Differentiation Of Mouse iPS Cells Research Articles

Identification of Novel Regulator Involved in Differentiation of Mouse iPS Cells into Odontoblast-like Cells.

Odontoblasts differentiate from dental papilla stem cells, but the genetic changes that occur during this process remain unclear. The aim of this study was to investigate gene expression patterns during differentiation of mouse iPS cells into odontoblast-like cells. Mouse iPS cells were cultured on a collagen type-1 scaffold with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results of immunofluorescence studies for dentin sialoprotein, dentin matrix protein 1 (DMP1), and nestin were positive. A qRT-PCR analysis revealed that mRNA expression levels of neural crest marker sex determining region Y box (Sox)-10, dentin sialophosphoprotein (Dspp), and Dmp1 were up-regulated, but that mRNA expression levels of the mineralization markers bone sialoprotein and osteocalcin were down-regulated. Microarray analysis showed that 2,597 entities were up-regulated and 1,327 down-regulated among a total of 15,330 investigated. Sox11 was among the up-regulated genes identified. The Sox11 mRNA expression level with odontoblast induction after day 11 was higher than that after day 2 (p<0.05). Gene knockdown using small interference RNA (siRNA) silencing was used to characterize the function of Sox11. The Dspp mRNA expression level in Sox11 siRNA-treated cells was significantly lower than that in the control (p<0.05). These results suggest that BMP4 and RA induce mouse iPS cells to differentiate into odontoblast-like cells. The differentiation efficiency is not high, however, and many stem cells remain. The results also suggest that Sox11 is an important factor in odontoblastic differentiation.

Effect of EDTA-treated dentin on the differentiation of mouse iPS cells into osteogenic/odontogenic lineages in vitro and in vivo.

To investigate the effect of EDTA-treated dentin on the differentiation of mouse induced pluripotent stem (iPS) cells. Dentin discs were prepared from bovine incisors and treated with 17% EDTA. Embryoid bodies (EBs) formed from mouse iPS cells were seeded on the dentin discs for the experiment. The roughness of the EDTA-treated dentin surface, Sa and Sdr, was higher and collagen fibrillike structures were observed by the scanning electron microscopy (SEM) in vitro. In RT-PCR, the mRNA levels of the osteoblast markers Bsp and Ocn were significantly higher in the experimental group. Expression of the DMP1, DSP, and BSP proteins were more notable in the experimental group by immunofluorescence (ICF) study. In vivo study, cartilage and bone-like tissue were observed adjacent to the EDTA-treated dentin. The study demonstrates that the dentin treated with 17% EDTA induces mouse iPS cells to differentiate into the osteo/odontogenesis.

Effects of muscarinic acetylcholine receptor stimulation on the differentiation of mouse induced pluripotent stem cells into neural progenitor cells.

Muscarinic acetylcholine receptors (mAchRs), which are expressed in various embryonic cells, may regulate neuronal differentiation. In the present study, we examined the effects of mAchR stimulation on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells (NPCs). Mouse iPS cells were cultured on ultra-low attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 3μmol/L) and/or pilocarpine (10 or 100μmol/L), a mAchR agonist, were added to EB cultures for 4days, following which the EBs were cultured on gelatin-coated plates for 7days. Subtype-specific antibody staining revealed that mouse iPS cells predominantly express m2 - and m4 -AchR. Treatment with pilocarpine alone did not affect the expression of Nestin (a specific marker for neural progenitor cells). However, additional treatment with pilocarpine significantly suppressed ATRA-induced Nestin expression. Pretreating EBs with either AF-DX116 (an antagonist of both m2 - and m4 -AchR) or forskolin (an activator of adenylate cyclase) significantly reversed the pilocarpine-induced suppression of Nestin expression. In addition, treatment with pilocarpine significantly suppressed ATRA-induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). These findings suggest that the stimulation of m2 - or m4 -AchR suppresses ATRA-induced differentiation of mouse iPS cells into NPCs by inhibiting the cAMP/protein kinase A pathway and CREB activation.

BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2.

Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2 This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

Differentiation of mouse iPS cells is dependent on embryoid body size in microwell chip culture

A microwell chip possessing microwells of several hundred micrometers is a promising platform for generating embryoid bodies (EBs) of stem cells. Here, we investigated the effects of initial EB size on the growth and differentiation of mouse iPS cells in microwell chip culture. We fabricated a chip that contained 195 microwells in a triangular arrangement at a diameter of 600μm. To evaluate the effect of EB size, four similar conditions were designed with different seeding cell densities of 100, 500, 1000, and 2000cells/EB. The cells in each microwell gradually aggregated and then spontaneously formed a single EB within 1d of culture, and EB size increased with further cell proliferation. EB growth was regulated by the initial EB size, and the growth ability of smaller EBs was higher than that of larger EBs. Furthermore, stem cell differentiation also depended on the initial EB size, and the EBs at more than 500cells/EB promoted hepatic and cardiac differentiations, but the EBs at 100cells/EB preferred vascular differentiation. These results indicated that the initial EB size was one of the important factors controlling the proliferation and differentiation of stem cells in the microwell chip culture.

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.

Expression of neuropeptide receptor mRNA during osteoblastic differentiation of mouse iPS cells

Various studies have shown a relationship between nerves and bones. Recent evidence suggests that both sensory and sympathetic nerves affect bone metabolism; however, little is known about how neuropeptides are involved in the differentiation of pluripotent stem cells into osteoblastic (OB) cells. To evaluate the putative effects of neuropeptides during the differentiation of mouse induced pluripotent stem (iPS) cells into calcified tissue-forming OB cells, we investigated the expression patterns of neuropeptide receptors at each differentiation stage. Mouse iPS cells were seeded onto feeder cells and then transferred to low-attachment culture dishes to form embryoid bodies (EBs). EBs were cultured for 4 weeks in osteoblastic differentiation medium. The expression of α1-adrenergic receptor (AR), α2-AR, β2-AR, neuropeptide Y1 receptor (NPY1-R), neuropeptide Y2 receptor (NPY2-R), calcitonin gene-related protein receptor (CGRP-R), and neurokinin 1-R (NK1-R) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Among these neuropeptide receptors, CGRP-R and β2-AR were expressed at all stages of cell differentiation, including the iPS cell stage, with peak expression occurring at the early osteoblastic differentiation stage. Another sensory nervous system receptor, NK1-R, was expressed mainly in the late osteoblastic differentiation stage. Furthermore, CGRP-R mRNA showed an additional small peak corresponding to EBs cultured for 3 days, suggesting that EBs may be affected by serum CGRP. These data suggest that the sensory nervous system receptor CGRP-R and the sympathetic nervous system receptor β2-AR may be involved in the differentiation of iPS cells into the osteoblastic lineage. It follows from these findings that CGRP and β2-AR may regulate cell differentiation in the iPS and EB stages, and that each neuropeptide has an optimal period of influence during the differentiation process.

Differentiation of mouse iPS cells into ameloblast-like cells in cultures using medium conditioned by epithelial cell rests of Malassez and gelatin-coated dishes.

Induced pluripotent stem (iPS) cells are generated from adult cells and are potentially of great value in regenerative medicine. Recently, it was shown that iPS cells can differentiate into ameloblast-like cells in cultures using feeder cells. In the present study, we sought to induce differentiation of ameloblast-like cells from iPS cells under feeder-free conditions using medium conditioned by cultured epithelial cell rests of Malassez (ERM) cells and gelatin-coated dishes. Two culture conditions were compared: co-cultures of iPS cells and ERM cells; and, culture of iPS cells in ERM cell-conditioned medium. Differentiation of ameloblast-like cells in the cultures was assessed using real-time RT-PCR assays of expression of the marker genes keratin 14, amelogenin, and ameloblastin and by immunocytochemical staining for amelogenin. We found greater evidence of ameloblast-like cell differentiation in the cultures using the conditioned medium. In the latter, the level of amelogenin expression increased daily and was significantly higher than controls on the 7th, 10th, and 14th days. Expression of ameloblastin also increased daily and was significantly higher than controls on the 14th day. The present study demonstrates that mouse iPS cells can be induced to differentiate into ameloblast-like cells in feeder-free cell cultures using ERM cell-conditioned medium and gelatin-coated dishes.

Effects of BMP4/7 on high-glucose-level induced suppression of osteogenic differentiation of mouse iPS cells

Effects of BMP4/7 on high-glucose-level induced suppression of osteogenic differentiation of mouse iPS cells

Stimulation of 5‐HT4 receptor enhances differentiation of mouse induced pluripotent stem cells into neural progenitor cells

Activation of serotonin (5-hydroxytryptamine; 5-HT) receptors plays a role in adult neurogenesis and differentiation of neural progenitor cells (NPC). Herein, we examined the involvement of 5-HT receptors in the differentiation of mouse induced pluripotent stem (iPS) cells into NPC. To induce embryoid body (EB) formation, mouse iPS cells were cultured on ultralow-attachment dishes. All-trans retinoic acid (ATRA; 1μmol/L) and/or 5-HT (0.03 or 0.1μmol/L) was added to the EB cultures for 4days and then EB plated on gelatin-coated plates were cultured for 7 or 14days. Immunofluorescence staining revealed that mouse iPS cells expressed both 5-HT2A and 5-HT4 receptors and, to a lesser extent, 5-HT1A receptors. Treatment with 5-HT significantly enhanced the ATRA-induced expression of nestin, a specific marker for NPC, and phosphorylation of cAMP response element-binding protein (CREB). Pretreatment of EB cultures with either 1μmol/L GR113808 (a selective 5-HT4 receptor antagonist) or 1μmol/L H89 (a protein kinase (PKA) inhibitor) significantly inhibited these effects of 5-HT. These findings suggest that stimulation of 5-HT4 receptors may enhance ATRA-induced neural differentiation of mouse iPS cells through activation of PKA and CREB.

The Characteristics of Murine iPS Cells and siRNA Transfection Under Hypoxia.

iPS cells are attractive for the regenerative medicine. The creation of pluripotent cells from somatic cells has great potential for basic and clinical research and application. Retroviral transduction of four or three transfection factors has been shown to initiate a reprogramming process. Here, we describe the effect of transcription factors regarding the growth and differentiation of mouse iPS cells in normoxia or hypoxia. Furthermore, we introduce the function of hypoxia-inducible factors (HIFs) in mouse iPS cells in hypoxia using RT-PCR and western blotting together with HIFs knockdown techniques.

Mouse-Induced Pluripotent Stem Cells Differentiate into Odontoblast-Like Cells with Induction of Altered Adhesive and Migratory Phenotype of Integrin

Methods for differentiating induced pluripotent stem (iPS) cells into odontoblasts generally require epithelial–mesenchymal interactions. Here, we sought to characterize the cells produced by a ‘hanging drop’ technique for differentiating mouse iPS cells into odontoblast-like cells that requires no such interaction. Cells were cultured by the hanging drop method on a collagen type-I (Col-I) scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4) without an epithelial–mesenchymal interaction. We evaluated the expression of odontoblast-related mRNA and protein, and the proliferation rate of these cells using reverse-transcription polymerase chain reaction, immunofluorescence staining, and BrdU cell proliferation enzyme-linked immunosorbent assay, respectively. The differentiated cells strongly expressed the mRNA for dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (Dmp-1), which are markers of mature odontoblasts. Osteopontin and osteocalcin were not expressed in the differentiated cells, demonstrating that the differentiated iPS cells bore little resemblance to osteoblasts. Instead, they acquired odontoblast-specific properties, including the adoption of an odontoblastic phenotype, typified by high alkaline phosphatase (ALP) activity and calcification capacity. The cell-surface expression of proteins such as integrins α2, α6, αV and αVβ3 was rapidly up-regulated. Interestingly, antibodies and siRNAs against integrin α2 suppressed the expression of DSPP and Dmp-1, reduced the activity of ALP and blocked calcification, suggesting that integrin α2 in iPS cells mediates their differentiation into odontoblast-like cells. The adhesion of these cells to fibronectin and Col-I, and their migration on these substrata, was significantly increased following differentiation into odontoblast-like cells. Thus, we have demonstrated that integrin α2 is involved in the differentiation of mouse iPS cells into odontoblast-like cells using the hanging drop culture method, and that these cells have the appropriate physiological and functional characteristics to act as odontoblasts in tissue engineering and regenerative therapies for the treatment of dentin and/or dental pulp damage.

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.

Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd.

Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) exhibit reduced efficiency and higher variability in neural differentiation compared to embryonic stem cells (ESCs). In this study, we showed that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation. We reported a novel approach which remarkably increases neural differentiation of mouse iPSCs. This novel approach initiated embryoid body (EB) formation directly from the whole cell clones isolated from the top of feeder cells. Compared to conventional neural induction methods such as single cell suspension or monolayer culture, the cell clone-derived EB method led to a pronounced increase in directed generation of various types of neural cells including neural stem cells, motoneurons and dopaminergic neurons in response to different inducers. Through gene expression microarray analysis, we identified 14 genes that were highly expressed in the cell clone-derived EBs. Among them, we found that Cdh2, also known as N-cadherin, played important roles in controlling the neural differentiation efficiency of mouse iPSCs. Forced expression of Cdh2 in iPSCs substantially enhanced the differentiation efficiency while knocking-down of Cdh2 by shRNA blocked the neural differentiation. Our results revealed a critical role of Cdh2 in the process of efficient neural differentiation of mouse iPS cells.

Differentiation of Induced Pluripotent Stem Cells into Male Germ CellsIn Vitrothrough Embryoid Body Formation and Retinoic Acid or Testosterone Induction

Generation of germ cells from pluripotent stem cells in vitro could have great application for treating infertility and provides an excellent model for uncovering molecular mechanisms controlling gametogenesis. In this study, we explored the differentiation potential of mouse induced pluripotent stem (iPS) cells towards male germ cells. Embryoid body formation and retinoic acid/testosterone induction were applied to promote differentiation of mouse iPS cells into male germ cells in vitro. Quantitative RT-PCR and immunoflourescence were performed to characterize the iPS cell differentiation process, and notably there were different temporal expression profiles of male germ cell-associated genes. The expression of proteins, including MVH, CDH1, and SCP3, was remarkably increased. mRNA expression of Stra8, Odf2, Act, and Prm1 was upregulated in iPS cells by retinoic acid or testosterone induction, whereas Oct-4 transcription was reduced in these cells compared to the controls. Hormones were also measured in the EB medium. DNA content analysis by flow cytometry revealed that iPS cells could differentiate into haploid cells through retinoic acid or testosterone treatment. Collectively, our results suggest that mouse iPS cells possess the potency to differentiate into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction.

In Vitro Modeling of Paraxial Mesodermal Progenitors Derived from Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α+/Flk-1− population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α+/KDR− population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α+/KDR− population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.

Involvement of MicroRNAs in Regulation of Osteoblastic Differentiation in Mouse Induced Pluripotent Stem Cells

BackgoroundMicroRNAs (miRNAs), which regulate biological processes by annealing to the 3′-untranslated region (3′-UTR) of mRNAs to reduce protein synthesis, have been the subject of recent attention as a key regulatory factor in cell differentiation. The effects of some miRNAs during osteoblastic differentiation have been investigated in mesenchymal stem cells, however they still remains to be determined in pluripotent stem cells.Methodology/Principal FindingsBone morphogenic proteins (BMPs) are potent activators of osteoblastic differentiation. In the present study, we profiled miRNAs during osteoblastic differentiation of mouse induced pluripotent stem (iPS) cells by BMP-4, in which expression of important osteoblastic markers such as Rux2, osterix, osteopontin, osteocalcin, PTHR1 and RANKL were significantly increased. A miRNA array analysis revealed that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a were significantly downregulated. Interestingly, miR-124a and miR-181a directly target the transcription factors Dlx5 and Msx2, both of which were increased by about 80-and 30-fold, respectively. In addition, transfection of miR-124a and miR-181a into mouse osteo-progenitor MC3T3-E1 cells significantly reduced expression of Dlx5, Runx2, osteocalcin and ALP, and Msx2 and osteocalcin, respectively. Finally, transfection of the anti-miRNAs of these six miRNAs, which are predicted to target Dlx5 and Msx2, into mouse iPS cells resulted in a significant increase in several osteoblastic differentiation markers such as Rux2, Msx2 and osteopontin.Conclusions/SignificanceIn the present study, we demonstrate that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a miRNAs, especially miR-124a and miR-181a, are important regulatory factors in osteoblastic differentiation of mouse iPS cells.

β1-Adrenoceptor stimulation enhances the differentiation of mouse induced pluripotent stem cells into neural progenitor cells

The cyclic AMP/protein kinase A signaling pathway is thought to be involved in neural differentiation of mesenchymal stem cells. In the present study, we examined the involvement of β-adrenoceptor signaling on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells. Mouse iPS cells were cultured on ultra-low-attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 1μM) and/or the β-adrenoceptor agonist l-isoproterenol (0.3 or 1μM) were added to the EB cultures for 4 days, then EBs were plated on gelatin-coated plates and cultured for 7 or 14 days. Subtype-specific antibody staining revealed that mouse iPS cells express β1-adrenoceptors predominantly. Although treatment with l-isoproterenol alone did not affect the expression of Nestin (a specific marker for neural progenitor cells), l-isoproterenol significantly enhanced ATRA-induced Nestin expression. Pretreatment of EBs with either atenolol (a selective β1-adrenoceptor antagonist) or H89 (a protein kinase A inhibitor) significantly inhibited the l-isoproterenol-enhancement of ATRA-induced Nestin expression. In addition, the l-isoproterenol treatment significantly enhanced ATRA-induced expression of NeuN (a neuron-specific nuclear protein). These findings suggest that β1-adrenoceptor stimulation enhances ATRA-induced neural differentiation of mouse iPS cells.

Differentiation of Induced Pluripotent Stem Cells Into Dental Mesenchymal Cells

Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited neural crest (NC) cell markers as evidenced by immunocytochemistry, flow cytometry, and real-time reverse transcription-polymerase chain reaction. Further, in recombination cultures of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies.