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Abstract

Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 ± 2.62%, ADCs 70.61 ± 15.4%, NSCs, 68.72 ± 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 ± 4.10 colonies, 0.13 ± 0.06%) and ADCs (118.20 ± 38.28 colonies, 1.14 ± 0.77%) when compared with the mEF control (3.17 ± 0.29 colonies, 0.03 ± 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Keywords

Induced pluripotent stem (iPS) cells Reprogramming Adipose-derived cells Neural stem cells

Introduction

Pluripotency describes the ability of a cell to differentiate into cells of the three germ lineages. This property is usually restricted to a few cell types, including cells of the inner cell mass of a preimplanted blastocyst and their in vitro counterparts, ES cells. While somatic cells have a restricted ability to differentiate during normal development, under certain experimental conditions they can be induced to revert to a pluripotent state. This process is known as reprogramming and results from the reactivation of pluripotent genes with concurrent inactivation of somatic genes (31). Reprogramming has been previously achieved via somatic cell nuclear transfer (SCNT) (38, 41) and cell fusion of somatic cells with pluripotent cells (6, 33). However, given the requirement for oocytes and ES cells, respectively, for each approach, ethical and logistical concerns have largely diminished their utility [reviewed in Hochedlinger and Jaenisch (11)].

In 2006, the pioneering work of Takahashi and Yamanaka showed that it was possible to directly repro-gram mouse adult fibroblasts into pluripotent-like cells, without the use of embryonic material (36). The cells were termed induced pluripotent stem (iPS) cells and were generated by the retroviral transduction of four transcription factors: Oct4, Sox2, c-Myc, and Klf4. iPS cells were subsequently shown to possess many hallmarks of ES cells, including the expression of endogenous pluripotency genes, ability to be differentiated both in vitro and in vivo into derivatives of the three germ layers, and form viable chimeric animals capable of germline transmission (19, 22). Subsequent generation of human iPS cells further strengthened a place for iPS technology in the reprogramming field (24, 35). The ability to generate iPS cells from differentiated adult somatic cells has widespread potential for in vitro disease modeling, drug screening, and regenerative cell therapy.

An important consideration for iPS technology is determining an appropriate starting somatic cell type. A majority of iPS studies use fibroblast cells, most likely due to their ease of derivation and extensive use in SCNT and fusion-based reprogramming studies (33, 39). Various other starting somatic cell populations have been used for iPS induction in the mouse, including adult stomach and liver cells (2), embryonic or newborn neural progenitor cells (9, 14), pancreatic β-cells (29), mature B lymphocytes (10), and bone marrow hematopoietic cells (15). An important observation from these studies is that the somatic cell type chosen had a significant influence on the efficiency of iPS generation and level of reprogramming. Aoi et al. also reported that stomach- and liver-derived iPS cells possessed fewer retroviral integrations than fibroblast-derived iPS cells (2). The omission of c-Myc during induction of these two cell types only decreased the incidence of iPS colony formation by 20–40% when compared to four-factor induction, whereas fibroblast iPS colony numbers drop by more than 90%.

In another study using whole brain-derived neural stem cells, Oct4 was sufficient to generate fully reprogrammed iPS cells, albeit at a significantly lower efficiency, by taking advantage of their endogenous expression of Sox2 (13). While these results are promising, the isolation of some of these somatic cell types may not be feasible in humans due to the invasive nature of tissue collection and limited donor samples available. There are reports of using easily accessible human somatic cells, keratinocytes, and peripheral blood cells to generate human iPS cell lines that provide viable options for therapy (1, 16). However, there are suggestions that differences exist in the range of cell types that human iPS cell lines are capable of differentiating into (17, 27). This may reflect an epigenetic memory retained by the iPS cells for the parental donor somatic cell type.

A recent report by Miura et al. indicates that mouse iPS cell lines generated from different somatic cell sources vary greatly in their subsequent differentiation characteristics (21). The study showed that mouse tail tip fibroblast-derived iPS cell lines, after being differentiated to neurons and transplanted into immunocompromised mice, resulted in a higher incidence of teratoma formation, when compared with mEF and hepatocyte-derived iPS or ES cell lines. A possible explanation was that iPS cells from tail tip fibroblasts were highly resistant to neuronal differentiation, therefore potentially harboring more pluripotent cells among the differentiated cells. This finding reemphasizes the need to search for various somatic cell types for inducing pluripotency to determine the most suitable for a particular application.

Here, we directly compare the efficiency of iPS generation and reprogramming ability of different adult somatic cells using retroviral induction with the four Yamanaka factors. Adult somatic cells were isolated from neural and adipose tissue of OG2 mice, which possess a green fluorescent protein (GFP) transgene under control of the Oct4 promoter (Oct4-GFP) (42). The comparison of different cell types derived from the same inbred transgenic mouse strain also negates any possible genotype effects present when trying to draw conclusions from different studies. While drug selection has been used extensively to generate iPS colonies, timing of drug selection has been reported to greatly influence iPS colony numbers and the level of reprogramming attained (4, 18, 19). Consequently, in this study, we examined the efficiency of iPS colony formation based on Oct4-GFP expression alone (32, 42).

Materials and Methods

All reagents were sourced from Invitrogen, Australia unless otherwise stated.

Tissue Culture

All cells were cultured in tissue culture coated flasks or plates (BD Falcon) at 37°C in a humidified incubator in 5% CO₂/95% air.

mEFs were derived from OG2 (42) Bl6 homozygous transgenic fetuses (for iPS induction) and Quakenbush (QS)/Rosa26 heterozygous transgenic fetuses (for feeders) as previously described (28). Briefly, fetuses were decapitated, internal organs removed, and the remaining carcass minced using a scalpel. TrypLE (stable trypsin-like enzyme) was added and the embryonic tissue was further dissociated by homogenizing several times through a 3-ml syringe (Terumo) fitted with a 15-mm-gauge needle. The homogenized cell mixture was plated at a density of 6 × 10⁴ cells/cm² in mEF medium consisting of high-glucose Dulbecco's minimum essential media (DMEM) supplemented with 10% fetal bovine serum (FBS from JRH Biosciences, Australia), 1x nonessential amino acids, 1x GlutaMAX, and 1x penicillin-streptomycin.

Adipose derived cells (ADCs) were obtained from adult OG2 homozygous males or females. The inguinal fat pads were dissected, washed, and put into digestion media that consisted of 0.1% collagenase A type I (Sigma Australia) and 10 ng/ml DNase I from bovine pancreas (Sigma, Australia) dissolved in Hank's balanced salt solution (Gibco). This digesting solution was placed in a 37°C water bath for 1-1.5 h. The entire suspension was filtered through a 70-μm cell strainer (Falcon) and then centrifuged (Eppendorf centrifuge 5702) at 400 × g for 10 min. The pellet was resuspended in ADC medium, which consists of DMEM/F12 media with glutaMAX, supplemented with 20% FBS and 1x penicillin-streptomycin.

Adult neural stem cells (NSCs) were derived from the subventrical zone (SVZ) of adult OG2 homozygous male brains as described by Rietze and Reynolds (26). Briefly, the SVZ of six adult mice were pooled, minced finely using a scalpel, and then digested with 0.25% trypsin-EDTA for 7 min in a 37°C water bath and then neutralized using trypsin inhibitor (Sigma-Aldrich). Digested cells were resuspended in complete NS media, which consisted of NS proliferation supplements (Neurocult), 20 ng/ml of epidermal growth factor (StemCell Technologies), 10 ng/ml of human β-fibroblast growth factor (StemCell Technologies), 0.2% bovine serum albumin fraction V (Gibco), and 10 μg/ml of heparin (Sigma-Aldrich). Cells were seeded on 0.1% gelatin-coated T-25 flasks. Neurospheres formed after 7 days of culture and these were disaggregated with 0.25% trypsin-EDTA and replated onto 0.1% gelatinized T-25 flasks. Adherent NSCs appeared after 2–3 days culture.

Induced Pluripotency

iPS induction was performed according to Takahashi et al. (34). Briefly, pMX-based retroviral vectors encoding the mouse DNA sequence of Oct4, Sox2, Klf4, and c-Myc (Addgene) were transfected into packaging PlatE cells using Fugene 6 transfection reagent (Roche). Twenty-four hours later, viral supernatants were collected, pooled, and filtered through a 0.45-μm cellulose acetate filter. Viral supernatants were then mixed with polybrene to a final concentration of 4 μg/ml. mEFs, ADCs, and NSCs were plated 1 day prior to induction at a density of 50,000 cells/well of a 0.1% gelatinized six-well plate. The cells were incubated overnight with the viral/polybrene supernatant containing either all four factors, pMX-GFP (at 1:4 dilution), or a no vector mock control. Infected cells were then cultured in ES medium, which consisted of high-glucose DMEM supplemented with 15% FBS, 1x nonessential amino acids, 1x GlutaMAX, 1x penicillin-streptomycin, 0.1 mM β-mercapto-ethanol, and 1,000 U/ml ESGRO LIF (Chemicon, Australia). Five days after iPS induction, cells were passaged onto mEFs (QS/Rosa26) inactivated with 4 μg/ml of mitomycin C and plated in a 60-mm tissue culture dish coated with 0.1% gelatin. Oct4-GFP-positive colonies were counted at day 12 postinduction and individually picked for clonal expansion using an inverted fluorescent microscope (1 × 71 Olympus, Melville, NY). The transduction efficiency of mEFs, ADCs, and NSCs were determined by extrapolation from the pMX-GFP vector control, which was conducted in parallel with the iPS induction experiments. Seventy-two hours after pMX-GFP induction, cells were photographed under a fluorescence microscope and the percentage of cells expressing GFP was quantified by flow cytometry. Mock transfections (no DNA vector control) were conducted for each cell type and this yielded no GFP-positive cells. The GFP-positive percentages for each cell type were averaged over four repeats with each repeat performed in duplicate.

Flow Cytometry

For cell cycle analysis, 2 × 10⁶ cells were trypsinized to a single cell suspension, fixed in 70% ethanol, and stored at −20°C until analyzed. For analysis, fixed cells were washed three times with ice-cold PBS- (calcium and magnesium free) with 1% fetal calf serum (JRH Biosciences, Australia) and then resuspended in PBS-at room temperature. RNase A and propidium iodide (PI) were added to a final concentration of 100 and 5 μg/ml, respectively. Cells were then covered and left on a rocking platform at room temperature for 30 min and then maintained at 4°C for 1 h before flow cytometric analysis using the MoFlo® cell sorter (DakoCytomation, Fort Collins, CO, USA).

For antibody staining, 0.5 × 10⁶ cells were washed two times in PBS-, resuspended in blocking solution (1% BSA in PBS-), and left for 15 min at room temperature. Cells were then stained for 30 min at 4°C with directly conjugated FITC or PE antibodies specific for mouse CD117, CD45, Sca-1, CD44, CD31, CD34 (BD Pharmingen), and CD90.1 (Thy-1) (eBiosciences). Cells were washed twice with blocking solution and kept on ice until flow analysis.

Determination of GFP Expression

GFP expression was examined in living cells in 60-mm tissue culture dishes with epifluorescence microscopy using an Olympus 1X70 microscope at 12 days postinduction. Both phase contrast and epifluorescence images were captured.

Statistical Analysis

Differences in the transfection efficiency, the number of Oct4-GFP-positive colonies observed, and the reprogramming efficiency among the experimental groups were analyzed by one-way analysis of variance (ANOVA) and then using Bonferroni's multiple comparison test using the GraphPad Prism version 4.03 software. A probability of p < 0.05 was considered to represent a statistically significant difference between groups.

PCR

GFP-positive colonies were picked using epifluorescence microscopy, replated on a feeder layer, and expanded to approximately 1 × 10⁶ cells. Cells were harvested with TrypLE and the cell pellet was stored at −80°C until analysis. For RT-PCR, RNA was isolated from cells using the RNeasy kit (Qiagen) following the manufacturer's instructions. RNA quality and concentrations were measured using the NanoDrop ND-1000 (NanoDrop Technologies, Australia). The isolated RNA was subjected to RQ1 DNase (Promega, Australia) treatment to remove any contaminating genomic DNA. cDNA was generated using the Superscript III enzyme following the manufacturer's instructions. Oligonucleotide primers have been described elsewhere (36). The PCR amplification included a total 30 cycles of denaturation at 94°C for 45 s followed by the appropriate annealing temperature for 45 s and extension at 72°C for 45 s with a first denaturation step at 94°C for 5 min and a final extension step at 74°C for 5 min. PCR products were run on a 2% agarose gel at 80 V for 2 h.

Genomic DNA was isolated using the DNeasy tissue and blood kit (Qiagen) as per the manufacturer's protocol. To detect genomic integration of the four factors, the same RT transgene primers were used for amplification. Genomic PCR conditions were the same as RT-PCR conditions except with the extension step increased to 1 min.

Immunostaining

iPS clones and mESCs were plated onto an OG2-xOG2 mEF (to act as a negative control) feeder layer in four-well glass culture slides. Cells were fixed in 4% paraformaldehyde for 10 min and washed three times with PBS. For SSEA-1 (Chemicon) immunostaining, fixed cells were incubated for 1 h at room temperature with blocking solution (5% goat serum, 1% BSA in PBS+). Cells were then incubated overnight with the primary antibodies diluted 1:100 in blocking solution at 4°C. Next, cells were washed three times with PBS+ and then incubated at room temperature for 1 h with secondary antibodies (goat α-mouse IgM Alexa 594, Invitrogen, Australia) diluted 1:1000 in blocking solution. Cells were washed three times with PBS+ and then mounted in Vectashield (containing DAPI) (Vector Laboratories, USA) with a coverslip. Cells were analyzed by epifluorescence microscopy using the IX71 Olympus microscope.

For alkaline phosphatase staining, cells were fixed as above and stained with the Chemicon alkaline phosphatase kit as per the manufacturer's instructions.

Bisulfite Analysis

Bisulfite conversion of genomic DNA was performed using MethylEasy Xceed™ (Human Genetic Signatures, NSW Australia) according to the manufacturer's instructions. The Oct4 promoter region was amplified using nested primers (first round forward: AAG TAT GGA TTA ATT TAT TAA GGT AGT T, reverse: AAA AAA CCC ACA CTC ATA TCA ATA TA; second round forward: AAG TAT GGA TTA ATT TAT TAA GGT AGT T, reverse: CAA CCA AAT CAA CCT ATC TAA AAA) and both rounds of PCR performed as follows: 4 min at 95°C; 30 cycles of 30 s at 95°C, 1.5 min at 57°C, 2 min at 95°C; and 10 min at 72°C. The Nanog promoter region was amplified using nested primers [primer sequences have been described elsewhere (43)] and both rounds of PCR performed as follows: 5 min at 94°C; 35 cycles of 30 s at 94°C, 45 s at 59°C, 3 min at 72°C; and 10 min at 72°C. PCR products were cloned into pGEM-T easy vectors. Nine randomly selected clones were individually sequenced by the Gandel Charitable Sequencing Trust (Monash Health Research Precinct, Clayton Australia). Data was analyzed using BiQ analyzer version 2.0 (Max-Planck-Institute for Informatics and Saarland University, Germany).

In Vitro Differentiation via Embryoid Body Formation

Approximately 1 × 10⁶ Oct4-GFP-positive iPS clones were harvested and resuspended in 10 ml of mEF media. The cell suspension was transferred to low adhesion pe-tri dishes (Falcon) and incubated at 37°C. At day 4, cultures were passaged in 1:4 ratio with medium changed every 2 days. Day 10 embryoid bodies (EBs) were collected for RT-PCR analysis of differentiation markers of the three germ layers [primer sequences have been described elsewhere (36)].

In Vivo Differentiation via Teratoma Formation

Oct4-GFP-positive iPS cells (2.5 × 10⁶) were injected into the hind leg muscle of a SCID mouse. Mice were sacrificed and teratomas excised between 4 and 5 weeks postinjection and fixed in HistoChoice® (Amresco). Tumors were sectioned and stained with hematoxylin and eosin. Histology was performed by the Monash Institute of Medical Research Histological Services Centre, Clayton Australia.

Results

Characterization of NSCs and ADCs

The morphology of NSCs and ADCs compared with mEFs are shown in Figure 1. RT-PCR analysis confirmed that NSCs expressed the neural lineage markers, Sox2 and nestin. ADCs were stained for CD surface markers common to other adult stem cells. CD44 was expressed on 90% of ADCs while 43% of the population was stem cell antigen-1 (Sca-1) positive. Both markers are common to mesenchymal stem cells (MSCs), suggesting similarities between the two cell types. However, CD117, which is expressed on many adult stem cells including MSCs and hematopoietic stem cell (HSCs), were not expressed by ADCs. Also, the HSCs markers CD45 and CD34 were not expressed by ADCs.

Figure 1.

Generation of iPS cells from four-factor induction of mEFs, NSCs, and ADCs. (a) Morphology of mEFs, NSCs, and ADCs in culture. P denotes passage number. (b) Flow cytometry cell cycle profiles. The x-axis denotes the intensity of propidium iodide (PI) fluorescence and the γ-axis denotes cell number. The different phases of the cell cycle are indicated by the different labeled peaks. ADCs show a higher proportion of cells at the G₂/M phase compared with NSCs and mEFs. ES cells show a higher proportion of cells at both the S and G₂/M phases. (c) Histogram showing the number of Oct4-GFP-positive colonies observed at day 12 postinduction with four factors. Columns indicate mean ± SD (n = 4). *Significantly different compared with mEF (p < 0.05, t-test). (d) Morphology of mEFs, NSCs, and ADCs at day 12 postinduction with Yamanaka's four factors. p0 indicates that Oct4-GFP-positive colonies have not been passaged since reactivation of GFP. (e) Phase and UV photographs of established iPS cell lines.

Generation of iPS Cell Lines

The pMX-GFP transduction efficiencies observed were not significantly different (p = 0.9663) between the three cell types: for mEFs, NSCs, and ADCs, the efficiencies were 68.70 ± 2.62%, 68.72 ± 3%, and 70.61 ± 15.40%, respectively. On day 12 postinduction, all groups were screened for Oct4-GFP-positive (+), iPS-like colonies. The colonies had a speckled, rough appearance and were not compact like mouse ES cell colonies (see Fig. 1d). Oct4-GFP⁺ colonies derived from mEFs, ADCs, and NSCs were individually picked and expanded on feeders. A homogenous Oct4-GFP⁺ cell population was obtained either by FACS sorting for GFP⁺ cells or subcloned by repicking and expanding individual GFP⁺ colonies. With increased time in culture, Oct4-GFP⁺ iPS clones became more ES cell-like in morphology, usually within one to two passages after initial picking (3–4 days between passages). Three iPS clonal lines were established for each somatic cell type and used for characterization (Fig. 1e). The reprogramming efficiencies of different cell types were calculated by correlating the pMX-GFP transduction efficiencies with the number of Oct4-GFP⁺ iPS colonies observed (Fig. 1c) as previously described (36). The reprogramming efficiencies were significantly higher for NSC-iPS (0.13 ± 0.06%) and ADC-iPS cell (1.14 ± 0.77%) generation when compared with mEF derived iPS cells (0.03 ± 0.005%) (p < 0.05) (Table 1). ADC-iPS cell generation was also significantly higher (p < 0.05) when compared with NSC-iPS cells. Genomic PCR confirmed the presence of all four transgenes in mEF-, ADC-, and NSC-derived iPS cell lines generated. Conversely, the transgenes were not present in the starting somatic cell populations prior to transduction (data not shown).

Table 1.

Reprogramming Efficiency of Generating Four-Factor iPS Cells From Different Cell Types

OG2 Cell Type	Average Oct4-GFP⁺ Colonies	Reprogramming Efficiency %^*
mEF p4	3.17 ± 0.29	0.03 ± 0.005
ADC p4	118.20 ± 38.28^†	1.14 ± 0.77^‡
NSC p12–18	13.50 ± 4.10	0.13 ± 0.06^†

Values are mean ± SD (n = 4).

Reprogramming efficiency % = average number of Oct4-GFP⁺ colonies observed/the number of cells expressing all four factors x 100. Calculations have been described previously (34).

†

Significance compared with mEF (p < 0.05, one-way ANOVA).

‡

Bonferroni's multiple comparison test was performed post ANOVA and showed significance between mEFs and NSCs.

Characterization of iPS Cells

Cell Cycle Analysis

Flow cytometric analysis showed that a majority of somatic cells were at G₀/G₁ stage of the cell cycle, while few cells are at S phase, suggesting that they are a slow cycling population of cells (Fig. 1b). On the other hand, OG2 ES cells were shown to have a high proportion of cells at the S phase, confirming that ES cells are rapidly cycling cells (Fig. 1b). Clonal iPS cell lines all possessed cell cycle profiles similar to ES cells, suggesting that they were more ES-like in their proliferation characteristics (Fig. 2a).

Figure 2.

Characterization of Oct4-GFP-positive iPS clonal cell lines. (a) Cell cycle flow cytometry histographs of iPS cell lines, as determined by PI staining. (b) iPS cell lines were stained for the early markers of pluripotency, alkaline phosphatase (pink) and immunostained SSEA-1 (red). (c) RT-PCR for the expression of endogenous Nanog, Rex-1, Klf4, c-Myc, Oct4, and Sox2. Exogenous expression of the four transgenes was also determined. β-Actin was used as a housekeeping gene while RT neg is a negative control of cDNA generation without superscript III. (d) Methylation analysis of the Oct4 and Nanog promoter regions. Open circles represent unmethylated cytosines of CpG dinucleotides while black filled circles represent methylated cytosines.

Protein and Gene Expression

All Oct4-GFP⁺ iPS clones examined were found to express the early markers of pluripotency, alkaline phosphatase, and stage-specific embryonic antigen 1 (SSEA-1) by immunostaining (Fig. 2b). Further, SSEA-1 was shown to correctly localize to the cell surface of iPS cells. Using RT-PCR, iPS clones were also shown to express the pluripotency markers, Nanog and Rex-1, albeit at varying levels, which are not expressed by the noninduced mEFs and NSCs. ADCs, however, appear to express basal levels of Nanog and Rex-1 (Fig. 2c). RT-PCR analysis using primers specific for the transgenic and endogenous sequences of Oct4, Sox2, Klf4, and c-Myc showed that the majority of iPS clones expressed all four factors endogenously, even when the corresponding transgene levels could not be detected. Expression of Oct4 was not detected in noninduced mEFs and NSCs. However, ADCs showed some low-level expression of Oct4. Conversely, Sox2 was not detected in noninduced ADCs but there was some low-level expression of Sox2 by noninduced mEFs, which is expected because mEFs are a heterogenous mixture of cells that could contain neural cells.

Methylation Analysis

Methylation patterns of the three types of iPS clonal lines were distinct from that of mEFs, NSCs, and ADCs but were highly similar to ES cells (Fig. 2d). A predominantly demethylated Oct4 and Nanog promoter region of the iPS cell lines suggests epigenetic remodeling occurred during the reprogramming process.

Pluripotency Potential

Embryoid bodies (EBs) were formed using the iPS clonal lines (Fig. 3a). At day 7 postformation, EBs were collected for RT-PCR analysis. Markers indicative of the three germ layers: brachyury (mesoderm), Foxa2 (endoderm), and nestin (ectoderm) were observed confirming the in vitro differentiation capability of iPS-derived EBs. All EBs tested showed expression of these differentiation markers (Fig. 3b).

Figure 3.

Pluripotency of iPS clonal cell lines. (a) iPS cell lines were spontaneously differentiated into EBs, shown here at day 5. (b) Day 7 EBs were collected for total RNA isolation and analyzed by RT-PCR for differentiation markers indicative of the three germ layers: Foxa2 (endoderm), Brachyury (mesoderm), and Nestin (ectoderm). (c) Teratoma formation. Hematoxylin and eosin staining showed tissue derivatives indicative of the three germ layers: keratinocyte-like structures and neuronal rosettes, adipocytic-and cartilage-like structures, and secretory epithelial structures.

Teratomas were formed for all iPS clonal lines tested (n = 2). Histological analysis using H&E staining revealed tissue structures indicative of the three germ layers (Fig. 3c). Keratinocytes and neural rosettes were observed, suggesting differentiation into ectoderm. Secretory epithelium and adipocytic- and cartilage-like structures were observed, suggesting differentiation into endoderm and mesoderm, respectively.

Discussion

When choosing the optimal somatic cell type for induced pluripotency, several key considerations need to be taken into account, including the ability of the cell to be reprogrammed, ease of access to the tissue, and ease of deriving and maintaining the cells in vitro. While neural cells and hepatic cells have shown to be more efficiently reprogrammed in mouse iPS studies than mEFs, they remain inaccessible cell types, particularly for human cell therapies as their derivation is highly invasive. While human iPS cells have been generated from easily accessible cells such as keratinocytes and peripheral blood, the need to search for other candidate somatic cell types that can be efficiently reprogrammed is not obviated. A recent report showed that mouse iPS cell lines derived from different somatic cell sources varied greatly with their ability to be differentiated into neuronal cells, even though they were all capable of forming teratomas and were germline transmission competent (21). The resistance of iPS cells generated from somatic cells from certain sources to neuronal differentiation led to a higher propensity to form teratomas upon transplantation, which raises some doubt over the safety of iPS cell lines for therapy. Additionally, it is well documented that different human ES cell lines have different capacities for differentiation (23).

Recent genome-wide microarray analysis shows that variations exist between different iPS cell lines. Therefore, it is likely that certain iPS cell lines will have a higher propensity to differentiate into certain lineages than other lines. Indeed, in this study different patterns of gene expression were detected between iPS cell lines generated from the different starting somatic cell types and even within the different clonal lines generated from the same starting somatic cell type. This variation may be due to different levels of reprogramming achieved. Such variablity between different cell lines highlights the fact that different somatic cell types need to be investigated so that many iPS cell lines will be available for specific lineage differentiation and different research purposes.

A cell's differentiation state is thought to correlate with a specific epigenetic profile, and ES cells are known to have loosely bound chromatin with many of the pluripotency genes being unmethylated at the promoter regions (3). Given that adult stem cells are multipotent, it is suggested that they have an epigenetic profile more similar to ES cells than terminally differentiated cells and therefore may encounter less roadblocks to reprogramming (12). Indeed, hematopoietic stem and progenitor cells have been found to be more easily reprogrammed than terminally differentiated T and B cells (8). Therefore, it is not surprising that NSCs can be more efficiently reprogrammed to pluripotency given their demonstrated plasticity. Furthermore, neuroectoderm is the default differentiation pathway of ES cells, so NSCs may have the closest semblance to a pluripotent-like state compared with other adult stem cells and it has been suggested they may be the most primed cell type for reprogramming (37).

Here we report the generation of iPS cell lines from NSCs derived from the subventricular zone (SVZ), a site of neurogenesis in adult brains, derived using the neurosphere assay (26). The neurosphere assay uses a defined, serum-free culture system to nurture the growth of EGF-responsive cells that typically form less than 0.1% of the brain (7). Neurospheres derived using this assay enable the maintenance of a population of NSCs, which are proliferative and self-renewable (more than 70 passages) while retaining the ability to differentiate into functional astrocytes, neurons, and oligodendrocytes (7). While iPS cell lines have been previously generated from neural cell types, in our study we examined a defined neural population rather than isolating cells from whole brains (14). The neurosphere assay used in this study may enrich for more true NSCs than using whole brains, which would have multiple cell types including progenitors and differentiated neurons. This may account for the higher reprogramming efficiency seen in our study (0.13 ± 0.06%) when compared to another study using neural cells where the reprogramming efficiency was reported at 0.0002–0.0008% (9).

Interestingly, we have shown that ADCs can be used to generate iPS cell lines at a higher efficiency than both mEFs (38-fold increase) and NSCs (8-fold increase). The higher passage number of NSCs could potentially have contributed to the lower reprogramming efficiency seen compared with ADCs. However, NSCs consistently yielded a higher reprogramming efficiency than mEFs, suggesting that the late passage of NSCs did not affect its reprogramming ability. Also, it was not possible to obtain sufficient NSC numbers for experiments at early passage as the derivation efficiency of neurospheres from adult SVZ is low and primary neurosphere cultures contain many cellular aggregates and debris that can only be eliminated after serial passaging (7). As it is also not possible to maintain mEFs beyond passage 6–8, comparisons could not be made across all somatic cell types at the same passage number.

Nonetheless, the reprogramming efficiency of ADCs was found to be 1.14 ± 0.77%, which is significantly higher than the mEF standard (0.03 ± 0.005%) used in our study and also in other studies 0.01–0.5% (22, 40). An explanation for the high reprogramming efficiency of ADCs could be the low-level expression of key pluripotency genes, Oct4, Nanog, and Rex-1. Furthermore, ADCs also share some surface markers with multipotent mesenchymal stem cells (CD44 and Sca-1) and show a similar phenotype of surface markers to the adipose stem cell side population (Sca1⁺, CD90⁺, CD117^-, CD45^-) reported recently, which has the potential to differentiate into osteogenic, adipocytic, chrondrogenic, and even hepatic cells (5, 25). Because the reprogramming process is believed to be the result of several stochastic events, it has been argued that the differentiation state of somatic cells may influence their ability to be reprogrammed (12). As ADCs are heterogenous in nature, low-level expression of some pluripotency markers coupled with their multipotent potential may result in a subpopulation of ADCs being more primed for reprogramming.

We believe that ADCs represent a more clinically relevant somatic cell type as fat tissue can be easily accessed, ADCs can be grown easily and rapidly in culture, with 100 ml of human adipose tissue reported to yield 1 million stem cells (20). Furthermore, expression of Oct4, Nanog, and Rex-1 has been reported for human adipose-derived stem cells and, therefore, the high reprogramming efficiency of mouse ADCs may also be true for human ADCs (44). While preparing this manuscript, Sun et al. showed that human iPS cells could be generated from primary human adipose stem cells at a faster rate and higher efficiency than the nonrelated IMR90 human fibroblast cell line, derived from fetal lung tissue (30). Our results confirm their finding using direct comparison of three cell types from genetically identical mice and demonstrate that the murine Oct4-GFP transgene reporter system is a useful determinant of a somatic cell's reprogramming potential.

Questions still remain about whether iPS cell lines derived from different somatic cells have a tendency to differentiate towards their original lineage of origin, or whether certain lines be more easily differentiated into specific cell types rather than others. There is also the issue of whether certain cell lines will have a greater resistance to differentiation and therefore be more susceptible to tumor formation. The generation of iPS cell lines from various somatic cell types would help answer these questions and bring us a step closer to clinical applications.

In summary, we have directly compared the efficiency of iPS cell generation between primary NSCs, ADCs, and mEFs derived from adult mice of the same genotype and have shown that both a defined population of NSCs and ADCs can be used to generate mouse iPS cell lines more efficiently than mEFs. ADCs showed the highest efficiency with an 8- and 38-fold greater efficiency than NSCs and mEFs, respectively. Using ADCs for generating iPS cells has many advantages over NSCs and mEFs. Firstly, the derivation of ADCs is relatively simple and does not require specialist anatomical knowledge that is pertinent for SVZ dissections. Secondly, they can be cultured for longer periods (more than 20 passages) than mEFs, which senescence at around passage 5. ADCs also proliferate more rapidly and sufficient cell numbers can be obtained 1 passage after initial isolation. ADCs can also be isolated from aged mice (>8 weeks) without loss of proliferative ability. These advantages, together with our findings of high efficiency reprogrammability, suggest that ADCs are an ideal somatic cell source for generating iPS cells and may decrease the time needed to establish iPS cell lines.

Footnotes

Acknowledgments

We thank Daniele Pralong for critical reading of the manuscript and Mary White, Maria Calderia, Loic Delerollye (Queensland Brain Institute, University of Queensland), and Brent Reynolds (McKnight Brain Centre, University of Florida) for guidance in primary neurosphere isolation and culture protocols. We also thank Andrew Bo-quest for help and advice in isolating primary adipose-derived cells. P.A.T. was the recipient of an Australian Stem Cell Centre, Post Graduate Bursary. The work was part funded by an Australian Stem Cell Centre Collaborative Stream Program grant and supported by the Victorian Government's Operational Infrastructure Support Program.

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The Efficient Generation of Induced Pluripotent Stem (iPS) Cells from Adult Mouse Adipose Tissue-Derived and Neural Stem Cells

Abstract

Keywords

Introduction

Materials and Methods

Tissue Culture

Induced Pluripotency

Flow Cytometry

Determination of GFP Expression

Statistical Analysis

PCR

Immunostaining

Bisulfite Analysis

In Vitro Differentiation via Embryoid Body Formation

In Vivo Differentiation via Teratoma Formation

Results

Characterization of NSCs and ADCs

Generation of iPS Cell Lines

Characterization of iPS Cells

Cell Cycle Analysis

Protein and Gene Expression

Methylation Analysis

Pluripotency Potential

Discussion

Footnotes

Acknowledgments

References