A model of cancer stem cells derived from mouse induced pluripotent stem cells

Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5'-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model.     

Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins

Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.     

A molecular view on pluripotent stem cells

Pluripotent stem cells are undifferentiated cells that are capable of differentiating to all three embryonic germ layers and their differentiated derivatives. They are transiently found during embryogenesis, in preimplantation embryos and fetal gonads, or as established cell lines. These unique cell types are distinguished by their wide developmental potential and by their ability to be propagated in culture indefinitely, without loosing their undifferentiated phenotype. This short review intends to give a general overview on the pluripotent nature of embryo-derived stem cells with a focus on human embryonic stem cells.     

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells

Background

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.

Methods

hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.

Results

Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.

Conclusion

hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.

General significance

Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.     

Generation of CD44 gene-deficient mouse derived induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) show good promise for the treatment of defects caused by numerous genetic diseases. Herein, we successfully generated CD44 gene-deficient iPSCs using Oct4, Sox2, Klf4, and vitamin C. The generated iPSCs displayed a characteristic morphology similar to the well-characterized embryonic stem cells. Alkaline phosphatase, cell surface (SSEA1, NANOG, and OCT4), and pluripotency markers were expressed at high levels in these cells. The iPSCs formed teratomas in vivo and supported full-term development of constructed porcine embryos by inter-species nuclear transplantation. Importantly, incubation with trichostatin A increased the efficiency of iPSCs generation by increasing the histone acetylation levels. Moreover, more iPSCs colonies appeared following cell passaging during colony picking, thus increasing the effectiveness of iPSCs selection. Thus, our work provides essential stem cell materials for the treatment of genetic diseases and proposes a novel strategy to enhance the efficiency of induced reprogramming.     

Artificial niche substrates for embryonic and induced pluripotent stem cell cultures

Stem cells possess the ability to self-renew and differentiate into other cell types. In vivo, stem cells reside in their own anatomic niches in a defined physiological environment, from which they are released to differentiate into a required cell type when deemed appropriate. While a resident within the niche, the stem cell receives signals that in turn maintain the cell in a pluripotent state. In addition, the niche also provides nourishment to the cell. Physically, the niche also serves to anchor the cell via various ECM components and cell-adhesion molecules. Therefore, in vitro models that replicate the in vivo niche will lead to a better understanding of stem cell fate and turnover. In turn, this will help inform attempts to culture stem cells in vitro on artificial niche-like substrates. In this review, we have highlighted recent studies describing artificial niche-like substrates used to culture embryonic and induced pluripotent stem cells in vitro.     

Mouse induced pluripotent stem cells

The recent discovery that it is possible to directly reprogramme somatic cells to an embryonic stem (ES) cell-like pluripotent state, by retroviral transduction of just four genes (Oct3/4, Sox2, c-Myc and Klf4), represents a major breakthrough in stem cell research. The reprogrammed cells, known as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells, and represent one of the most promising sources of patient-specific cells for use in regenerative medicine. While the ultimate goal is the use of iPS cells in the treatment of human disease, much of the research to date has been carried out with murine cells, and improved mouse iPS cells have been shown to contribute to live chimeric mice that are germ-line competent. Very recently, it has been reported that iPS cells can be generated by three factors without c-Myc, and these cells give rise to chimeric mice with a reduced risk of tumour development.     

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     

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.     

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.     

Disease-specific induced pluripotent stem cells

Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.     

Treatment of multiple sclerosis by transplantation of neural stem cells derived from induced pluripotent stem cells

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), with focal T lymphocytic infiltration and damage of myelin and axons. The underlying mechanism of pathogenesis remains unclear and there are currently no effective treatments. The development of neural stem cell (NSC) transplantation provides a promising strategy to treat neurodegenerative disease. However, the limited availability of NSCs prevents their application in neural disease therapy. In this study, we generated NSCs from induced pluripotent stem cells (iPSCs) and transplanted these cells into mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. The results showed that transplantation of iPSC-derived NSCs dramatically reduced T cell infiltration and ameliorated white matter damage in the treated EAE mice. Correspondingly, the disease symptom score was greatly decreased, and motor ability was dramatically rescued in the iPSC-NSC-treated EAE mice, indicating the effectiveness of using iPSC-NSCs to treat MS. Our study provides pre-clinical evidence to support the feasibility of treating MS by transplantation of iPSC-derived NSCs.     

X-chromosome epigenetic reprogramming in pluripotent stem cells via noncoding genes

Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally ‘naïve’, with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy.