Differentiation Of iPS Cells Research Articles

Novel three-dimensional micro vibration actuator for imposing dynamic stimulations to promote differentiation of iPS cells

In recent advancement of regenerative medicine, it is strongly needed to develop devices to support active dynamic actuation of minute living cells. In this study, with the aid of finite element method analysis, simple but effective cantilever-type three-dimensional micro vibration actuator to be used within limited space and environment on inverted phase-contrast microscope system is newly developed to stimulate cultured living iPS cells colony directly to promote their differentiation into objective cells. The developed actuator enables us to excite three-dimensional vibrations in its micro-probe with total amplitude of displacement up to 30 µm in each X, Y and Z direction. In order to estimate effect of the actuator, mouse iPS cells colony is stimulated with 10 µm indenting as well as shearing vibrations with frequencies of 1, 5, 10, 15 Hz. Effects of these dynamic stimulations on the differentiation process of the mouse iPS cells are investigated by measuring the immunostained areas of βIII-tubulin and F-actin of the differentiated neural cells with or without the dynamic stimulation. It is found that there is significant difference in differentiation efficiency of the mouse iPS cells into neurons between the statically cultured cells and the dynamically stimulated ones. Also, it is confirmed that the mode of the dynamic stimulation has a decisive influence to promote the differentiation of mouse iPS cells into neurons.

Transplantation of neurons derived from human iPS cells cultured on collagen matrix into guinea-pig cochleae.

The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd.

Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection

The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can be potential cell sources for tooth regeneration. However, these previous methods still have problems, such as usage of other cell types, heterogeneity of differentiated cells, and tumorigenicity. In the present study, a novel method to differentiate iPS cells into odontoblast-like cells without tumorigenicity using gene transfection was established. It is an important advance in the establishment of efficient methods to generate homogeneous functional odontogenic cells derived from iPS cells.

MicroRNA-199a induces differentiation of induced pluripotent stem cells into endothelial cells by targeting sirtuin 1.

The ability to reprogram induced pluripotent stem (iPS) cells from somatic cells may facilitate significant advances in regenerative medicine. MicroRNAs (miRNAs) are involved in a number of core biological processes, including cardiogenesis, hematopoietic lineage differentiation and oncogenesis. An improved understanding of the complex molecular signals that are required for the differentiation of iPS cells into endothelial cells (ECs) may allow specific targeting of their activity in order to enhance cell differentiation and promote tissue regeneration. The present study reports that miR‑199a is involved in EC differentiation from iPS cells. Augmented expression of miR‑199a was detected during EC differentiation, and reached higher levels during the later stages of this process. Furthermore, miR‑199a inhibited the differentiation of iPS cells into smooth muscle cells. Notably, sirtuin 1 was identified as a target of miR‑199a . Finally, the ability of miR‑199a to induce angiogenesis was evaluated in vitro, using Matrigel plugs assays. This may indicate a novel function for miR‑199a as a regulator of the phenotypic switch during vascular cell differentiation. The present study provides support to the notion that with an understanding of the molecular mechanisms underlying vascular cell differentiation, stem cell regenerative therapy may ultimately be developed as an effective treatment for cardiovascular disease.

Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells.

Epigenetic modification in congenital heart diseases by using stem cell technologies

Congenital heart diseases are the most common birth defects, occurring in more than 1% of newborns. The gene regulatory network of cardiac development has been revealed and some cases of congenital heart diseases have been shown to be associated with cardiac-specific gene mutations or related chromosomal abnormalities; however, almost all cases of congenital heart diseases are sporadic and the pathogenesis of heart malformations still remains unknown. Recently, studies of epigenetic regulation have been making progress in the field of cardiac development and the accumulated knowledge helps us understand more about cardiogenesis and congenital heart diseases. Interestingly, pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have attracted attention as tools to dissect epigenetic regulation during differentiation. Reprogramming and differentiation of ES cells and iPS cells can be induced by changes of gene expression patterns and epigenetic regulation, not by changing DNA sequences. We can also modify histone patterns and chromatin structures using chemical reagents. Hence, pluripotent stem cells enable us to dissect and modify epigenetic regulation during cardiogenesis in vitro. Moreover, in terms of iPS cells, they have become disease models because they can be generated from the somatic cells of patients. In this review, we summarize the recent findings of epigenetic regulation in cardiac development and congenital heart diseases. We also review the epigenetic modification during the reprogramming and cardiac differentiation of ES cells and iPS cells, and introduce our study showing that the disease-specific iPS cells of hypoplastic left heart syndrome, one of the severest congenital heart diseases with single ventricular circulation, exhibit unique epigenetic regulation during differentiation. Furthermore, we briefly focus on modifications of epigenetic regulation in cardiac development using chemical reagents, which suggest the possibility of treating congenital heart diseases using drugs. Lastly, we discuss the epigenetic memory of iPS cells, a specific feature that often complicates or prevents study of the differentiation of iPS cells.

MicroRNA-199b Modulates Vascular Cell Fate During iPS Cell Differentiation by Targeting the Notch Ligand Jagged1 and Enhancing VEGF Signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration. Methods and Results: In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen‐coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR‐199b is involved in EC differentiation. A step‐wise increase in expression of miR‐199 was detected during EC differentiation. Notably, miR‐199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA‐mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR‐199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR‐199b‐mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR‐199b also regulated VEGF expression and angiogenesis. Conclusions: This study indicates a novel role for miR‐199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405–1418

Thyroid cell differentiation from murine induced pluripotent stem cells.

Here, we demonstrate the successful differentiation of induced pluripotent stem (iPS) cells into functional thyroid cells indicating the therapeutic potential of this approach when applied to individuals with thyroid deficiency. Using embryonic murine fibroblasts, we generated iPS cells with a single lentiviral "stem cell cassette" vector and then differentiated these iPS cells into thyroid cells after transfection with PAX8 and NKX2-1 by Activin A and TSH stimulation. The generated iPS cells expressed pluripotent stem cell markers as assessed using both reverse transcription quantitative PCRs and immunofluorescence staining with ~0.5% reprograming efficiency. Compared to control cells, the expression of thyroid-specific genes NIS, TSHR, Tg, and TPO were greatly enhanced in PAX8(+)NKX2-1(+) iPS cells after differentiation. On stimulation with TSH, these differentiated iPS cells were also capable of dose-dependent cAMP generation and radioiodine uptake indicative of functional thyroid epithelial cells. Furthermore, the cells formed three-dimensional follicles in culture, and "thyroid organoids" formed after PAX8(+)NKX2-1(+) iPS cells transplanted into nude mice, and all expressed Tg protein as judged immunohistochemically. Taken together, thyroid epithelial cells differentiated from iPS cells, which were themselves derived from murine fibroblasts, exhibited very similar properties to thyroid cells previously developed from traditional murine embryonic stem cells. Thyroid cells differentiated from iPS cells offer the opportunity to examine the detailed transcriptional regulation of thyroid cell differentiation and may provide a useful future source for individualized regenerative cell therapy.

Manipulation of Induced Pluripotent Stem Cell Differentiation to Endothelial Cells

With the potential use of induced pluripotent stem cells (iPSCs) in regenerative medicine, strategies must be implemented to guide iPS cell differentiation. Without guidance, the pluripotent nature may yield tumorigenic and/or undesired cell types. Previously we observed that the endothelial cell secretome (EC‐S) derived from conditioned media (EC‐CM) guided iPS cell differentiation to an endothelial cell phenotype. Specifically, the EC‐S induced iPS cell differentiation as evidenced by LDL scavenging and tube formation in vitro. Of keen interest is the question of what product(s) of the EC‐S induce the differentiation of iPS cells. To determine this, we first confirmed that treatment of IPS cells with conditioned media from normoxic or hypoxic (1‐2% O2) endothelial cells produced differentiation of iPS cells into endothelial cells. To determine if a protein factor is responsible for the differentiation, we subjected the conditioned media to heat denaturation (10 min @ 70 C). In media obtained from normoxic endothelial cells, heat denaturation greatly reduced the differentiation of iPS cells into endothelial‐like morphology. In fact the vast majority of cells in culture retained an IPS cell phenotype. Conversely, the heat denatured conditioned media from hypoxic endothelial cells did reduce the differentiation of IPS cells into an endothelial phenotype. These results suggest that the products of the secretome that induce differentiation of iPS cells into endothelial cells are changed by hypoxia. The results also suggest that during normoxia, the active component of the secretome are proteins (the activity is lost with heat denaturation), but this changes during hypoxia to a factor (or factors) that is resistant to heat denaturation.

The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression.

Long non-coding RNAs (lncRNAs) regulate diverse biological processes, including cell lineage specification. Here, we report transcriptome profiling of human endoderm and pancreatic cell lineages using purified cell populations. Analysis of the data sets allows us to identify hundreds of lncRNAs that exhibit differentiation-stage-specific expression patterns. As a first step in characterizing these lncRNAs, we focus on an endoderm-specific lncRNA, definitiveendoderm-associated lncRNA1 (DEANR1), and demonstrate that it plays an important role in human endoderm differentiation. DEANR1 contributes to endoderm differentiation by positively regulating expression of the endoderm factor FOXA2. Importantly, overexpression of FOXA2 is able to rescue endoderm differentiation defects caused byDEANR1 depletion. Mechanistically, DEANR1 facilitates FOXA2 activation by facilitating SMAD2/3 recruitment to the FOXA2 promoter. Thus, our studynot only reveals a large set of differentiation-stage-specific lncRNAs but also characterizes a functional lncRNA that is important for endoderm differentiation.

Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Fluid driving system for a micropump by differentiating iPS cells into cardiomyocytes on a tent-like structure

A number of recent studies have exploited the sizes and functional properties of microdevices and cellular mechanical components to construct bio-microactuators. We previously developed bio-micropumps powered by cardiomyocytes that utilizes glucose in the medium as chemical energy. To fabricate the pump, however, primary neonatal rat cardiomyocytes are indispensable. The operation of harvesting primary cells is inconvenient and ethically not adequate due to the need for animals sacrifice. In contrast, induced pluripotent stem (iPS) cells are obtained from subcutaneous tissue. Their most significant properties are that they proliferate indefinitely and can be differentiated into many kinds of cells, including cardiomyocytes, and also have no ethical issue differently from ES cells. By exploiting these properties of iPS cells, the above issues will be addressed. Based on this concept, we constructed a system for driving fluids as a principal component of a micropump by differentiating iPS cells into spontaneously beating cardiomyocytes. Cellular contractile force was transmitted to fluid in a microchannel by a tent-like thin membrane. The microchip was irradiated with O2 plasma and coated with gelatin to attach the cells. Embryoid bodies (EBs) of mouse-derived iPS cells were seeded on the microchip and incubated at 37°C without Leukemia Inhibitory Factor (LIF) to differentiate them into cardiomyocytes. About 2 weeks later, EB beating and periodical oscillation of fluid in a microchannel connected to a diaphragm chamber was observed. The theoretical flow rate assuming the use of ideal check valves (Q) was 6.9nL/min. Our device presents a reasonable alternative to normal cardiomyocytes for preliminary investigations requiring bio-actuating pumps.

The differentiation of rat-induced pluripotent stem cells into alveolar type II epithelial cells with a three-step induction protocol

Induced pluripotent stem (iPS) cells derive from autologous somatic cells, the application prospect of iPS cells for regenerative medicine and tissue engineering is better than embryonic stem cells (ESCs) to some extent. Alveolar type II (AT II) epithelial cells play key role in the injured lung tissue regeneration and function recovery. The differentiation of iPS cells into AT II cells could provide available source for injured lung treatment. In this study, rat iPS (riPS) cells were resuscitated and proliferated for 14 days before differentiation. A modified three-step induction protocol similar to the reported ESCs inducing procedure was used in this study for the differentiation groups. Routine cell culture was done to the riPS cell control group (riPS-con). At stage 3, cells of day 7 (Diff. 7) and day 14 (Diff. 14) were collected for the real-time polymerase chain reaction tests for gene expressions of Oct4, Nanog, SPA, SPB, SPC, SPD, and CC10. Immunofluorescence staining of SPC and SSEA-1 was conducted. At the end of the differentiation, cell morphology became outstretched and epithelium-like. Cells of the Diff. 14 group positively expressed SPC and negatively expressed SSEA-1, which is contrary to the riPS-con group. In the Diff. 7 and the Diff. 14 groups, the expression of Oct4, Nanog, and SPB decreased (P < 0.05), whereas the expression of SPA, SPC, SPD (P < 0.05), and CC10 (P > 0.05) increased. This study indicated that riPS cells can successfully differentiate into AT II epithelial cells with the three-step induction protocol and may be further applied to implanting in decellularized rat lung scaffolds and building a bio-artificial lung.

J0420306 音響共鳴現象と超音波パルスエコー法を利用したソフトマテリアルの膜厚と音速の同時計測

We propose a new measurement method for simultaneously measuring thickness and sound velocity for soft materials such as a conductive polymer film and stacked induced pluripotent stem cells (iPS cells). Using the 75 MHz underwater-type ultrasound transducer, simultaneous thickness and sound velocity measurement has been carried out by combining the acoustic resonant spectroscopy and pulse-echo measurement. The advantage of this method is that the thickness can be derived without using the known value of sound velocity. The thickness of the conductive polymer film and the stacked iPS cells on metal substrate are measured at 10.4 μm and 16.0 μm, respectively. The thickness of conductive polymer and the staked iPS cells agrees well with the reference measured by the confocal laser microscope. T These results indicate that the developed measurement method can be one of the potential candidates for the real-time thickness measurement for the soft materials. For future work, the thickness of living iPS cells should be measured in real time. Using this method, we would like to figure out the relationship between the stacked state and the differentiation of iPS cells.

Determination of the potential of induced pluripotent stem cells to differentiate into mouse nucleus pulposus cells in vitro.

We determined the potential for induced pluripotent stem (iPS) cells to differentiate into nucleus pulposus (NP)-like cells in mice. iPS cells were generated from tail-tip fibroblasts. We used a pellet culture model with the aim of determining the applicability of iPS cell-based therapy to intervertebral disc degeneration (IVD). The cell pellet was cultured in an NP cell basal medium comprising Dulbecco's modified Eagle's medium supplemented with transforming growth factor beta 1, dexamethasone, ascorbate-2-phosphate, and 1% ITS-Premix. The pellet was evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemical staining, and biochemical composition. The differentiation of iPS cells into NP cells was demonstrated by the protein and mRNA expression levels of proteoglycan, collagen II, aggrecan, and CD24. Furthermore, increased hydroxyproline content and dimethylmethylene blue staining demonstrated that the collagen II and glycosaminoglycan content in the NP cells increased with time. We have shown that cultured mouse iPS cells can be induced to differentiate into NP cells. Such proof-of-concept opens up the possibility of producing patient-specific NP cells in a relatively simple and straightforward manner with high efficiency. We are confident that such cells could be immediately useful for the study of IVD disease.

Differentiation of Human Induced Pluripotent Stem Cells to Chondrocytes.

Human induced pluripotent stem (iPS) cells are relevant tools for modeling human skeletal development and disease, and represent a promising source of patient-specific cells for the regeneration of skeletal tissue, such as articular cartilage. Devising efficient and reproducible strategies, which closely mimic the physiological chondrogenic differentiation process, will be necessary to generate functional chondrocytes from human iPS cells. Our previous study demonstrated the generation of chondrogenically committed human iPS cells via the enrichment of a mesenchymal-like progenitor population, application of appropriate high-density culture conditions, and stimulation with bone morphogenetic protein-2 (Bmp-2). The differentiated iPS cells showed temporal expression of cartilage genes and the accumulation of a cartilaginous extracellular matrix in vitro. In this chapter, we provide detailed methodologies for the differentiation of human iPS cells to the chondrogenic lineage and describe protocols for the analysis of chondrogenic differentiation.

Thymic derived iPs cells can be differentiated into cardiomyocytes.

Ventricular septal defect (VSD) is one of the common congenital heart malformations. Several factors lead to the development of VSD, including familial causes, exposure to certain drugs, infectious agents, and maternal metabolic disturbances. We considered that induced pluripotent stem (iPS) cells derived from VSD patients can be used to study the origin and pathogenesis of the VSD. Here, we show generation and cardiomyocyte differentiation potential of iPS cells from thymic epithelial cells of a patient with VSD (TECs-VSD) by overexpressing the four factors: OCT4, SOX2, NANOG, and LIN28 with lentiviral vectors. The self-renewal and pluripotency of the VSD-iPS cells was verified in iPS cells by in vitro expression of pluripotency markers and formation of teratoma in vivo. iPS cell lines from VSD patients differentiated into functional cardiomyocytes can serve as a model system for studying the pathophysiology and identifying etiology of VSD.

IPS細胞を用いた喉頭・気管の上皮再生

Reconstruction of the upper airway after the resection of malignancies or stenotic inflammatory lesions is thought to be difficult. Postoperative scarring or granulation with stenosis is sometimes observed. Therefore, the regeneration of the respiratory tract with full functionality is vital after surgical treatment for cancer or inflammatory lesions.ES cell-like pluripotent cells, also known as induced pluripotent stem（iPS）cells, were generated from mouse skin fibroblasts by the introduction of 4 transcription factors in 2006. These cells are capable of unlimited symmetrical self-renewal, and thus provide an unlimited source of cells for tissue-engineering applications. In addition, the use of iPS cells obtained from patient-derived somatic cells can prevent transplant rejection.In this paper, using a technique for the differentiation of iPS cells, respiratory epithelium-like tissue was histologically observed, and the ciliary epithelium was immunohistologically confirmed. The transcript expression levels of CK14 were significantly increased in hiPS hVFF cultures. The cellular morphology was clearly cohesive and displayed a degree of nuclear polarity, which was suggestive of epithelial differentiation.Our results suggest that iPS cells could be a new source cells for use in the regeneration of the tracheal and laryngeal epithelium.

Differentiation of iPS Cells to Cochlear Cells are Regulated Depending on the Part of Cocultured Organs

Background: Induced pluripotent stem (iPS) cells aremultipotentstem cellsthat can be producedfrom adult cells such as fibroblastsby the induction ofartificial reprogramming. Since such cells can be differentiated to endoderm, mesodermand ectoderm asembryonic stem (ES) cells, theirpotential utility for the regenerative therapy of various organs is expected. The inner ear is composed of various types of functional cells (hair cells, supporting cells, spiral ganglion, cochlear fibrocytes, striavascularis, etc.), and each cell type. Although the degeneration of these cells leads directly to severe hearing loss, there are few reports concerning thedifferentiation of iPS cells to various types of inner ear cells for regenerative therapy. Results: In this study, we co-cultured iPS-derived cells with three different regions (spiral ganglion, the organ of Corti with spiral limbus, lateral wall) of the cochlear tissue and tod attempted to induce their differentiation into various types of inner ear cells. The number of positive colonies (Nanogpositive colony) with reporter GFP controlled by Nanogpromotor was counted to assess the undifferentiated level of the cells. In the co-cultured cells with spiral ganglion, many Nanog-positive colonies were observed, and many cells with neurite-like protrusions were observed among the colonies. In the co-culture with the organ of Cortiand spiral limbus (OC/SL), neuron-like cells were observed on the cell layer with tight junction-like adhesions. In the co-culture with the cochlear lateral wall, many cell layers with tight junction-like adhesions were observed, while undifferentiated Nanogpositive colonies were not observed. Conclusion: In this study, we demonstrated that differentiation of iPS cells to cochlear cells was regulated depending on the part of the co-cultured organs. Co-culture with cochlea tissue and the induced pluripotent stem cells may enable regenerative therapy of various types of inner ear cells.

Dual gene expression in embryoid bodies derived from human induced pluripotent stem cells using episomal vectors.

Transcription factors are essential for the differentiation of human induced pluripotent stem cells (iPS) into specialized cell types. Embryoid body (EB) formation promotes the differentiation of iPS cells. We sought to establish an efficient method of transfection and rotary culture to generate EBs that stably express two genes. The pMetLuc2-Reporter vector was transfected using FuGENE HD (FuGENE), Lipofectamine LTX (LTX), X-tremeGENE, or TransIT-2020 transfection reagents. The media was analyzed using a Metridia luciferase (MetLuc) assay. Transfections were performed on cells adherent to plates/dishes (adherent method) or suspended in the media (suspension method). The 201B7 cells transfected with episomal vectors were selected using G418 (200 μg/mL) or hygromycin B (300 μg/mL). Rotary culture was performed at 2.5 or 9.9 rpm. Efficiency of EB formation was compared among plates and dishes. Cell density was compared at 1.6×10(3),×10(4), and×10(5) cells/mL. The suspended method of transfection using the FuGENE HD reagent was the most efficient. The expression of pEBMulti/Met-Hyg was detected 11 days posttransfection. Double transformants were selected 6 days posttransfection with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg. Both EGFP and CherryPicker were expressed in all of the surviving cells. EBs were formed most efficiently from cells cultured at a density of 1.6×10(5) cells/mL in six-well plates or 6 cm dishes. The selected cells formed EBs. FuGENE-mediated transfection of plasmids using the suspension method was effective in transforming iPS cells. Furthermore, the episomal vectors enabled us to perform a stable double transfection of EB-forming iPS cells.