Establishment of induced pluripotent stem cells from centenarians for neurodegenerative disease research

Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimer's disease and familial Parkinson's disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.     

Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells

Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.     

iPSC-Derived Natural Killer Cells for Cancer Immunotherapy

The discovery of human pluripotent stem cells (PSCs) at the turn of the century opened the door to a new generation of regenerative medicine research. Among PSCs, the donors available for induced pluripotent stem cells (iPSCs) are greatest, providing a potentially universal cell source for all types of cell therapies including cancer immunotherapies using natural killer (NK cells). Unlike primary NK cells, those prepared from iPSCs can be prepared with a homogeneous quality and are easily modified to exert a desired response to tumor cells. There already exist several protocols to genetically modify and differentiate iPSCs into NK cells, and each has its own advantages with regards to immunotherapies. In this short review, we detail the benefits of using iPSCs in NK cell immunotherapies and discuss the challenges that must be overcome before this approach becomes mainstream in the clinic.     

Immunological considerations for embryonic and induced pluripotent stem cell banking

Recent advances in stem cell technology have generated enthusiasm for their potential to study and treat a diverse range of human disease. Pluripotent human stem cells for therapeutic use may, in principle, be obtained from two sources: embryonic stem cells (hESCs), which are capable of extensive self-renewal and expansion and have the potential to differentiate into any somatic tissue, and induced pluripotent stem cells (iPSCs), which are derived from differentiated tissue such as adult skin fibroblasts and appear to have the same properties and potential, but their generation is not dependent upon a source of embryos. The likelihood that clinical transplantation of hESC- or iPSC-derived tissues from an unrelated (allogeneic) donor that express foreign human leucocyte antigens (HLA) may undergo immunological rejection requires the formulation of strategies to attenuate the host immune response to transplanted tissue. In clinical practice, individualized iPSC tissue derived from the intended recipient offers the possibility of personalized stem cell therapy in which graft rejection would not occur, but the logistics of achieving this on a large scale are problematic owing to relatively inefficient reprogramming techniques and high costs. The creation of stem cell banks comprising HLA-typed hESCs and iPSCs is a strategy that is proposed to overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. Estimates have shown that a stem cell bank containing around 10 highly selected cell lines with conserved homozygous HLA haplotypes would provide matched tissue for the majority of the UK population. These simulations have practical, financial, political and ethical implications for the establishment and design of stem cell banks incorporating cell lines with HLA types that are compatible with different ethnic populations throughout the world.     

Generation of Induced Pluripotent Stem Cells with High Efficiency from Human Umbilical Cord Blood Mononuclear Cells

Human induced pluripotent stem cells （iPSCs） hold great promise for regenerative med- icine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononu- clear cells （UCBMCs） is of great significance. Here we report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors. We generated these cells by optimizing the existing iPSC induction protocol. The UCBMC-derived iPSCs （UCB-iPSCs） have characteristics that are identical to pluripotent human embryonic stem cells （hESCs）. This study highlights the use of UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases.     

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.     

HLA homozygous stem cell lines derived from human parthenogenetic blastocysts

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S. population, and is shared by different racial groups. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. G-banded karyotyping demonstrates a normal 46,XX karyotype in lines hpSC-Hhom-1 and hpSC-Hhom-4, and chromosomal anomalies in lines hpSC-Hhom-2 and hpSC-Hhom-3, both derived from the same donor. HLA genotyping of all four hpSC-Hhom lines demonstrates that they are HLA homozygous. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. Single-nucleotide polymorphism (SNP) data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by SNP analysis. The protocol used for deriving these HLA homozygous stem cell lines minimizes the use of animal-derived components, which makes them more appealing for potential clinical application.     

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.     

Efficient feeder-free episomal reprogramming with small molecules

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.     

Five-locus HLA typing of hematopoietic stem cell donor volunteers using PCR sequence specific primers

We have developed a strategy for five-locus human leukocyte antigen (HLA) typing of hematopoeitic stem cell (HSC) donors using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The PCR-SSP method is robust, reproducible, and accurate. New PCR-SSP mixtures can be added as required and all reactions are carried out under the same conditions, which can easily be applied to the typing of other loci, e.g., ABO blood groups. Initially, 127 PCR-SSP reactions were used to detect simultaneously HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles, differentiated generally to the level of the first two digits of the allele name, essentially equivalent to the serological split specificity. Approximately 40% of subjects were tested against a further 29 HLA-A, -B SSP mixtures to exclude rare alleles and unambiguously assign a two-digit HLA allele family. This gave an overall typing resolution equivalent to or greater than the split specificity level and covered all HLA-A, -B, -C, -DRBland DQB1 alleles listed in the WHO's Nomenclature for Factors of the HLA System, 2000. The Welsh Bone Marrow Donor Registry has used this strategy to HLA type over 35,000 HSC donors over 9 years. Comprehensive and accurate five-locus HLA typing allows confident and rapid identification of potential matched HSC donors for patients requiring stem cell transplantation generally without the need for typing additional loci. This allows resources to be focused directly on allele level typing of DRB1 and other loci. This strategy decreases overall donor work-up time, which is a major benefit to patients.     

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.     

Skeletal muscle generated from induced pluripotent stem cells-induction and application

Human induced pluripotent stem cells(hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patientiPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle diseaseiPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage:(1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease;(2) setting up a highthroughput drug screening system; and(3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.     

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.     

Distinct Recognition of Non-Clade B Human Immunodeficiency Virus Type 1 Epitopes by Cytotoxic T Lymphocytes Generated from Donors Infected in Africa

We present detailed studies of human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses to clade A or C HIV type 1 in three donors infected in East Africa. We define several novel non-clade B CTL epitopes, including some restricted by HLA alleles common in Africans. Although cross-clade CTL recognition of these epitopes does occur, recognition can also be highly clade specific.     

The human brain is a detector of chemosensorily transmitted HLA-class I-similarity in same- and opposite-sex relations

Studies on subjective body odour ratings suggest that humans exhibit preferences for human leucocyte antigen (HLA)-dissimilar persons. However, with regard to the extreme polymorphism of the HLA gene loci, the behavioural impact of the proposed HLA-related attracting signals seems to be minimal. Furthermore, the role of HLA-related chemosignals in same- and opposite-sex relations in humans has not been specified so far. Here, we investigate subjective preferences and brain evoked responses to body odours in males and females as a function of HLA similarity between odour donor and smeller. We show that pre-attentive processing of body odours of HLA-similar donors is faster and that late evaluative processing of these chemosignals activates more neuronal resources than the processing of body odours of HLA-dissimilar donors. In same-sex smelling conditions, HLA-associated brain responses show a different local distribution in male (frontal) and female subjects (parietal). The electrophysiological results are supported by significant correlations between the odour ratings and the amplitudes of the brain potentials. We conclude that odours of HLA-similar persons function as important social warning signals in inter- and intrasexual human relations. Such HLA-related chemosignals may contribute to female and male mate choice as well as to male competitive behaviour.     

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

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.