Cells derived from iPSC can be immunogenic — Yes or No?

The induced pluripotent stem cells (iPSCs), derived by ectopic expression of reprogramming factors in somatic cells, can potentially provide unlimited autologous cells for regenerative medicine. In theory, the autologous cells derived from patient iPSCs should be immune tolerant by the host without any immune rejections. However, our recent studies have found that even syngeneic iPSC-derived cells can be immunogenic in syngeneic hosts by using a teratoma transplantation model (Nature 474:212–215, 2011). Recently two research groups differentiated the iPSCs into different germ layers or cells, transplanted those cells to the syngeneic hosts, and evaluated the immunogenicity of those cells. Both of the two studies support our conclusions that some certain but not all tissues derived from iPSCs can be immunogenic, although they claimed either “negligible” or “lack of” immunogenicity in iPSC derivatives (Nature 494:100–104, 2013; Cell Stem Cell 12:407–412, 2013). To test the immunogenicity of clinically valuable cells differentiated from human iPSCs are emergently required for translation of iPSC technology to clinics.     

Molecular Imaging of Induced Pluripotent Stem Cell Immunogenicity with In Vivo Development in Ischemic Myocardium

Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.     

Application of induced pluripotent stem cells to hematologic disease

Induced pluripotent stem cells (iPSC) were first generated from somatic cells via the transduction of four ‘Yamanaka’ factors, Oct4, Sox2, Klf4 and c-Myc. Because iPSC are similar to embryonic stem cells (ESC) and can be differentiated into any cell type of choice, iPSC have the potential to become a platform for personalized medicine by allowing a patient's own cells to become a source of therapeutic tissue. This review describes the main challenges in iPSC technology by focusing on its application to hematologic diseases. The explosive interest in improving iPSC technology has generated numerous genetic and chemical methods for iPSC derivation, but these methods must be evaluated comparatively for their safety and efficacy because there are risks of genetic abnormalities and oncogenesis. Competent iPSC will need to be selected carefully based on physical, genetic and functional criteria, and differentiated efficiently into hematopoietic stem cells via modulation of several signaling pathways before they prove valuable in the clinic.     

Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, α-fetoprotein; ectoderm, βIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 × 10⁵ newborn marmoset skin fibroblasts, we obtained ～ 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy.     

应用诱导多能干细胞治疗的进展与挑战

诱导多能干细胞(Induced pluripotent stem cells,i PSCs)是利用特定的转录因子诱导体细胞获得的,像胚胎干细胞一样,可以进行无限的自我更新,并具有分化成三个胚层的能力。iPSC有可能提供无限的自体细胞治疗,目前研究已经证实,不同种类疾病的患者提供的成体细胞诱导后可产生种类繁多的iPSC,这项技术给目前无有效治疗手段的多类疾病带来了治疗的希望,并有可能避免利用胚胎干细胞(embryonic stem cells,ESCs)治疗面临的伦理问题和免疫排斥反应。本文回顾iPSC技术优化过程,着重关注应用i PSC建立细胞模型、进行细胞治疗的进展,并探讨iPSC在基础研究及临床应用中遇到的挑战。     

Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca(2+) transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) 'Ca(2+)-release units', which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.     

Neurodegenerative disease-specific induced pluripotent stem cell research

Neurodegenerative disease-specific induced pluripotent stem cell (iPSC) research contributes to the following 3 areas; “Disease modeling”, “Disease material” and “Disease therapy”.“Disease modeling”, by recapitulating the disease phenotype in vitro, will reveal the pathomechanisms. Neurodegenerative disease-specific iPSC-derived non-neuronal cells harboring disease-causative protein(s), which play critical roles in neurodegeneration including motor neuron degeneration in amyotrophic lateral sclerosis, could be “Disease material”, the target cell(s) for drug screening. These differentiated cells also could be used for “Disease therapy”, an autologous cellular replacement/neuroprotection strategy, for patients with neurodegenerative disease.Further progress in these areas of research can be made for currently incurable neurodegenerative diseases.     

Generation of functional neutrophils from a mouse model of X-linked chronic granulomatous disorder using induced pluripotent stem cells

Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils.     

Recognition of cellular implants by the brain's innate immune system

Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.     

The use of patient-specific stem cells in different autoimmune diseases

Autoimmune diseases are developed when the immune system mistakenly attacks the body’s cells. These inflammatory disorders can be inherited or triggered by external forces, such as type 1 diabetes, which is caused by the immune system's destruction of pancreatic beta cells. So far, stem cells such as hESC and iPSC have been used to treat autoimmune disorders such as type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE), although these procedures have certain ethical concerns. On the other hand, bone marrow-derived mesenchymal stem cells (BM-MSC) are thought to be the best source of stem cells. Later, it was shown that mesenchymal stem cells produced from autologous adipose tissues have a great potential for producing huge volumes of stem cells. In-vitro and in-vivo investigations using autologous hematopoietic stem cells and autologous mesenchymal stem cells have been carried out on various rodent and human models, while clinical trials for inflammatory diseases such as multiple sclerosis and diabetes mellitus have yielded promising results. We attempted to summarise the usage of diverse stem cells in the therapy of various autoimmune disorders in this review. Shortly, we expect that the use of autologous stem cells will provide a new perspective on the treatment of autoimmune disorders.     

iPS cells: a game changer for future medicine

The induced pluripotent stem cell (iPSC) technology is instrumental in advancing the fields of disease modeling and cell transplantation. We herein discuss the various issues regarding disease modeling and cell transplantation presented in previous reports, and also describe new iPSC‐based medicine including iPSC clinical trials. In such trials, iPSCs from patients can be used to predict drug responders/non‐responders by analyzing the efficacy of the drug on iPSC‐derived cells. They could also be used to stratify patients after actual clinical trials, including those with sporadic diseases, based on the drug responsiveness of each patient in the clinical trials. iPSC‐derived cells can be used for the identification of response markers, leading to increased success rates in such trials. Since iPSCs can be used in micromedicine for drug discovery, and in macromedicine for actual clinical trials, their use would tightly connect both micro‐ and macromedicine. The use of iPSCs in disease modeling, cell transplantation, and clinical trials could therefore lead to significant changes in the future of medicine.     

Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation

Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.     

A risk-based approach for cell line development,manufacturing and characterization of genetically engineered,induced pluripotent stem cell–derived allogeneic cell therapies

Advances in cellular reprogramming and gene-editing approaches have opened up the potential for a new class of ex vivo cell therapies based on genetically engineered, induced pluripotent stem cell (iPSC)-derived allogeneic cells. While these new therapies share some similarities with their primary cell-derived autologous and allogeneic cell therapy predecessors, key differences exist in the processes used for generating genetically engineered, iPSC-derived allogeneic therapies. Specifically, in iPSC-derived allogeneic therapies, donor selection and gene-editing are performed once over the lifetime of the product as opposed to as part of the manufacturing of each product batch. The introduction of a well-characterized, fully modified, clonally derived master cell bank reduces risks that have been inherent to primary-cell derived autologous and allogeneic therapies. Current regulatory guidance, which was largely developed based on the learnings gained from earlier generation therapies, leaves open questions around considerations for donor eligibility, starting materials and critical components, cell banking and genetic stability. Here, a risk-based approach is proposed to address these considerations, while regulatory guidance continues to evolve.     

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.     

A poor imitation of a natural process: A call to reconsider the iPSC engineering technique

Reprogramming somatic cells into a pluripotent state is expected to initiate a new era in medicine. Because the precise underlying mechanism of reprogramming remains unclear, many efforts have been made to optimize induced pluripotent stem cell (iPSC) engineering. However, satisfactory results have not yet been attained. In this review, we focus on recent roadblocks in iPSC reprogramming engineering, such as the inefficiency of the process, tumorigenicity and heterogeneity of the generation. We conclude that cell reprogramming is a naturally occurring phenomenon rather than a biological technique. We will only be able to mimic the natural process of reprogramming when we fully understand its underlying mechanism. Finally, we highlight the alternative method of direct conversion, which avoids the use of iPSCs to generate cell materials for patient-specific cell therapy.     

Application of biomaterials to advance induced pluripotent stem cell research and therapy

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation.     

Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients

Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.     

Heated tumour cells of autologous and allogeneic origin elicit anti-tumour immunity

Vaccination with established tumour cell lines may circumvent the problem of obtaining autologous tumour cells from patients, but may also need immunological adjuvants. Up-regulation of heat shock proteins within tumour cell vaccines has resulted in increased immunogenicity in some models, but this has yet to be demonstrated for allogeneic (MHC-disparate) cell vaccines. This was investigated here using a rat model for prostate tumour cell vaccination. Heating of tumour cells (42°C, 1 h) elicited significant increases in HSP70 expression. Vaccination with heated autologous PAIII cells elicited protection against PAIII challenge in 60% of rats >50 days compared to 0% with unheated vaccine and was associated with an increased Th1 (IFN) immune response. Heated allogeneic MLL cells elicited significant protection against PAIII challenge, in contrast to unheated cells. The principle was confirmed in two mouse models, although the allogeneic melanoma vaccine K1735 elicited the best protection when heated and administered mixed with autologous dendritic cells. Thus, while heating of vaccine cells in some models is highly beneficial, and is a means of enhancing immunogenicity without genetic modification or inclusion of potentially toxic adjuvants, additional immune enhancement may be required.     

A poor imitation of a natural process

Reprogramming somatic cells into a pluripotent state is expected to initiate a new era in medicine. Because the precise underlying mechanism of reprogramming remains unclear, many efforts have been made to optimize induced pluripotent stem cell (iPSC) engineering. However, satisfactory results have not yet been attained. In this review, we focus on recent roadblocks in iPSC reprogramming engineering, such as the inefficiency of the process, tumorigenicity and heterogeneity of the generation. We conclude that cell reprogramming is a naturally occurring phenomenon rather than a biological technique. We will only be able to mimic the natural process of reprogramming when we fully understand its underlying mechanism. Finally, we highlight the alternative method of direct conversion, which avoids the use of iPSCs to generate cell materials for patient-specific cell therapy.