Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells

PurposeTo compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs).ResultsGeneration of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle‐like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single‐cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP‐derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time‐consuming due to an intrinsically faster timing of retinal differentiation.ConclusionsRetinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.     

Immunogenicity of mAbs in non-human primates during nonclinical safety assessment

The immunogenicity of biopharmaceuticals used in clinical practice remains an unsolved challenge in drug development. Non-human primates (NHPs) are often the only relevant animal model for the development of monoclonal antibodies (mAbs), but the immune response of NHPs to therapeutic mAbs is not considered to be predictive of the response in humans because of species differences. In this study, we accessed the drug registration files of all mAbs registered in the European Union to establish the relative immunogenicity of mAbs in NHPs and humans. The incidence of formation of antidrug-antibodies in NHPs and patients was comparable in only 59% of the cases. In addition, the type of antidrug-antibody response was different in NHP and humans in 59% of the cases. Humanization did not necessarily reduce immunogenicity in humans. Immunogenicity interfered with the safety assessment during non-clinical drug development when clearing or neutralizing antibodies were formed. While important to interpret the study results, immunogenicity reduced the quality of NHP data in safety assessment. These findings confirm that the ability to compare relative immunogenicity of mAbs in NHPs and humans is low. Furthermore, immunogenicity limits the value of informative NHP studies.     

Placental-derived stem cells: Culture,differentiation and challenges

Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice.     

The non-human primate oocyte and embryo as a model for women, or is it vice versa?

The role of the non-human primate (NHP) oocyte and embryo in translational research is considered here including both in vitro activities directly involving oocytes or embryos as well as animal studies that impact reproductive function. Reasons to consider NHPs as animal research models along with their limitations are summarized. A case is made that in limited instances, such as in the development and application of the assisted reproductive technologies or in the study of embryonic stem cells, the human oocyte and embryo have acted as models for the monkey. The development of strategies for the preservation of fertility is used as an example of ongoing research in the non-human primate that cannot be conducted in women for ethical reasons. In animal studies, monitoring reproductive potential, responses to embryonic stem cell transplantation, along with translational research in the field of contraceptive development for women are considered as subjects that benefit from the availability of a NHP model.     

UNCERTAIN TRANSLATION,UNCERTAIN BENEFIT AND UNCERTAIN RISK: ETHICAL CHALLENGES FACING FIRST‐IN‐HUMAN TRIALS OF INDUCED PLURIPOTENT STEM (IPS) CELLS

The discovery of induced pluripotent stem (iPS) cells in 2006 was heralded as a major breakthrough in stem cell research. Since then, progress in iPS cell technology has paved the way towards clinical application, particularly cell replacement therapy, which has refueled debate on the ethics of stem cell research. However, much of the discourse has focused on questions of moral status and potentiality, overlooking the ethical issues which are introduced by the clinical testing of iPS cell replacement therapy. First‐in‐human trials, in particular, raise a number of ethical concerns including informed consent, subject recruitment and harm minimisation as well as the inherent uncertainty and risks which are involved in testing medical procedures on humans for the first time. These issues, while a feature of any human research, become more complex in the case of iPS cell therapy, given the seriousness of the potential risks, the unreliability of available animal models, the vulnerability of the target patient group, and the high stakes of such an intensely public area of science. Our paper will present a detailed case study of iPS cell replacement therapy for Parkinson's disease to highlight these broader ethical and epistemological concerns. If we accept that iPS cell technology is fraught with challenges which go far beyond merely refuting the potentiality of the stem cell line, we conclude that iPS cell research should not replace, but proceed alongside embryonic and adult somatic stem cell research to promote cross‐fertilisation of knowledge and better clinical outcomes.     

Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics

Induced pluripotent stem cells (iPSCs) and their differentiated derivatives can potentially be applied to cell-based therapy for human diseases. The properties of iPSCs are being studied intensively both to understand the basic biology of pluripotency and cellular differentiation and to solve problems associated with therapeutic applications. Examples of specific preclinical applications summarized briefly in this minireview include the use of iPSCs to treat diseases of the liver, nervous system, eye, and heart and metabolic conditions such as diabetes. Early stage studies illustrate the potential of iPSC-derived cells and have identified several challenges that must be addressed before moving to clinical trials. These include rigorous quality control and efficient production of required cell populations, improvement of cell survival and engraftment, and development of technologies to monitor transplanted cell behavior for extended periods of time. Problems related to immune rejection, genetic instability, and tumorigenicity must be solved. Testing the efficacy of iPSC-based therapies requires further improvement of animal models precisely recapitulating human disease conditions.     

Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment

Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.     

Illuminating the Regenerative Properties of Stem Cells In Vivo with Bioluminescence Imaging

Preclinical animal studies are essential to the development of safe and effective stem cell therapies. Bioluminescence imaging (BLI) is a powerful tool in animal studies that enables the real-time longitudinal monitoring of stem cells in vivo to elucidate their regenerative properties. This review describes the application of BLI in preclinical stem cell research to address critical challenges in producing successful stem cell therapeutics. These challenges include stem cell survival, proliferation, homing, stress response, and differentiation. The applications presented here utilize bioluminescence to investigate a variety of stem and progenitor cells in several different in vivo models of disease and implantation. An overview of luciferase reporters is provided, along with the advantages and disadvantages of BLI. Additionally, BLI is compared to other preclinical imaging modalities and potential future applications of this technology are discussed in emerging areas of stem cell research.     

Neural regeneration therapies for Alzheimer's and Parkinson's disease-related disorders

Neurodegenerative diseases are devastating mental illnesses without a cure. Alzheimer's disease (AD) characterized by memory loss, multiple cognitive impairments, and changes in personality and behavior. Although tremendous progress has made in understanding the basic biology in disease processes in AD and PD, we still do not have early detectable biomarkers for these diseases. Just in the United States alone, federal and nonfederal funding agencies have spent billions of dollars on clinical trials aimed at finding drugs, but we still do not have a drug or an agent that can slow the AD or PD disease process. One primary reason for this disappointing result may be that the clinical trials enroll patients with AD or PD at advances stages. Although many drugs and agents are tested preclinical and are promising, in human clinical trials, they are mostly ineffective in slowing disease progression. One therapy that has been promising is ‘stem cell therapy’ based on cell culture and pre-clinical studies. In the few clinical studies that have investigated therapies in clinical trials with AD and PD patients at stage I. The therapies, such as stem cell transplantation – appear to delay the symptoms in AD and PD. The purpose of this article is to describe clinical trials using 1) stem cell transplantation methods in AD and PD mouse models and 2) regenerative medicine in AD and PD mouse models, and 3) the current status of investigating preclinical stem cell transplantation in patients with AD and PD.     

Mesenchymal stem cells and immunomodulation: current status and future prospects

The unique immunomodulatory properties of mesenchymal stem cells (MSCs) make them an invaluable cell type for the repair of tissue/ organ damage caused by chronic inflammation or autoimmune disorders. Although they hold great promise in the treatment of immune disorders such as graft versus host disease (GvHD) and allergic disorders, there remain many challenges to overcome before their widespread clinical application. An understanding of the biological properties of MSCs will clarify the mechanisms of MSC-based transplantation for immunomodulation. In this review, we summarize the preclinical and clinical studies of MSCs from different adult tissues, discuss the current hurdles to their use and propose the future development of pluripotent stem cell-derived MSCs as an approach to immunomodulation therapy.     

Conventional and molecular cytogenetic diagnostic methods in stem cell research: a concise review

Regenerative medicine and cell therapy are emerging clinical disciplines in the field of stem cell biology. The most important sources for cell transplantation are human embryonic and adult stem cells. The future use of these human stem cell lines in humans requires a guarantee of exhaustive control with respect to quality control, safety and traceability. Genetic instability and chromosomal abnormalities represent a potential weakness in basic studies and future therapeutic applications based on these stem cell lines, and may explain, at least in part, their usual tumourigenic properties. So, the introduction of the cytogenetic programme in the determination of the chromosomal stability is a key point in the establishment of the stem cell lines. The aim of this review is to provide readers with an up-to-date overview of all the cytogenetic techniques, both conventional methods and molecular fluorescence methods, to be used in a stem cell bank or other stem cell research centres. Thus, it is crucial to optimize and validate their use in the determination of the chromosomal stability of these stem cell lines, and assess the advantages and limitations of these cutting-edge cytogenetic technologies.     

Bioreactor design for clinical-grade expansion of stem cells

The many clinical trials currently in progress will likely lead to the widespread use of stem cell-based therapies for an extensive variety of diseases, either in autologous or allogeneic settings. With the current pace of progress, in a few years' time, the field of stem cell-based therapy should be able to respond to the market demand for safe, robust and clinically efficient stem cell-based therapeutics. Due to the limited number of stem cells that can be obtained from a single donor, one of the major challenges on the roadmap for regulatory approval of such medicinal products is the expansion of stem cells using Good Manufacturing Practices (GMP)-compliant culture systems. In fact, manufacturing costs, which include production and quality control procedures, may be the main hurdle for developing cost-effective stem cell therapies. Bioreactors provide a viable alternative to the traditional static culture systems in that bioreactors provide the required scalability, incorporate monitoring and control tools, and possess the operational flexibility to be adapted to the differing requirements imposed by various clinical applications. Bioreactor systems face a number of issues when incorporated into stem cell expansion protocols, both during development at the research level and when bioreactors are used in on-going clinical trials. This review provides an overview of the issues that must be confronted during the development of GMP-compliant bioreactors systems used to support the various clinical applications employing stem cells.     

Stem/progenitor cells in liver injury repair and regeneration

Morbidity and mortality from cirrhosis is increasing rapidly in the world. Currently, orthotopic liver transplantation is the only definitive therapeutic option. However, its clinical use is limited, because of poor long‐term graft survival, donor organ shortage and high costs associated with the procedure. Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair and regeneration. In this review we discuss recent preclinical and clinical investigations that explore the therapeutic potential of stem cells in repair of liver injuries. Several types of stem cells. including embryonic stem cells, haematopoietic stem cells and mesenchymal stem cells, can be induced to differentiate into hepatocyte‐like cells by defined culture conditions in vitro. Stem cell transplantation has been shown to significantly improve liver function and increase animal survival in experimentally‐induced liver‐injury models. Moreover, several pilot clinical studies have reported encouraging therapeutic effects in patients treated with stem cells. Although there remain many unresolved issues, the available data support the notion that stem cell technology may lead to the development of effective clinical modalities for human liver diseases.     

Adult stem cell transplantation in stroke: its limitations and prospects

A growing number of studies have demonstrated stem cell-based therapy provides a feasible, realistic approach to the restoration of lost brain function after stroke. Moreover, adult stem cells may provide more appropriate clinical strategies. Leading candidate sources include bone marrow, peripheral blood, adipose tissue, skeletal muscle, and olfactory mucosa, which act as central repositories for multipotent stem cells that can repopulate neural tissues. The medical society is currently enthusiastic concerning the clinical applications of autologous adult stem cells in stroke, based on promising results obtained during experimental studies and initial clinical trials. However, before embracing clinical applications, several essential precautions must be properly addressed. For example, the regenerative potentials of adult stem cells decline with age, and stem cells isolated from aged patients may retain dysfunctional characteristics. Are the natures and amounts of available autologous cells appropriate for therapeutic application in stroke? Do transplanted cells remain functional in the diseased brain, and if so what are the optimal injection routes, cell doses, and timings? Thus, we believe that success in future clinical trials will depend on careful investigation at the experimental level, to allow us to understand not only the practicalities of stem cell use, but also the underlying biological principles involved. Here, we review the advantages and disadvantages of the different adult stem cell sources and discuss the challenges that must be negotiated to achieve transplantation success.     

Localized immunomodulation technologies to enable cellular and organoid transplantation

Localized immunomodulation technologies are rapidly emerging as a new modality with the potential to revolutionize transplantation of cells and organs. In the past decade, cell-based immunomodulation therapies saw clinical success in the treatment of cancer and autoimmune diseases. In this review, we describe recent advances in engineering solutions for the development of localized immunomodulation techniques focusing on cellular and organoid transplantation. We begin by describing cell transplantation and highlighting notable clinical successes, particularly in the areas of stem cell therapy, chimeric antigen receptor (CAR)-T cell therapy, and islet transplantation. Next, we detail recent preclinical studies centered on genome editing and biomaterials to enhance localized immunomodulation. We close by discussing future opportunities to improve clinical and commercial success using these approaches to facilitate long-term immunomodulation technologies.     

Human adipose stem cells: current clinical applications

Adipose-derived stem cells are multipotent cells that can easily be extracted from adipose tissue, are capable of expansion in vitro, and have the capacity to differentiate into multiple cell lineages, which have the potential for use in regenerative medicine. However, several issues need to be studied to determine safe human use. For example, there are questions related to isolation and purification of adipose-derived stem cells, their effect on tumor growth, and the enforcement of U.S. Food and Drug Administration regulations. Numerous studies have been published, with the interest in the potential for regenerative medicine continually growing. Several clinical trials using human adipose stem cell therapy are currently being performed around the world, and there has been a rapid evolution and expansion of their number. The purpose of this article was to review the current published basic science evidence and ongoing clinical trials involving the use of adipose-derived stem cells in plastic surgery and in regenerative medicine in general. The results of the studies and clinical trials using adipose-derived stem cells reported in this review seem to be promising not only in plastic surgery but also in a wide variety of other specialties. Nevertheless, those reported showed disparity in the way adipose-derived stem cells were used. Further basic science experimental studies with standardized protocols and larger randomized trials need to be performed to ensure safety and efficacy of adipose-derived stem cells use in accordance with U.S. Food and Drug Administration guidelines.     

c-kit~+心脏干细胞在心脏再生修复中的应用

心血管疾病的流行是一个全球性的现象,在我国,其患病率也不断增长。目前尚无有效的治疗方法以解决心肌细胞损失这一关键问题,而干细胞移植很可能成为新的治疗方法。C-kit+心脏干细胞(cardiac stem/progenitor cell,CSCs)的发现证实了CSCs的存在,并为心脏的再生和修复治疗带来了新的曙光。C-kit+CSCs在心肌梗死、心力衰竭等心脏疾病中的作用得到了多项体内外实验及临床试验的证实,但由于目前尚缺乏直接的充分的证据证明内源性或外源性的c-kit+CSCs可分化为相当数量的成熟的具有功能的心肌细胞,其治疗机制尚存争议,同时,将其应用于临床仍面临多个问题。     

非人灵长类肿瘤模型研究进展(英文)

癌症是人类第二大致死的疾病。将体外细胞模型获得的癌症研究结果向临床转化过程中,动物活体实验是必不可少的一个环节。现在的肿瘤活体实验绝大部分采用啮齿类实验动物如小鼠和大鼠,这是因为它们具有个体小、繁殖迅速、遗传背景清楚、转基因技术成熟等优势。但是啮齿类和人的亲缘关系比较远,许多从啮齿类动物模型获得的研究结果不能在人体重现。非人灵长类动物在遗传进化、免疫、生理和代谢等诸多方面与人类高度近似,理论上更加适合癌症研究。本文对现有的非人灵长类肿瘤研究做一综述,主要集中介绍用化学和生物致癌剂在不同的非人灵长类动物诱导肿瘤的研究,为将来用非人灵长类动物研究人类癌症奠定基础。     

Cell transplantation for cardiac regeneration: where do we stand?

During the last decade transplantation of cells into the heart has emerged as a novel therapy for the prevention and treatment of heart failure. Although various cell types have been used, most experience has been obtained with the progenitor cells of skeletal muscle, also called myoblasts, and a wide array of bone marrow-derived cell types. The first preclinical studies demonstrated an improvement in global and regional heart function that was attributed mainly to a direct contractile effect of the transplanted cells. Furthermore, it was suggested that multiple cell types are able to form true cardiomyocytes and truly ‘regenerate’ the myocardium. More recent studies have questioned these early findings. Other mechanisms such as paracrine effects on the infarct and remote myocardium, a reduction in adverse remodelling and improvement of mechanical properties of the infarct tissue likely play a more important role. On the basis of encouraging preclinical studies, multiple early-phase clinical trials and several randomised controlled trials have been conducted that have demonstrated the feasibility, safety and potential efficacy of this novel therapy in humans. This review summarises the available evidence on cardiac cell transplantation and provides an outlook on future preclinical and clinical research that has to fill in the remaining gaps. (Neth Heart J 2008; 16:88-95.)     

The role of stem cells for treatment of cardiovascular disease

Cardiovascular disease is a global cause of mortality and morbidity. Current treatments fail to address the underlying scarring and cell loss, which are the causes of ischaemic heart failure. Cellular transplantation can overcome these problems and new impetus has been injected into this field following the isolation of human embryonic and adult stem cells. These cells have shown remarkable ability to produce cardiomyocytes and vascular cells in vitro and in vivo. Initial transplantation studies have demonstrated functional benefits and it is hoped further randomised clinical trials will concur with initial findings. Much basic science remains to be unearthed, such as the signals for homing, differentiation and engraftment of transplanted cells. Further matters of concern are the role of cell fusion and the mechanisms by which transplanted cells improve cardiac function. In spite of initial progress made in stem cell therapy there is still much to be done and we are some way off from achieving the goal of effective cellular regeneration.