Human iPS cell-based therapy: Considerations before clinical applications

Generation of induced pluripotent stem (iPS) cells has revolutionized the field of regenerative medicine. With the exponential increase in iPS cell research in the past three years, human iPS cells have been derived with different technologies and from various cell types. From a translational perspective, however, a number of issues must be addressed before safe and high quality patient-specific iPS cells can be derived for clinical applications. In addition, iPS cell-based therapies also need to be thoroughly evaluated in pre-clinical animal models before they can be applied to human subjects.     

Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a promising therapeutic tool for many diseases, including aged tissues and organs at high risk of failure. However, the intrinsic self-renewal and pluripotency of ES and iPS cells make them tumorigenic, and hence, the risk of tumor development hinders their clinical application. Here, we present a novel approach to limit their tumorigenicity and increase their safety through increased copy number of tumor suppressors. iPS containing an extra copy of the p53 or Ink4a/ARF locus show normal pluripotency, as determined by in vitro and in vivo differentiation assays. Yet, while retaining full pluripotency, they also possess an improved engagement of the p53 pathway during teratocarcinoma formation, which leads to a reduced tumorigenic potential in various in vitro and in vivo assays. Furthermore, they show an improved response to anticancer drugs, which could aid in their elimination in case tumors arise with no adverse effects on cell function or aging. Our system provides a model for studying tumor suppressor pathways during reprogramming, differentiation, and cell therapy applications. This offers an improved understanding of the pathways involved in tumor growth from engrafted pluripotent stem cells, which could facilitate the use of ES and iPS cells in regenerative medicine.     

Human-Induced Pluripotent Stem Cells: In Quest of Clinical Applications

In the field of regenerative medicine, the development of induced pluripotent stem (iPS) cells may represent a potential strategy to overcome the limitations of human embryonic stem cells (ESCs). iPS cells have the potential to mimic human disease, since they carry the genome of the donor. Hypothetically, with iPS cell technology it is possible to screen patients for a genetic cause of disease (genetic mutation), develop cell lines, reprogram them back to iPS cells, finally differentiate them into one or more cell types that develop the disease. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations. Finally, direct reprogramming strategies can provide rapid production of models of human ??diseases in a dish??, without first passing the cells through a pluripotent state, so avoiding the challenges of time-consumming and labor-intensive iPS cell line generation. This review will overview methods to develop iPS cells, current strategies for direct reprogramming, and main applications of iPS cells as human disease model, focusing on human cardiovascular diseases, with the aim to be a potential information resource for biomedical scientists and clinicians who exploit or intend to exploit iPS cell technology in a range of applications.     

共表达外源OCT4/SOX2能够激活人胚肾细胞中内源NANOG的转录

自2006年诱导多能干细胞（iPS）技术诞生以来,采用病毒等载体进行的诱导方法已取得了成功,但是其致瘤性的影响限制了病毒载体的推广与应用,而采用非病毒载体诱导iPS细胞成为研究的热点. 本研究通过两个启动子的独立启动,构建了带有绿色荧光标记的OCT4/SOX2共表达诱导载体(pOct4/Sox2-EGFP). 将该载体转染HEK 293FT 细胞后,阳性克隆明显表达绿色荧光,并通过RT-PCR,免疫荧光等方法证明其中的转录因子OCT4和SOX2能在转染细胞中高效表达,同时诱导受体细胞中内源NANOG的转录表达. 本研究说明OCT4/SOX2共表达载体能激活NANOG基因的内源表达,暗示着非病毒不整合载体pOct4/Sox2-EGFP本身或与其它转录因子和小分子结合可用于诱导成体细胞的重编程. 因此,本研究为下一步应用质粒载体诱导体细胞重编程为iPS细胞的研究奠定了工作基础.     

Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons

The generation of induced pluripotent stem (iPS) cells represents a promising approach for innovative cell therapies. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity. Transposition of reprogramming cassettes represents a recent alternative to viral approaches. Since binary transposons can be produced as common plasmids they provide a safe and cost-efficient alternative to viral delivery methods. Here, we compared the efficiency of two different transposon systems, Sleeping Beauty (SB) and piggyBac (PB), for the generation of murine iPS. Murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, and fibroblasts derived from outbred NMRI mice were employed for reprogramming. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-Myc was compatible with iPS cell line derivation, albeit with reduced reprogramming efficiencies. The transposon-derived iPS cells featured typical hallmarks of pluripotency, including teratoma growth in immunodeficient mice. Thus SB and PB transposons represent a promising non-viral approach for iPS cell derivation.     

Generating a Non-Integrating Human Induced Pluripotent Stem Cell Bank from Urine-Derived Cells

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine, drug discovery, and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency, offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study, we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research, facilitating future applications of human iPS cells.     

Mouse meningiocytes express Sox2 and yield high efficiency of chimeras after nuclear reprogramming with exogenous factors

Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application.     

Human parthenogenetic embryonic stem cells: one potential resource for cell therapy

Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem (iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies. However, the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells. Embryonic stem cells (ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy. Recent studies on human parthenogenetic embryonic stem cells (hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics, but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions. To generate various pluripotent stem cells, diverse reprogramming techniques and approaches will be developed and integrated. This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology, and ultimately benefit cell therapy and regenerative medicine. Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA02A101).     

Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.     

Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming, and integration of viral transgenes, in particular the oncogenes c-Myc and Klf4, may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA), a histone deacetylase inhibitor, enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4. The two factor-induced human iPS cells resemble human ES cells in pluripotency, global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means, which would make therapeutic use of reprogrammed cells safer and more practical.     

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum- and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells.     

Labeling stem cells with ferumoxytol, an FDA-approved iron oxide nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes. A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo. Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM(-1) sec(-1) strength. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure. Our group has applied this agent in an "off label" use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.