Reprogramming of differentiated cells of various mammalian species to apluripotent state is possible due to the overexpression of four transcriptionfactors. These factors are Oct4, Sox2, Klf4, and c-Myc (OSKM). The genes areevolutionarily conserved in mammals [5,6], and their products have asubstantially overlapping set of key target genes, and thus genetic constructsexpressing human or mouse OSKM can be frequently applied for cell reprogrammingin different mammalian species [7-10]. To date, iPSCs of mouse, human, macaque,rat, dog, many farm animals and some other mammalian species, including prairievole Microtus ochrogaster, have been obtained. The conditionsfor induction and maintenance of pluripotency vary among the species [11-19].This is partly due to the species-specificity of the signaling cascadesinvolved in the activation and maintenance of an undifferentiated statein vitro, and due to various requirements for the compositionof the culture medium, e.g. the presence or absence of bovine serum in themedium. Induction and maintenance of pluripotency are facilitated by thepresence in the medium of inhibitors of various signaling pathways, inhibitorsof histone deacetylases, histone and DNA methyltransferases, as well asantioxidants.
In the first experiment, two media were used for the induction of pluripotencyin M. levis M. arvalis hybrid cells,which were previously used to obtain M. ochrogaster iPSCs[16]. The media were prepared from Advanced DMEM/F12 (1:1)(Gibco) containing 15% KSR (Knockout Serum Replacement; Gibco), 1 NEAA,0.1 mM 2-mercaptoethanol, 1 Pen Strep, and 1 GlutaMAX. The firstmedium was supplemented with three inhibitors of signaling pathways (3iRconditions): CHIR99021 (3 μM, StemRD), PD0325901 (1 μM, StemRD),A83-01 (0.5 μM, Stemgent), and ROCK inhibitor (Y-27632, 10 μM,StemRD); the second medium did not contain any inhibitor. Both media contained1,000 u/ml mouse LIF (mLIF, Invitrogen) and 2 μg/ml doxycycline (Sigma),which was added only at the initial stages of cultivation. Mouse embryonicfibroblasts mitotically inactivated by a mitomycin C solution (10 mg/ml, Sigma)for 2 h were used as a feeder substrate for the generation and cultivation ofvole iPSCs. Transfer of individual primary colonies at the first passage wasperformed with a glass capillary, and further using TrypLE (Gibco).
Siberian mouse m 41
Three plasmids were used in the experiments: 1) TetO- FUW-OSKM (Addgene Plasmid20321) encoding mouse reprogramming factors OSKM; 2) FUdeltaGWrtTA (AddgenePlasmid 19780) carrying tetracycline transactivator cDNA necessary forregulation of the transcriptional activity of the construct with OSKM bysupplementing the growth media with doxycycline; 3) pGpur expressing a greenfluorescent protein (EGFP) gene for monitoring transduction efficiency.Lentiviruses were obtained by cotransfection of the plasmids into 293FT cellswith pxPAX2 (Addgene Plasmid 12260) and pMD2.G (Addgene Plasmid 12259) plasmidsencoding the proteins required for packaging of viral particles [34, 35]. Human embryo kidney cells, 293FT, were seeded at adensity of 6 106 into T75 culture flasks and incubatedovernight. Transfection was performed by the calcium phosphate method [36]. The medium with viral particles washarvested and filtered (0.45 μm; Millipore) 48, 72, and 96 h aftertransfection. Viruses were concentrated using an ultracentrifuge (BeckmanCoulter, Optima XE-90 Ultracentrifuge) for 90 min at 70, 000g. A viral pellet was dissolved in 200 μl of F12/DMEM andkept in aliquots at -70oC.
The fibroblasts and cells isolated from the brain were seeded in 12-well platesat a density of 50 103 to 75 103 cells perwell, respectively, 24 h before transduction. The cells were transduced for 4-6passages. On the day of transduction, the medium with lentiviruses obtainedusing TetO-FUW-OSKM, FUdeltaGW-rtTA, or pGpur plasmids was added for 14-16 h,with a titer of about 3 107 infectious units per 1 ml (MOI5-7.5) for each of the lentiviruses and 4 μg/ml polybrene (Hexadimethrinebromide, Sigma). After 4 days, cells transduced by lentiviruses withreprogramming factors and tetracycline transactivator were passaged usingTrypLE at a dilution of 1:10 to 1:20 (depending on cell density in a well) onmitotically inactivated mouse embryonic fibroblasts in culture media varying incomposition. For determination of transduction efficiency, cells transducedwith lentiviruses containing pGpur were used, and the assessment of thepercentage of green cells was performed using a fluorescent microscope and/orflow cytometry 4 days after transduction.
Cells were fixed with 4% formaldehyde for 10-15 min at room temperature, washedwith PBS, and incubated with 0.1% Triton X-100 and 2.5% FBS (or BSA) dissolvedin PBS for 30 min at room temperature. Immunoprecipitation was performed byusing primary antibodies overnight at 4oC. A list of the primary antibodiesused in this study is providedin Table 1.Localization of primary antibodies was visualized using secondary anti-rabbitor -mouse antibodies conjugated to the fluorescent dyes Alexa 488 and Alexa568 (Life Technologies). Nuclei were stained with DAPI (Vector Laboratories).
RNA was isolated with Trizol Reagent (Ambion). Samples were treated with DNaseI (Turbo DNA-free, Ambion) in order to prevent DNA contamination; cDNA wassynthesized using Super-ScriptIII (Invitrogen). Primer sequences and reactionconditions for RT-PCR are shownin Table 2. For thetranscription analysis of the exogenous lentiviral cDNA of OSKM in mouse cells,we used the primers and RT-PCR conditions listed in[34]. A negative control reaction (RT-),with the reaction solution containing all the components necessary for cDNAsynthesis, except for reverse transcriptase, was performed for every primer pair.
Common vole iPSCs were obtained using lentiviruses expressing cDNA of four keymouse reprogramming factors OSKM under a doxycycline-regulated promoter.Transcription from this promoter can be activated by adding an antibiotic inthe culture medium; however, the cells should be previously transduced withlentiviruses expressing cDNA of a tetracycline-dependant transactivator. Thissystem, where mouse OSKM expression is modulated by the addition ofdoxycycline, was successfully utilized previously for obtaining human and mouseiPSCs [34]. Mice and voles belong to thesame family of rodents (Muridae), order Rodentia, and exhibit high similarityof OSKM genes [40]. The advantage ofthis system, in our opinion, is the fact that all four reprogramming factorsare delivered into a cell by the same viral particle.
Pluripotency in common vole cells was induced in two types of media that hadpreviously been used for obtaining iPSCs from the fibroblasts of prairievole M. ochrogaster [16]. The first type of media included Advanced DMEM/F12 with15% KSR and mLIF, the second medium contained the same components, but it alsoincluded an inhibitor cocktail called 3iR. The cocktail 3iR contains inhibitorsof kinases MEK/ERK and GSK3b (PD0325901 and CHIR99021, respectively), anantagonist of the IL-type receptor TGF-s (A83-01) used for obtaining mouse andrat PSCs and maintaining them in undifferentiated state, and a ROCK inhibitorthat enhances the survival of single cells in culture [41-46].
Four days after transduction, M. levis M. arvalishybrid cells s were transferred on mitotically inactivated mouse embryonicfibroblasts and the medium with or without 3iR for obtaining iPSCs was added.The first morphological changes appeared 24 h after cell transfer, and anincrease in cell proliferation was noted. After 5-8 days, the transduced cellsthat were isolated from the brain began to merge into primary homogeneouscolonies (Fig. 1D),whereas the transduced fibroblasts did notgive rise to such PSC-like primary colonies(Table 3).
Primary colonies obtained during brain cell reprogramming were transferred intoindividual wells by disaggregation with a glass capillary on day 10-13 afterthe start of the experiment. Cell morphology remained the same as in the caseof mESCs: threedimensional dense colonies with tight intercellular contacts(Fig. 1E,F).The cells in these colonies had a highnuclear/cytoplasmic ratio, which is a characteristic of ESCs. After transfer ofprimary colonies, most cells underwent differentiation on the first passage.However, about 40% of the cells retained an ESC-like morphology. The differencein the level of endogenous AP was detected in selected colonies by histochemicalstaining (Fig. 1G).Cell lines with no detectable AP activity were excluded from further analysis.Thus, a total of 11 cell lines obtained in the presence of inhibitors (viBr; voleinhibitor Brain) and 10 lines obtained in their absence (vBr) were selected basedon the results of an AP activity analysis at early passages. It should be notedthat most of the cells did not express one of the major early markers of pluripotencyspecific to mouse PSCs, surface antigen SSEA1. The iPSC-like cells underwentrapid proliferation in the medium containing doxycycline without altering themorphology for approximately 40 passages (more than 120 days). The cells werepassaged every 2-3 days at a dilution of 1:8 to 1:10: they resisted repeatedfreezing in liquid nitrogen with further thawing without changing theirphenotypic characteristics.
Our experience shows that mouse OSKM, which is used for the induction ofpluripotency in somatic cells of different types and in different species, canbe effective in obtaining common vole iPSCs. However, the presence of LIFcytokine in the medium, an important factor in the formation and maintenance ofundifferentiated state in ESCs and iPSCs in rodents (mouse, rat, as well asprairie vole) [16, 57-59], is notsufficient for the induction and maintenance of pluripotency in common volecells in vitro. This result is consistent with the reports ofunsuccessful attempts to obtain ESCs of common vole group species from theinner cell mass of blastocysts in the presence of mouse or M. levisLIF [31]. Taken together, thesedata allow one to suggest that the signaling pathway triggered by cytokine LIF,due to some species-specific features of the common vole, cannot independentlyprovide pluripotency in in vitro conditions. 2ff7e9595c
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