Basal Expression of Pluripotency-Associated Genes Can Contribute to Stemness Property and Differentiation Potential
Pluripotency and stemness is believed to be associated with high Oct-3/4, Nanog, and Sox-2 (ONS) expression. Similar to embryonic stem cells (ESCs), high ONS expression eventually became the measure of pluripotency in any cell. The threshold expression of ONS genes that underscores pluripotency, stemness, and differentiation potential is still unclear. Therefore, we raised a question as to whether pluripotency and stemness is a function of basal ONS gene expression. To prove this, we carried out a comparative study between basal ONS expressing NIH3T3 cells with pluripotent mouse bone marrow mesenchymal stem cells (mBMSC) and mouse ESC. Our studies on cellular, molecular, and immunological biomarkers between NIH3T3 and mBMSC demonstrated stemness property of undifferentiated NIH3T3 cells that was similar to mBMSC and somewhat close to ESC as well. In vivo teratoma formation with all three germ layer derivatives strengthen the fact that these cells in spite of basal ONS gene expression can differentiate into cells of multiple lineages without any genetic modification. Conclusively, our novel findings suggested that the phenomenon of pluripotency which imparts ability for multilineage cell differentiation is not necessarily a function of high ONS gene expression.
Ability to isolate and establish pluripotent or multipotent stem/precursor population is a major objective for the biotechnological industry and clinical translation of regenerative medicine. A major impediment for using stem cells in a clinical setup is poor availability of cells, especially those obtained with noninvasive procedures without raising much ethical issues. These limitations greatly restrict the usage of stem cells in clinics, disabling treatments of many degenerative diseases. This lacuna can be filled if any tissue-specific cells can be verifiably demonstrated to possess pluripotent or multipotent capacity. This may elevate the hope to find a well-suited stem cell-like cell line that can serve as an autologous, noncontroversial, and renewable source for cell therapy without ethical and immunological concerns, which are usually associated with embryonic stem cells (ESCs).
Numerous gene and protein expression criteria have been set for recognizing a cell as pluripotent. Microarray analyses have demonstrated a set of various transcripts that are associated with stemness as in the case of ESCs . Notably, it has been demonstrated by Yamanaka and colleague that the combinations of four major transcription factors, Oct-3/4, Nanog, Sox-2, and Klf-4, is indispensable for the cells to maintain pluripotency as in the case of ESCs [2,3]. However, there are some other reports which indicate that expressions of not all the four genes are essential to maintain the stemness. According to NIH and ISSCR guidelines, teratoma formation is one of the major criteria for classifying a cell to be pluripotent . Apart from this, the presence of alkaline phosphatase (ALP) is another reliable property shown for pluripotent cells such as ESCs [1,5].
In principle, a cell that is able to differentiate into cell types of all three germinal layers is considered pluripotent. On the other hand, a multipotent cell can give rise to cells originating from the same germ layer . A classic example of a stem cell with pluripotent and multipotent potential is mesenchymal stem cells (MSC). MSCs can differentiate into various cell types. Depending on isolation procedure and tissue source, both pluripotent and multipotent type of stem cells can be isolated . Mesenchymal cells display fibroblastic cell morphology and express vimentin protein as a characteristic marker along with high Oct-3/4, Nanog, and Sox-2 (ONS) expression . Bone marrow MSC (BMSC) isolated from crude bone marrow are reported to possess pluripotent gene expression and also show tri-lineage differentiation [9–12]. Therefore, it appears that BMSC can serve as a model that is used for screening various cell types for their degree of stemness. Hence, cells which portray specific features that are in accordance with the parameters mentioned earlier can be designated as pluripotent cells.
In a very recent report, Wang et al. exemplify that ONS genes are the core regulators of pluripotency. This group showed that Oct4 regulates and interacts with the BMP4 pathway to specify four developmental fates. High levels of Oct4 enable self-renewal in the absence of BMP4; Nanog represses embryonic ectoderm differentiation but has little effect on other lineages, whereas Sox-2 is redundant and represses mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates . Numerous cell types isolated from tissues and organs such as heart, kidney, liver, lungs, brain, pancreas, spleen, muscles, adipose tissue, dental pulp, placenta, and amnion are now being tested on the parameters mentioned earlier with an aim of generating patient-specific pluripotent stem cell lines for treating various incurable degenerative diseases.
With numerous published reports, it becomes a general belief that pluripotency, stemness, and differentiation potential into trigerminal lineages are associated with high levels of ONS genes, but this does not explain the fact that many low ONS expressing cells can also demonstrate multilineage differentiation potential, high ALP staining, and a teratoma-like structure formation and, hence, generate valid questions. (1) What should be the threshold of ONS gene expression for any cell to be pluripotent? (2) Is pluripotency really a function of high ONS gene expression or can basal level expressing cells also show lower but sustained pluripotency? Attempts are made in the present study to answer these questions scientifically using NIH3T3 as a model and are compared with high ONS expressing cells.
NIH3T3 cells, isolated from Swiss albino mice , are not known as stem cells due to the reduced expression of pluripotency-associated genes despite being embryonic in nature. These cells have been extensively used as a model system for various molecular, cellular, and toxicological studies [15–18] and are considered differentiated dermal fibroblast cells. Many groups used NIH3T3 as control cells that fail to differentiate [19–21]. However, their differentiation into adipocytes, myocytes, and neural cell types has been reported either after transforming genetically or if differentiated under defined media conditions [19–21]. Piestun et al. reported that NIH3T3 cells after being transfected with Nanog show induced pluripotency and expressed markers that are specific to various differentiating cell types . In 2006, Yamanaka and colleagues also compared pluripotent ESCs and untransformed NIH3T3 by a microarray analysis and concluded that although these cells did not express pluripotency genes, especially the four crucial ones, that is, Oct3/4, Nanog, Sox-2, and Klf4; they expressed c-Myc . However, this group neither examined nor commented on the multipotent nature of these cells. Later, in 2008 and 2009, Deng and colleagues published two reports demonstrating differentiation of untransformed NIH3T3 cells into oesteocytes and neuronal cell types in bone and spinal cord injury mouse models [1,23,24]. Two other groups also reported that NIH3T3 can give rise to neural cells and oesteoblasts, respectively [21,25]. Recently, our group also reported pancreatic islet cell differentiation of untransformed NIH3T3 with appropriate induction .
To answer these questions and to understand whether pluripotency can also be associated with basal ONS gene expression, we carried out a comparative study of basal ONS expressing NIH3T3 cells with pluripotent mouse BMSC (mBMSC) and mouse ESC (mESC). We carefully examined NIH3T3 cells for pluripotent and multipotent stem cell specific markers and compared them with those of mBMSC and mESC with regard to morphological features, expression profile of stemness related genes, ability to undergo multilineage differentiation, and in vivo teratoma formation.
Materials and Methods
Male Balb/c mice, 3–4 weeks old, weighing around 25–30 g, were used for transplantation and bone marrow cell isolation experiments. All animal experiments were performed in accordance with our institutional ‘‘Ethical Committee for Animal Experiments’’ and CPCSEA guidelines and regulations.
Chemicals and cell culture media
All chemicals, media, and antibodies used in this study were purchased from Sigma Aldrich. Molecular biology reagents and cDNA and PCR kits were procured from Fermentas Inc. All plasticwares were purchased from Nunc, Inc.
Isolation of marrow-derived MSCs
Bone marrow-derived mononuclear cells were isolated from 4 week-old Balb/c mice according to Zhu et al. [10,27]. Mice were sacrificed after proper anaesthesia. Briefly, tibia and femur bones were dissected under sterile conditions. The metaphyseal ends of the bones were cut, and the marrow plugs were flushed out by passing low glucose Dulbecco’s modified Eagle’s medium (L-DMEM) (Sigma) through a needle inserted into one end of the bone. Bone marrow cells from mice were collected; big tissue debris were removed with the help of a cell strainer and were then seeded in low-glucose RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) for 2 days.
Purification and propagation of MSCs
MSCs were purified as described earlier [10,27]. Briefly, the cultured cells were seeded at a density of 10,000 cells/mL in complete medium (high glucose DMEM supplemented with 10% FBS (Sigma), penicillin (100 IU/mL), streptomycin (100 IU/mL; MP Biological, Inc.) in polystyrene T25 culture flasks and incubated in a humidified 5% CO2 incubator at 37°C for 48 h. Loosely attached hematopoietic cells were discarded on trypsinizing adherent cells for 5 min. Cells that remained adhered were trypsinized for another 7 min  and then cultured in complete medium for 10 days. The cell media was replaced with fresh one every third day. The cells were trypsinized using Trypsin-EDTA (Sigma-Aldrich, USA) and passaged in T75 flasks until the second generation became 70%–90% confluent. Cultured mBMSC were then characterized and used for a comparative study with NIH3T3 after ensuring their pluripotency.
Cell culture and maintenance
NIH3T3 cells were obtained from three different sources (Cell Repository, National Centre for Cell Science, Pune, India, ATCC procured cells from Zydus Pharmaceuticals, Ahmedabad, India and a generous gift from Dr. Girish M. Shah’s lab, Laval University. Quebec, Canada) and were maintained in high-glucose DMEM supplemented with 10% FBS. Cells were split into a 1:1 ratio in new tissue culture flasks on being about 90% confluent. Culture media was changed after every 2 days.
Cell count and growth curve
Fully confluent mBMSC and NIH3T3 cells were trypsinized with 0.1% Trypsin EDTA solution and counted under an inverted phase-contrast microscope (Nikon TE2000). One hundred thousand cells were seeded into each well of 24-well plates for growth curve studies. Cells were eventually trypsinized and counted at different time points (0, 24, 48, 72, 96, and 120 h). Cell counts were then plotted versus time to establish the growth curve of cells. Growth curve of NIH3T3 cells was compared with that of mBMSC. Doubling time of both the cells were also determined using the algorithm ln (Nt−N0) ln (t), where Nt and N0 were number of cells at final time point and at initial seeding point, respectively, and t was time period in hours for which cell counts were recorded.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] assay
Cell proliferation rate and effect of cytotoxic agent [dimethyl sulphoxide (DMSO)] were determined by MTT assay (Roche). NIH3T3 cells were seeded into 96-well plates at a density of 50,000 cells per well in complete medium. MTT reagent was added in culture in each well after 24 h of incubation with different concentrations of DMSO. The optical density values were analyzed 4 h after the MTT reaction using Multiskan PC (Thermo Lab), and cell survival curves were then plotted against time in culture.
Cells were trypsinized and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) solution. A histological examination was carried out by standard histological techniques. Fixed cells were stained with H&E stain, and observations were recorded under a light microscope.
Transfection and GFP labeling
Before carrying out an in vivo teratoma assay, we transfected NIH3T3 cells with GFP harboring plasmid p-eGFP-N1 (Clontech), so that the cells could be differentiated from those of the host. One hundred thousand cells were transfected with 1 μg p-eGFP-N1 plasmid DNA using lipofectamine 2000 (Invitrogen) as a transfecting agent in a 1:3 volume ratio in a 3.5 cm2 dish, according to the manufacturer’s protocol. Following the transfection, stably GFP expressing clones were selected by growing them in media containing G418 at 300 μg/mL concentration for the first 2 days and thereafter in 900 μg/mL G418 for the next 7 days. Clones were purified from isolated colonies using clonal discs and scaled up for subcutaneous transplants with agarose plugs in Balb/c mice.
For each graft, ∼0.2 million GFP expressing NIH3T3 cells, washed and resuspended in 300 μL DMEM complete medium, were injected subcutaneously into Balb/c mice (maintained in MSU in-house animal house facility) in 1.5% melted agarose [28–30]. Visible tumors, after 2 weeks of transplantation, were dissected and fixed overnight with 4% PFA solution. The tissues were then paraffin embedded, sectioned, stained with H&E, and, the sections were examined for the presence of cells that were representatives of all three germ layers produced by NIH3T3-eGFP cells .
ALP staining of spheroids of both NIH3T3 and mBMSC origin was carried out with BCIP/NBT stain. Cell clusters were mechanically isolated, and cells were dissociated with trypsin EDTA digestion for 5 min at 37°C. After washing with 10% FBS and then with PBS, the cells were fixed with 2% PFA solution for 5 min and stained with NBT/BCIP . 3T3L-1 cells served as controls.
An immunocytochemical analysis was performed for various stem/progenitor markers in both cell types as previously described [32,33]. Cells were seeded on glass coverslips in high-glucose DMEM complete medium and allowed to adhere for 3–4 h. After attachment, cells were fixed with 4% PFA solution for 10 min at room temperature. After permeabilizing them with 0.1% Triton X-100 in PBS for 5 min, the cells were incubated in blocking buffer (1% bovine serum albumin and 4% FBS in PBS) for 1 h at room temperature. Primary antibodies were added in blocking solution, and coverslips were incubated overnight at 4°C. A list of the antibodies used and their dilutions are given in Table 1. Cells were then washed thrice with washing buffer (1:10 dilution of blocking buffer in PBS) and incubated with secondary antibodies conjugated to FITC and TRITC flurophores (Sigma Aldrich) in the dark for 30 min at room temperature. For negative controls, cells were incubated with normal IgG. Cells were mounted with Vectashield mounting medium containing 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories, Inc.). Fluorescence imaging was carried out by using a Nikon TES2000 microscope. All images were analyzed with the help of NIH element software supplied by Nikon Japan.
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