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Keywords:

  • ES cell;
  • cell-free extract;
  • somatic cell;
  • reprogramming;
  • pluripotent genes

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Reprogramming of somatic cells was induced by ES cell-free extract. The system relied on the transient uptake of regulatory components from a nuclear and cytoplasmic extract derived from ES cells by the nucleus of a reversibly permeabilized NIH3T3 cell. NIH3T3 cells were permeabilized by streptolysin O (SLO). Reprogramming cell-free extracts were prepared by repeatedly freezing and thawing ES cells in liquid nitrogen. After incubation in the extract for 1 hr, permeabilized NIH3T3 cells were resealed by CaCl2 and continually cultured for weeks to assess expression of ES cell specific markers. As we observed using FACS and fluorescence microscope, the optimal SLO concentration for permeabilizing NIH3T3 cells was 25 U. After 2 weeks of culture, the treated NIH3T3 cells began to express Nanog, c-Myc, Klf4, and 6 weeks later Oct4 was detectable. However, Sox2 was detected only after 8 weeks of culture. Differentiated somatic cells could be reprogrammed in ES extract in vitro, which provides a new approach to decreasing differentiation levels in somatic cells without disturbing the DNA sequences. Anat Rec, 292:1229–1234, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Recently, it was reported that overexpression of defined transcription factors can induce fibroblasts to become pluripotent stem cells (named induced pluripotent stem (iPS) cells. Four factors, Oct4, Sox2, Klf4, and c-Myc, were able to induce pluripotency in mouse embryonic and adult fibroblast cells (Takahashi K, 2006). To induce pluripotency, combinations of defined transcription factors expressed in ES cells were cotransduced in the target fibroblasts using retroviral vectors that each carried one transgene. This procedure was used in human fibroblast cells to obtain human iPS cells (Yu, 2007; Takahashi K, 2007). As reactivation of the c-Myc retrovirus increases tumorigenicity in chimeras and progeny mice, a modified protocol for the generation of iPS cells that does not require the c-Myc retrovirus has been reported (Nakagawa, et al.2008). Recently, a micromolecular substance, BIX-01294, was used as a substitute for Sox2 and c-Myc (Shi, et al.,2008). The current protocols for generating iPS cells seem simpler for clinical application, but the necessary use of lentivirus involves random genomic integration and risk of insertional mutagenesis.

The epigenetic state of a somatic nucleus can be heritably reprogrammed to become a pluripotential stem cell using hybridization of the two cell types. Following fusion with an undifferentiated ES cell, a differentiated somatic cell nucleus can be epigenetically reprogrammed (Tada et al.,2001; Cowan et al.,2005). As shown in this system, the hybrids display morphology, growth rate, and surface antigen expression patterns characteristic of ES cells. Whole-genome gene expression changes, allele specific gene expression, and DNA demethylation in the Oct4 locus provides evidence for near-complete reprogramming of the fibroblast genome. The occurrence of epigenetic changes in somatic cell-ES cell hybrids indicates that fusion was likely to provide a valuable system for investigating the mechanisms underlying nuclear reprogramming. However, a major hurdle remains. Although the somatic genome is reprogrammed, the tetraploid state of the reprogrammed cell makes epigenetic analyses of the reprogrammed somatic genome challenging.

In recent years, nuclear and cytoplasmic extracts from several cell types have been shown to elicit changes in cell fate. The procedure involves reversible permeabilization of a somatic cell, exposure of the permeabilized cells to the reprogramming extract, and resealing of the cells. Using this approach, extracts of regenerated newt limbs have been shown to induce some dedifferentiation of cultured C2C12-derived mouse myotubes into myoblasts capable of replicating DNA (McGann et al.,2001). Extracts of Xenopus eggs can also induce the expression of pluripotency gene Oct4 and Sox2 in mammalian somatic cells (Miyamoto et al.,2007). Transient exposure of reversibly permeabilized fibroblasts to an extract of undifferentiated mouse ES cells results in Oct4 biphasic activation, with the first transient rise of Oct4 upregulation being necessary for the second longterm activation of Oct4 (Taranger et al.,2005). However, expression and precise timing of other genes involved in cell-free extract induced reprogramming have not been addressed.

Here, transient exposure of reversibly permeabilized NIH3T3 cells to extracts of mouse ES cells induced partial reprogramming of NIH3T3 cell nucleus. We found that pluripotential genes were expressed in a time order. When compared with cell fusion or iPS, our method does not introduce ES cell chromosomes or virus into somatic cells. Therefore, reprogrammed somatic cells avoided chromosome aploid or mutagenesis. The method provides a new approach to studying mechanisms of reprogramming and may be useful for therapeutic purposes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Cell Lines and Reagents

All reagents were purchased from Sigma Chemical Company unless stated otherwise. Mouse ES cell line, GD3, and mouse fibroblast cell line, NIH3T3, purchased from ATCC Company.

Seeding Cells

NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn bovine serum (NBS). Cells were seeded to reach a confluence of 70% on the day of reprogramming. GD3 cells were cultured on mitomycin-treated mouse embryonic fibroblast feeder layers in ES cell medium (DMEM, 15% fetal bovine serum FBS, 1% nonessential amino acids, 0.1% β-mercaptoethanol, 100 U/L penicillin, and 100 U/L streptomycin) supplemented with 1,000 U/mL leukemia inhibitory factor (LIF). Before harvesting, GD3 cells were passaged and cultured on gelatin-coated plates in order to eliminate feeders. After treatment with ES cell extract, NIH3T3 cells were cultured in ES cell medium without feeder layers.

Cell Extract Preparation

To prepare GD3 and NIH3T3 extracts, cells were washed in DPBS and centrifuged at 1000 rpm for 5 min at 4°C. Cell pellet volume was estimated, and cells were resuspended into the same volume of ice-cold DPBS containing 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM protease inhibitor cocktail. Sedimented cells were resuspended in cold DPBS and incubated for 3 h on ice. Cells were repeatedly frozen and thawed in liquid nitrogen. The lysate was sedimented at 25,000 rpm for 15 min at 4°C, and the supernatant was collected in a new tube and frozen in liquid nitrogen.

Cell Permeabilization

NIH3T3 cells were washed in cold Ca2+, Mg2+-free phosphate buffered saline (DPBS) three times. Approximately 500,000 cells were resuspended in 500 μL DPBS, and centrifuged at 1,000 rpm for 5 min at 4°C in a swing-out rotor. Sedimented cells were suspended in cold DPBS, tubes were warmed for 2 min at 37°C, and SLO was added to a final concentration of 10 U, 25 U, and 50 U. Cells were incubated in a water bath for 50 min at 37°C with occasional agitation. 500 μL cold DPBS was added to dilute the samples, and cells were sedimented at 1,000 rpm for 5 min at 4°C. Permeabilization was assessed by fluorescent dye uptake under fluorescence microscope or by FACS. Uptake of 10,000-Mr dextran Oregon green 488 (50 μg/mL; Invitrogen) and PI were monitored 2 hr after cell membrane resealing in 2 mM CaCl2.

In Vitro Reprogramming of NIH3T3 Cells

After permeabilization, cells were suspended at 1,000 cells/μL in 100 μL NIH3T3 (control) or ES extract which contained an ATP-regenerating system (1 mM ATP, 10 mM creatine phosphate, 25 μg/mL creatine kinase, 100 μM GTP), and 1 mM of each nucleotide triphosphate (NTP; Roche Diagnostics, Mannheim, Germany). The tube containing cells was incubated for 1 hr at 37°C with occasional agitation. To reseal plasma membranes, cells were cultured with medium containing 2 mM CaCl2 for 24 hr, and cells were seeded at 100,000 cells per well on a 48-well plate. Reprogrammed NIH3T3 cells were continually cultured in ES medium for further study.

RT-PCR

Total RNA was extracted from cells with Trizol reagent (Invitrogen) according to the manufacturer's protocol. 1 μg RNA was loaded to reverse transcribe cDNA and amplified by the RT-PCR system (Promega). The primer sequences and amplified fragment lengths are listed in Table 1.

Table 1. Primer sequence and annealing temperature
GenePrimer sequenceAmplified fragment length/bpAnnealing temperature/°C
Oct4sense5′-CTGTAGGGAGGGCTTCGGGCACTT-3′21658
anti5′-CTGAGGGCCAGGCAGGAGCACGAG-3′  
NanogSense5′-ATGAGTGTGGGTCTTCCTGGTCC-3′91862
anti5′-GACTCCACCAGGTGAAATATGA-3′  
Sox2sense5′-GGCAGCTACAGCATGATGCAGGAGC-3′12555
anti5′-CTGGTCATGGAGTTGTACTGCAGG-3′  
KIf-4sense 5′-TGCCAGACCAGATGCAGTCAC-3′23355
anti5′-GTAGTGCCTGGTCAGTTCATC-3′  
c-Mycsense 5′-CCCAGCGAGGACATCTGGAAGAA-3′21355
anti5′-GAGAAGCCGCTCCACATGCAGTC-3′  
LaminAsense5′-CTCCTCCCACTCATCCCA-3′46955
Anti5′-CCTCGTCGTCATCCTCATT-3′  
GAPDHsense5′-TGAAGGTCGGTGAACGGAT-3′98065
anti5′-CAGGGGGGCTAAGCAGTTGGT-3′  

Immunofluorescence

Cells were seeded onto cover slips, fixed with 4% paraformaldehyde for 20 min at 4°C, washed with PBS containing 0.1% Triton X-100 for 45 min at room temperature, and blocked in PBS supplemented with 10% goat serum for 30 min. After washing with PBS, the cells were incubated at room temperature for 1 hr with the anti-Oct4 monoclonal antibody (1:200, Santa Cruz Biotechnology) or anti-LaminA monoclonal antibody (1:100, Santa Cruz Biotechnology). The cells were incubated with anti-mouse IgG secondary antibody (1:200, Santa Cruz Biotechnology) for 1 hr. Nuclei of the cells were stained with Hoechst 33342 (1:20, Santa Cruz Biotechnology).

Data Analysis

Two-sample t-tests were used to compare the protein level of Oct4 and LaminA and analysis of variance (ANOVA) and q-test were used to make multiple comparison.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Cell-Free Extract

We tried three different methods to prepare the cell extracts. Cells were disrupted by a sonicator, sonicator with lysis buffer, or liquid nitrogen. Protein concentration and osmotic pressure of the extracts were measured. We found that the optimal method was repeated freezing and thawing of the cells in liquid nitrogen. Using this method, we obtained extracts with a high protein concentration (∼46.1 mg/mL) and comparable osmotic pressure (∼279 mmol/kg) (Table 2).

Table 2. Comparison of different methods for ES cell extract preparation
MethodProtein concentration (mg/mL)Osmotic pressure (mmol/kg)
  1. Note: Data are mean values from seven independent experiments.

Sonicator2.949.5
Sonicator with cell lysis buffer3.255
Freezing and thawing in LN246.1279

Permeabilization of Somatic Cells

Here, 10 mM DTT was determined as the most suitable reagent to activate SLO. The result as determined by fluorescence microscope and FACS was that the higher the concentration of SLO the more dead cells were observed. Immediately after permeabilization, fluorescence microscope observation showed that 25 U SLO could successfully permeabilize the NIH3T3 cells (44.58%) (Fig. 1A–D). However, FACS results indicated that 10 U SLO had a better effect on permeabilization (38.32%) than that of other concentrations (Fig. 1E–I). Considering both fluorescent microscope and FACS results, we selected 25 U SLO to permeabilize NIH3T3 cells.

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Figure 1. Optimal permeabilization concentration of SLO. (A–D) The result was observed by fluorescence microscope: A partly NIH3T3 cells were successfully permeabilized by SLO with a concentration of 25U, judging by the dextran Oregon green 488, 10,000 MW uptake. (C) The control of A. (B, D) Light microscope photographs of A and C, respectively. (E–I) The result was detected by FACS: UL the cells died a natural death, not by SLO. UR the cells died because of SLO. LR the cells were successfully permeabilized by SLO. (E) NIH3T3 cells were permeabilized by SLO with a concentration of 10U. (F) SLO with a concentration of 25U. (G) SLO with a concentration of 50U. (H) SLO with a concentration of 0U as a control. (I) More cells were dead when treated with a higher concentration of SLO. (Bar = 50 μm).

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ES Cell Extract Partly Reprogrammed NIH3T3 Cells and Induced Expression of Pluripotent Factors

Some transcription factors, such as Oct4, Sox2, and Nanog are expressed in early stage embryos and ES cells to maintain pluripotency. Some tumor correlated genes, for instance, stat3, E-ras, c-Myc, and Klf4, could maintain the phenotype and increment characteristics of ES cells longterm. Two independent experiments revealed that 2 weeks after treatment with ES cell extract, NIH3T3 cells (named N-ES cells) started to express Nanog, c-Myc, and Klf4 genes. Oct4 was detectable after 6 weeks of culture. Moreover, after 8 weeks of culture, there was no significant difference in the expression of c-Myc and Klf4 between N-ES cells and ES cells. The expression of Sox2, Oct4, and Nanog were exceptions, and were less expressed in N-ES cells at 8 weeks of culture than that in ES cells. LaminA, a marker for differentiated cells in mammals, showed no visible differences in expression in NIH3T3 cells, NIH3T3 cells treated with extracts of NIH3T3 cells (N-N cells) and N-ES cells 2–8 weeks after extract treatment. However, in ES cells it was lower than in the others (Fig. 2).

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Figure 2. RT-PCR analysis of pluripotent gene expression in N-ES cells. (A) After treatment with ES cell extracts, Nanog, c-Myc, and Klf4 were detected after 2 weeks of culture, and Oct4 was detected after 6–8 weeks of culture in N-ES cells. After 8 weeks, there was no significant difference in the expression of c-Myc and Klf4 between N-ES cells and ES cells. The expression of Sox2 in N-ES cells was observed after 8 weeks of culture and Sox2, Oct4, Nanog were less expressed in N-ES cells at 8 weeks of culture than that in ES cells. There were no visible differences in the expression of LaminA in NIH3T3 cells, N-N cells, N-ES cells between 2–8 weeks after culture, but in ES it was lower than the others. (B) Gray scale value (mean ± SD) of Nanog, c-Myc, Klf4, Sox2, Oct4, Nanog, and LaminA in NIH3T3 cells, N-N cells, N-ES cells, ES cells. *P < 0.05 when compared with NIH3T3 cells (ANOVA, q test); **P < 0.05 when compared with N-ES 8W cells (ANOVA, q test).

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At 8 weeks after treatment, pluripotent marker OCT4 and differentiated marker LAMINA were detected in N-ES cells by immunochemistry. The results revealed that some N-ES cells were OCT4 positive, meanwhile some cells still expressed LAMINA constantly (Fig. 3). These results were confirmed by RT-PCR.

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Figure 3. Immunofluorescence analysis of pluripotent genes in N-ES cells. (A) 8 weeks after culture, some of the N-ES cells expressed OCT4, while some still expressed LAMINA. In N-ES cells, reprogramming of nuclear function had partly taken place in vitro. ES cells just expressed OCT4, and NIH3T3 cells just expressed LAMINA. (Bar in ES cell is 50 μm, and in NIH3T3 cells is 30 μm). (B) Proportions (mean ± SD) of NIH3T3 cells, N-ES cells, and ES cells expressing OCT4, LAMINA. Three sets of 200 cells were examined for each marker. *P < 0.05 when compared with NIH3T3 cells (t test), **P < 0.05 when compared with N-ES cells (t test), ***P < 0.05 when compared with NIH3T3 cells (t test).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Cell-Free Extract Preparation

The most optimal method for preparing the cell-free extracts was repeated freezing and thawing of cells in liquid nitrogen. Using this method, we obtained ES cell-free extract with a protein concentration of 46.1 mg/mL, and osmotic pressure close to physiological conditions. In other studies, high-speed centrifugation and sonication were used to prepare cell extracts from Xenopus eggs (Miyamoto et al.,2007), mitotic bovine kidney cells (Eddie et al., 2004), and cadiocytes (Gaustad et al.,2004). These methods obtained similar protein concentration and osmotic pressure in cell-free extracts. However, the freezing and thawing of cells in liquid nitrogen is much easier to perform and requires less equipment.

Permeabilization of Somatic Cells

Although some cell types, such as adipose tissue stem cells, readily endocytose (Gaustad et al.,2004), most cell types require efficient permeabilization for components from the extract to be taken up. Permeabilization should be reversible as cells are expected to be cultured after exposure to the extract. Permeabilization could be accomplished with SLO, a cholesterol-binding cytolysin, forming pores of about 30 nm in the surface membrane (Walev et al.,2002). This permeabilization could be reversed by subsequent incubation with fetal bovine serum or Ca2+. Although the result obtained by fluorescence microscope and FACS was not concordant, we decided to use 25 U SLO to permeabilize NIH3T3 cells. The reason was that in FACS, we found some cells which were successfully permeabilized with 25 U SLO but not fully recovered after resealing for a short time. These cells were stained by PI and show up as dead cells but were able to survive after overnight culture. The sensitivity to SLO appears to vary in many species and cell types because of the different cholesterol content of the cell membrane (Bhakdi et al.,1996). It was recommended to test a range of SLO concentrations varying between 3.57–35.7 U/mL (100–1,000 ng/mL) (Collas, www.collaslab.com) on any given cell type. In our study, the SLO concentration used to permeabilize NIH3T3 cells was 25 U/mL, which was in the recommended range.

Pluripotent Factors Expressed in a Particular Order in NIH3T3 Cells After Reprogramming by ES Cell-Free Extract

RT-PCR results showed that Nanog, c-Myc, and Klf4 expressed earlier than Oct4 and Sox2 in N-ES cells. The precise mechanism for this expression order needs further study. In the mammalian genome, there may be up to 25,000 c-Myc binding sites (Cawley et al.,2004), and C-MYC protein may induce global histone acetylation (Fernandez et al.,2003), thus allowing Oct4 and Sox2 to bind to their specific target loci. Klf4 has been shown to repress p53 directly (Rowland et al.,2005), and P53 protein has been explored as a suppressor of Nanog during ES cell differentiation (Lin et al.,2004). Therefore, Klf4 might contribute to the activation of Nanog and other ES cell-specific genes through p53 repression (Takahashi and Yamaka,2006). This might partly explain why Nanog, c-Myc, and Klf4 express earlier than Oct4 and Sox2 in N-ES cells.

Oct4 and Sox2 both showed up late in N-ES cell development, but were not expressed simultaneously. Oct4 expression started after 6 weeks of reprogramming while Sox2 reactivated 2 weeks later. Both transcription factors have been shown to cooperate in the transcriptional activation of target genes in maintaining pluripotent state. However, Oct4 is expressed in the inner cell mass (ICM), epiblast, and later in germ cells (Pesce and Scholer,2000). It is essential for establishing and maintaining pluripotency of the ICM. Sox2 also marks the pluripotent lineage of the early mouse embryo, but more specifically in the multipotential cells of the extraembryonic ectoderm (ExE). It is possible that Sox2 and Oct4 function together to maintain a pluripotent state throughout early development, but Oct4 functions earlier than Sox2 (Avilion et al.,2003). This finding is consistent with our results that Oct4 is expressed earlier than Sox2 in N-ES cells, and Oct4 might be a prerequisite for Sox2 expression.

Collas et al. reported that ES cell extract induced a biphasic wave of Oct4 transcription and translation in fibroblast cells. In their study, Oct4 was first detected as early as 1 hr after recovery from ES cell extract treatment, the level peaked at 24 hr, and then Oct4 expression decreased and was barely detected at 48 hr. However, by 72 hr, a second wave of Oct4, similar or even higher than the first wave, showed up and lasted for 120 hr (Taranger, 2005). We did not check Oct4 transcription soon after treatment, because we presumed the early Oct4 expression in somatic cells might have derived from the ES extract. We started Oct4 detection from the 2nd week, and found that Oct4 was detectable 6 weeks after treatment. This late expression suggests Oct4 reactivation in reprogrammed NIH3T3 cells. Whether Oct4 expression persists at a stable level or varies at different time points needs further study.

There were no visible differences in the expression of LaminA in N-ES cells between 2–8 weeks after extract treatment, but LaminA was lower in ES cells. The reason might be that after treatment with ES cell extract, not all NIH3T3 cells were reprogrammed, and some nonreprogrammed cells remained, which contributed to LaminA expression after PCR amplification. In ES cells, there was still weak LaminA expression, which might have derived from a few feeder layer cells (MEF) mixing in ES cells.

Extracts of nondifferentiated cells contain regulatory factors that induce pluripotency of differentiated cells. Sox2 was detected in permeabilized and nonpermeabilized pig fibroblasts after treatment with Xenopus egg extracts, but Nanog was not detected (Miyamoto et al.,2007). These results suggest that the nuclei of pig somatic cells could be partially reprogrammed by Xenopus egg extracts but not to a pluripotent state as ES cells. So, there are variations in the extent of transcriptional reprogramming which might reflect incomplete reprogramming or progressive deregulation of the new program. Functional reprogramming in vivo also remains to be demonstrated. Ways to enhance stability of extract-induced gene expression may include repeated extract exposure, destabilization of the epigenome with chromatin modifying agents, or induction of a dedifferentiation step before triggering a new target cell-specific program.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The authors are grateful to Xi-Mei Zhang and Qiu-Ming Li at Harbin Medical University for their valuable discussions.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
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