In Vivo Generation of Neural Stem Cells Through Teratoma Formation

Abstract

Pluripotent stem cells have the potential to differentiate into all cell types of the body in vitro through embryoid body formation or in vivo through teratoma formation. In this study, we attempted to generate in vivo neural stem cells (NSCs) differentiated through teratoma formation using Olig2-GFP transgenic embryonic stem cells (ESCs). After 4 to 6 weeks of injection with Olig2-GFP transgenic ESCs, Olig2-GFP⁺ NSCs were identified in teratomas formed in immunodeficient mice. Interestingly, 4-week-old teratomas contained higher percentage of Olig2-GFP⁺ cells (∼11%) than 6-week-old teratomas (∼3%). These in vivo-derived NSCs expressed common NSC markers (Nestin and Sox2) and differentiated into terminal neuronal and glial lineages. These results suggest that pure NSC populations exhibiting properties similar to those of brain-derived NSCs can be established through teratoma formation.

Introduction

Pluripotent stem cells (PSCs) are derived from their in vivo pluripotent counterpart, the inner cell mass of the preimplantation blastocyst, or through reprogramming processes, such as nuclear transfer [1 –4] and direct reprogramming by forced expression of pluripotency factors [5 –11]. PSCs possess the potential to differentiate into cells of all three germ layers and maintain unlimited self-renewal ability. This potential makes PSCs a valuable resource for replacement therapy against incurable diseases. Therefore, the establishment of efficient protocol for differentiation of PSCs into specific somatic cell lineages is a pivotal step for stem cell therapy using PSCs. More importantly, PSC-derived somatic cells should recapitulate the in vivo system on both a functional and molecular level; however, in vitro differentiation systems are not compatible with in vivo tissue development during embryogenesis. Therefore, there were various efforts for recapitulation of in vivo developmental environment. In this way, three-dimensional (3D) organogenesis technique is on rise to overcome shortage of in vitro differentiation. Organ is developed during embryogenesis by spatiotemporal regulation of diverse factors and interaction of adjacent cells. In vitro-derived 3D organ bud could be spontaneously formed in coculture with endothelial cells and mesenchymal stem cells in proper condition, indicating the importance of cellular environment for PSC differentiation [12,13].

One method for testing cell pluripotency is teratoma formation; pluripotent cells should form teratomas containing various cell types, including endodermal, ectodermal, and mesodermal cells. Therefore, teratomas could provide an in vivo PSC differentiation environment. In line with this, recent reports have shown that fully functional and engraftable hematopoietic stem cells could be obtained from induced PSCs (iPSCs) through teratoma formation [14], and in vivo hematopoietic stem cells could be differentiated into functional B- and T-cell lineages [15]. These results suggest that an in vivo environment, even non-niche specific enables pluripotent cells to recapitulate in vivo development into specific cell lineages.

In this study, we developed an in vivo neural stem cell (NSC) differentiation system through teratoma formation using pluripotent embryonic stem cells (ESCs). These in vivo-derived NSCs were similar to brain-derived NSCs in both gene expression profiles and the potential to differentiate into neurons and glial cells. Based on these results, we suggest that teratomas or an in vivo environment, although not niche specific, endow pluripotent cells with the ability to recapitulate the required NSC niche.

Materials and Methods

The methods were carried out in accordance with the approved guidelines, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Konkuk University.

Cell culture

Olig2-GFP transgenic ESCs (Olig2 ESCs) containing the GFP transgene under the control of the Olig2 promoter [16] were used for our experiments. The GFP-neo cassette was integrated into the Olig2 gene translational start site. Olig2 ESCs were grown on mitomycin C-treated mouse embryo fibroblast (MEF) feeders with standard mouse ESC culture medium: Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco), 1× penicillin/streptomycin/glutamine, 1 mM nonessential amino acids (NEAA; Gibco), 0.1 mM β-mercaptoethanol (Gibco), and 1,000 U/mL leukemia inhibitory factor (ESGRO; Chemicon).

Directed in vitro differentiation into NSCs

Directed differentiation of Olig2 ESCs into NSCs was carried out using a previously described method [17]. Olig2 ESCs were cultured for 2–3 days with MEF medium supplemented with 15% FBS (Gibco), 1× penicillin/streptomycin/glutamine (Gibco), 0.1 mM NEAA (Gibco), and 1 mM β-mercaptoethanol (Gibco) in DMEM (Gibco). Subsequent to 2–3 days culturing, the ESCs were cultured for 2 days without feeder cells in suspension culture dishes containing N2B27 medium (1:1 mixture of DMEM/F-12 medium; Gibco, Neurobasal medium (Invitrogen) supplemented with 10 ng/mL epidermal growth factor (EGF; Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), N2 supplement, B27 supplement (Gibco), and 1× penicillin/streptomycin/glutamine (Gibco). Then, they were plated onto 0.1% gelatin-coated dishes containing NSC expansion medium: DMEM/F12 (Gibco) supplemented with N2 supplement (Gibco), 10 ng/mL EGF (Invitrogen), 10 ng/mL bFGF (Invitrogen), 50 mg/mL bovine serum albumin (BSA, BSA fraction, Gibco), and 1× penicillin/streptomycin/glutamine (Gibco); this was followed by culture for 3 days.

Teratoma formation

Immunodeficient Balb/c Nude (5 weeks, male) mice were purchased from Orient Bio (Gyeonggi-do, Korea) to carry out experimental procedures. In this study, 1 × 10⁶ cells Olig2 ESCs were injected into the testis capsule of immunodeficient mice. Teratomas were harvested surgically from mice at 4 or 6 weeks postinjection. Dissected teratomas were fixed in 4% paraformaldehyde (Sigma), processed through graded ethanol, and embedded in paraffin, followed by hematoxylin/eosin (Endoderm), PAS (Ectoderm), and Alcian blue (Mesoderm) staining. Immunohistochemistry staining of Nestin and Musashi was performed with an anti-Nestin and anti-Musashi antibody (Nestin, monoclonal, 1:100; Millipore), Musashi (monoclonal, 1:50; Millipore).

Flow cytometry analysis and sorting

Teratomas were enzymatically dissociated in 0.25% trypsin (Gibco) for 15 min at 37°C with mechanical dissociation by pipetting every 5 min. Dissociated cells were passed through a 70 μm nylon mesh (Falcon) to remove large cell clusters. The cells were then centrifuged at 1,500 rpm for 5 min and resuspended in phosphate-buffered saline (PBS; Gibco). Flow cytometry analysis and sorting were performed using a FACSAria (Becton Dickinson).

In vivo NSC derivation

FACS-sorted Olig2-GFP⁺ cells were plated on suspension dishes containing NSC culture medium [DMEM/F12 (Gibco) supplemented with N2 supplement (Gibco), B27 supplement (Gibco), 1× penicillin/streptomycin/glutamine (Gibco), 1M HEPES (Gibco), 45% Glucose (Sigma), 20 ng/mL EGF (Invitrogen), and 20 ng/mL bFGF (Invitrogen)] and then cultured for 2 to 3 weeks. Neurospheres were then replated on gelatin-coated tissue culture dishes containing NSC expansion medium and cultured for 2–3 days. Expanded neurospheres were dissociated into single cells by 0.25% trypsin (Gibco) treatment and passaged every 2–3 days in NSC expansion medium.

Differentiation of NSCs into neurons and glial cells

NSCs were differentiated into neurons and glial cells by culturing for 3 weeks in neuronal differentiation medium [1:1 mixture of DMEM/F12 medium (Gibco), Neurobasal medium (Gibco) supplemented with 1% FBS (Gibco), N2 supplement (Gibco), B27 supplement (Gibco), 1 × penicillin/streptomycin/glutamine (Gibco)].

Immunocytochemistry

NSCs were stained for markers of the NSCs: Nestin, Sox2, and Musashi. For immunocytochemistry, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min at 4°C. After washing with PBS (Gibco), cells were treated with PBS containing 3% Bovine albumin serum and 0.03% Triton X-100 (Sigma) for 45 min at room temperature. Primary antibodies used were anti-Nestin (Nestin, monoclonal, 1:500; Millipore), anti-Sox2 (Sox2, polyclonal, 1:500; Millipore), and anti-Musashi-1 (Musashi-1, polyclonal, 1:500; Millipore). For detection of primary antibodies, fluorescence-labeled (Alexa fluor 488 or 568; Molecular Probes, Eugene, OR) secondary antibodies were used according to the specifications of the manufacturer.

RNA isolation and real-time quantitative reverse transcription-polymerase chain reaction analysis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands, http://www.qiagen.com) and was treated with DNase to remove genomic DNA contamination. One microgram of total RNA was reverse transcribed with Super-Script III Reverse Transcriptase Kit (Invitrogen) and oligo (dT)₂₀-primer (Invitrogen) according to the manufacturer's instructions. Quantitative Real-Time polymerase chain reaction (qRT-PCR) reactions were set up in duplicate with the Power SYBR Green Master Mix (Takara) and analyzed with the Roche LightCycler5480 (Roche) [18]. The primers for qRT-PCR used were as follows: Oct4(endo) sense 5′-CCA ATC AGC TTG GGC TAG AG-3′, Oct4 (endo) antisense 5′-CTG GGA AAG GTG TCC CTG TA-3′; Nanog (endo) sense 5′-AGG CTG ATT TGG TTG GTG TC-3′, Nanog (endo) anti-sense 5′-CCC AGG AAG ACC CAC ACT CAT-3′.

Statistical analysis

All experiments were performed in triplicate, and data represented as mean ± SD. Significance of differences was assessed by an unpaired t-test at P < 0.05.

Results

Generation of NSCs from ESCs in vitro

Olig2-GFP transgenic ESCs (Olig2 ESCs) containing the GFP transgene under the control of the Olig2 promoter [16] were used to generate NSCs. We first tested whether Olig2 ESCs are efficiently differentiated into NSCs in vitro with activation of the Olig2-GFP marker by examining neurosphere formation; Olig2-GFP was not active in the pluripotent state, but was activated when the ESCs differentiated and formed neurospheres from which monolayered NSCs were established (Fig. 1A). Immunocytochemistry analysis also showed that ESC-derived NSCs expressed NSC markers, including Nestin, Sox2, and Musashi (Fig. 1B). These results indicate that NSCs could be efficiently derived from Olig2 ESCs through the in vitro differentiation system, and Olig2-GFP marker could be a selection marker for NSCs differentiated from ESCs.

FIG. 1.

In vitro differentiation of Olig2 ESCs into NSCs. (A) Olig2 ESCs are GFP negative in the pluripotent state (top panels); scale bar = 50 μm. Olig2-GFP was expressed when the Olig2 ESCs differentiated and formed neurospheres from which monolayered NSCs were established (lower panels); scale bar = 100 μm. (B) Immunofluorescence staining analysis for NSCs markers (Nestin, Sox2, and Musashi) in NSCs differentiated from Olig2 ESCs; nuclei were counterstained with DAPI, scale bar = 50 μm. ESCs, embryonic stem cells; NSCs, neural stem cells.

Generation of NSCs from Olig2 ESCs in vivo

We next set out to generate NSCs from Olig2 ESCs through in vivo environment, or teratoma formation (Fig. 2A). Olig2 ESCs were injected into the testis capsule of BALB c/Nude or NOD/SCID mice, and the testes were harvested 4 to 6 weeks postinjection. Teratoma formation was observed in the testes of mice injected with Olig2 ESC (Fig. 2B). Teratoma sections were stained with tissue-specific antibodies, which showed that all three germ layer tissues were present in the teratoma (Fig. 2C). Staining of teratoma sections with the Nestin or Musashi antibody revealed numerous Nestin⁺ or Musashi⁺ cells in the teratomas (Fig. 2D), confirming the presence of NSCs in the teratoma. Next, the teratomas were dissociated into small clumps or single cells, in which many Olig2-GFP⁺ cells were observed (Fig. 3A). These observations suggest that the teratomas formed from Olig2 ESCs contain a high percentage of Nestin⁺ and Olig2⁺ cells.

FIG. 2.

In vivo differentiation of Olig2 ESCs through teratoma formation. (A) Schematic diagram of in vivo NSCs generation through teratoma formation. (B) Teratoma formation in testes of immunocompromised mice subsequent to injection of Olig2 ESCs. (C) In vivo differentiation potential of Olig2 ESCs through teratoma formation. The tissues of all three germ layers were detected in sections of Olig2 ESC-derived teratomas; scale bar = 100 μm. (D) Expression of Nestin and Musashi in Olig2 ESC-derived teratoma sections; original magnification 100 × .

FIG. 3.

Flow cytometry analysis of teratomas. (A) Olig2-GFP-positive populations in Olig2 ESC-derived teratoma tissue; scale bar = 100 μm. (B) FACS analysis of dissociated teratomas for Olig2-GFP⁺ cells. The proportion of Olig2-GFP⁺ cells was ∼11% in 4-week-old teratomas and ∼3% in 6-week-old teratomas. (C) Sox2 expression in Olig2-GFP⁺ cells. Approximately 94% of the Olig2-GFP⁺ cells were also positive for the NSC marker, Sox2, indicating that the sorted Olig2-GFP⁺ cells constituted a pure population of NSCs; scale bar = 50 μm.

Next, Single cells dissociated from teratomas were FACS sorted for Olig2-GFP⁺ cells. Since the proportion of Olig2-GFP⁺ cells is different in each teratoma sample, separate cells from each teratoma were independently analyzed by FACS. Interestingly, the proportion of Olig2-GFP⁺ cells was much higher in 4-week-old teratomas (∼11%) than in 6-week-old teratomas (∼3%; Fig. 3B). This result could be explained by the fact that neural lineage specification is the one of the earliest events during embryo development [19]. Thus, neural lineage might be formed at the earlier stage of differentiation in teratoma environment. Olig2-GFP⁺ cells from 4-week-old teratomas tended to form neurospheres more efficiently than those from 6-week-old teratomas (data not shown). Therefore, we attempted to obtain NSCs from earlier stage of teratoma, but we could not observe detectable teratomas at 3 weeks after ESC injection. These results indicate that 4-week-old teratomas provide the best option for the derivation of in vivo NSCs through teratomas. To confirm whether the GFP⁺ cells are NSCs, we stained the GFP⁺ cells with an NSC marker, Sox2. Nearly all the Olig2-GFP⁺ cells were also positive for Sox2 (Fig. 3C), indicating that the sorted Olig2-GFP⁺ cells are mostly a pure population of NSCs.

Sorted GFP-positive cells were subjected to neurosphere formation in EGF and bFGF-containing medium. The Olig2-GFP⁺ cells began to form small clumps subsequent to one day of culturing and became large neurospheres at day 5 of culturing. In addition, the Olig2-GFP⁺ neurospheres stained positive for the NSC marker, Sox2 (Fig. 4A). These neurospheres were plated onto gelatin-coated dishes on which adherent bipolar NSCs were propagated (Fig. 4B). These adherent NSCs (in vivo-t-NSCs) expressed the common NSC markers, Nestin, Sox2, and Musashi (Fig. 4C). The NSCs remained Olig2-GFP⁺ for over 20 passages and their proliferation rate was similar to brain-derived NSCs, which is derived from normal brain tissue of 13.5 embryos (Fig. 4D). Furthermore, adherent NSCs (in vivo-tNSCs) also form neurospheres on nonadherent conditions (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/scd). When the Olig2-GFP⁺ NSCs were induced to differentiate by withdrawing bFGF and EGF, they differentiated into neurons and glial cells (Fig. 4E and Supplementary Fig. S2A). These in vivo-t-NSCs did not express Oct4 and Nanog, indicating that there was no contamination by undifferentiated pluripotent cells (Fig. 4F). This was confirmed using a teratoma formation assay with the established in vivo-t-NSCs (Fig. 4G); no teratoma formation was detected in the testes postinjection of in vivo-t-NSCs into the testis capsule. Since the in vivo-t-NSCs are derived from teratomas, which are a type of tumor, the cells could become prone to acquiring chromosomal anomalies. However, karyotype analysis revealed that the in vivo NSCs retained a normal karyotype (Fig. 4H). These results suggest that in vivo-t-NSCs consist of a pure population of nontumorigenic NSCs with a normal karyotype.

FIG. 4.

In vivo generation of NSCs by teratoma formation. (A) Expression of Olig2-GFP and Sox2 in neurospheres derived from Olig2-ESC teratomas; scale bar = 100 μm. (B) Establishment of adherent bipolar NSCs from Olig2-ESC teratomas (in vivo-t-NSCs). These in vivo-t-NSCs were indistinguishable from brain-derived NSCs; scale bar = 100 μm. (C) Immunofluorescence staining of NSCs markers (Nestin, Sox2, and Musashi) in in vivo-t-NSCs; scale bar = 50 μm. (D) Proliferation rate of in vivo-t-NSCs and control brain-derived NSCs (brain-NSCs). (E) In vivo-t-NSCs could differentiate into neurons (Tuj1⁺) and glial cells (GFAP⁺) in vitro; nuclei were counterstained with DAPI, scale bar = 50 μm. (F) Real-time reverse transcription-polymerase chain reaction analysis for endogenous Oct4 and Nanog. Relative expression was calculated by normalizing to β-actin levels; data represent mean ± SEM. Student's t-test: ***P < 0.001. (G) In vivo-t-NSCs did not form teratomas subsequent to injection into the testes of immunocompromised mice. (H) Karyotype analysis of in vivo-t-NSCs. Forty diploid chromosomes were observed; DNA was stained with Giemsa.

Discussion

In this study, we demonstrated that NSCs could be directly derived from teratomas formed subsequent to the injection of ESCs into immunocompromised mice. These results suggest that teratomas could provide a permissive niche for neurogenesis from PSCs. In addition, we found that in vivo NSCs derived from teratomas retain a normal karyotype, suggesting that although teratomas constitute a type of tumor, chromosomal anomalies do not occur in the NSCs grown in teratoma environment.

Directed in vitro differentiation into specialized cell types used for naive pluripotent ESCs is applicable to iPSCs and primed PSCs only by using a modified protocol. For example, different stem cell types require different protocols for NSC derivation. EpiSCs at early passages can be differentiated into NSCs using a medium with a high concentration of bFGF [20]. In vitro differentiation of iPSCs into NSCs is a more time-consuming process compared with ESCs; subsequent to the induction of neural differentiation in vitro, neural tube-like rosettes formed at day 10 from ESCs but at day 30 from iPSCs [17]. In particular, efficiency of NSC derivation was as low as 1% in the in vitro system, but was 11% in the in vivo system (using teratomas harvested 4 weeks post-ESC injection). Thus, in vivo differentiation protocol has an advantage over the in vitro system, at least when it comes to the efficiency of NSC derivation from PSCs.

Teratomas are benign tumors formed by injection of PSCs into immunodeficient mice [14,15,21]. Since teratomas contain many different types of cells derived from all three germ layers, teratoma formation is one of the most stringent assays for confirming the pluripotency of naive and primed PSCs. An in vivo differentiation method was first reported for the generation of hematopoietic lineage cells; Amabile et al. reported the derivation of in vivo human hematopoietic cells from iPSCs through teratomas [14,15,21]. These hematopoietic cells could colonize murine hematopoietic tissues and differentiate into functional B- and T-cell lineages. Recently, Suzuki et al. also derived fully functional and engraftable hematopoietic stem cells from iPSCs through teratoma formation [14]. Although the stem cell lines were not established, Choi et al. initially found that a hepatocyte population of varying maturity was present in the teratomas subsequent to the injection of ESCs [21]. These findings suggest that many other somatic stem cell types in addition to NSCs and hematopoietic stem cells that could also be isolated and established as cell lines may be present in the teratomas. Therefore, the in vivo differentiation approach could contribute to the development of a differentiation protocol for other cell types, not established thus far using in vitro systems.

One of the most interesting findings of the present study is that teratomas harvested 4 weeks post-ESC injection contain approximately a four-fold greater population of Olig2-GFP⁺ cells than those harvested 6 weeks post-ESC injection (Fig. 3A). It is possible that late-stage (6 week) Olig2-GFP⁺ cells are more differentiated than early-stage (4 week) NSCs. However, considering the property of NSCs, Olig2-GFP⁺ cells, which were established in the early stage, might be differentiated afterward and lose Olig2-GFP expression. Determining the exact timeframe of neural specification during teratoma development is essential, since harvesting a pure population of NSCs with high differentiation efficiency is critical for clinical application. The optimal time point for neural specification is as early as 4 weeks; ∼11% of all cells in these teratomas are Olig2⁺ cells. This is due the fact that neural specification is one of the earliest events to occur during embryonic development [19]. This timeframe may be also useful for deriving NSCs from cells lacking selection markers, such as Olig2-GFP cells. Differentiation using in vivo environments may guarantee efficient derivation of NSCs from teratomas formed 4 weeks postcell injection. The in vivo differentiation method could be combined with specific chemicals or with coinjection of supportive cells to obtain a higher ratio of NSCs in the teratoma. For example, coinjection of OP9 stromal cells with iPSCs enhances hematopoietic specification during teratoma development [15]. This approach requires further improvement for the derivation of human PSCs such as nuclear transfer ESCs and human iPSCs. An efficient in vivo differentiation protocol following cellular reprogramming in combination with gene or epigenetic correction [22] could therefore provide a tool for drug screening and the desirable cell types for tissue replacement therapy for incurable diseases.

We developed an in vivo NSC differentiation system through teratoma formation using ESCs. These in vivo teratoma-derived NSCs were similar to brain-derived NSCs in both NSC marker expression patterns and the potential to differentiate into neurons and glial cells. Moreover, in vivo NSCs maintained normal karyotype and were not tumorigenic, although they were derived through tumor tissue, teratoma. Therefore, this in vivo differentiation system that mimics 3D in vivo environment will be a powerful method for generation of specific cell types.

Footnotes

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (grant no. 2015R1A2A2A01003604 and 2015R1A5A1009701) and the Next-Generation BioGreen 21 Program (grant no. PJ01133802) funded by the Rural Development Administration, Republic of Korea.

Author Disclosure Statement

No competing financial interests exist.

References

Yang

and Smith

. (2007). ES cells derived from cloned embryos in monkey—a jump toward human therapeutic cloning. Cell Res, 17:969–970.

Tachibana

, Amato

, Sparman

, Gutierrez

, Tippner-Hedges

, Ma

, Kang

, Fulati

, Lee

, et al. (2013). Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 153:1228–1238.

Chung

, Eum

, Lee

, Shim

, Sepilian

, Hong

, Lee

, Treff

, Choi

and Kimbrel

. (2014). Human somatic cell nuclear transfer using adult cells. Cell Stem Cell, 14:777–780.

Yamada

, Johannesson

, Sagi

, Burnett

, Kort

, Prosser

, Paull

, Nestor

, Freeby

, et al. (2014). Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells. Nature, 510:533–536.

Takahashi

and Yamanaka

. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.

Zhao

X-Y

, Li

, Lv

, Liu

, Tong

, Hai

, Hao

, Guo

C-L

, Ma

Q-W

and Wang

. (2009). iPS cells produce viable mice through tetraploid complementation. Nature, 461:86–90.

Kim

, Sebastiano

, Wu

, Araúzo-Bravo

, Sasse

, Gentile

, Ko

, Ruau

, Ehrich

and van den Boom

. (2009). Oct4-induced pluripotency in adult neural stem cells. Cell, 136:411–419.

Warren

, Manos

, Ahfeldt

, Loh

Y-H

, Li

, Lau

, Ebina

, Mandal

, Smith

and Meissner

. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 7:618–630.

Anokye-Danso

, Trivedi

, Juhr

, Gupta

, Cui

, Tian

, Zhang

, Yang

, Gruber

and Epstein

. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, 8:376–388.

10.

Miyoshi

, Ishii

, Nagano

, Haraguchi

, Dewi

, Kano

, Nishikawa

, Tanemura

, Mimori

and Tanaka

. (2011). Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell, 8:633–638.

11.

Hou

, Li

, Zhang

, Liu

, Guan

, Li

, Zhao

, Ye

, Yang

and Liu

. (2013). Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science, 341:651–654.

12.

Takebe

, Sekine

, Enomura

, Koike

, Kimura

, Ogaeri

, Zhang

, Ueno

, Zheng

, et al. (2013). Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature, 499:481–484.

13.

Takebe

, Enomura

, Yoshizawa

, Kimura

, Koike

, Ueno

, Matsuzaki

, Yamazaki

, Toyohara

, et al. (2015). Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell, 16:556–565.

14.

Suzuki

, Yamazaki

, Yamaguchi

, Okabe

, Masaki

, Takaki

, Otsu

and Nakauchi

. (2013). Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Mol Ther, 21:1424–1431.

15.

Amabile

, Welner

, Nombela-Arrieta

, D'Alise

, Di Ruscio

, Ebralidze

, Kraytsberg

, Ye

, Kocher

, et al. (2013). In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells. Blood, 121:1255–1264.

16.

Xian

, McNichols

, St Clair

and Gottlieb

. (2003). A subset of ES-cell-derived neural cells marked by gene targeting. Stem Cells, 21:41–49.

17.

Choi

, Kim

, Choi

, Hong

, Kim

, Seo

and Do

. (2014). Neural stem cells differentiated from iPS cells spontaneously regain pluripotency. Stem Cells, 32:2596–2604.

18.

Hwang

, Kang

, Ham

, Yoo

, Lee

, Paek

, Park

, Kim

, Lim

and Seo

. (2012). Activation of peroxisome proliferator-activated receptor gamma by rosiglitazone inhibits lipopolysaccharide-induced release of high mobility group box 1. Mediators Inflamm, 2012:352807.

19.

Brown

and Storey

. (2000). A region of the vertebrate neural plate in which neighbouring cells can adopt neural or epidermal fates. Curr Biol, 10:869–872.

20.

Jang

, Kim

, Choi

, Jeon

, Choi

, Kim

, Song

and Do

. (2014). Neural stem cells derived from epiblast stem cells display distinctive properties. Stem Cell Res, 12:506–516.

21.

Choi

, Oh

H-J

, Chang

U-J

, Koo

, Jiang

, Hwang

S-Y

, Lee

J-D

, Yeoh

, Shin

H-S

and Lee

J-S

. (2002). In vivo differentiation of mouse embryonic stem cells into hepatocytes. Cell Transplant, 11:359–368.

22.

Kim

, Choi

, Jang

, Chung

, Arauzo-Bravo

, Scholer

and Do

. (2013). Conversion of genomic imprinting by reprogramming and redifferentiation. J Cell Sci, 126:2516–2524.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.21 MB