miPS Cells Research Articles

The effect of BMP4, FGF8 and WNT3a on mouse iPS cells differentiating to odontoblast-like cells.

We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells.

MEK1/2 is a bottleneck that induces cancer stem cells to activate the PI3K/AKT pathway

Cancer stem cells (CSCs) are responsible for cancer initiation, drug resistance, and aggressive tumor phenotypes. Our lab has established a novel method to induce CSCs from induced pluripotent stem (iPS) cells in a microenvironment mimicking chronic inflammation. The converted cells acquired CSC characteristics and developed malignant tumors. Recently, we demonstrated that nonmutagenic chemical inhibitors accelerated the conversion of mouse iPS (miPS) cells into CSCs. Here, we investigated the effects of AZD-6244, a MEK1/2-specific inhibitor, on the conversion of iPS cells into CSCs. The miPS cells were cultured for one week in the presence of the conditioned medium (CM) of Lewis lung carcinoma (LLC) cells and AZD-6244, PD0325901, a pan-MEK inhibitor, or GDC-0879, a B-Raf inhibitor. As a result, AZD-6244 enhanced the conversion of iPS cells into CSCs and upregulated AKT phosphorylation as same as GDC-0879 and PD0325901. The converted cells maintained their self-renewal ability and stemness gene expression. The expression of the CSC markers CD24, CD44 and CD133 was higher in the cells cultured with MAPK inhibitors than in those cultured without MAPK inhibitors. Moreover, converted cells gained migration and invasion abilities assessed by in vitro assays. Therefore, the inhibition of MEK1/2 was found to be critical for the conversion of normal stem cells into CSCs in the tumor-inducing microenvironment.

Magnetic Targeted Delivery of Induced Pluripotent Stem Cells Promotes Articular Cartilage Repair.

Cartilage regeneration treatments using stem cells are associated with problems due to the cell source and the difficulty of delivering the cells to the cartilage defect. We consider labeled induced pluripotent stem (iPS) cells to be an ideal source of cells for tissue regeneration, and if iPS cells could be delivered only into cartilage defects, it would be possible to repair articular cartilage. Consequently, we investigated the effect of magnetically labeled iPS (m-iPS) cells delivered into an osteochondral defect by magnetic field on the repair of articular cartilage. iPS cells were labeled magnetically and assessed for maintenance of pluripotency by their ability to form embryoid bodies in vitro and to form teratomas when injected subcutaneously into nude rats. These cells were delivered specifically into cartilage defects in nude rats using a magnetic field. The samples were graded according to the histologic grading score for cartilage regeneration. m-iPS cells differentiated into three embryonic germ layers and formed teratomas in the subcutaneous tissue. The histologic grading score was significantly better in the treatment group compared to the control group. m-iPS cells maintained pluripotency, and the magnetic delivery system proved useful and safe for cartilage repair using iPS cells.

Differentiation of mouse-induced pluripotent stem cells into cardiomyocytes in vitro

In order to investigate the effective method to induce mice-induced pluripotent stem (miPS) cells into cardiomyocytes in vitro and to investigate the effect of vitamin C on cardiomyocyte differentiation from miPS cells to find a highly efficient and clinically safe method. MiPS cells were isolated and expanded to form embryoid bodies (EBs) using the hanging drop way. EBs were induced using differentiation medium containing vitamin C (10−4 mmol/ml). The control group did not receive any form of inducer. The time and frequency at which beating cardiomyocytes appeared and the percentage of beating colonies were determined to investigate the function of vitamin C on cardiac myocytes differentiation from miPS cells. Beating cell areas were found in (62.5 ± 1.7%) of EBs when using differentiation medium containing vitamin C, which was at a significantly greater frequency than in the control group (7.6 ± 2.6%). Beating cardiomyocytes within the two groups were positive for troponin (cTnT) staining. Vitamin C markedly increased the productivity of miPS cell differentiation into cardiomyocytes, as supported by expression of the unique cardiac protein cTnT. The vitamin C is suitable candidate for the induction of miPS cell differentiation into cardiomyocytes in vitro.

Motor neurons derived from ALS-related mouse iPS cells recapitulate pathological features of ALS.

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by the loss of motor neurons in the spinal cord and brain. Mutations in Cu/Zn superoxide dismutase 1 (SOD1) are known to induce ALS. Although many research models have been developed, the exact pathological mechanism of ALS remains unknown. The recently developed induced pluripotent stem (iPS) cell technology is expected to illuminate the pathological mechanisms and new means of treatment for neurodegenerative diseases. To determine the pathological mechanism of ALS, we generated mouse iPS (miPS) cells from experimental ALS transgenic mice and control mice and characterized the cells using molecular biological methods. The generated miPS cells expressed many pluripotent genes and differentiated into three germ layers in vitro and in vivo. Motor neurons derived from ALS-related miPS cells recapitulated the pathological features of ALS. The ALS-model motor neurons showed SOD1 aggregates, as well as decreased cell survival rate and neurite length compared with wild-type motor neurons. Our study will be helpful in revealing the mechanism of motor neuronal cell death in ALS.

Abstract 2510: Iron control is a novel therapeutic target of cancer stem cells

Abstract Background: Iron overload is known to cause cancer. Iron depletion treatment has been reported to have an anti-cancer effect. However, it is unclear whether iron depletion treatment is also effective in cancer stem cells (CSCs) or not. Recently, new CSCs model, miPS-LLCcm, was epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama university (Chen at el. PLOS ONE 2012). In this model, Green Fluorescent Protein (GFP) was designed under Nanog promoter lesion. Therefore, GFP positive cells indicated the undifferentiated cancer progenitor cells. The tumor tissue derived from miPS-LLCcm cells involved glandular structure, angiogenesis, and cytokeratin positive lesion in immunostaining. We hypothesized that CSCs depended on iron in proliferation and differentiation. Thus, we also hypothesized iron depletion treatment might have a novel therapeutic effect. Methods: We used miPS-LLCcm cells as CSCs, colon26 and 4T1 cells as differentiated cancer cells. Iron depleted condition was simulated by iron free medium with 1% fetal bovine serum. Transferrin (Holo) and iron chelators (Deferoxamine and Deferasirox) additional examinations were done to reveal the dependency of iron. Cell viability was examined by XTT assay 48 hours after administration of transferrin and iron chelators. Western blot analysis was done to examine the effect of iron depletion on iron related markers and stemness markers including TfR-1, DMT-1, Nanog, SOX2, c-Myc, Oct3/4, Klf-4, E-cadherin. Subcutaneous tumor model of miPS-LLCcm cells was used in vivo. Deferoxamine (30mg/kg/day) and deferasirox (30mg/kg/day) were administrated directly into the tumor. Tumors were collected for immunostaining. Results: Transferrin strongly promoted cell proliferation of miPS-LLCcm in iron depleted condition. However, Transferrin did not promote proliferation of colon26 and 4T1 in the condition. Iron depletion by iron chelators suppressed miPS-LLCcm proliferation and the expression of stemness markers including Nanog, SOX2, c-Myc, Oct3/4, Klf-4, and E-cadherin in vitro study. Deferasirox more strongly suppressed the expression of stemness markers than deferoxamine. Iron depletion by iron chelators suppressed subcutaneous tumor growth. The average tumor volume of the control group was 1270.8 ± 411.2 mm3 while that of deferoxamine treatment group was 502.5 ± 207.5 mm3 (p<0.05) and deferasirox treatment group was 360.2 ± 148.8 mm3 (p<0.01) on day 19. The expression of stemness markers was also more strongly suppressed in the deferasirox treatment group than deferoxamine treatment group. Conclusions: Iron is a key element of the proliferation and stemness in CSCs model. Iron control therapy including iron chelators is a novel therapeutic target of CSCs. Citation Format: Toshiaki Ohara, Takayuki Ninomiya, Kazuhiro Noma, Hajime Kashima, Yuki Katsura, Takuya Kato, Yasuko Tomono, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Iron control is a novel therapeutic target of cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2510.

Surface modified alginate microcapsules for 3D cell culture

Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core–shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

Abstract 4243: Iron control is a novel therapeutic target of cancer stem cells

Abstract Background: Iron overload is known to cause cancer in animal models. Several studies have shown that iron deprivation treatment has a strong anti-cancer effect. However, it is unclear whether iron deprivation treatment suppresses cancer stem cells or not. A cancer stem cell model, miPS-LLCcm, was recently epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama University. In this model, the green fluorescent protein (GFP) and the puromycin resistant gene were inserted into the 59-bp untranslated region of the Nanog gene of miPS cells. By this procedure, undifferentiated cancer progenitor cells are recognized as GFP positive cells. We then examined the iron dependency of these cancer stem cells both in in vitro and in vivo studies and we used this model to examine the possibility of cancer stem cells by iron deprivation. Materials and Methods: In in vitro studies, the miPS-LLCcm cells were used as cancer stem cells and colon26 and 4T1 cells were used as differentiated murine cancer cells. Puromycin was used to purify the cancer stem cells before seeding of the miPS-LLCcm cells. The dependency on iron for cell proliferation was examined following transferrin addition. Transferrin (Holo) was used to simulate an iron rich condition. Iron free medium and the iron chelators, Deferasirox and Deferoxamine, plus 1% fetal bovine serum (FBS) were used to simulate iron depletion conditions. Cell proliferation assays and flow cytometric analyses were performed 48 hours after adjustment of the iron concentration level. In in vivo studies, an iron depleted diet was used to simulate iron depleted conditions. Nude mice were divided into normal diet and iron depleted diet groups. The mice were fed with these diets for three weeks and then a suspension of miPS-LLCcm cells was injected into the backs of the nude mice. Tumor size was measured and the tumors were immunohistologically examined. Results: In the in vitro studies, transferrin strongly promoted the proliferation of cancer stem cells under iron depletion conditions compared to no transferrin(p<0.001). However, transferrin did not promote the proliferation of the differentiated 4T1 and colon26 cancer cells. The number of GFP-expressing cancer stem cells decreased as the iron concentration was decreased. In the vivo studies, iron depletion significantly suppressed the tumor growth of the cancer stem cells (p<0.05). Immunohistological analysis indicated that Nanog expression was also decreased in the tumors of the iron depletion diet group. Conclusions: Iron is a key element for the proliferation and differentiation of cancer stem cells. Iron controlling therapy including iron chelators is a novel therapeutic target of cancer stem cells. Citation Format: Takayuki Ninomiya, Toshiaki Ohara, Hajime Kashima, Ryoichi Katsube, Kazuhiro Noma, Yasuko Tomono, Akifumi Mizutani, Tomonari Kasai, Masaharu Seno, Shinji Kuroda, Hiroyuki Kishimoto, Hiroshi Tazawa, Yasuhiro Shirakawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Iron control is a novel therapeutic target of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4243. doi:10.1158/1538-7445.AM2015-4243

Abstract 3016: Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles

Abstract It is widely accepted that the microenvironment/niche exert genetic and/or epigenetic effects on stem cells. Current studies have demonstrated that an aberrant microenvironment may contribute to the genesis of cancer stem cells (CSCs). In the case of cancer, growing evidence indicate a cancerous niche can play an active role in the regulation of tumor cell maintenance and progression through exosomes/microvesicles-based intercellular communication. It has not been reported, however, whether this vesicle-mediated communication induces the malignant transformation of normal stem cells/differentiating progenitors, result in CSC generation. Recentry, we have reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into CSCs. Here, we investigated the contribution of tumor-derived exosomes/microvesicles (TEMVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. The differentiating miPSCs were exposed to TEMVs derived from LLC cells for 4 weeks. We used Nanog-GFP miPS, in which GFP expression is under control of Nanog promoter, indicative of undifferentiation state. During this period, we observed the reemergence of the GFP-positive colonies, whereas most of cells were differentiated and GFP-negative without TEMV-treatment. The resultant TEMVs treated cells (miPS-LLCemv) expressed Nanog and Oct3/4 proteins comparable to miPSCs assessed by Western blotting. The sphere formation of the miPS-LLCemv cells in suspension culture indicated the selfrenewal capacity of the miPS-LLCemv cells. When the miPSLLCemv cells were subcutaneously allografted into immunodeficient mice, malignant tumors with extensive angiogenesis developed. Phathological features such as vementin, Ki67, PPAR gamma expression, and accumulation of lipid droplets in tumor revealed these tumors were liposarcomas. Tumor cells established from liposarcomas showed the capacity of spheroid formation in suspension culture and the capacity of differntiation into adipocytes and endothelial cells. These results indicate that miPS-LLCemv cells and established tumor cells possess CSC properties. Interestingly, these cells expressed Sox2 and Klf4 genes significantly higher than parental miPS cells, might be related to selfrenewal and addipogenesis. Thus, we concluded that TEMVs should have the potential to induce CSC properties in normal stem cells. Citation Format: Yan Ting, Junko Masuda, Akifumi Mizutani, Ling Chen, Tsukasa Shigehiro, Shuichi Matsuda, Tomonari Kasai, Takayuki Kudoh, Hiroshi Murakami, Mary J.C. Hendrix, Luigi Strizzi, David S. Salomon, Li Fu, Masaharu Seno. Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2014-3016

Flk1+ and VE-Cadherin+ Endothelial Cells Derived from iPSCs Recapitulates Vascular Development during Differentiation and Display Similar Angiogenic Potential as ESC-Derived Cells

RationaleInduced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1+ and Vascular Endothelial (VE)-cadherin+ ECs derived identically from mouse (m)ES and miPS cells. Methods and ResultsNaive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1+ VE-cadherin+ cells from both mES and miPS cells. Emergence of Flk1+VE-cadherin+ cells entailed expression of the vascular developmental transcription factor Er71, which bound identically to Flk1, VE-cadherin, and CD31 promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures in vitro and incorporated into CD31+ neovessels in matrigel plugs implanted in nude mice in vivo.ConclusionThus, iPS cell-derived Flk1+VE-cadherin+ cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31+ neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.

Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation

Embryoid bodies (EBs) are large (>100 μm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 μm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.

Abstract 2647: In vitro niche created by cancer stem-like cells derived from mouse induced pluripotent stem cell.

Abstract Cancer stem cell (CSC) is a cell within tumor, which is considered to possess the capacity of self-renewal and differentiation for providing heterogeneous lineages of cancer cells in the tumor. Previously, we converted mouse iPS (miPS) cells into CSCs by treatment with conditioned medium prepared from Lewis Lung Carcinoma culture (miPS-LLCcm)1. RT-PCR and FACS analysis revealed that miPS-LLCcm could give rise to endothelial cells. When miPS-LLCcm were cultured on MatrigelTM, they exhibited the tubular structure containing CD31 positive cells. In contrast, undifferentiated cells enriched population, which were resistant to puromycin (puro) as parental miPS cell2, failed to form tubes. After puro was removed to allow the differentiate cells to survive, the tube formation was observed again, indicating that the differentiated cells were likely to contribute to tube formation. To confirm the stability of this tube formation capacity, we repeated further two cycles of puro selection and releasing differentiation. As the result, the tube formation ability of the differentiated cells decreased. In parallel, we added the conditioned medium of miPS-LLCcm culture into sphere-forming assay, observed the increase of size and number of spheroids. This effect was partly depended on Notch signaling pathway. These results raise the possibility that the differentiated cells from miPS-LLCcm including endothelial cells or their progenitor cells should be required for the maintenance of miPS-LLCcm to differentiate towards vascular endothelial linage. Collectively, we concluded that the differentiated cells from miPS-LLCcm create a niche in vitro for the self-renewal of CSC in the aspect of not only proliferation but also differentiation lineage. 1. Chen, L., et al. PLoS ONE 7, e33544 (2012). 2. Okita, K., et al. Nature 448, 313-317 (2007). Citation Format: Akifumi Mizutani, Shuichi Matsuda, Tin Yan, Tatsuyuki Sota, Tomonari Kasai, Takayuki Kudoh, Hiroshi Murakami, Masaharu Seno. In vitro niche created by cancer stem-like cells derived from mouse induced pluripotent stem cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2013-2647

Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium

Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.

Hydrogels as feeder-free scaffolds for long-term self-renewal of mouse induced pluripotent stem cells

Expanding undifferentiated induced pluripotent stem (iPS) cells in vitro is a basic requirement for application of iPS cells in both fundamental research and clinical regeneration. In this study, we intended to establish a simple, low cost and efficient method for the long-term self-renewal of mouse induced pluripotent stem (miPS) cells without using feeder-cells and adhesive proteins. Three scaffolds were selected for the long-term subculture of miPS cells over two months starting from passages 14 to 29: 1) a gelatin coated polystyrene (Gelatin-PS) that is a widely used scaffold for self-renewal of mouse embryonic stem (mES) cells; 2) a neutral hydrogel poly(N,N-dimethylacrylamide) (PDMAAm); and 3) a negatively charged hydrogel poly(2-acrylamido-2-methyl-propane sulfonic acid sodium salt) (PNaAMPS). Each passaged miPS cells on these scaffolds were cryopreserved successfully and the revived cells showed high viability and proliferation. The passaged miPS cells maintained a high undifferentiated state on all three scaffolds and a high level of pluripotency by expressing differentiation markers in vitro and forming teratomas in SCID mice with derivatives of all three germ layers. Compared to Gelatin-PS, the two hydrogels exhibited much better self-renewal performance in terms of high proliferation rate and level of expression of undifferentiated gene markers as well as efficiency in pluripotent teratoma formation. Furthermore, the PNaAMPS hydrogel demonstrated a slightly higher efficiency and simpler operation of cell expansion than the PDMAAm hydrogel. To conclude, PNaAMPS hydrogel is an excellent feeder-free scaffold because of its simplicity, low cost and high efficiency in expanding a large number of miPS cells in vitro.

Application of induced pluripotent stem cells in generation of a tissue-engineered tooth-like structure.

Stem cells, such as adult stem cells or embryonic stem cells, are the most important seed cells employed in tooth tissue engineering. Even though dental-derived stem cells are a good source of seed cells for such procedures, they are not often used in clinical applications because of the limited supply. Induced pluripotent stem (iPS) cells, with their high proliferation and differentiation ability, are now considered a promising alternative. The objectives of this study were to assess the role of iPS cells in tooth tissue engineering. We used real-time polymerase chain reaction to confirm that mouse iPS (miPS) cells can be induced to express both odontogenic and osteogenic gene profiles. We then established a tooth germ model and transplanted the recombinant tooth germ into a mouse subrenal capsule for 4 weeks to reproduce early-tooth organogenesis. After 4 weeks, hematoxylin and eosin staining results showed newly formed bone-like and dental pulp-like areas. Further immunohistochemical staining confirmed that osteopontin was present in the apical part of the tooth-like structure. These results demonstrate that miPS cells have the potential to differentiate into odontogenic cells, confirming that they could be a new source of seed cells for use in tooth tissue engineering.

Characterization and neural differentiation of mouse embryonic and induced pluripotent stem cells on cadherin-based substrata

A suitable culture condition using advanced biomaterials has the potential to improve stem cell differentiation into selective lineages. In this study, we evaluated the effects of recombinant extracellular matrix (ECM) components on the mouse embryonic stem (mES) and induced pluripotent stem (miPS) cells' self-renewal and differentiation into neural progenitors, comparing conventional culture substrata. The recombinant ECMs were established by immobilizing two chimera proteins of cadherin molecules, E-cadherin-Fc and N-cadherin-Fc, either alone or in combination. We report that the completely homogeneous population of mES and miPS cells could be maintained on E-cadherin-based substrata under feeder- and serum-free culture conditions to initiate neural differentiation. Using defined monolayer differentiation conditions on E-cadherin and N-cadherin (E-/N-cad-Fc) hybrid substratum, we routinely obtained highly homogeneous population of primitive ectoderm and neural progenitor cells. Moreover, the differentiated cells with higher expression of βIII-tubulin, Pax6, and tyrosine hydroxylase (TH) in absence of GFAP (a glial cell marker) expression suggesting the presence of a lineage restricted to neural cells. Our improved culture method should provide a homogeneous microenvironment for differentiation and obviate the need for protocols based on stromal feeders or embryoid bodies.

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.

Feeder Cells Support the Culture of Induced Pluripotent Stem Cells Even after Chemical Fixation

Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis.

Recurrent trisomy and Robertsonian translocation of chromosome 14 in murine iPS cell lines

Induced pluripotent stem (iPS) cells have greatly provoked people's interest due to their enormous potential of clinical applications. Increasing care is taken with the genetic safety of iPS cells. However, up to now, the chromosomal integrity of murine iPS (miPS) cells has been largely unknown. We have observed recurrent trisomy and/or Robertsonian translocation (Rb) of chromosome 14 in six out of nine independent miPS cell lines from three laboratories by G-banding, fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) analyses, while all the miPS cell lines were derived from mouse embryonic fibroblasts (MEFs) or neural precursor cells (NPCs) with a normal karyotype. The miPS cells with trisomy and/or Rb of chromosome 14 showed growth advantage over the miPS cells with a normal karyotype. We found a significantly higher frequency of Rbs in the miPS cell lines induced with c-Myc than those without c-Myc. Our findings demonstrate that miPS cell lines have the propensity for chromosomal aberrations and there is an obvious correlation between the extent of chromosomal aberrations in miPS cells and the transcriptional factors used for their reprogramming. Therefore, our study raises awareness of the need for improvements of the induction conditions of miPS cells in order to avoid the chromosomal aberrations and ensure future safe applications.

Agathisflavone Enhances Retinoic Acid-Induced Neurogenesis and Its Receptors α and β in Pluripotent Stem Cells

Flavonoids have key functions in the regulation of multiple cellular processes; however, their effects have been poorly examined in pluripotent stem cells. Here, we tested the hypothesis that neurogenesis induced by all-trans retinoic acid (RA) is enhanced by agathisflavone (FAB, Caesalpinia pyramidalis Tull). Mouse embryonic stem (mES) cells and induced pluripotent stem (miPS) cells growing as embryoid bodies (EBs) for 4 days were treated with FAB (60 μM) and/or RA (2 μM) for additional 4 days. FAB did not interfere with the EB mitotic rate of mES cells, as evidenced by similar percentages of mitotic figures labeled by phospho-histone H3 in control (3.4% ± 0.4%) and FAB-treated groups (3.5% ± 1.1%). Nevertheless, the biflavonoid reduced cell death in both control and RA-treated EBs from mES cells by almost 2-fold compared with untreated EBs. FAB was unable, by itself, to induce neuronal differentiation in EBs after 4 days of treatment. On the other hand, FAB enhanced neuronal differentiation induced by RA in both EBs of mES and miPS. FAB increased the percentage of nestin-labeled cells by 2.7-fold (mES) and 2.4 (miPS) and β-tubulin III-positive cells by 2-fold (mES) and 2.7 (miPS) in comparison to RA-treated EBs only. FAB increased the expression of RA receptors α and β in mES EBs, suggesting that the availability of RA receptors is limiting RA-induced neurogenesis in pluripotent stem cells. This is the first report to describe that naturally occurring biflavonoids regulate apoptosis and neuronal differentiation in pluripotent stem cells.