pluripotent stem cells (hiPSCs), can provide an unlimited T cell source for CAR-T cell development with the potential of generating off-the-shelf T cell products. T-iPSCs (iPSC-derived T cells) are phenotypically defined, expandable, and as functional as physiological T cells. The combination of iPSC and CAR technologies provides an exciting opportunity to oncology and greatly facilitates cell-based therapy for cancer patients. However, T-iPSCs, in combination with CARs, are at the early stage of development and need further pre-clinical and clinical studies. This review will critically discuss the progress made in iPSC-derived T cells and provides a roadmap for the development of CAR iPSC-derived T cells and off-the-shelf T-iPSCs.

Hypoparathyroidism: State of the Art on Cell and Tissue Therapies.

Hypoparathyroidism is an endocrine disorder characterized by low serum calcium levels, high serum phosphorus levels, and by inappropriate or absent secretion of the parathyroid hormone (PTH). The most common therapeutic strategy to treat this condition is hormone replacement therapy with calcium and vitamin D but, unfortunately, in the long term this treatment may not be sufficient to compensate for the loss of endocrine function. Glandular autotransplantation is considered the most effective technique in place of replacement therapy. Although it leads to excellent results in most cases, autotransplantation is not always possible. Allograft is a good way to treat patients who have not been able to undergo autograft, but this technique has limited success due to side effects related to tissue rejection. This therapy is supported by systemic immunosuppression, which leads to the onset of serious side effects in patients, with a risk of endocrine toxicity. Today, research on endocrine disorders is focused on discovering alternative graft therapies that can allow optimal results with the fewest possible side effects. In this review, we will make an update on the current state of the art about the cell and tissue therapy as treatment for hypoparathyroidism, to identify which type of therapeutic strategy could be valid for a future clinical use.

The Next Generation of Molecular and Cellular Therapeutics for Inherited Retinal Disease.

Inherited retinal degenerations (IRDs) are a diverse group of conditions that are often characterized by the loss of photoreceptors and blindness. Recent innovations in molecular biology and genomics have allowed us to identify the causative defects behind these dystrophies and to design therapeutics that target specific mechanisms of retinal disease. Recently, the FDA approved the first in vivo gene therapy for one of these hereditary blinding conditions. Current clinical trials are exploring new therapies that could provide treatment for a growing number of retinal dystrophies. While the field has had early success with gene augmentation strategies for treating retinal disease based on loss-of-function mutations, many novel approaches hold the promise of offering therapies that span the full spectrum of causative mutations and mechanisms. Here, we provide a comprehensive review of the approaches currently in development including a discussion of retinal neuroprotection, gene therapies (gene augmentation, gene editing, RNA modification, optogenetics), and regenerative stem or precursor cell-based therapies. Our review focuses on technologies that are being developed for clinical translation or are in active clinical trials and discusses the advantages and limitations for each approach.

Modern Approaches to Acellular Therapy in Bone and Dental Regeneration.

An approach called cell-free therapy has rapidly developed in regenerative medicine over the past decade. Understanding the molecular mechanisms and signaling pathways involved in the internal potential of tissue repair inspires the development of new strategies aimed at controlling and enhancing these processes during regeneration. The use of stem cell mobilization, or homing for regeneration based on endogenous healing mechanisms, prompted a new concept in regenerative medicine: endogenous regenerative medicine. The application of cell-free therapeutic agents leading to the recruitment/homing of endogenous stem cells has advantages in overcoming the limitations and risks associated with cell therapy. In this review, we discuss the potential of cell-free products such as the decellularized extracellular matrix, growth factors, extracellular vesicles and miRNAs in endogenous bone and dental regeneration.

Characteristics and Therapeutic Potential of Human Amnion-Derived Stem Cells.

Stem cells including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells (ASCs) are able to repair/replace damaged or degenerative tissues and improve functional recovery in experimental model and clinical trials. However, there are still many limitations and unresolved problems regarding stem cell therapy in terms of ethical barriers, immune rejection, tumorigenicity, and cell sources. By reviewing recent literatures and our related works, human amnion-derived stem cells (hADSCs) including human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial stem cells (hAESCs) have shown considerable advantages over other stem cells. In this review, we first described the biological characteristics and advantages of hADSCs, especially for their high pluripotency and immunomodulatory effects. Then, we summarized the therapeutic applications and recent progresses of hADSCs in treating various diseases for preclinical research and clinical trials. In addition, the possible mechanisms and the challenges of hADSCs applications have been also discussed. Finally, we highlighted the properties of hADSCs as a promising source of stem cells for cell therapy and regenerative medicine and pointed out the perspectives for the directions of hADSCs applications clinically.

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells.

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.

Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research.

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

Cell therapy for Parkinson's disease is coming of age: current challenges and future prospects with a focus on immunomodulation.

Parkinson's disease (PD) is a neurodegenerative disease that affects more than 1% of people over the age of 60. The principal feature of this disease is the progressive loss of dopaminergic neurons (DAn) within the nigrostriatal system, causing the motor symptoms observed in these patients. At present, there is no therapeutic approach with a cytoprotective effect that can prevent DAn cell death or disease progression. Cell replacement therapy began 30 years ago with the objective to compensate for the loss of DAn by transplantation of dopamine-producing cells. The results from these trials have provided proof of concept of safety and efficacy of cell replacement. However, a major limiting factor of this strategy has been the poor survival rate of grafted DAn. An important factor that could cause cell death of DA precursors is the host response to the graft. In this review, we discuss the factors that affect the outcome of cell therapy in PD, with focus on the cell types used and the functional effects of the host immune response on graft survival and differentiation. We also discuss the strategies that may increase the efficacy of cell replacement therapy which target the host immune response.

Regulatory T cell therapy: Current and future design perspectives.

Regulatory T cells (Tregs) maintain immune equilibrium by suppressing immune responses through various multistep contact dependent and independent mechanisms. Cellular therapy using polyclonal Tregs in transplantation and autoimmune diseases has shown promise in preclinical models and clinical trials. Although novel approaches have been developed to improve specificity and efficacy of antigen specific Treg based therapies, widespread application is currently restricted. To date, design-based approaches to improve the potency and persistence of engineered chimeric antigen receptor (CAR) Tregs are limited. Here, we describe currently available Treg based therapies, their advantages and limitations for implementation in clinical studies. We also examine various strategies for improving CAR T cell design that can potentially be applied to CAR Tregs, such as identifying co-stimulatory signalling domains that enhance suppressive ability, determining optimal scFv affinity/avidity, and co-expression of accessory molecules. Finally, we discuss the importance of tailoring CAR Treg design to suit the individual disease.