Generation of induced pluripotent stem cells from human nasal epithelial cells using a sendai virus vector

The generation of induced pluripotent stem cells (iPSCs) by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs)-derived iPSCs by gene transduction with Sendai virus (SeV) vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08-0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes.     

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.     

Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.     

Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors

The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood. Specifically, successful reprogramming was demonstrated from a single vial of blood or less using cells expressing the early lineage marker CD34 as well as from unpurified peripheral blood mononuclear cells. From these experiments, we also show that proliferation and cell identity play a role in the number of iPSCs per input cell number. Resulting iPSCs were further characterized and deemed free of transfected DNA, integrated transgene DNA, and lack detectable gene rearrangements such as those within the immunoglobulin heavy chain and T cell receptor loci of more differentiated cell types. Furthermore, additional improvements were made to incorporate completely defined media and matrices in an effort to facilitate a scalable transition for the production of clinic-grade iPSCs.     

Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.     

Hematopoietic cells as sources for patient-specific iPSCs and disease modeling

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.     

Hematopoietic cells as sources for patient-specific iPSCs and disease modeling

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.Key words: induced pluripotent stem cells (iPSCs), blood, B lymphocytes, hematopoietic differentiation, hepatic differentiation, disease modeling, drug testing     

Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures

To identify accessible and permissive human cell types for efficient derivation of induced pluripotent stem cells (iPSCs), we investigated epigenetic and gene expression signatures of multiple postnatal cell types such as fibroblasts and blood cells. Our analysis suggested that newborn cord blood (CB) and adult peripheral blood (PB) mononuclear cells (MNCs) display unique signatures that are closer to iPSCs and human embryonic stem cells (ESCs) than age-matched fibroblasts to iPSCs/ESCs, thus making blood MNCs an attractive cell choice for the generation of integration-free iPSCs. Using an improved EBNA1/OriP plasmid expressing 5 reprogramming factors, we demonstrated highly efficient reprogramming of briefly cultured blood MNCs. Within 14 days of one-time transfection by one plasmid, up to 1000 iPSC-like colonies per 2 million transfected CB MNCs were generated. The efficiency of deriving iPSCs from adult PB MNCs was approximately 50-fold lower, but could be enhanced by inclusion of a second EBNA1/OriP plasmid for transient expression of additional genes such as SV40 T antigen. The duration of obtaining bona fide iPSC colonies from adult PB MNCs was reduced to half (～14 days) as compared to adult fibroblastic cells (28-30 days). More than 9 human iPSC lines derived from PB or CB blood cells are extensively characterized, including those from PB MNCs of an adult patient with sickle cell disease. They lack V(D)J DNA rearrangements and vector DNA after expansion for 10-12 passages. This facile method of generating integration-free human iPSCs from blood MNCs will accelerate their use in both research and future clinical applications.     

Derivation of Induced Pluripotent Stem Cells from Human Peripheral Blood T Lymphocytes

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (“TiPS”) retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.     

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner, representing a unique model to study reprogramming and an important step toward establishing a safe, clinical grade of cells for therapeutic use. In this study, we provide a detailed protocol for isolation, preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in <1 week, with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks, respectively.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patient specific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patientspecific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.Key words: induced pluripotent stem cells (iPSCs), hepatic differentiation, liver ngraftment, disease modeling, drug testing, alpha-1 antitrypsin, liver cirrhosis, hepatocellular carcinoma, cell therapy     

DNA methylation dynamics in human induced pluripotent stem cells over time

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.     

Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation

Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells.     

Short-term immunosuppression promotes engraftment of embryonic and induced pluripotent stem cells

Embryonic stem cells (ESCs) are an attractive source for tissue regeneration and repair therapies because they can be differentiated into virtually any cell type in the adult body. However, for this approach to succeed, the transplanted ESCs must survive long enough to generate a therapeutic benefit. A major obstacle facing the engraftment of ESCs is transplant rejection by the immune system. Here we show that blocking leukocyte costimulatory molecules permits ESC engraftment. We demonstrate the success of this immunosuppressive therapy for mouse ESCs, human ESCs, mouse induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells, and more differentiated ESC/(iPSCs) derivatives. Additionally, we provide evidence describing the mechanism by which inhibition of costimulatory molecules suppresses T cell activation. This report describes a short-term immunosuppressive approach capable of inducing engraftment of transplanted ESCs and iPSCs, providing a significant improvement in our mechanistic understanding of the critical role costimulatory molecules play in leukocyte activation.