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Identification of Lactoferrin as a Human Dedifferentiation Factor Through the Studies of Reptile Tissue Regeneration MechanismsS
Identification of Lactoferrin as a Human Dedifferentiation Factor Through the Studies of Reptile Tissue Regeneration MechanismsS
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 869-878
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : February 05, 2014
  • Accepted : March 13, 2014
  • Published : June 30, 2014
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About the Authors
Kil Soo Bae
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
Sun Young Kim
Research Center for Integrative Cellulomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Soon Yong Park
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
Ae Jin Jeong
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
Hyun Hee Lee
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
Jungwoon Lee
Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Yee Sook Cho
Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Sun-Hee Leem
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
Tae-Hong Kang
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
Kwang-Hee Bae
Research Center for Integrative Cellulomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Jae Ho Kim
Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan 626-870, Republic of Korea
Yong Woo Jung
Department of Pharmacy, Korea University, Sejong 339-700, Republic of Korea
Woojin Jun
Department of Food and Nutrition, Chonnam National University, Gwangju 500-757, Republic of Korea
Suk Ran Yoon
Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Sang-Chul Lee
Research Center for Integrative Cellulomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Jin Woong Chung
Department of Biological Science, Dong-A University, Busan 604-714, Republic of Korea
jwchung@dau.ac.kr

Abstract
In this study, we performed two-dimensional electrophoresis with protein extracts from lizard tails, and analyzed the protein expression profiles during the tissue regeneration to identify the dedifferentiation factor. As a result, we identified 18 protein spots among total of 292 spots, of which proteins were specifically expressed during blastema formation. We selected lactoferrin as a candidate because it is the mammalian homolog of leech-derived tryptase inhibitor, which showed the highest frequency among the 18 proteins. Lactoferrin was specifically expressed in various stem cell lines, and enhanced the efficiency of iPSC generation upto approximately 7-fold relative to the control. Furthermore, lactoferrin increased the efficiency by 2-fold without enforced expression of Klf4 . These results suggest that lactoferrin may induce dedifferentiation, at least partly by increasing the expression of Klf4 .
Keywords
Introduction
The generation of induced pluripotent stem cells (iPSCs) is one of the most promising techniques in the area of stem cell biology. iPSCs were first discovered by Shinya Yamanaka [20 , 21] . He and his colleagues found that somatic cells can be reprogrammed to a pluripotent state by forced expression of defined transcription factors, including Oct4 , Sox2 , Klf4 , and c-Myc . Since its first identification, the iPSC has been considered a highly promising cellular agent that can overcome the ethical problems of using embryonic stem cells (ESCs). However, to achieve the eventual goal of clinical application, several limitations of the current iPSC technique must be overcome, such as low reprogramming efficiency, use of oncogenes, and genomic alterations due to viral integration [12 , 16 , 17 , 19] . Therefore, it is important to identify novel dedifferentiation factors that improve the safety and efficiency of the iPSC technique.
Dedifferentiation can occur naturally during tissue regeneration in several lower animals, including coelenterates, amphibians, and reptiles [9 , 23] . Among the animals that can regenerate their tissues in nature, lizards are the evolutionarily closest to mammals. When threatened, many lizards can voluntarily autotomize their tails to escape from the attack. After the autotomy, tail regeneration starts with the aggregation of cells to form a blastema [1 , 4 , 15] . The blastema possesses the characteristics of stem cells and can eventually redifferentiate into many tissues that were present in the original tail, including muscle, skin, bone, and blood vessels, during the regeneration process. However, the detailed mechanisms of blastema formation have not been elucidated. Since blastema formation resembles the dedifferentiation of normal cells into stem cells, identification of the key factors in blastema formation may provide a clue to the dedifferentiation factors in mammalian cells.
In this study, we selected the lizard as an animal model to study tissue regeneration mechanisms, and thus to identify human dedifferentiation factors by analyzing protein expression profiles using proteomic tools. As a result, we found that leech-derived tryptase inhibitor (LDTI) was specifically expressed in the blastema. However, LDTI was expressed only in the lower animals. Therefore, to apply this finding to a mammalian iPSC technique, we searched for mammalian homologs, and learned that lactoferrin (LF) is a functional homolog of LDTI [6] . Functional studies with LF showed that it was specifically expressed in ESC and iPSC lines, and the addition of LF promoted the efficiency of iPSC generation up to approximately 7-fold compared with the conventional method.
Materials and Methods
- Animals and Housing Conditions
Outbred geckos ( Eublepharis macularius , 20 males, 4-6 weeks old) were purchased from Mowgli Pet (Seoul, Korea). All lizards were kept in a controlled environment with the temperature maintained at 25-28℃, and an artificial photoperiod (4 h daily on ultraviolet lights) was imposed. Lizards were housed individually in standard plastic cages with sterilized gauze as a nesting material and provided with mealworms ( Tenebrio molitor ) and purified water. Every 4 days, the animals were placed in clean cages with fresh gauze.
- Tissue Collection
Tail tips of lizards (20 males, 4-6 weeks old) were amputated using a surgical knife. After amputation, tissues were collected from the regenerating tail of the animals on days 0, 3, 6, and 12 and stored at -20℃ until use.
- 2D Gel Electrophoresis
Tissues were lysed in 500 μl of buffer containing 150 mM NaCl, 1.0% NP-40, 50 mM Tris-HCl (pH 8.0), and 1× protease inhibitor mix (Calbiochem). Tissues were kept on ice and sonicated with an ultrasonic processor (Fisher Scientific) 20 times for 2 min. The homogenate was centrifuged (16,000 × g , 10 min, 4℃) to remove cellular debris, and the resultant supernatant was ultracentrifuged at 25,000 × g for 1 h 30 min. The supernatant was stored at 20℃ until use. Total protein concentration was determined using the Bradford Protein Assay Kit (Bio-Rad) according to the manufacturer’s instructions. Total proteins (120 mg) were precipitated by the acetone method and dissolved in rehydration solution (7 M urea, 2 M thiourea, CHAPS [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate], 1% bromophenol blue, 140 mg/ml DTT, and 2% immobilized pH gradient (IPG) buffer). The eluted solution was applied onto pre-cast IPG strips with a nonlinear pH range of 3-10. For separation of proteins by their isoelectric point (first dimension), nonlinear pH 3-10 7 × 7 cm IPG gel strips were rehydrated for 14 h in rehydration solution. A three-phase program was used for isoelectric focusing: phase 1, 300 V/2 mA/5 W/1 vh; phase 2, 3,500 V/2 mA/5 W/5,250 vh; and phase 3, 3,500 V/2 mA/5 W/45,500 vh. Focused IPG strips were briefly equilibrated for 15 min with equilibration solution (50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, and 30% glycerol) containing 1% DTT, and it was equilibrated again with the same solution containing 5% iodoacetamide instead of DTT for 15 min. The second-dimensional separation was performed in 10% SDSpolyacrylamide gel electrophoresis (PAGE) gels. The gels were run at 100 V until the proteins were separated (~2 h) and the twodimensional gel electrophoresis gel was visualized by silver staining according to the manufacturer’s instructions (Bio-Rad).
- Staining and Image Analysis
After electrophoresis, gels were fixed and protein spots were visualized by silver staining (PlusOne Silver Staining Kit, GE Healthcare). The two-dimensional electrophoresis (2DE) images were scanned and processed with Progenesis SameSpots ver. 3.0 software (Nonlinear Dynamics). To confirm the variations, at least three gels were prepared for every case. Spot volumes were normalized based on the total spot volume of each gel. Protein spot intensity was defined as the normalized spot volume; that is, the ratio of the single spot volume to the total of the spot volumes on the 2DE gel (total spot normalization n). Computer analysis facilitated the automatic detection and quantification of protein spots, as well as matches between gels of controls and dysplasia or HCA samples. Spots displaying reliable and significant differences (over 2-fold, p < 0.05) were selected for MS analysis.
- In-Gel Digestion and Identification by LC-MS/MS
Spots of interest were manually excised from 2DE gels and destained with chemical reducers to remove the silver. Briefly, 50–100 ml of the freshly prepared reducing solution (1:1 mixture of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate) was added to the gel plugs and mixed. After the brown color disappeared, the gel plugs were rinsed with water and incubated in 200 mM ammonium bicarbonate for 20 min. Subsequently, the gel plugs were cut into small pieces, washed with water, and dehydrated repeatedly with acetonitrile (ACN) until the pieces appeared opaque and white. Next, the gel pieces were dried in a vacuum centrifuge for 30 min, and the proteins were digested with 20 ng/ml of sequencing-grade modified trypsin (Promega) for 16–24 h at 37℃. Digested peptides were extracted with extraction solution (50% ACN and 5% trifluoroacetic acid), and the extracted peptides were dried using a vacuum drier. Samples were then subjected to MS analysis. Peptides were analyzed using a Synapt High Definition Mass Spectrometer (Waters, Manchester, UK) equipped with a nano ACQUITY Ultra Performance LC system (Waters, Milford, MA, USA). In brief, 2 ml of peptide solution was injected onto a 75 m × 100 mm Atlantis dC18 column (Waters, USA). Solvent A consisted of 0.1% formic acid in water, and Solvent B was composed of 0.1% formic acid in ACN. Peptides were initially separated using 100 min gradients and electrosprayed into the mass spectrometer (fitted with a nano Lock- Spray source) at a flow rate of 300 nl/min. Mass spectra were acquired from m/z 300 to 1,600 for 1 sec, followed by four data-dependent MS/MS scans from m/z 50 to 1,900 for 1 sec each. The collision energy used to perform MS/MS was varied according to the mass and charge state of the eluting peptide. (Glu1)-Fibrinopeptide B was infusedat a rate of 350 nl/min, and an MS scan was acquired for 1 sec every 30 sec throughout the run. A database search was performed with MASCOT (Matrix Science, London, UK) using the following parameters: NCBInr.08.03.26 database, Mus musculus species, and maximum number of missed cleavage by trypsin at 1. Mass tolerance ranged from 750 to 7,100 ppm. The peptide modification allowed was carbamidomethylation in the fixed modification mode.
- Western Blot Analysis
Cell extracts (40 μg) were separated an 10% SDS-PAGE gels at 100 V until proteins were separated (~2 h) and transferred to polyvinylidene difluoride membranes (Millipore) for 30 min at 15 V by semi-dry transfer (Bio-Rad). The membranes were washed in TBS (0.2 M trizma base, 1.37 M NaCl) and blocked in 5% milk in TBS plus 0.05% Tween 20 for 30 min. The membranes were incubated with antibodies against β-actin (Bethyl) and LF (Santa Cruz Biotechnology) overnight at 4℃, washed, and incubated with horseradish-peroxidase-conjugated anti-rabbit antibodies for 1 h at room temperature. Immunoblots were visualized using ECL reagent (Thermo).
- Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted using Tri-reagent (MRC) in accordance with the manufacturer’s instructions. Aliquots (500 μg) of total RNA were transcribed into cDNA at 37℃ for 1 h in a total volume of 20 μl with 2.5 U MMLV (ELIPIS Biotech). Reverse-transcribed cDNA samples were added to a PCR mixture consisting of 10× PCR buffer, 0.2 mM dNTP, 0.5 U Taq DNA polymerase (RBC), and 10 pmol of primers specific for each gene. The primer sequences were as follows: β-actin, 5’-cctaaggccaaccgtgaa-3’ and 5’-ccgctcgttgccaatagt-3’; Oct-4, 5’-agctgctgaagcagaagagg-3’ and 5’-tgggaaaggtgtccctgtag-3’; Sox-2, 5’-agaaccccaagatgcacaac-3’ and 5’-atgtaggtctgcgagctggt-3’; c-Myc, 5’-gcccagtgaggatatctgga-3’ and 5’-ctgaggggtcaatgcactc-3’; Klf-4, 5’-cagcttcatcctcgtcttcc-3’ and 5’-cgcctcttgcttaatcttgg-3’; and mLF, 5’-aaacaagcatcgggattccag-3’ and 5’-acaatgcagtcttccgtggtg-3’. PCR analysis of genomic integration and real-time quantitative PCR (qPCR) were performed as described previously [13] . Briefly, genomic DNA or total RNA samples were extracted from LF-iPSC clones #2 and #3 using the DNeasy kit (Qiagen) or RNeasy kit (Qiagen). Total RNA (3 μg) was reverse transcribed using a First Strand Synthesis kit (Invitrogen). PCR primers were used as previously described [13 , 20] , and listed in the Supplementary Fig. S3.
- Cell Culture
J1mESC or established mouse induced pluripotent stem cell (miPSC) lines were maintained on irradiated mouse embryonic fibroblast (MEF) feeder cells in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 15% fetal bovine serum (FBS) (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen), 1% non-essential amino acids (NEAAs; Invitrogen), 1% sodium pyruvate, 1 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), and 1,000 U/ml leukemia inhibitory factor (LIF; Millipore). MEFs and GP2-293 cells were grown in DMEM-high glucose (Invitrogen) with 10% FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 1% NEAA (Invitrogen), and 0.1 mM β-mercaptoethanol (Sigma).
- Retrovirus Production and Lactoferrin-Induced miPSC Generation
The reprogramming procedure was performed according to previously reported protocols with a slight modification [11 , 20] . Briefly, Moloney-based retroviral vectors (pMXs) containing cDNAs for Oct4 , Sox2 , Klf4 , and c-Myc were obtained from Addgene. These plasmids were transfected into GP2-293 cells with packaging vector pCMV-VSVG using a CalPhos transfection kit (Clontech). The viral supernatants were collected 24 and 48 h after transfection. MEFs were derived from E13.5 embryos of a wild-type CF1 mouse, and were plated at 1 × 10 5 cells per well in 6-well plates1 day before transduction. Two rounds of infection were performed during a 48 h period and the viral supernatants were removed. The infected MEFs from one well were reseeded on a 6-well plate coated with 0.2% gelatin 3 days after transduction and cultured in mESC culture medium supplemented with a designated concentration of lactoferrin (LF; Sigma). The media were changed every other day. To establish stable LF-induced miPSC lines, single ESC-like colonies were picked approximately 3 weeks post-infection, seeded in individual wells of a 4-well plate, and expanded on irradiated MEF feeder layers for characterization. When required, neutralizing antibody to LF (OriGene) was added to the culture medium.
- Immunofluorescence Staining
Cells were fixed in PBS containing 4% paraformaldehyde for 15 min at RT. After washing with PBS, the cells were incubated in PBS containing 1% bovine serum albumin and 0.1% Triton X-100 for 30 min at RT. The primary antibodies used were specific for Oct4 (sc-9081; Santa Cruz Biotechnology), Sox2 (sc-17320; Santa Cruz Biotechnology), SSEA1 (MC-480; R&D Systems), α-SMA (A5228; Sigma), Tuj1 (PRB-435P; Convance), and Sox17 (MAB1924; R&D). Alexa546- or Alexa488-conjugated secondary antibodies were used. The DNA was stained with 0.3 μg/ml DAPI (Invitrogen).
- In Vitro Embryoid Body Formation
LF-miPSC clones #2 and #3 were trypsinized and transferred to bacterial culture dishes in mES medium without LIF. After 5 days, aggregated cells were plated onto gelatin-coated 4-well plates and incubated for another 5 days.
- Teratoma Formation
LF-miPS cells (1 × 10 7 cells) were trypsinized and injected subcutaneously into 5-week-old SPF/VAF immunodeficient mice (Orientbio). After 25 days, teratomas were fixed overnight in 4% paraformaldehyde/phosphate buffered saline solution and embedded in paraffin. Sectioned teratomas were stained with hematoxylin and eosin.
Results and Discussion
- Identification of Differentially Expressed Proteins During Blastema Formation
Many lower animals have the capacity for spontaneous tissue regeneration after injury, a property that is lacking in humans except for limited liver regeneration [7] . The lower the evolutionary status, the higher the regeneration ability; for example, Planaria is known to be able to reproduce its whole body from as little as one tenth of its original parts [18] . Newts, lizards, and zebra fish can also regenerate virtually every tissue and organ [2] , whereas humans and other mammals cannot. Thus, it is thought that such regeneration abilities were lost during evolution. Every case of tissue regeneration in lower animals involves cellular dedifferentiation to form a blastema that has characteristics of stem cells. Therefore, we initially thought that we could translate this property into mammalian systems if we identified the key factors in blastema formation in lower animals. As a regeneration model, we selected the lizard with the hope of isolating genes that are shared with mammals because reptiles are evolutionarily closest to mammals.
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Lager Image
Representative 2DE images of tail tip samples from tail-tip tissues on days 0, 3, 6, and 12. Total protein lysates were separated on pH 3–10 nonlinear immobilized pH gradient (IPG) strips in the first dimension, followed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis in the second dimension and subsequent visualization by silver staining. Protein spots that were differentially expressed compared with control are indicated with circles. Spots were identified by liquid chromatography–mass spectrometry/mass spectrometry, as outlined in Table 1.
List of proteins identified from comparative proteomics using mass spectrometry.
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Lager Image
Expression profiles of selected proteins during tail regeneration period.
A model of tail regeneration in Eublepharis macularius has previously been established [15] . The earlier report divided regeneration into seven stages, and found that the blastema was well established in stage III (4-8 days post-autotomy (dpa)) in Eublepharis macularius . Therefore, we collected tail-tip tissues from 20 animals at 6 dpa (stage III: dedifferentiation period) as the blastema. Samples from 0, 3, and 12 dpa were also included as representative samples of stage I (control), stage II (wound healing period), and stage IV (regenerating period), respectively (Fig. S1). Proteins were extracted from each sample and subjected to 2DE. Among the total of 292 spots ( Fig. 1 ), we initially selected 18 proteins after further analysis of these proteins by mass spectrometry, which showed at least a 2-fold change in expression at 6 dpa compared with the day zero control ( Table 1 ). These include the proteins involved in cellular metabolism, cytoskeleton, nucleic acid transporter, and protease inhibitors. Among them, we first focused on LDTI based on its high frequency and expression pattern, which indicated increased expression during the dedifferentiation period and decreased expression during redifferentiation ( Fig. 2 , Figs. S2A and S2B).
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Lager Image
Protein expressions during tail regeneration. Protein expressions on designated days during the regeneration period were analyzed, and the normalized volume of each spot is shown (name of each protein is listed in Table 1).
- Confirmation of Lactoferrin Expression in Human and Mouse ESCs and iPSCs
Tryptase is a trypsin-like serine proteinase in the secretory granules of mast cells, and has been implicated in a variety of inflammatory diseases such as asthma and rheumatoid arthritis [5 , 8 , 10] . Unfortunately, LDTI is present only in lower animals and is absent in mammals. We therefore searched for homologs of LDTI in mammals. Although a sequence homolog of LDTI was not found in mammalian databases, we learned that lactoferrin is a functional homolog of LDTI and serves as a potent tryptase inhibitor in mammals [6] . LF is the only natural tryptase inhibitor, and is known to regulate various inflammatory diseases [6 , 14] . In fact, LF is a multifunctional protein involved in various processes associated with wound healing, including cellular proliferation, migration, and survival [22 , 24 , 25] . Since wound healing is the key event in tissue regeneration, we hypothesized that LF might be involved in dedifferentiation of mammalian cells, and performed further functional studies. To evaluate the feasibility of LF as a dedifferentiation factor, we determined the expression of LF in ESC and iPSC lines. RT-PCR and western blot analysis showed that LF was specifically expressed in human ESC and iPSC lines but was not expressed in the control fibroblast cells (Figs. S2A and S2B). The specific expression of LF in mouse stem cell lines was also confirmed by RT-PCR ( Fig. 3 C). The fact that LF was specifically expressed in human and mouse stem cells and iPS cell lines but absent in the parent fibroblasts suggests a possible role of LF in mammalian dedifferentiation.
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Lager Image
Specific expression of lactoferrin in ESC and iPSC lines. RNA and protein expressions of lactoferrin in human cells were analyzed by RT-PCR (A) and western blot assay (B), respectively. For RT-PCR, Oct4 and Sox2 were included as stem cell specific markers. Lactoferrin expressions in mouse cell lines in RNA levels were also confirmed by RT-PCR (C). CRL2097, human fibroblast cell line; H9, human ES cell line; h-iPS, human iPS cell line; MEF, mouse embryonic fibroblast; mES, mouse ES cell; m-iPS, mouse iPS cell line. Results are representatives of three independent experiments.
- Specific Effect of Lactoferrin on iPSC Generation
We next investigated the functional effects of LF on iPSC generation. As expected, addition of LF to the culture medium during in vitro dedifferentiation increased the efficiency of iPSC generation by upto approximately 700% in a dosedependent manner, compared with the control in which only four genes ( Oct4 , Sox2 , Klf4 , and c-Myc ) were introduced ( Fig. 4 A). The effect was inhibited by addition of neutralizing antibody to LF, whereas control antibody did not show any inhibitory effect, thereby proving that LF exerted a specific effect on iPSC generation. Interestingly, LF also increased the efficiency approximately 2-fold compared with the control even without forced expression of Klf4 ( Fig. 4 B). To analyze the mechanisms of LF-induced iPSC generation, we next investigated the effect of LF on the expression of Yamanaka’s factors in MEFs by RT-PCR and found that LF increased the expression of Klf4 but did not affect the expression of the other factors ( Fig. 4 C). These results suggest that LF may directly increase the expression of endogenous Klf4 , thus promoting dedifferentiation. Although this iPSC technology is considered an innovative tool in stem cell therapeutics, it has several problems such as low efficiency and safety [2] . Thus, since the first identification of dedifferentiation factors, many attempts have been made to improve the safety and efficiency of the technology. In particular, use of oncogenes such as Klf4 and c-Myc in iPSC generation raised concerns about the safety of iPSCs for practical applications. Although Thomson and colleagues found that Klf4 and c-Myc could be substituted with Nanog and Lin28 [27] , these oncogenes may be required to improve the efficiency of reprogramming [16 , 26] . In fact, until recently these genes have been routinely included in iPSC generation procedures by many researchers. However, our results suggest that the forced induction of Klf4 in the iPSC generation procedure can be omitted in the presence of lactoferrin.
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Lager Image
Induction of iPSC generation and Klf4 expression by lactoferrin. (A) MEFs were transduced with OSKM (Oct4, Sox2, Klf4, and c-Myc) and incubated with or without the designated concentration (μg/ml) of lactoferrin for 14 days. For inhibition of lactoferrin, neutralizing antibody (50 μg/ml) to lactoferrin was co-treated with lactoferrin (200 μg/ml). (B) MEFs were transduced with OSKM (Oct4, Sox2, Klf4, and c-Myc: 4G) or OSM (3G), and incubated with or without 200 μg/ml of lactoferrin for 14 days. ES-like colonies were stained with AP, and the numbers of AP-positive colonies were counted to determine reprogramming efficiency. *p < 0.05. (C) MEFs were treated with or without lactoferrin (200 μg/ml) for 12 h, and the RNA expressions of Yamanaka factors were analyzed by RT-PCR.
- Pluripotency of Lactoferrin-Induced miPSCs
We randomly picked two miPSC (mouse induced pluripotent stem cell) colonies derived from reprogramming by OSKM ( Oct4 , Sox2 , Klf4 , and c-Myc ) in the presence of LF based on their mESC-like morphology at 3 weeks after transduction. To further characterize their pluripotency, those two miPSC lines were expanded in conventional mESC culture medium. LF-induced miPSCs were morphologically similar to normal mouse ESCs and positive for alkaline phosphatase, an ESC-specific pluripotent marker ( Fig. 5 A). LF-induced miPSCs also expressed high levels of pluripotency markers, including Oct4, Sox2, and SSEA1, as determined by immunofluorescence staining ( Fig. 5 B). In parallel, real-time quantitative PCR analysis confirmed increased expression of pluripotency markers, including Oct4 , Sox2 , Nanog , and Fgf4 , at the mRNA level compared with the parental MEFs ( Fig. 5 C). We used specific primers that amplified only the endogenous, but not the transgenic, transcripts. Genomic integration of retroviral transgenes for these four factors was confirmed by PCR analysis ( Fig. 5 D). In vitro differentiation potential of the LF-miPSCs was confirmed by embryoid body formation in suspension culture ( Fig. 5 E), and generation of all three embryonic germ layers following embryoid body differentiation was demonstrated by immunofluorescent staining with anti-α-SMA (mesoderm), anti-Tuj1 (ectoderm), and anti-Sox17 (endoderm; Fig. 5 F). We also examined the pluripotency of LF-miPSCs by teratoma formation. Histological examination revealed that LF-miPSCs differentiated into tissue representative of all three embryonic germ layers, including gut (endoderm), muscle and cartilage (mesoderm), and neural rosette (ectoderm; Fig. 5 G). Thus, we conclude that LF-miPSCs maintain features of pluripotency.
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Lager Image
Characterization of LF-induced miPSCs. (A) Morphology of MEFs and LF-miPSCs on irradiated MEF feeder cells. LF-miPSCs were stained positive for an alkaline phosphatase (ALP). Scale bars = 100 μm (B) Immunofluorescence staining of pluripotency markers (Oct4, Sox2, and SSEA1) in LF-miPSCs. Scale bars = 100 μm. (C) The qPCR analysis of pluripotency marker genes (Oct4, Sox2, Nanog, and Fgf4) in MEFs and LF-miPSCs. Endogenous RNA levels were determined using specific primers. Shown are the averages ± SD of three independent experiments. The value of MEFs was set to 1 in each experiment. (D) The qPCR analysis revealed integration of all the four retroviruses (Oct4, Sox2, Klf4, and c-Myc). (E) In vitro embryoid body (EB) formation of LFmiPSCs. (F) Immunofluorescence staining of the differentiation markers for the mesodermal (SMA), ectodermal (Tuj1), and endodermal (Sox17) germ layers in LF-miPSCs. Scale bar = 50 μm. (G) Hematoxylin and eosin staining of LF-miPSCs-derived teratomas from showing endoderm (gut)-, mesoderm (muscle and cartilage)-, and ectoderm (neural rosette)-like structures.
In summary, LF was identified as a novel dedifferentiation factor through the studies of reptile tissue regeneration mechanisms using the lizard as a model. Although the detailed mechanisms are still to be elucidated, our results showed that LF can be used as an effective exogenous factor to promote the dedifferentiation of normal cells into the pluripotent state, at least in part by inducing endogenous expression of Klf4 . Further studies of the detailed mechanisms in LF-induced dedifferentiation, such as involvement of protease inhibitory activity, will prove a valuable contribution to the development of more efficient and safer iPSC technologies. Furthermore, our results provide new insights into the evolutionary strategies of the lower animals in tissue regeneration that may contribute to the development of iPSC technology and various fields of stem cell biology.
Acknowledgements
This work was partially supported by the National Research Foundation of Korea (NRF-20100009086, NRF-2012R1A1A2039992, and 2012M3A9C7050093) and the KRIBB/KRCF research initiative program (NAP-09-3).
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