Published online before print May 15, 2008, doi: 10.1161/CIRCRESAHA.108.176115
© 2008 American Heart Association, Inc.
UltraRapid Communication |
Cell-Free Embryonic Stem Cell Extract–Mediated Derivation of Multipotent Stem Cells From NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair
From the Feinberg Cardiovascular Research Institute (J.R., E.L., H.H., E.B., T.T., I.G., P.K., D.A., G.Q., D.W.L., R.K.), Feinberg School of Medicine, Northwestern University, Chicago, Ill; and Division of Neurology Research (K.M.R.) and Cardiovascular Research (Y.Z.), Caritas St. Elizabeth’s Medical Center. Tufts University School of Medicine, Boston, Mass.
Correspondence to Raj Kishore, PhD, Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, 303 E Chicago Ave, Chicago IL 60611. E-mail r-kishore{at}northwestern.edu
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Abstract |
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The oocyte-independent source for the generation of pluripotent stem cells is among the ultimate goals in regenerative medicine. We report that on exposure to mouse embryonic stem cell (mESC) extracts, reversibly permeabilized NIH3T3 cells undergo dedifferentiation followed by stimulus-induced redifferentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 control and ESC extract–treated NIH3T3 cells including the reactivation of ESC-specific transcripts. Epigenetically, ESC extracts induced CpG demethylation of Oct4 promoter, hyperacetylation of histones 3 and 4, and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically induced hindlimb ischemia or acute myocardial infarction transplantation of reprogrammed NIH3T3 cells significantly improved postinjury physiological functions and showed anatomic evidence of engraftment and transdifferentiation into skeletal muscle, endothelial cell, and cardiomyocytes. These data provide evidence for the generation of functional multipotent stem-like cells from terminally differentiated somatic cells without the introduction of retroviral mediated transgenes or ESC fusion.
Key Words: Somatic Cell Dedifferentiation • ES cells • Nuclear reprogramming • Epigenetics • Tissue Repair • Myocardial infarction
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Introduction |
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The available evidence demonstrating improvement in myocardial function following transplantation of autologous bone marrow–derived stem/progenitor cells, both in preclinical and in available clinical trials, remains a potent force driving discovery and clinical development simultaneously and has provided new hope for patients with debilitating heart diseases.1,2 However, certain potential limitations of autologous bone marrow– or peripheral blood–derived stem/progenitor cells have also been identified. Risk factors for coronary artery disease are reported to be associated with a reduced number and impaired functional activity of endothelial progenitor cells in the peripheral blood of patients.3–6
Dedifferentiation of adult somatic cells into multipotent cells may provide an attractive source of stem cells for regenerative medicine, including postinfarct cardiac and other ischemic tissue repair. Recent experimental evidence has revealed that nuclear reprogramming of terminally differentiated adult mammalian cells leading to their dedifferentiation is possible.7–10 Recently, 2 breakthrough studies have reported the generation of embryonic stem cells (ESCs) from terminally differentiated human fibroblasts by the retroviral transduction of defined ESC-specific transcription factors.11,12 The best example of somatic cell reprogramming to totipotent stage comes from reproductive and therapeutic cloning experiments using somatic nuclear transfer (SNT), wherein transplantation of somatic nuclei into enucleated oocyte cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression, new pathways of cell differentiation, and successful generation of ESCs and birth of cloned animals.13–17 Therapeutic cloning by SNT for clinical application, although conceptually attractive, is, to date, not practical, given the technical difficulties, extremely low efficiency, oocyte dependence, ethical and legal concerns, and prohibitive cost associated with the process. It, therefore, becomes imperative to develop alternative strategies for somatic cell reprogramming. One strategy would be to develop oocyte-independent systems, for instance, exposure of somatic cell nuclei to ESC-derived cell-free factors/proteins to drive somatic cell dedifferentiation and nuclear reprogramming. Indeed, alterations in the fate of 1 kind of differentiated somatic cells by cell-free extracts from another, leading to the acquirement of donor cell characteristics and functions by recipient cells, has been previously reported.18–24
In the present study, we report that exposure to mouse ESC (mESC) extracts induces marked epigenetic reprogramming in NIH3T3 fibroblasts including reactivation of ESC-specific gene expression. These reprogrammed cells possess multipotent stem cell–like characteristics including multilineage differentiation potential and, more importantly, therapeutic efficacy for improvement in physiological functions and anatomic tissue repair in mouse models of surgically induced hindlimb ischemia and acute myocardial infarction (AMI).
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Materials and Methods |
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Cell Culture
NIH3T3 Swiss albino fibroblasts (American Type Culture Collection) were cultured in DMEM (Sigma-Aldrich) with 10% FCS, L-glutamine, and 0.1 mmol/L β-mercaptoethanol. The D3 mouse mESCs were obtained from American Type Culture Collection and cultured as described earlier.25
Cell Extracts
The mESC and 3T3 cell extracts were prepared as described previously.24 Briefly, the cells were washed in PBS and in cell lysis buffer (100 mmol/L HEPES, pH 8.2, 50 mmol/L NaCl, 5 mmol/L MgCl2, 1 mmol/L dithiothreitol, and protease inhibitors), sedimented at 10 000 rpm, resuspended in 1 volume of cold cell lysis buffer, and incubated for 30 to 45 minutes on ice. Cells were sonicated on ice in 200-µL aliquots using a sonicator fitted with a 3-mm-diameter probe until all cells, and nuclei were lysed, as judged by microscopy. The lysate was sedimented at 12 000 rpm for 15 minutes at 4°C to pellet the coarse material. The supernatant was aliquoted, frozen in liquid nitrogen, and stored at –80°C. Protein concentration of the mESC and NIH3T3 cell extracts were determined by Bradford assay.
Streptolysin O–Mediated Permeabilization and Cell Extract Treatment
NIH3T3 cells were washed in cold PBS and in cold Ca2+- and Mg2+-free Hank’s balanced salt solution (HBSS) (Invitrogen, Carlsbad, Calif). Cells were resuspended in aliquots of 100 000 cells/100 µL of HBSS or multiples thereof; placed in 1.5-mL tubes; and centrifuged at 1500 rpm for 5 minutes at 4°C. After sedimentation, cells were suspended in 97.7 µL of cold HBSS, tubes were placed in a H2O bath at 37°C for 2 minutes, and 2.3 µL of streptolysin O (SLO) (Sigma-Aldrich) (100 µg/mL stock diluted 1:10 in cold HBSS) was added to a final SLO concentration of 230 ng/mL. Samples were incubated horizontally in a H2O bath for 50 minutes at 37°C with occasional agitation and set on ice. Samples were diluted with 200 µL of cold HBSS, and cells were collected by sedimentation at 1500 rpm for 5 minutes at 4°C. Permeabilization efficiency of >80% was obtained, as assessed by monitoring uptake of a Mr 70 000 Texas red–conjugated dextran (50 µg/mL; Invitrogen). After permeabilization, NIH3T3 cells were suspended at 2000 cells/µL in 100 µL of mESC extract or control NIH3T3 cells extract containing an ATP-regenerating system (1 mmol/L ATP, 10 mmol/L creatine phosphate, and 25 µg/mL creatine kinase; Sigma-Aldrich), 100 µmol/L GTP (Sigma-Aldrich), and 1 mmol/L each nucleotide triphosphate (NTP) (Roche Diagnostics, Indianapolis, Ind). The cells were incubated for 1 hour at 37°C in a H2O bath with occasional agitation. To reseal plasma membranes, the cell suspension was diluted with complete DMEM medium containing 2 mmol/L CaCl2, antibiotics and cells were seeded at 100 000 cells per well on a 48-well plate. After 2 hours, floating cells were removed, and cells were cultured in D3 maintenance medium.
Determination of Dedifferentiation
Dedifferentiation of NIH3T3 following D3 cell extract treatment (referred to as 3T3/D3) was determined by the induction of ESC markers both at mRNA level by quantitative real-time PCR and at protein level by immunostaining. Total cellular RNA was harvested at various time points, and the quantitative real-time RT-PCR was performed to determine mRNA expression of selected ESC markers in self extract (referred to as 3T3/3T3) and 3T3/D3 cells, as described previously.25,26 Relative mRNA expression of target genes was normalized to the endogenous 18S control gene (Applied Biosystems). Induction of ESC specific mRNAs was further corroborated by immunofluorescence protein staining of induced specific stem cell markers in 3T3/D3 cells. For immunostaining, 3T3/3T3 and 3T3/D3 cells were cultured in medium in the absence and presence of leukemia inhibitory factor (LIF) for 10 days. Then, the cells were harvested and cultured 1x104 cells per well in 4-well slides coated with 0.5% gelatin for another 2 days. The slides were stained with specific antibodies to stem cell markers, c-Kit, SSEA1 and Oct-4. Dedifferentiation was also determined by the lamin B and lamin A/C (markers of soma) protein expression.
Cardiomyocyte and Endothelial Cell Lineage Differentiation of Reprogrammed Cells
To determine the redifferentiation potency of dedifferentiated 3T3/D3 cells into cardiomyocytes (CMCs), cells were cultured in complete DMEM containing 5 ng/mL LIF and 3 ng/mL bone morphogenetic protein 2 in 6-well culture plates (1x106 cells per well) and 4-well chamber slides (1x104 cells per well) coated with 0.5% gelatin for 7 days.25 Total cellular RNA was harvested from 6-well culture plate and used to analyze quantitative mRNA expression of the CMC-specific markers cardiac troponin (CT)I and CTT, connexin 43, GATA4, Mef2c, Nkx2.5, and Tbx5. The expression was normalized to that of 18S RNA. The protein expression of was determined by immunochemical staining. For endothelial cell (EC) lineage differentiation, cells were cultured in endothelial differentiation medium (10% FBS/EBM-2; Clonetics) medium containing supplements (SingleQuot Kit; Clonetics) for 7 days. mRNA expression for the EC markers CD31 and Flk1 was determined by real-time PCR and by incubation with DiI-acetylated LDL (DiI-acLDL) (Biomedical Technologies) for 1 hour, followed by isolectin B4 staining. The dual-stained cells were considered ECs.
Induction of Neuronal and Adipogenic Differentiation
The neuronal differentiation was performed as described earlier.27 Briefly, cells were seeded in complete DMEM medium at 5x105 cells per 90-mm sterile culture dish. Suspension cultures were maintained for 24 hours before adding 10 µmol/L all-trans-retinoic acid (Sigma-Aldrich). Cells were cultured for 3 weeks in retinoic acid, replacing the medium every 2 to 3 days. Subsequently, cell aggregates were washed in complete DMEM medium and plated onto poly-L-lysine–coated (10 µg/mL; Sigma-Aldrich) plates in complete DMEM medium containing the mitotic inhibitors fluorodeoxyuridine (10 µmol/L; Sigma-Aldrich), cytosine arabinoside (1 µmol/L; Sigma-Aldrich), and uridine (10 µmol/L; Sigma-Aldrich). The culture dishes were stained for the neuronal markers nestin and β-tubulin-III. The adipogenic differentiation was performed as described elsewhere.28 Briefly, the cells were cultured for 21 days in complete DMEM/Ham’s F-12 medium containing dexamethasone, insulin, and indomethacin. Cells were fixed with 4% paraformaldehyde, washed in 5% isopropanol, and stained for 15 minutes with oil red O (Sigma Aldrich).
Immunochemical Staining
For immunochemical staining, cells under different culture conditions were cultured on 4-well slides for indicated time, rinsed once with PBS, and fixed with 4% paraformaldehyde (Sigma) in PBS for 30 minutes. The slides were again rinsed three times with PBS and then permeabilized with 0.3% of Triton X-100 (Sigma) in PBS for 5 minutes. After 2 washes with PBS, specific primary antibodies diluted in PBS containing 1% FBS were added and incubated overnight at 4°C. After 3 washes with PBS, the slides were incubated with respective secondary antibodies for 1 hour at 37°C. The excess secondary antibodies on the slides were rinsed off with PBS 3 times. Finally, to visualize nuclei, slides were stained with DAPI for 5 minutes, washed 3 times with PBS, allowed to dry for 5 minutes and then mounted on Vectashield mounting medium for fluorescence imaging. The photographs were taken in a Nikon TE200 Digital Imaging system.
Determination of Oct4 Promoter Methylation, Bisulfite Genomic Sequencing, and Chromatin Immunoprecipitation
Genomic DNA prepared from mESCs (D3) and 3T3/D3 and 3T3/3T3 cells was amplified for the Oct4 promoter and the PCR product was digested with HpyCH4IV restriction enzymes that only cleave at methylated CpG sites. The digested products were analyzed on agarose gels. For genomic bisulphite sequencing, genomic DNA from cells was digested with EcoR1 and was used for bisulphite treatment using an EZ DNA methylation Gold kit essentially following the instructions of the manufacturer. The treated DNA was ethanol-precipitated and resuspended in water and then amplified by PCR using mouse methylation–specific Oct4 primers. PCR products were digested with the HpyCH4IV (New England Biolabs) restriction enzyme. Because only unmethylated cytosine residues were changed to thymines by the sodium bisulphite reaction, PCR fragments from nonmethylated genomic DNA were resistant to HpyCH4IV, and those from methylated DNA were digested by the enzymes. The resultant products of restriction mapping were assessed by agarose gel electrophoresis. The remaining PCR products were purified through the Wizard DNA Clean-Up system (Promega, Madison, Wis) and were directly sequenced to determine the methylation status of all 10 CpG residues present in the amplified promoter region. Chromatin immunoprecipitation (ChIP) assays were performed essentially as described in our recent publication.29 Anti-acetyl H3, anti–acetyl H4, and anti–dimethyl K9H3 antibodies were purchased from Upstate Biotech and Santa Cruz Biotechnology.
Genome-Wide Expression Profiling and Gene Expression Analyses
Affymetrix mouse genome A2 GeneChips were used for hybridization. Using a poly(dT) primer with an incorporated T7 promoter, double-stranded cDNA was synthesized from 5 µg of total RNA using a double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, Calif). Double-stranded cDNA was purified with the Affymetrix sample cleanup module (Affymetrix, Santa Clara, Calif). Biotin-labeled cRNA was generated from the double-stranded cDNA template though in vitro transcription with T7 polymerase and a nucleotide mix containing biotinylated UTP (3'-Amplification Reagents for IVT Labeling Kit; Affymetrix). The biotinylated cRNA was purified using the Affymetrix sample cleanup module. For each sample, 15 µg of IVT product was digested with fragmentation buffer (Affymetrix) for 35 minutes at 94°C to an average size of 35 to 200 bases. Ten micrograms of the fragmented, biotinylated cRNA, along with hybridization controls (Affymetrix), were hybridized to a Mouse 430A 2.0 GeneChip for 16 hours at 45°C and 60 rpm. Arrays were washed and stained according to the standard Antibody Amplification for Eukaryotic Targets protocol (Affymetrix). The stained arrays were scanned at 532 nm using an Affymetrix GeneChip Scanner 3000.
During analysis and for quality control, GeneChip arrays were first inspected using a series of quality control steps. Present call rates were consistent across the arrays, ranging from 56% to 63%. The hybridization controls (BioB, BioC, Cre) were found to be present 100% of the time. Images of all arrays were examined, and no obvious scratches or spatial variation was observed. A visual inspection of the distribution of raw perfect match probe values for the 12 arrays showed no outlying arrays. Similarly, digestion curves describing trends in RNA degradation between the 5' end and the 3' end of each probe set were generated, and all 12 proved comparable. Probe sets with no present calls across the 12 arrays as well as Affymetrix control probe sets were excluded from further analyses. Raw intensity values for the remaining 17 213 probe sets were processed first by RMA (Robust Multi-Array Average) using the R package affy.26 Specifically, expression values were computed from raw CEL files by first applying the RMA model of probe-specific correction of perfect match probes. These corrected probe values were then normalized via quantile normalization, and a median polish was applied to compute one expression measure from all probe values. Resulting RMA expression values were log2-transformed (see the affy manual, available at www.bioconductor.org). Distributions of expression values processed via RMA of all arrays were very similar, with no apparent outlying arrays. Pearson correlation coefficients and Spearman rank coefficients were computed on the RMA expression values (log2-transformed) for each set of biological triplicates. Spearman coefficients ranged from 0.990 to 0.996; Pearson coefficients ranged between 0.991 and 0.997. Differential expression of genes was determined by 1-way ANOVA on the RMA expression values of each probe set, using the R package limma.30 A multiple testing adjustment31 was performed on the resulting statistics to adjust the false discovery rate. Differentially expressed probes with adjusted probability values <0.01 and a fold change of >2 (absolute log fold change of >1) were extracted for further inspection. Hierarchical clustering was obtained by using the Pearson correlation coefficient and an average agglomeration method, and the heat maps were generated using z-scored probes, in which z-scores (subtraction of mean and division by SD of normalized values) were computed for each probe across all arrays.
Green Fluorescent Protein Transduction and DiI Labeling of Cells for Transplantation
For tracking of transplanted cells in the AMI model, cells were transduced with a lentivirus–green fluorescent protein (GFP) construct.25 For tracking of transplanted cells in hindlimb ischemic tissues, the cells were labeled with DiI before the transplantation.27
Hindlimb Ischemia, Cell Transplantation, and Laser Doppler Imaging and Histology
All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. The hindlimb ischemia was established by the excision of femoral artery in the left hindlimb in 10 male 8-week- old FVB mice (The Jackson Laboratory, Bar Harbor, Me) essentially as described in our previous publication.29 The animals were grouped into 2 (n=15/group), each receiving either a total of DiI-labeled 3T3/3T3 cells or 3T3/D3 cells (1x105) at multiple sites into the ischemic muscle. Laser Doppler imaging to determine blood flow was performed immediately after surgery (day 0) and at days 7, 10, and 14 after cell injections. Fourteen days after cell transplantation, the tissues were harvested and assayed by histochemical/immunofluorescence staining for isolectin B4, CD31 (EC identity), desmin, 
-SMA (muscle), and DiI, followed by fluorescence microscopy. In some experiments, animals were perfused with fluorescein isothiocyanate (FITC)-labeled Bandeiraea simplicifolia (FITC-BS-1) lectin to identify capillaries before euthanasia and tissue retrieval.
Establishment of Acute Myocardial Infarction
The study involved 8-week-old male C57BL/6J mice (n=30; The Jackson Laboratories). Mice underwent surgery to induce AMI by ligation of the left anterior descending coronary artery, as described before.32,33 Animals were subdivided into 3 groups and received intramyocardial injection of 5x104 lentiviral-GFP transduced D3 extract–treated cells, 3T3 fibroblast control cells, and saline, respectively, in a total volume of 10 µL at 5 sites (basal anterior, mid-anterior, mid-lateral, apical anterior, and apical lateral) in the periinfarct area.
Physiological Assessments of Left Ventricular Function and Histology
Mice underwent echocardiography just before MI (base level) and 1, 2, and 4 weeks after AMI as described before.31,33 Briefly, transthoracic echocardiography was performed with a 6- to 15-MHz transducer (SONOS 5500, Hewlett Packard). Two-dimensional images were obtained in the parasternal long and short axis and apical 4-chamber views. M-mode images of the left ventricular short axis were taken just below the level of the midpapillary muscles. Left ventricular end-diastolic and end-systolic dimensions were measured and functional shorting was determined according to the modified guideline recommendations of the American Society of Echocardiography. A mean value of 3 measurements was determined for each sample. On day 28 post-AMI, mice were euthanized and the aortas were perfused with saline. The hearts were sliced into 4 transverse sections from apex to base and fixed with 4% paraformaldehyde, methanol, or frozen in OCT compound and sectioned into 5-µm thickness. Immunofluorescence staining was performed to determine CMC and EC differentiation of transplanted cells. For the measurement of fibrosis, tissues sections were frozen in OCT compound and sectioned for elastic tissue/trichrome to measure the average ratio of the external circumference of fibrosis area to left ventricular area.
Statistical Analyses
All experiments were carried out at least 3 times with similar results. Results are presented as means±SEM. Comparisons were performed by ANOVA (GB-STAT; Dynamic Microsystems Inc) or 
2 test for percentages. All tests were 2-sided, and a probability value of <0.05 was considered statistically significant.
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Exposure of NIH3T3 Fibroblasts to mESC Extracts Leads to the Induction of ESC-Specific Genes
Reversibly permeabilized (by streptolysin O) NIH3T3 cells were exposed to either whole cell extracts from NIH3T3 cells ("self" as control) or D3 cells in an ATP-regenerating reaction (see Materials and Methods). Plasma membranes were sealed by including 2 mmol/L calcium chloride in medium, and cells were further cultured in D3 maintenance medium (complete DMEM+10 ng/mL LIF). NIH3T3 cells treated with the self extracts (referred to as 3T3/3T3) did not show any morphological changes up to day 10 posttreatment (Figure 1A, a), whereas NIH3T3 cells treated with D3 extracts (referred to as 3T3/D3) showed noticeable changes in cell morphology as early as day 3 posttreatment, forming colonies resembling ESC morphology by day 10 (Figure 1A, b through d). Quantitative mRNA expression of mESC-specific transcripts Oct4, Nanog, SSEA1, SCF, and c-Kit was induced in 3T3/D3 cells, whereas 3T3/3T3 cells did not express measurable mRNA for any of these stem cell markers (Figure 1B). Enhanced mRNA expression of stem cell–specific genes was further corroborated by immunofluorescence staining for selected proteins Oct4 and SSEA1 (Figure 1C and 1D) Further corroboration of NIH3T3 dedifferentiation was evident by the loss of lamin A protein expression, a specific marker of somatic cells, in 3T3/D3 cells (Figure 2A and 2B). Taken together, these data suggest that as an alternative to ESC–somatic cell fusion and SNT, cell-free ESC extracts could provide the necessary regulatory components required to induce somatic nuclear reprogramming and alter the differentiation status of nonembryonic cells. We further confirmed whether the D3 proteins and not the contaminating nucleic acids drove 3T3/D3 dedifferentiation. Inactivation of proteins from D3 extracts before reaction by either temperature, trypsin, or proteinase K digestion failed to induce any morphological changes or to the activation of Oct4 mRNA (Figure 2C), whereas inactivation of either DNA or RNA had no such effect.
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D3 Extract–Induced Epigenetic Changes in 3T3/D3 Cells Involve Oct4 Promoter Demethylation and Posttranslational Histone Modifications
DNA methylation of CpG residues leading to the silencing of pluripotent embryonic genes, including that of Oct4, is known as an integral step governing differentiation and development. Because our data indicated that D3 extract exposure leads to the induction of Oct4 mRNA and protein expression in 3T3/D3 cells, we performed CpG methylation status analysis of Oct4 promoter. As depicted in Figure 3A, on bisulphite treatment (see Materials and Methods), all 10 CpG sites in D3 cells were unmethylated (open circles). All 10 sites were methylated in 3T3/3T3 cells (filled circles); however, treatment of 3T3 cells with D3 extracts led to demethylation at 8 of 10 CpG residues in 3T3/D3 cells. The D3 extract–induced Oct4 promoter demethylation in the 3T3/D3 cells was independently corroborated by restriction enzyme digestion. In the Oct4 promoter region, there is 1 HpyCH4IV (methylated CpG-specific restriction enzyme) site at –202. We analyzed the DNA methylation status of the –202 site by HpyCH4IV restriction digestion analysis of PCR-amplified Oct4 promoter in D3, 3T3/3T3, and 3T3/D3 cells. As shown in Figure 3B, the PCR product was not digested with HpyCH4IV in D3 cells, indicating that the genomic DNA of D3 cells was unmethylated at this particular Oct4 promoter site. In contrast, the PCR product was readily digested in 3T3/3T3 cells, indicating methylation of the Oct4 –202 CpG site. Interestingly, the PCR product from 3T3/D3 cells was resistant to digestion by HypCH4IV, suggesting that treatment of 3T3 cells with D3 extracts induced demethylation of CpG sites, thereby reversing the repression of Oct4 mRNA expression, observed in 3T3/3T3 cells.
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DNA methylation/demethylation dependent gene suppression/activation is coupled with posttranslational modifications to the histone proteins, which, together, lead to chromatin remodeling and new patterns of gene expression.30,31 Therefore, we further confirmed the D3 extract–induced epigenetic changes by assessing the acetylation of histones (H)3 and -4 and methylation status of lysine 9 (K9) residue in H3 protein within Oct4 promoter by ChIP assays29 using specific antibodies. The Oct4 promoter was amplified from immunoprecipitated chromatin DNA by PCR. ChIP analyses showed that the promoter of Oct4 had increased acetylation of H3 and H4 (Figure 3C) and decreased dimethylation of K9-H3 (Figure 3D) in 3T3/D3 cells compared with 3T3/3T3 cells. Together, these data suggest that D3 extract–induced dedifferentiation and nuclear reprogramming of 3T3/D3 cells is mediated, at least in part, by chromatin remodeling, leading to the activation of Oct4. Considering that DNA methylation and histone modifications are involved in various biological phenomena, such as tissue-specific gene expression, cell differentiation, X chromosome inactivation, genomic imprinting, changes in chromatin structure, and tumorigenesis,35–43 it is conceivable that the changes in Oct4 promoter CpG methylation and histone modifications by exposure of 3T3 cells to ESC extracts, may be 1 of the principal epigenetic events underlying dedifferentiation and activation of ESC-specific genes in 3T3 cells.
D3 Extract Treatment Induces Genome-Wide Gene Expression Profile Changes in Reprogrammed 3T3/D3 Cells
To gain further insights into the changes in gene expression patterns in reprogrammed 3T3/D3 cells, we performed global gene expression profiles of D3, 3T3/3T3, and 3T3/D3 using Affymetrix mouse genome 2A gene chips.44–45 Differentially expressed probes with an adjusted probability value of <0.001 and fold change of >2 (absolute log fold change of >1) were extracted for further inspection. This resulted in 3286 probes with statistically significant differential expression between cell types 3T3/3T3 and 3T3/D3, including the significant upregulation of ESC-specific genes and downregulation of somatic genes. The heat map of z-scored probes illustrating this clustering, and the expression pattern of a subset of 99 significantly upregulated genes in 3T3/D3 and D3 cells, including Oct4 and nanog, is shown in Figure IA and IB in the online data supplement, and the functional grouping of all 3286 genes found to be differentially expressed between 3T3 and 3T3/D3 cell types is shown in supplemental Figure IC. The expression levels of representative upregulated and downregulated genes observed in gene chip experiments were independently confirmed by real-time RT-PCR, which confirmed the expression pattern observed in gene-profiling experiments (Figure 4). A list of selected genes, their relative expression level, and functional description is depicted in the Table.
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Reprogrammed 3T3/D3 Cells Redifferentiate into Cells of Multiple Lineages
We next determined the redifferentiation potential of dedifferentiated 3T3/D3 cells into multiple cell types, in vitro. Under culture conditions conducive for cell type–specific differentiation, 3T3/D3 cells acquired morphology (supplemental Figure II), mRNA and protein expression of CMC-specific (Figure 5A and 5B) and EC-specific (Figure 5C and 5D) genes. Furthermore, under specific culture conditions (see methods), 3T3/D3 cells differentiated into neuronal and adipocyte phenotypes (supplemental Figure III).
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Transplantation of Reprogrammed 3T3 Cells Into Surgically Induced Mouse Hindlimb Ischemia Model Improves Functional and Anatomic Repair
To ascertain the functional efficacy of reprogrammed cells in ischemic tissue repair, we conducted cell transplantation studies in a well-established mouse hindlimb ischemia model described in our previous publication.28 Immediately following the surgery, mice were assigned to 2 groups (n=15 each) and 3T3/D3 or 3T3/3T3 cells (1x105), labeled with DiI for tracing purposes, were injected into the ischemic muscles at 3 different sites. Physiological blood flow recovery was assessed by laser Doppler perfusion imaging on days 7, 10, and 14 (n=5 each time point) postsurgery, in both groups of mice. As shown in representative perfusion images in Figure 6A and quantified as the ratio of blood flow in ischemic to nonischemic limb, in Figure 6B, mice transplanted with 3T3/D3 cells displayed significantly improved perfusion in the ischemic limb at all time points compared with mice treated with 3T3/3T3 cells (P<0.01). Enhanced blood flow recovery in mice transplanted with 3T3/D3 cells was further corroborated by increased capillary density in the ischemic hindlimbs (Figure 6C). Histological analyses on retrieved tissues indicated both EC and skeletal muscle differentiation of transplanted 3T3/D3 cells (supplemental Figure IV).
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Intracardiac Transplantation of 3T3/D3 Cells Leads to Cardiomyocyte Differentiation and Improves Left Ventricular Functions in a Mouse AMI Model
Functional efficacy of 3T3/D3 cells in tissue repair was also investigated in a model of AMI. Mice underwent surgery to induce AMI by ligation of the left anterior descending coronary artery, as described.32,34 Animals were subdivided into 3 groups (10 each) and received intramyocardial injection of 5x104 lentiviral-GFP–transduced 3T3/D3 or 3T3/3T3 cells or saline, respectively. Echocardiography data revealed that left ventricular end-diastolic areas were similar in the 3T3/3T3 cell and saline groups before and at all time points after AMI (Figure 7A, red and gray lines, respectively). In contrast, however, mice treated with the 3T3/D3 cells revealed less ventricular dilation (Figure 7A, blue line, P<0.01 in 3T3/D3 versus control groups). Additionally, fractional shortening (FS), an indicator of contractile function, was consistently depressed in mice receiving saline and 3T3/3T3 cells (Figure 7B). However, treatment with 3T3/D3 cells significantly improved FS at all time points tested (at 4 weeks postsurgery, P<0.05 versus 3T3/3T3 cell treated group). Gains in post-AMI physiological functions in mice transplanted with 3T3/D3 cells were further corroborated by histological evaluation of hearts from each group of mice. As shown in Figure 7C, the percentage of fibrosis areas in mouse hearts receiving either saline or 3T3/3T3 cells were significantly larger than in mice that received 3T3/D3 cells (P<0.001). Tissue sections were also stained with BS-1 lectin to determine the capillary density at the border zone of the infarcted myocardium. Significantly higher capillary density was observed in the mice receiving 3T3/D3 cells than in mice receiving 3T3/3T3 cells or saline (Figure 7D, P<0.01).
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Immunofluorescence staining on myocardial sections was performed to determine CMC and EC differentiation of the transplanted (GFP-positive) cells. EC differentiation of transplanted cells was investigated by coexpression of GFP plus CD31 by transplanted cells. As shown in Figure 8A, GFP plus CD31 double-positive cells were observed in myocardial sections obtained from mice transplanted with 3T3/D3 cells, whereas sections obtained from 3T3/3T3 transplanted hearts did not show the evidence for EC differentiation. CMC differentiation of the transplanted cells in the myocardium was determined by the coexpression of GFP (green) and CMC-specific CTI (red). As shown in Figure 8B, GFP plus CTI double-positive cells (yellow fluorescence in merged image) were observed in mice treated with 3T3/D3 cells, suggesting that some of the transplanted cells differentiated into CMC lineage in vivo, whereas no evidence of CMC differentiation was observed for transplanted 3T3/3T3 cells. Transplanted 3T3/D3 cells also showed the evidence of proliferation in vivo (day 28), as determined by colocalization of GFP-positive cells with nuclear proliferation antigen, ki67 (supplemental Figure V), whereas no GFP plus Ki67 double-positive cells were observed in 3T3/3T3 cell–transplanted myocardial sections.
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The goal of therapeutic cloning is to produce pluripotent stem cells with the nuclear genome of the patient and induce the cells to differentiate into replacement cells, for example, CMCs for repairing damaged heart tissue. Reports on the generation of pluripotent stem cells14–16 or histocompatible tissues41 by nuclear transplantation, and on the correction of a genetic defect in cloned ESCs,46,47 suggest that therapeutic cloning could, in theory, provide a source of cells for regenerative therapy. Recent evidence on the efficacy of human therapeutic cloning, however, underscores the difficulties associated with the generation of human ESC lines for therapeutic purposes. Moreover, a number of limitations may hinder the strategy of therapeutic cloning for future clinical applications. Extremely low efficiency of SNT is a major concern.48 Analysis of the literature on mouse SNT-derived ESC lines raises concerns about the feasibility and relevance of therapeutic cloning, in its present embodiment, for human clinical practice.48–50 This limitation may be alleviated with oocytes from other species,41 but mitochondrial genome differences between species are likely to pose a problem. It is, therefore, desirable to develop alternative strategies to oocyte-dependent autologous stem cell generation. Our data indicating mESC extract–mediated reverse dedifferentiation of terminally differentiated murine fibroblasts and multilineage redifferentiation of these reprogrammed cells not only support the feasibility of such an approach but, more importantly, provide evidence that the stem-like cells obtained using this methodology are functionally competent for tissue repair.
Oocyte-independent epigenetic reprogramming of somatic cell by somatic cell–ESC fusion reported by several recent studies has generated much enthusiasm9,10 However, this strategy, although excellent for mechanistic studies, retains certain drawbacks that are associated with oocyte-dependent therapeutic cloning. Firstly, the 2 cells used to generate hybrid cells are not derived from autologous source; secondly, the efficiency of fusion remains low (1 to 3/1000); thirdly, the genetic stability of heterokaryon hybrids remains to be established (one will have to devise the technological innovations to delete additional set of chromosomes); and finally, the efficacy of reprogrammed cell to retain the embryonic stem (ES)-like properties if ESC-derived nucleus is removed remains to be elucidated. Recently, a major breakthrough was reported, whereby forced expression of transcription factors Oct4, Sox2, c-Myc, and Klf4 was shown to induce pluripotency in primary mouse fibroblasts51 and in human fibroblasts using either an Oct4, Sox2, c-myc, and Klf4 combination11 or an Oct4, nanog, Sox2, lin28 combination.12 Although these studies did provide the evidence that overexpression of these ES-specific genes leads to the derivation of ES-like cells from primary human fibroblasts, several critical questions were not answered by these studies. Firstly, it appears that the requirement of these factors or their combination is varied. These studies used different combinations of transcription factors in similar human cells and achieved similar results indicating that some of these factors may be dispensable. Secondly, these studies did not answer the question that how these transcription factors reactivated epigenetically silent endogenous pluripotent genes (eg, that of Oct4) because chromatin changes would be required to make transcriptionally silent promoters of these pluripotent genes to bind these transcription factors. Finally, 4 transcription factors were transduced by constitutively expressed retroviral vectors. It is unclear why the cells could be induced to differentiate and whether continuous vector expression was required for the maintenance of the pluripotent state. Despite these drawbacks, these studies did provide the identification of certain transcription factors, which, in combination, drive the dedifferentiation of somatic cells, leading to reactivation of pluripotent genes and ES-like phenotype. mESC-free extracts express all of the above proteins besides other putative chromatin modifying factors. Our data suggest that similar changes in somatic cell fate reported in these studies can be achieved without the introduction of transgenes or viral vectors.
Our observations demonstrating that mESC protein extracts can reprogram somatic cells toward multipotency would argue that multipotent epigenome could be activated in somatic cells without fusion and forced expression of nucleic acids. More importantly, our study is the first to demonstrate that transplantation of dedifferentiated somatic cells can repair ischemic tissue and mediate gain in physiological functions in relevant models of tissue injury. We, however, recognize certain limitations that our data do not sufficiently clarify. Firstly, in vitro differentiation of reprogrammed 3T3/D3 cells did not reveal any "beating cell." This may either reflect incomplete in vitro differentiation or, more likely, a reflection on in vitro culture conditions used for CMC differentiation. We only used 2 factors (LIF plus bone morphogenetic protein 2 for 4 to 7 days) for CMC differentiation, conditions that we have demonstrated previously lead to CMC precommitment of mESCs.25 Inclusion of additional cytokine as well as coculture with rat neonatal CMCs may enhance the CMC differentiation potential of 3T3/D3 cells. Secondly, only 2% to 3% of transplanted 3T3/D3 cells were retained in the myocardium by day 28, although 3T3/D3 cells were retained in higher number than control 3T3/3T3 cells. Thus, it appears that our reprogrammed cells share the similar low retention as has been observed with the transplantation of other adult stem cells. Finally, limited CMC and EC differentiation of transplanted 3T3/D3 cells may not be solely responsible for gains in the physiological functions. Indeed, it is quite possible that these reprogram cells may also participate in the ischemic tissue repair by secreting paracrine growth factors, resulting in enhanced neovascularization and/or resident CMC protection/salvage. Limited in vivo differentiation may also reflect the fact that the cells used in this study represented pooled cells, many of which were not completely reprogrammed. We have recently obtained clonal derivatives from reprogrammed cells that display long-term expression of mESC-specific genes. Future studies using these cells will help clarify these and other issues.
Taken together, our biochemical, molecular, and functional data provide an oocyte- independent approach for the generation of functional autologous multipotent cells from terminally differentiated somatic cells. The refinement of techniques and additional experimental data to elucidate applicability of this approach in primary somatic cells of different lineages and derivation of single-cell clones displaying stable, long-term reprogramming may hold significant promise for future use of such generated cells in regenerative medicine, including cardiac repair and regeneration.
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Acknowledgments |
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Sources of Funding
This work was supported, in part, by American Heart Association Grant 0530350N and NIH grant AA014575 (to R.K.) and by NIH grant HL63414 (to D.W.L.).
Disclosures
None.
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Footnotes |
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This manuscript was sent to James T. Willerson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received March 20, 2008; revision received April 30, 2008; accepted May 1, 2008.
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