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Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia
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Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia Ayako Kitazawa2 and Norio Shimizu1,2 1 2
Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan Bio-Nano Electronics Research Center, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan
Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 mm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 mm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.
Introduction Mouse induced pluripotent stem (iPS) cells were directly generated from mouse embryonic fibroblasts by introducing the four transcription genes, Oct3/4, Sox2, c-Myc and Klf4 [1]. By contrast, mouse embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of 3.5-day-old blastocysts of preimplantation mouse embryos [2,3]. IPS cells exhibit the morphology and growth properties of ES cells, differentiate into all three germ layers and contribute to the production of chimeric mice [4]. These cells have the pluripotent ability to differentiate in vitro into various cell lineages including neurons. Therefore, iPS cells may be used as patient-specific pluripotent stem cells for the studies of disease pathogenesis, drug discovery and transplantation therapy. Karumbayaram et al. reported that human iPS cells could differentiate into motor neurons with a similar efficiency as human ES cells by Corresponding author: Shimizu, N. ([email protected])
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treatment with retinoic acid [5]. Dimos et al. reported that iPS cells generated from patients with ALS were successfully directed to differentiate into motor neurons [6]. The synergistic action of two inhibitors of SMAD signaling induced human iPS cell differentiation into midbrain dopaminergic neurons and spinal motor neurons [7], and resveratrol facilitated osteogenic differentiation in both iPS and ES cells [8]. These findings suggest that iPS cells can differentiate into various cell lineages in a manner similar to ES cells. We have reported that a chick dorsal root ganglion (DRG)conditioned medium (CM) promoted the differentiation of mouse ES cells into neurons [9]. We also demonstrated that the percentage of neurons differentiated from mouse ES cells was approximately 50. The 40–60% of neurons that differentiated from the ES cells were primarily motor neurons as target cells of DRG neurons (sensory neurons) [10]. In this study, to demonstrate whether mouse iPS cells are able to differentiate into neural lineages to
1871-6784/$ - see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.03.011
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form motor neurons, we investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM in a manner similar to mouse ES cells. The in vitro differentiation of iPS cells may provide new perspectives for studying the cellular and molecular mechanisms of early embryonic development as well as the generation of donor cells for transplantation therapies.
tin-coated 24-well culture plate (353847; Becton Dickinson) with gelatin-coated coverslips. After DRG-CM was added to six replicate wells, colonies were cultured for 12 days at 378C in humidified 5% CO2. Half of the medium was changed with a fresh medium containing DRG-CM every three days.
Materials and methods Cultivation and colony formation of mouse iPS cells or ES cells
Mouse iPS cells were grown on a mitotically inactivated mouse embryonic fibroblast feeder layer in a 24-well culture plate with gelatin-coated coverslips. By contrast, iPS cell colonies were detached from nonadhesive 100-mm plastic dishes and placed on coverslips. Alkaline phosphatase (AP) staining was performed as follows. After washing three times in phosphate-buffered saline (PBS), iPS cells and cell colonies on the coverslips were fixed with 4% paraformaldehyde phosphate buffer solution for 1 min at room temperature. After washing three times in 20 mM Tris– HCl, pH 7.4 containing 0.15 M NaCl and 0.05% Tween-20, Naphthol AS-BI phosphate solution (90234, Chemicon International) and Fast Red Violet solution (90239, Chemicon International) were added. The iPS cells and cell colonies were incubated for 15 min at room temperature. After washing three times in 20 mM Tris–HCl, pH 7.4 containing 0.15 M NaCl and 0.05% Tween-20, iPS cells and cell colonies expressing AP were observed using an inverted microscope (IX50; Olympus, Tokyo, Japan).
Preparation of DRG-CM The DRGs (dorsal root ganglia) were dissected from 8-day-old chick embryos as described previously [11]. Sixty DRGs per dish were plated on gelatin-coated 100-mm culture dishes with DMEM/F12K medium containing 10 ng/ml nerve growth factor (NGF; 2256X; Techne, Minneapolis, MN, USA) and were cultured for two days at 378C in humidified 5% CO2. The DMEM/F-12K medium consisted of 49% DMEM (R-SLM-220-B; Dainippon Pharmaceutical) and 49% F-12 nutrient mixture (21127-022; Gibco BRL), which contained 1% N-2 supplement (17502-048; Gibco BRL) instead of serum and 1% penicillin/streptomycin (15140-122; Gibco BRL). The supernatant of the culture medium was obtained by centrifugation and was filtered through a 0.22-mm filter (SLGV033RS, Millipore, Bedford, MA, USA). This filtrate was designated as DRG-CM.
Differentiation of mouse iPS cells and ES cells To investigate the effects of chick DRG-CM on iPS cell or ES cell differentiation, the undifferentiated colonies (approximately 200 mm in diameter) were detached from the nonadhesive 100mm plastic dishes using a 200-ml siliconised pipette tip attached to a sterile pipette, and one colony per well was plated with the DMEM/F-12K medium on a gelatin-coated 96-well assay plate (353948; Becton Dickinson, Franklin Lakes, NJ, USA) or a gela-
Preparation of slice of colony Twenty colonies (approximately 200 mm in diameter) were washed in PBS and fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at room temperature. After washing three times in PBS, the colonies were incubated with 20% sucrose solution at 48C overnight and were sliced into 12-mm sections using a LEICA CM 1100-cryostat (Leica, Nussloch, Germany). The sliced colonies were fixed on an MS-coat micro slide grass (S-9441; Matsunami Grass, Osaka, Japan) and were dehydrated.
Immunofluorescence analysis Mouse iPS cell or ES cell colonies were cultivated in a 24-well culture plate with gelatin-coated coverslips or a gelatin-coated 96well assay plate, washed three times for 5 min each in PBS and fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at room temperature and 90% methanol for 30 min at 48C. After washing three times for 5 min in PBS, the cells were incubated for 3 min at 808C. The cells were washed again three times for 5 min in PBS and incubated with primary antibodies at 48C overnight. The following primary antibodies were used for labeling: anti-nanog (RCAB0001P; Cosmo Bio, Tokyo, Japan), anti-bIIItubulin (sc-9935; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Lim-3 (LB-7033; Cosmo Bio) and anti-Brn-3 (SC6026; Santa Cruz Biotechnology) antibodies. After washing three times for 5 min each in PBS, the cells were incubated for 15 min at room temperature with the Alexa-Fluor-488-labeled secondary antibody (A11055 or A11008; Molecular Probes, Eugene, OR, USA). After washing three times for 5 min each in PBS, the cells were incubated with Hoechst 33258 for 15 min at room temperature for nuclear staining. After washing two times in PBS, we measured the fluorescence intensity of the cells using a fluoro-image analyzer (FLA3000R; Fujifilm, Tokyo, Japan) and observed the immunofluoreswww.elsevier.com/locate/nbt
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Mouse iPS cells (iPS-MEF-Ng-20D-17; APS0001; RIKEN Cell Bank, Saitama, Japan) were grown on a mitotically inactivated mouse embryonic fibroblast (R-PMEF-H; Dainippon Pharmaceutical, Osaka, Japan) feeder layer in DMEM (R-SLM-220-B; Dainippon Pharmaceutical) supplemented with 15% fetal bovine serum (1614-063; GIBCO BRL, Grand Island, NY, USA), 1% nonessential amino acids (R-TMS-001C; Dainippon Pharmaceutical), 1% 2-mercaptoethanol (R-ES-007E; Dainippon Pharmaceutical), 2 mM L-glutamine (R-TMS-002C; Dainippon Pharmaceutical), 0.5% penicillin/streptomycin (15140-122; Gibco BRL) and 1000 U/ml leukemia inhibitory factor (LIF) (ESG1106; Chemicon International, Temecula, CA, USA or 125-05603; Wako Pure Chemical Industries, Osaka, Japan). We used 0.1% gelatin (52100325; Wako Pure Chemical Industries)-coated 100-mm culture dishes (430167; Corning, NY, USA). Mouse ES cells (129SV; Dainippon pharmaceutical) were grown as described previously [9]. To form iPS cell or ES cell colonies, approximately 4 105 cells were plated on a nonadhesive 100-mm plastic dish (AU2010; Eikenkizai, Tokyo, Japan) with DMEM and cultured for 11 days at 378C in humidified 5% CO2. The number of colonies was counted everyday. Half of the medium was changed with fresh medium every four days.
Alkaline phosphatase staining analysis of iPS cells and iPS cell colonies
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cence images using a confocal laser scanning microscope (LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany). We measured the types of neurons differentiated from iPS cells or ES cells as follows. Differentiated iPS cell or ES cell colonies cultivated with two gelatin-coated 96-well culture plates were collected and washed three times in cold PBS. The colonies were incubated with a trypsin/EDTA solution (SM-2003-C; Chemicon International) for 2 min at room temperature followed by DMEM/ F-12K medium. After removing the supernatant by centrifugation, we washed the colonies in cold PBS. The cells were dispersed by pipetting. After washing two times in cold PBS, the cells were incubated with 2% paraformaldehyde phosphate buffer solution for 10 min at 378C and were cooled for 1 min on ice. The cells were incubated with a cold 90% methanol solution on ice for 30 min. After removing the supernatant by centrifugation, we incubated the cells for 10 min at 808C. After washing two times in cold PBS, approximately 1 106 cells/tube were transferred to a 1.5-ml tube. The cells were incubated with the primary antibodies overnight at 48C. After washing three times for 5 min in cold PBS, the cells were incubated with the secondary antibodies for 30 min at room temperature. After washing the cells for 5 min five times in cold PBS, the cells were incubated with Hoechst 33258 for 15 min at room temperature to visualize the nuclei. After washing two times in PBS, we measured the number of fluorescence-activated cells using ten randomly chosen immunofluorescence images using a confocal laser scanning microscope.
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Results Proliferation of undifferentiated iPS cell colony Mouse iPS cells were grown on a feeder layer in DMEM supplemented with LIF for the preparation of cell mass. We investigated whether iPS cells were undifferentiated cells using AP staining and nanog staining. Nanog is a marker for undifferentiated cells. The iPS cells of 17 passages were stained by AP, as shown in Fig. 1a. In addition, the iPS cells of 19 passages were labeled with the antibody against nanog, as shown in Fig. 1b. These results show that the iPS cells within 19 passages were undifferentiated cells. However, there were several differentiated iPS cells over 20 passages (data not shown). By contrast, ES cells were undifferentiated even after 30 passages (data not shown). To form undifferentiated iPS cell or ES cell colonies, iPS cells or ES cells were transferred to nonadhesive 100-mm plastic dishes with DMEM supplemented with LIF and cultured. We measured the number of iPS cell or ES cell colonies. Figure 2 shows that the number of iPS cell colonies (approximately 200 mm in diameter) increased until six days of cultivation and decreased thereafter. Although we observed clear ES cell colonies that were approximately 200 mm in diameter after nine days of cultivation, many iPS cell colonies at 11 days of cultivation were deformed colonies, and were over 200 mm in diameter as shown in Fig. 2. Although the iPS cell colonies were stained red by AP staining at six days of cultivation, some of them were not stained red after seven days of cultivation (data not shown). ES cell colonies that were 200 mm
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FIGURE 1
Alkaline phosphatase (AP) staining and nanog staining of iPS cells. IPS cells were grown on a feeder layer in DMEM until 17 and 19 passages. IPS cells could be maintained in an undifferentiated state until 19 passages. (a) AP staining of iPS cells after 17 passages. (b) Nanog staining and nuclear staining of iPS cells after 19 passages. IPS cells were labeled with an antibody against nanog and the nuclei of iPS cells were stained with Hoechst 33258 dye. 328
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FIGURE 2
Formation of iPS cell colonies and ES cell colonies. IPS cells or ES cells were transferred to nonadhesive 100-mm plastic dishes with DMEM supplemented with LIF and cultured in vitro. Symbols: circles, iPS cell colonies; triangles, ES cell colonies.
in diameter started forming from seven days of cultivation. The highest number of ES cell colonies with 200 mm in diameter was achieved at ten days of cultivation. These results indicate that iPS cells proliferate and differentiate faster than ES cells. We investigated the insides of the iPS cell colony using slices (12 mm thickness) of fixed iPS cell colonies. Figure 3 shows the fluorescence and optical micrographs of slices of iPS cell colonies. The inside of an iPS cell colony at six days of cultivation was labeled with an antibody against nanog (Fig. 3a), but the inside of an iPS cell colony at ten days of cultivation was not labeled (Fig. 3b). Although ES cell colonies did not form hollow spaces inside the colonies, hollows were observed in the iPS cell colonies at ten days of cultivation. These results show that iPS cells differentiate easily, and differentiated iPS cell colonies form hollows in these colonies.
Effect of DRG-CM on differentiation of iPS cells or ES cells into neurons We investigated whether DRG-CM promoted the differentiation of iPS cells into neurons in a manner similar to ES cells. Figure 4 shows the effect of DRG-CM volume on the differentiation of iPS cells and ES cells into neurons. We used iPS cell colonies of 19 passages and ES cell colonies of 18 passages that were approximately 200 mm in diameter for the neuron differentiation studies. ES cells and iPS cells were labeled with an antibody against bIIItubulin (a marker of postmitotic neurons). The fluorescence intensity of iPS cells without DRG-CM addition was represented as 100%. The iPS cells effectively differentiated into neurons in a manner similar to ES cells. Figure 4a shows that 20% DRG-CM or
1% DRG-CM effectively induced the differentiation of iPS cells or ES cells into neurons, respectively, at 12 days of cultivation. However, Fig. 4b shows that 2.5% DRG-CM or 1% DRG-CM effectively induced the differentiation of iPS cells or ES cells into neurons, respectively, at 12 days of cultivation. Figure 4c shows that various morphological types of cells differentiated from iPS cells were expanded around the colonies. By contrast, ES cell colonies clearly exhibited neurite outgrowth, as shown in Fig. 4d. It seems that, compared to ES cells, iPS cells differentiate into various cell lineages by the addition of DRG-CM. Figure 5 shows the fluorescence and optical micrographs of an iPS cell colony with neurite outgrowth at seven days of cultivation. Differentiated iPS cells and neurite outgrowth were labeled with the antibody against bIII-tubulin. However, there were many cells around the neurons which were not labeled with the antibody against bIII-tubulin. These results showed that differentiated iPS cell colonies might contain both neurons and various other types of cells.
Characterization of neurons differentiated from iPS cells and ES cells We characterized neurons differentiated from iPS cells or ES cells by DRG-CM using immunofluorescence analysis. Figure 6 shows the fluorescence intensities of types of neurons. We used the antibodies against bIII-tubulin, Lim-3 (a marker of cranial motor neurons), and Brn-3 (a marker of sensory neurons). The fluorescence intensity of iPS cells labeled with the antibody against bIIItubulin was defined to be 100%. Although ES cells predominantly differentiated into motor neurons labeled with the anti-Lim-3 www.elsevier.com/locate/nbt
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Nanog staining and nuclear staining of iPS cell colonies. IPS colonies fixed with 4% paraformaldehyde phosphate buffer solution were incubated with 20% sucrose solution at 48C overnight and sliced in 12-mm sections using a LEICA CM 1100-cryostat. (a) Inside of an iPS cell colony after six days of cultivation. (b) Inside of iPS cell colony after ten days of cultivation. The arrows indicate the hollows inside the iPS cell colony.
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FIGURE 4
Effect of various DRG-CM concentrations on the differentiation of iPS cells and ES cells into neurons. DRG-CM was added at a range of 1–20% to DMEM/F-12K medium. IPS cell or ES cell colonies after 12 days of cultivation were labeled with an antibody against bIII-tubulin. The fluorescence intensity of iPS cells without DRG-CM supplementation was defined to be 100%. The values indicate the means of six replicates S.E. (a) Open bars, ES cell (18 passages) colonies formed after seven days of cultivation; solid bars, iPS cell (19 passages) colonies formed after seven days of cultivation. (b) Open bars, ES cell (18 passages) colonies formed after eight days of cultivation; solid bars, iPS cell (19 passages) colonies formed after eight days of cultivation. (c) Optical micrograph of iPS cell colony with neurite outgrowth after 12 days of cultivation. Arrows indicate neurite outgrowth. (d) Optical micrograph of ES cell colony with neurite outgrowth after 12 days of cultivation. 330
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FIGURE 5
bIII-Tubulin staining and nuclear staining of iPS cell colonies with neurite outgrowth after 12 days of cultivation. IPS cell colonies were cultured with DMEM/F-12K medium containing 5% DRG-CM. (a) IPS cells labeled with an antibody against bIII-tubulin. (b) IPS cells stained with Hoechst 33258 dye. (c) Bright field.
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FIGURE 6
FIGURE 7
Fluorescence intensities of types of neurons at 12 days of cultivation measured using a fluoro-image analyzer. Differentiated iPS cell and ES cell colonies were labeled with an antibody against bIII-tubulin, Lim-3 or Brn-3. The fluorescence intensity of the iPS cell colonies (5% DRG-CM) labeled with the antibody against bIII-tubulin was designated as 100%. The values indicate the means of six replicates S.E. Open bars, ES cell colonies cultured with 5% DRG-CM. Solid bars, iPS cell colonies cultured with 5% DRG-CM.
Number of types of neurons after 12 days of cultivation. After differentiated iPS cell and ES cell colonies were dispersed with trypsin, the cells were labeled with an antibody against Lim-3 or Brn-3. The number of fluorescenceactivated cells was measured for ten randomly selected immunofluorescence fields using a confocal laser scanning microscope. The values represent the means of ten fields S.E.
antibody as reported previously [10], iPS cells differentiated into sensory neurons labeled with the anti-Brn-3 antibody and motor neurons by the addition of 5% DRG-CM. Figure 7 shows the number of types of motor and sensory neurons. The population of types of neurons differentiated from ES cell colonies was predominantly composed of motor neurons. But, those of neurons differentiated from iPS cell colonies were mainly composed of motor and sensory neurons. These results indicate that the differentiation properties of iPS cells by the addition of DRG-CM may differ from those of ES cells.
cells. Because the iPS cells were labeled with the antibody against nanog after 19 passages, they were defined as a pure undifferentiated cell population. However, iPS cells contained some differentiated cells after over 20 passages. By contrast, ES cells were maintained in an undifferentiated state even after 30 passages. When iPS cells were cultured under floating culture conditions with DMEM supplemented with LIF to encourage the formation of undifferentiated iPS cell colonies, iPS cell colonies of 200 mm in diameter attained to the maximal colony number at six days of cultivation and were maintained in an undifferentiated state. However, ES cell colonies of 200 mm in diameter were maximal in number at ten days of cultivation and remained undifferentiated even after 11 days of cultivation. Differentiated iPS cell colonies after ten days of cultivation had hollows. Undifferentiated ES cell colonies at 11 days of cultivation did not form hollows inside the colonies. Kawamorita et al. reported that cavity formation was observed inside embryoid bodies (EBs) after induction with retinoic acid [12]. EBs formed by culturing ES cells in DMEM without LIF
Discussion To demonstrate whether mouse iPS cells are able to differentiate into the neural lineages, we investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM in the same manner as mouse ES cells. The maintenance of an undifferentiated state by the repeated subculturing of mouse iPS cells was compared to that of mouse ES
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differentiated into various cell lineages in vitro [12–15]. The formation of hollows inside iPS cell colonies may indicate their ability to differentiate into several cell types. Kim et al. reported that global gene expression profiles revealed a high degree of similarity between human iPS cells and human ES cells [16]. However, we found that the proliferation characteristics of iPS cells were different from those of ES cells. Compared to ES cells, iPS cells proliferated easily and could differentiate into various cell lineages. We investigated the effect of DRG-CM on the differentiation of iPS cells and ES cells into neurons. The addition of 1% DRG-CM effectively induced the differentiation of ES cell colonies into neurons. The optimal values of DRG-CM were 2.5% and 20% for the differentiation of iPS cell colonies into neurons in two independent experiments. Although we used undifferentiated iPS cell colonies for differentiation into neurons, the quality of the colonies may be slightly different for each experiment. We characterized the types of neurons and measured the number of types of neurons differentiated from iPS and ES cells by the addition of 5% DRG-CM. IPS cells differentiated into motor neurons (34% of total cells) and sensory neurons (32%). By contrast, ES cells differentiated into motor neurons (42%) and sensory neurons (13%). We previously reported that the addition of 5% DRG-CM induced the differentiation of ES cells into motor neurons (25%) and sensory neurons (1%) [10]. Although the differentiation propensities among human ES cell lines are different [17], the mouse ES cell line used in these experiments gave highly reproducible results. DRG-CM may contain DRG-derived signaling molecules that induce the differentiation of iPS cells into sensory neurons. Signals from sensory neurons in DRGs may determine the direction of iPS cell differentiation. Karumbayaram et al. reported that both human ES and human iPS cells can be directed to form comparable neural progenitors and are equally efficient at generating motor neurons (58–59% of neural progenitors) in the presence of retinoic acid, a Sonic Hedgehog pathway agonist and neurotrophic factors [5]. Dimos et al. reported that iPS cells generated from patients with ALS differentiated into motor neurons (20% of total cells) by the addition of retinoic acid [6]. These results showed the possibility of generating motor neurons from iPS cells after induction with retinoic acid, and we have described the generation of motor and sensory neurons from iPS cells using
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DRG-CM. Tokumoto et al. reported that the difference in oligodendrocyte differentiation rates between iPS cells and ES cells occurred in the terminal differentiation step from oligodendrocyte precursor cells to oligodendrocytes [18]. Polo et al. recently reported [19] that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Although early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, late-passage iPSCs (16 passages) are eliminated differences in the differentiation potentials of iPSCs. In addition, Kim et al. reported that embryonic tissues are the most efficiently reprogrammed, producing iPS cells that are nearly identical to ES cells [20]. Because we used iPS cells of 17–19 passages for iPS cell differentiation, signals from sensory neurons in DRGs may determine the direction of iPS cell differentiation. But, it is not clear whether the difference of differentiation efficiencies and patterns between iPS cells and ES cells is involved in the origin of iPS cells from embryonic fibroblast. Recently, some researchers [21–24] reported that the reprogramming process and subsequent culture of iPS cells in vitro can induce genetic and epigenetic abnormalities in these cells. These abnormalities may influence the cell growth and differentiation of iPS cells. We found that the differentiation efficiencies and patterns of iPS cells using DRG-CM were different from those of ES cells. The origin and genetic and epigenetic abnormalities of iPS cells may determine the differentiation pattern of iPS cells.
Acknowledgements This work was supported by a Grant-in-Aid for high-tech research centers organized by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), by a grant from MEXT’s the 21st century COE program to one of the authors (N.S.), and by a grant from JSPS Research Fellowships for Young Scientist and by a grant from the Inoue Enryo Memorial Foundation for Promoting Sciences to one of the authors (A.K.). Mouse iPS cells (APS0001) were provided by the RIKEN BRC. Author contributions: A.K. contributed to the collection and assembly of data, data analysis and/or interpretation, manuscript writing. N.S. contributed to the concept and design, manuscript writing, final approval of manuscript.
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Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia Ayako Kitazawa2 and Norio Shimizu1,2 1 2
Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan Bio-Nano Electronics Research Center, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan
Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 mm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 mm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.
Introduction Mouse induced pluripotent stem (iPS) cells were directly generated from mouse embryonic fibroblasts by introducing the four transcription genes, Oct3/4, Sox2, c-Myc and Klf4 [1]. By contrast, mouse embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of 3.5-day-old blastocysts of preimplantation mouse embryos [2,3]. IPS cells exhibit the morphology and growth properties of ES cells, differentiate into all three germ layers and contribute to the production of chimeric mice [4]. These cells have the pluripotent ability to differentiate in vitro into various cell lineages including neurons. Therefore, iPS cells may be used as patient-specific pluripotent stem cells for the studies of disease pathogenesis, drug discovery and transplantation therapy. Karumbayaram et al. reported that human iPS cells could differentiate into motor neurons with a similar efficiency as human ES cells by Corresponding author: Shimizu, N. ([email protected])
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treatment with retinoic acid [5]. Dimos et al. reported that iPS cells generated from patients with ALS were successfully directed to differentiate into motor neurons [6]. The synergistic action of two inhibitors of SMAD signaling induced human iPS cell differentiation into midbrain dopaminergic neurons and spinal motor neurons [7], and resveratrol facilitated osteogenic differentiation in both iPS and ES cells [8]. These findings suggest that iPS cells can differentiate into various cell lineages in a manner similar to ES cells. We have reported that a chick dorsal root ganglion (DRG)conditioned medium (CM) promoted the differentiation of mouse ES cells into neurons [9]. We also demonstrated that the percentage of neurons differentiated from mouse ES cells was approximately 50. The 40–60% of neurons that differentiated from the ES cells were primarily motor neurons as target cells of DRG neurons (sensory neurons) [10]. In this study, to demonstrate whether mouse iPS cells are able to differentiate into neural lineages to
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form motor neurons, we investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM in a manner similar to mouse ES cells. The in vitro differentiation of iPS cells may provide new perspectives for studying the cellular and molecular mechanisms of early embryonic development as well as the generation of donor cells for transplantation therapies.
tin-coated 24-well culture plate (353847; Becton Dickinson) with gelatin-coated coverslips. After DRG-CM was added to six replicate wells, colonies were cultured for 12 days at 378C in humidified 5% CO2. Half of the medium was changed with a fresh medium containing DRG-CM every three days.
Materials and methods Cultivation and colony formation of mouse iPS cells or ES cells
Mouse iPS cells were grown on a mitotically inactivated mouse embryonic fibroblast feeder layer in a 24-well culture plate with gelatin-coated coverslips. By contrast, iPS cell colonies were detached from nonadhesive 100-mm plastic dishes and placed on coverslips. Alkaline phosphatase (AP) staining was performed as follows. After washing three times in phosphate-buffered saline (PBS), iPS cells and cell colonies on the coverslips were fixed with 4% paraformaldehyde phosphate buffer solution for 1 min at room temperature. After washing three times in 20 mM Tris– HCl, pH 7.4 containing 0.15 M NaCl and 0.05% Tween-20, Naphthol AS-BI phosphate solution (90234, Chemicon International) and Fast Red Violet solution (90239, Chemicon International) were added. The iPS cells and cell colonies were incubated for 15 min at room temperature. After washing three times in 20 mM Tris–HCl, pH 7.4 containing 0.15 M NaCl and 0.05% Tween-20, iPS cells and cell colonies expressing AP were observed using an inverted microscope (IX50; Olympus, Tokyo, Japan).
Preparation of DRG-CM The DRGs (dorsal root ganglia) were dissected from 8-day-old chick embryos as described previously [11]. Sixty DRGs per dish were plated on gelatin-coated 100-mm culture dishes with DMEM/F12K medium containing 10 ng/ml nerve growth factor (NGF; 2256X; Techne, Minneapolis, MN, USA) and were cultured for two days at 378C in humidified 5% CO2. The DMEM/F-12K medium consisted of 49% DMEM (R-SLM-220-B; Dainippon Pharmaceutical) and 49% F-12 nutrient mixture (21127-022; Gibco BRL), which contained 1% N-2 supplement (17502-048; Gibco BRL) instead of serum and 1% penicillin/streptomycin (15140-122; Gibco BRL). The supernatant of the culture medium was obtained by centrifugation and was filtered through a 0.22-mm filter (SLGV033RS, Millipore, Bedford, MA, USA). This filtrate was designated as DRG-CM.
Differentiation of mouse iPS cells and ES cells To investigate the effects of chick DRG-CM on iPS cell or ES cell differentiation, the undifferentiated colonies (approximately 200 mm in diameter) were detached from the nonadhesive 100mm plastic dishes using a 200-ml siliconised pipette tip attached to a sterile pipette, and one colony per well was plated with the DMEM/F-12K medium on a gelatin-coated 96-well assay plate (353948; Becton Dickinson, Franklin Lakes, NJ, USA) or a gela-
Preparation of slice of colony Twenty colonies (approximately 200 mm in diameter) were washed in PBS and fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at room temperature. After washing three times in PBS, the colonies were incubated with 20% sucrose solution at 48C overnight and were sliced into 12-mm sections using a LEICA CM 1100-cryostat (Leica, Nussloch, Germany). The sliced colonies were fixed on an MS-coat micro slide grass (S-9441; Matsunami Grass, Osaka, Japan) and were dehydrated.
Immunofluorescence analysis Mouse iPS cell or ES cell colonies were cultivated in a 24-well culture plate with gelatin-coated coverslips or a gelatin-coated 96well assay plate, washed three times for 5 min each in PBS and fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at room temperature and 90% methanol for 30 min at 48C. After washing three times for 5 min in PBS, the cells were incubated for 3 min at 808C. The cells were washed again three times for 5 min in PBS and incubated with primary antibodies at 48C overnight. The following primary antibodies were used for labeling: anti-nanog (RCAB0001P; Cosmo Bio, Tokyo, Japan), anti-bIIItubulin (sc-9935; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Lim-3 (LB-7033; Cosmo Bio) and anti-Brn-3 (SC6026; Santa Cruz Biotechnology) antibodies. After washing three times for 5 min each in PBS, the cells were incubated for 15 min at room temperature with the Alexa-Fluor-488-labeled secondary antibody (A11055 or A11008; Molecular Probes, Eugene, OR, USA). After washing three times for 5 min each in PBS, the cells were incubated with Hoechst 33258 for 15 min at room temperature for nuclear staining. After washing two times in PBS, we measured the fluorescence intensity of the cells using a fluoro-image analyzer (FLA3000R; Fujifilm, Tokyo, Japan) and observed the immunofluoreswww.elsevier.com/locate/nbt
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Mouse iPS cells (iPS-MEF-Ng-20D-17; APS0001; RIKEN Cell Bank, Saitama, Japan) were grown on a mitotically inactivated mouse embryonic fibroblast (R-PMEF-H; Dainippon Pharmaceutical, Osaka, Japan) feeder layer in DMEM (R-SLM-220-B; Dainippon Pharmaceutical) supplemented with 15% fetal bovine serum (1614-063; GIBCO BRL, Grand Island, NY, USA), 1% nonessential amino acids (R-TMS-001C; Dainippon Pharmaceutical), 1% 2-mercaptoethanol (R-ES-007E; Dainippon Pharmaceutical), 2 mM L-glutamine (R-TMS-002C; Dainippon Pharmaceutical), 0.5% penicillin/streptomycin (15140-122; Gibco BRL) and 1000 U/ml leukemia inhibitory factor (LIF) (ESG1106; Chemicon International, Temecula, CA, USA or 125-05603; Wako Pure Chemical Industries, Osaka, Japan). We used 0.1% gelatin (52100325; Wako Pure Chemical Industries)-coated 100-mm culture dishes (430167; Corning, NY, USA). Mouse ES cells (129SV; Dainippon pharmaceutical) were grown as described previously [9]. To form iPS cell or ES cell colonies, approximately 4 105 cells were plated on a nonadhesive 100-mm plastic dish (AU2010; Eikenkizai, Tokyo, Japan) with DMEM and cultured for 11 days at 378C in humidified 5% CO2. The number of colonies was counted everyday. Half of the medium was changed with fresh medium every four days.
Alkaline phosphatase staining analysis of iPS cells and iPS cell colonies
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cence images using a confocal laser scanning microscope (LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany). We measured the types of neurons differentiated from iPS cells or ES cells as follows. Differentiated iPS cell or ES cell colonies cultivated with two gelatin-coated 96-well culture plates were collected and washed three times in cold PBS. The colonies were incubated with a trypsin/EDTA solution (SM-2003-C; Chemicon International) for 2 min at room temperature followed by DMEM/ F-12K medium. After removing the supernatant by centrifugation, we washed the colonies in cold PBS. The cells were dispersed by pipetting. After washing two times in cold PBS, the cells were incubated with 2% paraformaldehyde phosphate buffer solution for 10 min at 378C and were cooled for 1 min on ice. The cells were incubated with a cold 90% methanol solution on ice for 30 min. After removing the supernatant by centrifugation, we incubated the cells for 10 min at 808C. After washing two times in cold PBS, approximately 1 106 cells/tube were transferred to a 1.5-ml tube. The cells were incubated with the primary antibodies overnight at 48C. After washing three times for 5 min in cold PBS, the cells were incubated with the secondary antibodies for 30 min at room temperature. After washing the cells for 5 min five times in cold PBS, the cells were incubated with Hoechst 33258 for 15 min at room temperature to visualize the nuclei. After washing two times in PBS, we measured the number of fluorescence-activated cells using ten randomly chosen immunofluorescence images using a confocal laser scanning microscope.
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Results Proliferation of undifferentiated iPS cell colony Mouse iPS cells were grown on a feeder layer in DMEM supplemented with LIF for the preparation of cell mass. We investigated whether iPS cells were undifferentiated cells using AP staining and nanog staining. Nanog is a marker for undifferentiated cells. The iPS cells of 17 passages were stained by AP, as shown in Fig. 1a. In addition, the iPS cells of 19 passages were labeled with the antibody against nanog, as shown in Fig. 1b. These results show that the iPS cells within 19 passages were undifferentiated cells. However, there were several differentiated iPS cells over 20 passages (data not shown). By contrast, ES cells were undifferentiated even after 30 passages (data not shown). To form undifferentiated iPS cell or ES cell colonies, iPS cells or ES cells were transferred to nonadhesive 100-mm plastic dishes with DMEM supplemented with LIF and cultured. We measured the number of iPS cell or ES cell colonies. Figure 2 shows that the number of iPS cell colonies (approximately 200 mm in diameter) increased until six days of cultivation and decreased thereafter. Although we observed clear ES cell colonies that were approximately 200 mm in diameter after nine days of cultivation, many iPS cell colonies at 11 days of cultivation were deformed colonies, and were over 200 mm in diameter as shown in Fig. 2. Although the iPS cell colonies were stained red by AP staining at six days of cultivation, some of them were not stained red after seven days of cultivation (data not shown). ES cell colonies that were 200 mm
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FIGURE 1
Alkaline phosphatase (AP) staining and nanog staining of iPS cells. IPS cells were grown on a feeder layer in DMEM until 17 and 19 passages. IPS cells could be maintained in an undifferentiated state until 19 passages. (a) AP staining of iPS cells after 17 passages. (b) Nanog staining and nuclear staining of iPS cells after 19 passages. IPS cells were labeled with an antibody against nanog and the nuclei of iPS cells were stained with Hoechst 33258 dye. 328
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[()TD$FIG]
FIGURE 2
Formation of iPS cell colonies and ES cell colonies. IPS cells or ES cells were transferred to nonadhesive 100-mm plastic dishes with DMEM supplemented with LIF and cultured in vitro. Symbols: circles, iPS cell colonies; triangles, ES cell colonies.
in diameter started forming from seven days of cultivation. The highest number of ES cell colonies with 200 mm in diameter was achieved at ten days of cultivation. These results indicate that iPS cells proliferate and differentiate faster than ES cells. We investigated the insides of the iPS cell colony using slices (12 mm thickness) of fixed iPS cell colonies. Figure 3 shows the fluorescence and optical micrographs of slices of iPS cell colonies. The inside of an iPS cell colony at six days of cultivation was labeled with an antibody against nanog (Fig. 3a), but the inside of an iPS cell colony at ten days of cultivation was not labeled (Fig. 3b). Although ES cell colonies did not form hollow spaces inside the colonies, hollows were observed in the iPS cell colonies at ten days of cultivation. These results show that iPS cells differentiate easily, and differentiated iPS cell colonies form hollows in these colonies.
Effect of DRG-CM on differentiation of iPS cells or ES cells into neurons We investigated whether DRG-CM promoted the differentiation of iPS cells into neurons in a manner similar to ES cells. Figure 4 shows the effect of DRG-CM volume on the differentiation of iPS cells and ES cells into neurons. We used iPS cell colonies of 19 passages and ES cell colonies of 18 passages that were approximately 200 mm in diameter for the neuron differentiation studies. ES cells and iPS cells were labeled with an antibody against bIIItubulin (a marker of postmitotic neurons). The fluorescence intensity of iPS cells without DRG-CM addition was represented as 100%. The iPS cells effectively differentiated into neurons in a manner similar to ES cells. Figure 4a shows that 20% DRG-CM or
1% DRG-CM effectively induced the differentiation of iPS cells or ES cells into neurons, respectively, at 12 days of cultivation. However, Fig. 4b shows that 2.5% DRG-CM or 1% DRG-CM effectively induced the differentiation of iPS cells or ES cells into neurons, respectively, at 12 days of cultivation. Figure 4c shows that various morphological types of cells differentiated from iPS cells were expanded around the colonies. By contrast, ES cell colonies clearly exhibited neurite outgrowth, as shown in Fig. 4d. It seems that, compared to ES cells, iPS cells differentiate into various cell lineages by the addition of DRG-CM. Figure 5 shows the fluorescence and optical micrographs of an iPS cell colony with neurite outgrowth at seven days of cultivation. Differentiated iPS cells and neurite outgrowth were labeled with the antibody against bIII-tubulin. However, there were many cells around the neurons which were not labeled with the antibody against bIII-tubulin. These results showed that differentiated iPS cell colonies might contain both neurons and various other types of cells.
Characterization of neurons differentiated from iPS cells and ES cells We characterized neurons differentiated from iPS cells or ES cells by DRG-CM using immunofluorescence analysis. Figure 6 shows the fluorescence intensities of types of neurons. We used the antibodies against bIII-tubulin, Lim-3 (a marker of cranial motor neurons), and Brn-3 (a marker of sensory neurons). The fluorescence intensity of iPS cells labeled with the antibody against bIIItubulin was defined to be 100%. Although ES cells predominantly differentiated into motor neurons labeled with the anti-Lim-3 www.elsevier.com/locate/nbt
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[()TD$FIG]
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Nanog staining and nuclear staining of iPS cell colonies. IPS colonies fixed with 4% paraformaldehyde phosphate buffer solution were incubated with 20% sucrose solution at 48C overnight and sliced in 12-mm sections using a LEICA CM 1100-cryostat. (a) Inside of an iPS cell colony after six days of cultivation. (b) Inside of iPS cell colony after ten days of cultivation. The arrows indicate the hollows inside the iPS cell colony.
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FIGURE 4
Effect of various DRG-CM concentrations on the differentiation of iPS cells and ES cells into neurons. DRG-CM was added at a range of 1–20% to DMEM/F-12K medium. IPS cell or ES cell colonies after 12 days of cultivation were labeled with an antibody against bIII-tubulin. The fluorescence intensity of iPS cells without DRG-CM supplementation was defined to be 100%. The values indicate the means of six replicates S.E. (a) Open bars, ES cell (18 passages) colonies formed after seven days of cultivation; solid bars, iPS cell (19 passages) colonies formed after seven days of cultivation. (b) Open bars, ES cell (18 passages) colonies formed after eight days of cultivation; solid bars, iPS cell (19 passages) colonies formed after eight days of cultivation. (c) Optical micrograph of iPS cell colony with neurite outgrowth after 12 days of cultivation. Arrows indicate neurite outgrowth. (d) Optical micrograph of ES cell colony with neurite outgrowth after 12 days of cultivation. 330
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[()TD$FIG]
FIGURE 5
bIII-Tubulin staining and nuclear staining of iPS cell colonies with neurite outgrowth after 12 days of cultivation. IPS cell colonies were cultured with DMEM/F-12K medium containing 5% DRG-CM. (a) IPS cells labeled with an antibody against bIII-tubulin. (b) IPS cells stained with Hoechst 33258 dye. (c) Bright field.
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FIGURE 6
FIGURE 7
Fluorescence intensities of types of neurons at 12 days of cultivation measured using a fluoro-image analyzer. Differentiated iPS cell and ES cell colonies were labeled with an antibody against bIII-tubulin, Lim-3 or Brn-3. The fluorescence intensity of the iPS cell colonies (5% DRG-CM) labeled with the antibody against bIII-tubulin was designated as 100%. The values indicate the means of six replicates S.E. Open bars, ES cell colonies cultured with 5% DRG-CM. Solid bars, iPS cell colonies cultured with 5% DRG-CM.
Number of types of neurons after 12 days of cultivation. After differentiated iPS cell and ES cell colonies were dispersed with trypsin, the cells were labeled with an antibody against Lim-3 or Brn-3. The number of fluorescenceactivated cells was measured for ten randomly selected immunofluorescence fields using a confocal laser scanning microscope. The values represent the means of ten fields S.E.
antibody as reported previously [10], iPS cells differentiated into sensory neurons labeled with the anti-Brn-3 antibody and motor neurons by the addition of 5% DRG-CM. Figure 7 shows the number of types of motor and sensory neurons. The population of types of neurons differentiated from ES cell colonies was predominantly composed of motor neurons. But, those of neurons differentiated from iPS cell colonies were mainly composed of motor and sensory neurons. These results indicate that the differentiation properties of iPS cells by the addition of DRG-CM may differ from those of ES cells.
cells. Because the iPS cells were labeled with the antibody against nanog after 19 passages, they were defined as a pure undifferentiated cell population. However, iPS cells contained some differentiated cells after over 20 passages. By contrast, ES cells were maintained in an undifferentiated state even after 30 passages. When iPS cells were cultured under floating culture conditions with DMEM supplemented with LIF to encourage the formation of undifferentiated iPS cell colonies, iPS cell colonies of 200 mm in diameter attained to the maximal colony number at six days of cultivation and were maintained in an undifferentiated state. However, ES cell colonies of 200 mm in diameter were maximal in number at ten days of cultivation and remained undifferentiated even after 11 days of cultivation. Differentiated iPS cell colonies after ten days of cultivation had hollows. Undifferentiated ES cell colonies at 11 days of cultivation did not form hollows inside the colonies. Kawamorita et al. reported that cavity formation was observed inside embryoid bodies (EBs) after induction with retinoic acid [12]. EBs formed by culturing ES cells in DMEM without LIF
Discussion To demonstrate whether mouse iPS cells are able to differentiate into the neural lineages, we investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM in the same manner as mouse ES cells. The maintenance of an undifferentiated state by the repeated subculturing of mouse iPS cells was compared to that of mouse ES
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differentiated into various cell lineages in vitro [12–15]. The formation of hollows inside iPS cell colonies may indicate their ability to differentiate into several cell types. Kim et al. reported that global gene expression profiles revealed a high degree of similarity between human iPS cells and human ES cells [16]. However, we found that the proliferation characteristics of iPS cells were different from those of ES cells. Compared to ES cells, iPS cells proliferated easily and could differentiate into various cell lineages. We investigated the effect of DRG-CM on the differentiation of iPS cells and ES cells into neurons. The addition of 1% DRG-CM effectively induced the differentiation of ES cell colonies into neurons. The optimal values of DRG-CM were 2.5% and 20% for the differentiation of iPS cell colonies into neurons in two independent experiments. Although we used undifferentiated iPS cell colonies for differentiation into neurons, the quality of the colonies may be slightly different for each experiment. We characterized the types of neurons and measured the number of types of neurons differentiated from iPS and ES cells by the addition of 5% DRG-CM. IPS cells differentiated into motor neurons (34% of total cells) and sensory neurons (32%). By contrast, ES cells differentiated into motor neurons (42%) and sensory neurons (13%). We previously reported that the addition of 5% DRG-CM induced the differentiation of ES cells into motor neurons (25%) and sensory neurons (1%) [10]. Although the differentiation propensities among human ES cell lines are different [17], the mouse ES cell line used in these experiments gave highly reproducible results. DRG-CM may contain DRG-derived signaling molecules that induce the differentiation of iPS cells into sensory neurons. Signals from sensory neurons in DRGs may determine the direction of iPS cell differentiation. Karumbayaram et al. reported that both human ES and human iPS cells can be directed to form comparable neural progenitors and are equally efficient at generating motor neurons (58–59% of neural progenitors) in the presence of retinoic acid, a Sonic Hedgehog pathway agonist and neurotrophic factors [5]. Dimos et al. reported that iPS cells generated from patients with ALS differentiated into motor neurons (20% of total cells) by the addition of retinoic acid [6]. These results showed the possibility of generating motor neurons from iPS cells after induction with retinoic acid, and we have described the generation of motor and sensory neurons from iPS cells using
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DRG-CM. Tokumoto et al. reported that the difference in oligodendrocyte differentiation rates between iPS cells and ES cells occurred in the terminal differentiation step from oligodendrocyte precursor cells to oligodendrocytes [18]. Polo et al. recently reported [19] that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Although early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, late-passage iPSCs (16 passages) are eliminated differences in the differentiation potentials of iPSCs. In addition, Kim et al. reported that embryonic tissues are the most efficiently reprogrammed, producing iPS cells that are nearly identical to ES cells [20]. Because we used iPS cells of 17–19 passages for iPS cell differentiation, signals from sensory neurons in DRGs may determine the direction of iPS cell differentiation. But, it is not clear whether the difference of differentiation efficiencies and patterns between iPS cells and ES cells is involved in the origin of iPS cells from embryonic fibroblast. Recently, some researchers [21–24] reported that the reprogramming process and subsequent culture of iPS cells in vitro can induce genetic and epigenetic abnormalities in these cells. These abnormalities may influence the cell growth and differentiation of iPS cells. We found that the differentiation efficiencies and patterns of iPS cells using DRG-CM were different from those of ES cells. The origin and genetic and epigenetic abnormalities of iPS cells may determine the differentiation pattern of iPS cells.
Acknowledgements This work was supported by a Grant-in-Aid for high-tech research centers organized by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), by a grant from MEXT’s the 21st century COE program to one of the authors (N.S.), and by a grant from JSPS Research Fellowships for Young Scientist and by a grant from the Inoue Enryo Memorial Foundation for Promoting Sciences to one of the authors (A.K.). Mouse iPS cells (APS0001) were provided by the RIKEN BRC. Author contributions: A.K. contributed to the collection and assembly of data, data analysis and/or interpretation, manuscript writing. N.S. contributed to the concept and design, manuscript writing, final approval of manuscript.
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