Peter Karagiannis

The fate of cell reprogramming

The ability to convert somatic cells to induced pluripotent stem cells has immense potential to further our understanding of development and disease mechanisms, and for cellular therapy. Before researchers can achieve these goals, they must expand current methodology to incorporate animal models and quantitative descriptions of the essential phenomena driving reprogramming.

Ten years of induced pluripotency: from basic mechanisms to therapeutic applications.

Ten years ago, the discovery that mature somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) redefined the stem cell field and brought about a wealth of opportunities for both basic research and clinical applications. To celebrate the tenth anniversary of the discovery, the International Society for Stem Cell Research (ISSCR) and Center for iPS Cell Research and Application (CiRA), Kyoto University, together held the symposium ‘Pluripotency: From Basic Science to Therapeutic Applications’ in Kyoto, Japan. The three days of lectures examined both the mechanisms and therapeutic applications of iPSC reprogramming. Here we summarize the main findings reported, which are testament to how far the field has come in only a decade, as well as the enormous potential that iPSCs hold for the future. Summary: This Meeting Review summarises the recent ISSCR/CiRA symposium on pluripotency in Kyoto, Japan, and the exciting progress that has been made in the ten years since iPSCs were first reported.

Manipulating megakaryocytes to manufacture platelets ex vivo

Historically, platelet transfusion has proven a reliable way to treat patients suffering from thrombocytopenia or similar ailments. An undersupply of donors, however, has demanded alternative platelet sources. Scientists have therefore sought to recapitulate the biological events that convert hematopoietic stem cells into platelets in the laboratory. Such platelets have shown good function and potential for treatment. Yet the number manufactured ex vivo falls well short of clinical application. Part of the reason is the remarkable gaps in our understanding of the molecular mechanisms driving platelet formation. Using several stem cell sources, scientists have progressively clarified the chemical signaling and physical microenvironment that optimize ex vivo platelets and reconstituted them in synthetic environments. Key advances in cell reprogramming and the ability to propagate self‐renewal have extended the lifetime of megakaryocytes to increase the pool of platelet progenitors.

Reprogramming away from the exhausted T cell state.

Induced pluripotent stem cells (iPSCs) describe somatic cells that have been reprogrammed to the pluripotent state from which they can then be differentiated into any cell type of the body. This ability has tremendous implications on a wide number of medical sciences and applications, including cancer treatments. In many cancer patients, tumor infiltrating lymphocytes (TILs) have reached an exhausted state and are unable to exert effector function despite detecting and localizing at the tumor. Although the isolation, ex vivo expansion and transplantation of TILs is effective in a significant group of patients, too many patients do not respond positively to this treatment, in part because the expanded TIL population does not include a sufficient number of cells with the naïve or memory phenotype. Cell reprogramming using iPSC technologies aims to overcome this problem by returning TILs to the pluripotent state from which they can be differentiated into a heterogeneous population of T cells that are best suited to combat the tumor.

New Models for Therapeutic Innovation from Japan

Medical innovation has an extraordinary attrition rate. More than the same time reported disastrous results for AMD therapy using autol95% of drug candidates fail to receive final approval for patient care, and to reachmarket the average drug requires a dozen years of research and development at a cost in the billions of dollars (Giri and Bader, 2015). Medical companies are responding by seeking strategies that lower these costs, including a reluctance to invest in less profitablemedications, such as those for rare or complex diseases. One reason costs remain stubbornly high is the dependence on animal models. For many diseases, animal models are the only option, because human samples are difficult to acquire pre-mortem. Even when human cells are available, they often express phenotypes of the disease at late stage. In the case of Parkinsons disease, for example, it has been estimated that approximately 50% of dopaminergic neurons are already lost when patients begin showing symptoms (Bezard et al., 2001). Most experimental therapies tested on human cells at this stage of the disease are unlikely to recover the lost cells, which is whymany Parkinsons patients still await effective treatments. Induced pluripotent stem cells (iPSCs) may provide an alternative model for cheaper and faster drug discovery. Human iPSCs, which were first reported in 2007, describe somatic cells that have been reprogrammed to the pluripotent state fromwhich they can be differentiated into three germ layers (Takahashi et al., 2007). iPSCs revolutionized our understanding of cell identity and revealed the epigenetic mechanism determining this identity. From a medical perspective, iPSCs also launched research into regenerative medicine that had been hamstrung by legislative limitations on the use of embryonic stem cells (ESCs). To date, there exists only one case study of iPSC-based therapy for human patients. In 2014, researchers in Japan transplanted autologous iPSC-derived retinal cells into the eye of a patient suffering from age-related macular degeneration (AMD). Observation one year later showed the transplanted sheet survived well without immune response or adverse proliferation (Mandai et al., 2017). This study was done conservatively, as the same operation on a second patient was cancelled when some irregularities were found in the iPSC clones. Because there is no international consensus on the criteria of safe iPSCs, the authors chose prudence. Comparatively, another article published

RNA-based gene circuits for cell regulation.

A major goal of synthetic biology is to control cell behavior. RNA-mediated genetic switches (RNA switches) are devices that serve this purpose, as they can control gene expressions in response to input signals. In general, RNA switches consist of two domains: an aptamer domain, which binds to an input molecule, and an actuator domain, which controls the gene expression. An input binding to the aptamer can cause the actuator to alter the RNA structure, thus changing access to translation machinery. The assembly of multiple RNA switches has led to complex gene circuits for cell therapies, including the selective killing of pathological cells and purification of cell populations. The inclusion of RNA binding proteins, such as L7Ae, increases the repertoire and precision of the circuit. In this short review, we discuss synthetic RNA switches for gene regulation and their potential therapeutic applications.

Cell reprogramming for skeletal dysplasia drug repositioning

Skeletal dysplasia describes a wide group of disorders that affect skeletal growth. In some, such as achondroplasia (ACH), patients have serious complications, while in others, such as thanatophoric dysplasia (TD), neonatal mortality is more common. The majority of skeletal dysplasias are associated with mutations, for example, ACH and TD are caused by gain-of-function mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3). FGFR3 is a transmembrane tyrosine receptor and activates STAT and MAPK pathways to suppress the proliferation and differentiation of cartilage cells, chondrocytes. Although it is known that several types of ligands activate FGFR3 and that the resulting signaling cascade both up-regulates and down-regulates genes accordingly to achieve the suppression of chondrocytes, the mechanism for the gain-of-function is poorly understood, which is one reason drug development for ACH and TD has been relatively ineffective. Because of the difficulty in identifying or designing inhibitors specific to the FGFR3 isoform, molecules that instead act on downstream or upstream targets have been sought. One molecule, C-type natriuretic peptide (CNP), counters the mutation effect in mice by inhibiting the MAPK pathway to correct extracellular matrix synthesis and rescue bone growth.1 Another molecule, soluble FGFR3, reduces the FGFR3 signal by acting as a decoy receptor to reduce the available number of ligands that bind to membrane-bound FGFR3 and thus prevent activation of the corresponding signaling pathways.2 Yet results from mouse models are sometimes difficult to extrapolate to humans, especially for systems that endure physical stresses, because of the significantly different sizes and proportions of mouse and human cartilage. For example, the density of chondrocytes in cartilage is much less in humans than it is in mice, suggesting that these cells handle physical stress differently. Thus, drug discovery using patient cells is preferred, however, chondrocytes are extremely difficult to acquire, especially from child patients, and even then are difficult to expand and maintain in culture. Like many other diseases, the study of skeletal dysplasia has benefited tremendously from the invention of induced pluripotent stem cells (iPSCs). One crucial advantage of iPSCs is that they can be generated from patient somatic cells. Differentiating these iPSCs into the desired cell type has provided a whole new source of cells for scientific study. Since the first human cells were reprogrammed into iPSCs, a long list of new disease models has emerged.3 Furthermore, because the cells of these models are human based, there is an expectation that they will significantly reduce the cost and time of drug discovery. For this same reason, it is expected that iPSC technology will be instrumental in drug repositioning, as it will provide an abundant source of cells on which existing drugs that have had their safety already measured can be tested. Indeed, in our iPSC-based disease model for skeletal dysplasia, we demonstrated potential drug repositioning of statin, as we found a positive effect on bone growth in diseased cells.4 Even though several studies have described auspicious effects by statins on human chondrocytes,5,6 we would not have considered statin as a candidate for ACH or TD because of the negligible number of patient cells available had it not been for iPSCs. Additionally, because iPSCs can be derived from humans, they are less likely to suffer from false positives or negatives in drug testing, an unfortunate and frustrating outcome too commonly seen when using animal models. Coincidently, one excellent example of a false negative comes from a statin study where cholesterol levels did not improve in rats.7 We therefore investigated the effects of several molecules, including statins, on chondrocytes differentiated from iPSCs, which were reprogrammed from ACH and TD patient-fibroblasts in culture.4 One concern about iPSCs is whether they adequately recapitulate cellular properties upon differentiation. We found that compared with those from healthy-iPSCs, chondrocytes derived from patient-iPSCs had defective cartilage tissue formation, which is consistent with ACH and TD phenotypes (Fig. 1). Statin enhanced the degradation of FGFR3 in these cells, which diminished FGFR3 signaling and its downstream targets including the MAPK pathway. Importantly, this discovery is a rare instance when drug testing was done at the tissue level. Additionally, statin was found to induce bone growth in model mice that bore the ACH mutation, whereas wild-type mice showed no significant response to treatment. Overall, this report is the first to describe cartilage tissue generated from a completely iPSC-based system and demonstrated that statin could not only retard mutant FGFR3 activity, but also potentially recover cartilage malformation. Figure 1. Fibroblasts taken from a healthy subject (top) or patient (bottom) are reprogrammed into iPSCs and then differentiated into chondrocytes. The iPSC-derived chondrocytes from the healthy subject go on to form healthy cartilage. However, iPSC-derived chondrocytes ... Despite these encouraging findings, statin may not be an ideal candidate for treating skeletal dysplasia, especially in children, because of its effects on cholesterol, which is essential for development. It would be interesting, therefore, to determine if the mechanism of the FGFR3 action is independent of that on cholesterol. Nevertheless, statin could be a promising paradigm for drug compounds that could be further tested using iPSC-based models and eventually reach the clinic.

Induced Pluripotent Stem Cells and Their Use in Human Models of Disease and Development

The discovery of somatic cell nuclear transfer proved that somatic cells can carry the same genetic code as the zygote, and that activating parts of this code are sufficient to reprogram the cell to an early developmental state. The discovery of induced pluripotent stem cells (iPSCs) nearly half a century later provided a molecular mechanism for the reprogramming. The initial creation of iPSCs was accomplished by the ectopic expression of four specific genes (OCT4, KLF4, SOX2, and c-Myc; OSKM). iPSCs have since been acquired from a wide range of cell types and a wide range of species, suggesting a universal molecular mechanism. Furthermore, cells have been reprogrammed to iPSCs using a myriad of methods, although OSKM remains the gold standard. The sources for iPSCs are abundant compared with those for other pluripotent stem cells; thus the use of iPSCs to model the development of tissues, organs, and other systems of the body is increasing. iPSCs also, through the reprogramming of patient samples, are being used to model diseases. Moreover, in the 10 years since the first report, human iPSCs are already the basis for new cell therapies and drug discovery that have reached clinical application. In this review, we examine the generation of iPSCs and their application to disease and development.

Considerations in hiPSC-derived cartilage for articular cartilage repair

BackgroundA lack of cell or tissue sources hampers regenerative medicine for articular cartilage damage.Main textWe review and discuss the possible use of pluripotent stem cells as a new source for future clinical use. Human induced pluripotent stem cells (hiPSCs) have several advantages over human embryonic stem cells (hESCs). Methods for the generation of chondrocytes and cartilage from hiPSCs have been developed. To reduce the cost of this regenerative medicine, allogeneic transplantation is preferable. hiPSC-derived cartilage shows low immunogenicity like native cartilage, because the cartilage is avascular and chondrocytes are segregated by the extracellular matrix. In addition, we consider our experience with the aberrant deposition of lipofuscin or melanin on cartilage during the chondrogenic differentiation of hiPSCs.Short conclusionCartilage generated from allogeneic hiPSC-derived cartilage can be used to repair articular cartilage damage.

When Myc's asleep, embryonic stem cells are dormant

Myc is one of the original reprogramming factors used to produce induced pluripotent stem cells. However, it is not necessary, instead its main role is to increase the efficiency of the reprogramming. The article by Scognamiglio et al ( ) helps clarify how. The authors show that Myc depletion leads to a reversible dormant state consistent with diapause. In this state, the cell sees its proliferation potential diminished but its pluripotency unchanged. The ability to coordinate the induction of this state should have important implications in cell differentiation.