KR20190040479A - Method of confirming that direct reprogramming cell is in sate pluripotent stem cell - Google Patents

Abstract

The present invention relates to a method for confirming whether or not a pluripotent state has been passed through during a production process of an induced neural stem cell. The present invention can be effectively used not only for disease modeling related to the nervous system, neural stem cell differentiation and studies on development mechanism, but also as a technique for establishing an induced neural stem cell that does not go through a pluripotent state in the development of a cell therapy agent using the induced neural stem cell.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for identifying a cross-differentiated cell using a pluripotent cell,

More particularly, the present invention relates to a method for confirming whether or not a pluripotent state has passed through the production process of an inducible neural stem cell.

Recently, research into the technique of transforming cell traits (hereinafter referred to as cross-differentiation) by introducing a systemic-specific transcription factor into somatic cells has been actively conducted. It is known that the cross-differentiation technique does not pass through the universal state, unlike the method in which an existing patient-specific therapeutic cell is differentiated into the desired target cell through an induced pluripotent stem cell state. Therefore, it is evaluated as a merit of cross-differentiation technology that the problem of tumor formation due to the incorporation of undifferentiated residual cells can be excluded from the clinical viewpoint, the time taken to produce specific cells is short, and the process is simple. Thus, studies have been conducted to induce somatic cells or progenitor cells of other lines using patient-specific fibroblasts or urine cells.

Neural stem cells are a type of precursor cells that can differentiate into three representative cells of the brain, neurons, glial cells, and oligodendrocyte. Can be continuously generated. Using the above-mentioned cross-differentiation technique, direct neural stem cells are produced from somatic cells, and preclinical and disease mechanism researches are actively conducted based on this.

We used a series of studies to replace Oct4, one of the genes used in the induction of pluripotent stem cells, with Brn4, and introduce Klf4, Sox2, and cMyc genes into somatic cells using retroviruses to cross somatic cells with neural stem cells And the developed induction neural stem cells showed very similar pattern and function to the neural stem cells derived from the body in various aspects such as morphology, gene expression pattern, womb genetic pattern, electrophysiological characteristics, in vitro and in vivo differentiation ability.

In 2015, other researchers applied the systematic tracking system to confirm that the induction neural stem cells produced by crossing differentiation were made through the universal state. In this study, we used a polycistronic vector to transduce the neural stem cell-specific transcription factors Brn4 and Klf4, Sox2, and cMyc (BKSM), which were used in this study, to produce inducible neural stem cells. All of the induction neural stem cells were reported to have passed through the universal state.

In this study, we observed that BKSM, which is introduced through poly-systolic vector and individual vector, induces the conversion of somatic cells into inducible neural stem cells. We applied two systematic tracing systems to induce pluripotent stem cells Respectively.

Accordingly, the inventors of the present invention have established a method for verifying the universal state of cross-differentiated cells and solved the problems of the prior art as described above.

Accordingly, the present invention provides a method for confirming whether or not a pluripotent state has passed through the production process of induction neural stem cells.

The objects of the present invention are not limited to the above-mentioned objects, and other objects and advantages of the present invention which are not mentioned can be understood by the following description, and will be more clearly understood by the embodiments of the present invention. It will also be readily apparent that the objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

One aspect of the present invention is a universal state dystrophic identification method of crossed differentiating cells comprising the steps of:

(Cre-recombinase) gene coding sequence operably linked to the Oct4 promoter, a loxP coding nucleic acid sequence removed by the CRE gene activation, and a somatic cell containing a fluorescent protein coding nucleic acid sequence expressed upon loxP removal Preparation phase;

A transfection step of introducing into said somatic cell each vector that individually codes a nucleic acid sequence encoding Brn4, Klf4, Sox2 and cMyc; And

And determining that the crossed differentiated cells are generated via the pluripotent state when the somatic cells are cultured and fluorescence is expressed.

The fluorescent protein may be EYFP (Enhanced yellow fluorescent protein).

The somatic cells may be fibroblasts.

The cross-differentiated cells may be induction neural stem cells.

According to the present invention as described above, not only can it be used for disease modeling related to the nervous system, neural stem cell differentiation and development mechanism studies, but also induction of inducible neural stem cells that do not go through a universal state in the development of a cell therapy agent using induction neural stem cells There is an effect that can be utilized as technology.

FIG. 1A is a schematic diagram showing a tracking system for directly crossing differentiation from fibroblasts to iNSCs using Oct4-CreER. FIG.
1B is a photograph showing the shape of the first iNSC cluster and the generated iNSC from the MEF transformed with pcOKSM and pcBKSM.
FIG. 1C is a graph showing the expression of NSC and fibroblast-specific markers in the iNSC line transformed with pcOKSM and pcBKSM through qPCR analysis.
Figure 1D is a photograph of immunofluorescent staining of iNSC lines transformed with pcOKSM and pcBKSM.
FIG. 1E is a FACS analysis result for confirming the number of iNSCs generated directly (Olig2 + / EYFP-) or indirectly (Olig2 + / EYFP +) on the iNSC line transformed with pcOKSM and pcBKSM.
1F is a heat map analysis showing gene expression patterns of iNSC line transformed with pcOKSM and pcBKSM. Hierarchical clustering analysis based on gene expression profiles is displayed at the top. FPKM = 1 was drawn from at least one sample and a differentially expressed gene with FC = 4 between MEFs and the cNSC control.
Figure 1G is a photograph of immunofluorescent staining of EYFP-pcOKSM and pcBKSM iNSC.
1 h is a graph showing the results of qPCR analysis of EYFP-iNSCs.
FIG. 2A is a photograph of the result of immunofluorescence staining for the iNSC line generated by clonal mcBKSM.
Figure 2B shows the hierarchical clustering heatmap of the global gene expression pattern for fibroblasts and NSC control against the dicrobial mcBKSM iNSC line.
FIG. 2C shows the DNA methylation status of the promoter region of Nestin's second intron and Col1a1 in fibroblasts, NSC control, and four mcBKSM-mediated iNSC clones by bisulfite sequencing PCR.
Figure 2d is a graph of qPCR analysis of expression levels of individual retroviral transgene genes in an established clonal mcBKSM-mediated iNSC line (passage 5).
Figure 2E is a FAC analysis showing the number of iNSCs generated directly (Olig2 + / EYFP-) and indirectly (Olig2 + / EYFP +) in the clonal mcBKSM iNSC line.
Figure 2f shows the results of FACS analysis after 25 days of infection to determine the mass population of mcOKSM- or mcBKSM-transferred MEFs of the clonal mcBKSM iNSC line.
Figure 3a is a graph of time course qPCR analysis of versatile markers in MEF transduced with pcOKSM, pcBKSM, mcOKSM or mcBKSM.
Figure 3b is a graph of time course qPCR analysis of NSC-markers in MEF transduced with pcOKSM, pcBKSM, mcOKSM or mcBKSM.
Figure 3c is a schematic diagram showing the generation of a series of reciprocal Oct4-Brn4 chimeras by domain exchange.
FIG. 3D is a graph of the number of GFP + colonies counted to determine the reprogrammed efficiency of MEFs with Oct4, Brn4 or Oct4-Brn4 chimeric proteins.
Figure 3e shows the results of FACS analysis of the whole cell population three weeks after transfection into MEF with mcOKSM or mcBKSM.
FIG. 3f is a graph showing the results of qPCR titration of the retroviral transgene genes Oct4, Brn4 and the retroviral vector expressing the chimeric construct.
Figure 3g is a representative image of Oct4-GFP MEF reprogrammed 8 or 14 days after induction with tet-inducible pcOKSM, pcBKSM or pOct1-KSM.
Figure 3h is a graph of reprogramming results of tet-inducible pPOU-KSM construct and Oct4-GFP MEFs using dox-induced high (H), intermediate (M), and low (L) levels.
Figure 3i is a schematic diagram illustrating branching pathways of somatic cells to iNSC achieved by reprogramming into a poly and monosystronic delivery system.
Figure 4A is a schematic diagram showing that the F2A self-cleaving peptide between the POU factor and Klf4 in the STEMCCA structure was replaced with a more efficient self-cleaving factor P2A, a mutable F2A mutant (F2Am) or a synthetic glycine linker (GL).
Figure 4B is a Western blot analysis of FLAG antibodies against MEF transduced with 3xFLAG and N-terminal labeled POU factor with Oct4 and Brn4-Klf4 cassettes isolated from Klf4 by wild-type P2A, F2A or non-cleavable F2Am peptide .
Figure 4c is a photograph of the clonal iPSC line generated by the POU3-F2Am-KSM cassette after 5 passages.
4D is a photograph of PCR result of the clonal iPSC line generated with the POU3-F2Am-KSM cassette.
Figure 4e shows the reprogramming efficiency of Oct4-GFP MEF with POU-Klf4 construct linked by different peptides with KSM was determined by counting GFP + colonies after 1 and 2 weeks of induction.
Figure 4f is a graph showing a combination of Oct4-GFP MEFs with fully cutable Oct4 and Brn4-P2A-KSM polystyrene cassettes and additional constructs attempting to restore Brn4-P2A-KSM deficient in reprogramming and reprogramming to be.
FIG. 4g is a graph showing chromatographic immunoprecipitation (ChIP) -qPCR analysis of anti-FLAG antibody of MEF reprogrammed on day 4 with 3xFlag-POU-Klf4 together with KSM.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Linear tracking system Fibroblast to iNSCs  Direct conversion

Whether or not an iNSC can be obtained through a direct differentiation process is questionable. Because we did not use genetic tracing in the known studies, we were able to miss the versatile state of reprogramming experiments. Polysystolic lentiviral vectors based on the widely used STEMCCA reprogramming cassettes were also used. Therefore, we decided to compare two gene delivery systems using genetic tracing for the expression of endogenous Oct4, a crucial indicator of versatility. In the first set of experiments, mouse embryonic fibroblasts (MEFs) carrying a tamoxifen-inducible Oct4-CreER allele with Rosa26-loxSTOPlox-diphtheria toxin A fragment allele (R26-ls1-DTA) were used.

All of the mice used were kept and kept in the mouse facility of Konkuk University (KU), and animal handling was carried out in accordance with the KU animal protection guidelines. Oct4-CreER mouse (stock number: 016829, Jackson Laboratory) was crossed with Rosa26-lox-STOP-lox-EYFP mouse (stock number: 006148). MEFs were obtained from Oct4-CreER X Rosa26-loxSTOPlox-EYFP mouse embryos at 13.5 days after fertilization, after careful removal of all internal organs including head and spinal cord. The correct genotype was maintained in 500 ml of MEF medium in DMEM (Biowest) containing 10% FBS (Biowest), 5 ml penicillin / streptomycin / glutamine (Invitrogen) and 5 ml MEM NEAA solution (Invitrogen). The MEF of Oct4-CreER x Rosa26-loxSTOPlox-DTA mouse was obtained from Prof. Konrad Hochedlinger (Harvard Stem Cell Institute).

The extracted somatic cells express Cre-recombinase by the Oct4 promoter when expressing Oct4. The produced Cre-recombinase is activated under 4-OHT treated conditions to recognize and remove loxP. Finally, the stop codon is removed together with loxP, and Diphtheria toxin A (DTA) gene is expressed. DTA inhibits the protein synthesis process of ribosome and induces the cell. In other words, this double gene control system leads to apoptosis when at least one expression of Oct4 is expressed.

5 x 10 ⁴ MEFs were infected with lentivirus encoding for pcOKSM or pcBKSM to generate iNSC using the polystronic vector. After 24 hours of incubation, the medium was replaced with NSC medium containing 3 ug / ml of dox. After 7-8 days of induction with pcOKSM or pcBKSM, cells were grown in NSC medium without dox treatment for 23-24 days to inflate the initial iNSC clusters. 1 uM of 4-hydroxy tamoxifen (4-OHT) was added successively to the culture medium.

To generate individual retroviral vector-mediated iNSCs, MEFs were transfected with individual retroviral particles encoding mcOKSM or mcBKSM and routinely cultured. Briefly, 5 × 10 ⁴ fibroblasts were plated on gelatin-coated 35 mm dishes and cultured with eco-friendly retroviruses. After 48 hours of incubation, the medium containing the retrovirus particles was replaced with NSC medium. To enrich iNSC, unreprogrammed fibroblasts or inappropriate cells were removed.

INSC bulk cultures were stained for SSEA1 to establish clone iNSC lines and SSEA1 + single cells were sorted using BD FACSAria (BD Biosciences) and plated on laminin / poly-D-lysine-coated 96-well plates Plated. DMEM / F-12 supplemented with 2% B27 (Gibco), 10 ng / ml EGF (Peprotech), 10 ng / ml bFGF (Peprotech) and 1% BFGF was maintained in NSC culture medium.

For differentiation into neurons, iNSCs were plated on 2.5 x 10 ⁴ cells / cm ² laminin / polylysine-coated plates in NSC medium. The following day, the medium was replaced with DMEM / F-12 supplemented with neural differentiation medium: 2% B27 (Gibco), 1% penicillin / streptomycin / glutamine (Invitrogen) and 10 ng / ml bFGF (Peprotech). On the fourth day of differentiation, the medium was further cultured for 8-10 days with neural differentiation medium containing 200 mM ascorbic acid (Sigma) without growth factor. For differentiation into astrocytes, iNSCs were cultured in gelatin-coated culture dishes for 5 days in DMEM / F-12 supplemented with 10% FBS and 1% penicillin / streptomycin / glutamine.

Finally, differentiated into oligodendrocytes, iNSCs were plated on laminin / polylysine-coated plates at 2.5 x 10 ⁴ cells / cm ² in NSC medium. The following day, the medium was replaced with DMEM / F-12 supplemented with 2% B27, 1% penicillin / streptomycin / glutamine, 10 ng / ml bFGF and 10 ng / ml PDGF (Sigma). On the fourth day of differentiation, the medium was further cultured for 4 days with a ridgeline glue cell differentiation medium containing 30 ng / ml of T3 (Sigma) and 200 mM ascorbic acid. Differentiation badges were replaced every other day.

No morphological changes were observed in the control MEF. This whole cell was expanded to a stable iNSC line expressing the NSC marker (hereinafter DTA-iNSCs, Fig. S1C-S1D). Analysis of this lineage genotype revealed that pcBKSM, Oct4-CreER and R26-lsl-DTA were integrated and rarely survived DTA-iNSC did not pass through the pluripotent state.

All genome transcript profiling results showed that the gene expression pattern of DTA-iNSCs was similar to the control NSC, but different from that of MEF-initiated gene expression. The in vitro potency of DTA-iNSC to produce neurons, astrocytes and rare dendritic glial cells was verified, as evidenced by immunostaining for Tuj1, GFAP and MBP, respectively.

Finally, DTA-iNSC labeled with 1 Х 10 ⁶ GFP was transplanted into the rat cerebral cortex to examine the in vivo function of the resulting cells. After 4 weeks of transplantation, the transplanted DTA-iNSCs were differentiated into three neuronal cell types: neurons (GFP + / DCX1 +), astrocytes (GFP + / GFAP +) and rare dendritic glial cells (GFP + / NG2 + . As in previous studies, no evidence of tumor formation was found in any of the cells of the transplanted rats.

Taken together, this suggests that MEFs can be converted directly into functionally mature iNSC without necessarily passing the Oct4 + iPSC similarity. However, considering that the conversion efficiency of the direct conversion process (2 iNSC lines in 10 individual infections) is relatively low, we exclude the possibility that a small number of Oct4 + cells, which were not activated due to incomplete Cre recombination, contributed to iNSC I can not.

Example 2: iPSC similarity induced by polysystronic BKSM

To quantify the number of induced neural stem cells (iNSCs) generated by direct or indirect reprogramming, a systematic tracking system carrying R26-IL-EYFP was used instead of DTA as in Figure 1A. The system examined the activation of the EYFP reporter and made it possible to distinguish between directly and indirectly produced iNSCs.

Specifically, by crossing a transgenic mouse (Oct4-CreER) carrying the Oct4-CreER allele with a gene-knocked mouse (R26-lsl-EYFP) carrying the Rosa26-loxP-stop-loxP- A double genetically engineered mouse bearing the Oct4-CreER allele and the Rosa26-lsl-EYFP gene is generated. The generated double-genetically engineered mice are isolated from the mother after 13.5 days of fertilization and extract normal somatic cells. The extracted somatic cells express EYFP (Enhanced yellow fluorescent protein) when Oct4 is expressed under 4-OHT treatment conditions as described above. That is, in this double gene control system, expression of EOFP is expressed when at least once Oct4 is expressed.

BKSM (Brn4 + Klf4 + Sox2 + cMyc) was introduced into MEFs (mouse embryonic fibroblats) for 48 hours via a retroviral individual vector or by using a poly-systic vector, and then neural stem cell specific And cultured in Neural stem cell culture medium (NSC). After about 3 weeks, it was confirmed that induction of neural stem cells with neuronal morphology was observed. During 5 to 7 weeks, EYFP was not expressed by flow cytometry, and only SSEA1-expressing cells were selectively isolated to induce inducible neural stem cell line Respectively.

As shown in Fig. 1b, typical NSC forms (scale bar, 50 [mu] m) after two weeks of pcOKSM (polyclonal, Oct4 + Klf4 + Sox2 + cMyc) and pcBKSM induction were observed.

Cells were fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature for immunofluorescence staining and then stained with Dulbecco's (Sigma) solution containing 0.3% Triton X-100 (Sigma) and 5% FBS gt; PBS < / RTI > (Biowest). The cells were then incubated with the primary antibody for 16 h at 4 째 C, washed three times with PBS and then incubated in the dark room for 2 h at room temperature with the appropriate fluorescent-conjugated secondary antibody. Nuclei were stained with Hoechst 33342 (Sigma) (mouse anti-Nestin (Millipore, 1: 200), goat anti-Sox2 (Santa Cruz Biotechnology, 1: 200), mouse anti- SSEA1 (Santa Cruz Biotechnology, , Rabbit anti-Tuj1 (Covance, 1: 500), rabbit anti-GFAP (DAKO, 1: 500), rat anti- MBP (Abcam, 1: 100) and rat anti-NANOG (eBioscience, eBioMLC-51, : 1000)).

As can be seen in Figures 1c and 1d, the first cell clusters (6-8 sites per individual infection) were observed to generate a stable iNSC line (scale bar, 50 um) representing a typical NSC gene expression profile.

Fluorescence activated cell sorting (FACS) was used to quantify the activation of EYFP transgene and Olig2 expression on pcOKSM or pcBKSM-producing iNSCs.

Specifically, the cells were dissociated with trypsin, washed once with PBS and resuspended in FACS buffer (PBS containing 5% FBS). 1 × 10 ⁶ cells were incubated with FITC-conjugated antibodies incubated for 15 min at 4 ° C against anti-SSEA1 (Santa Cruz Biotechnology, 1:10). Cells were washed once with FACS buffer and resuspended in PBS for analysis using BD FACSAria (BD Biosciences).

As shown in FIG. 1e, in both cases, the majority of iNSCs were found to be positive for EYFP (96.1 and 97.9%, respectively) and Olig2 (both 99% or more for Olig2 +). In particular, the small recombinant EYFP-iNSC population (Olig2 + / EYFP-) was consistently observed in both reprogramming conditions, indicating that it could represent directly transformed cells.

As shown in FIG. 1F, the iNSC line transformed with pcOKSM and pcBKSM was analyzed by heatmap for fibroblast and NSC control. As a result, the identity of iNSC was confirmed to be close to that of NSC by whole genome transcription profiling. FC = 4 between MEFs and cNSCs, and FPKM = 1 in at least one of the samples.

As can be seen in Figures 1g and 1h, EYFP-iNSC cells obtained with pcOKSM or pcBKSM expressed NSC marker genes as well as EYFP + iNSC. Thus, few pcOKSMs and pcBKSM-iNSCs may have been directly metastasized (scale-bar, 50 μM) in MEFs without going through pluripotent states.

However, it could not be reliably excluded that such EYFP-iNSC cells exhibited incomplete excision by Cre.

Example 3: Individually transduced BKSM can not induce multipotency.

With reference to the results of the poly-systolic gene delivery system, it was decided to repeat the phylogenetic tracing experiments using monocystronic retroviral vectors encoding Brn4, Klf4, Sox2 and cMyc (mcBKSM).

The first iNSC cluster after infection for 2 to 3 weeks was observed (~ 10 clusters per individual infection). By classifying SSEA1 + cells from mcBKSM-transfected MEFs, we established four clone iNSC lines from four independent experiments.

As can be seen in FIG. 2a, iNSCs showed a similar pattern to NSC but differed from fibroblasts (scale bar, 50 .mu.m).

As can be seen in Figure 2b, FC = 4 between MEFs and cNSCs, and FPKM = 1 in at least one of the samples. iNSCs showed gene expression similar to NSC but different from fibroblasts.

Genomic DNA was treated with sodium bisulfite to determine the DNA methylation status of iNSCs and all non-methylated cytosine residues were converted to uracil residues using the EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer's instructions. Briefly, PCR amplification was performed using a SuperTaq polymerase (Ambion) at a total volume of 25 μl and a denaturing 40 cycle protocol annealed at the appropriate temperature for each target site for 30 seconds at 94 ° C, 5 minutes at 94 ° C Denatured at 72 ° C for 10 minutes, and lengthened at 72 ° C for 30 seconds. For each primer set, 3 ul products from the first round of PCR were used as templates in the second round of PCR. The amplified product was confirmed by electrophoresis on 1% agarose gel. The PCR products were subcloned using PCR 2.1-TOPO vector (Invitrogen). The reconstituted plasmid was purified using QIAprep Spin Miniprep Kit (QIAGEN) and sequenced of individual clones (Macrogen, Korea). Clones were analyzed using QUMA software.

As can be seen in FIG. 2C, promoter methylation of Nestin and Col1a1 similar to gene expression and NSC was shown, but it was different from fibroblasts. Open or filled circles showed unmethylated or methylated CpG, respectively.

As can be seen in Figure 2d, the retroviral transgene was found to be silenced in fully reprogrammed iPSCs and in iNSCs produced by reprogramming by pcOKSM or pcBKSM, whereas early mcBKSM iNSC had largely active retroviral transgenes. This result is consistent with previous studies showing that the retroviral transgene is stochastically silent in the iNSC line established after only two months of culture. This level was normalized to the level of MEF 5 days after transduction. The data are expressed as means ± SD, n = 3.

The iNSCs generated by FACS were analyzed to determine whether the MEFs induced by mcBKSM were converted directly or to the iNSCs via the Oct4 + intermediate step.

As can be seen in Figure 2E, surprisingly, the iNSC clones extracted from mcBKSM did not detect EYFP + cells but found Olig2 + in most cells. This indicates that all iNSCs derived by mcBKSM are the result of direct programming.

To eliminate the possibility that Olig2 + / EYFP-clone mcBKSM iNSC lines were selectively enriched from a mixture of directly and indirectly generated iNSC, bulk cell populations were analyzed after 25 days of reprogramming with mcBKSM.

As can be seen in Figure 2f, again all cells were found to be EYFP-. Taken together, it shows that iNSC can be obtained either directly by mcBKSM or indirectly through the multifunctional state by pcBKSM.

To exclude the possibility that contaminated neuronal crest cells or other residual neuronal populations are enriched by our exogenous reprogramming factors and contribute to the production of iNSCs, we next used Thy1 + (fibroblasts) and Thy1- (non-fibroblast- ) Cells were introduced and mcBKSM was introduced. After three weeks of infection, the Thy1 + population slightly reduced the number of iNSC clusters, but iNSC clusters expressing Olig2 could be found in both Thy1 + and Thy1- populations. This indicates that iNSCs can be generated from pure fibroblasts through a direct conversion strategy.

Example 4: Reprogramming path to iNSC

We decided to explore the reprogramming kinetics of each system to elucidate molecular reactions mediated by lentiviral pcBKSM versus retroviral mcBKSM reprogramming. To achieve the maximal induction level of transcription factors in both systems, expression was titrated using MEFs transduced with pcBKSM or pcOKSM and different volumes of dox of intensive retroviruses encoding mcBKSM or mcOKSM. Lentiviral expression levels of MEFs transduced with the polycistronic vector increased in a dose dependent manner and stagnated at 3 ug / ml of dox. On the other hand, higher concentrations of dox resulted in widespread apoptosis induced by overexpression. The induction level of retroviral transgenes was saturated at a volume of 10 ul. Using 3 μg / ml dox in polycistronic lentiviral vectors (pcBKSM and pcOKSM) and 10 ul of each retrovirus (mcBKSM or mcOKSM) for kinetic analysis, taking into account the induction level of the transgene and the survival of transduced MEFs Respectively. We also confirmed similar induction levels of Brn4 and Oct4 in both the polycistronic lentiviral and the monocistronic retroviral vector at the protein level.

Fibroblast markers were rapidly inhibited in MEFs after 1-2 weeks of induction, regardless of the system used.

As can be seen in FIG. 3A, the MEFs transduced with mcBKSM in the R26-IL-EYFP lineage tracing system, as already confirmed using Oct4-CreER, are endogenous, unlike pcBKSM-, mcOKSM- and pcOKSM- transduced MEFs The activity marker was not activated. This level was normalized to the level of non-transgenic ESCs and was expressed as mean ± SD, n = 3.

As can be seen in Figure 3b, mcBKSM-transduced MEFs strongly activated NSC markers such as Blbp and Olig2 after two weeks, but MEFs transfected with pcBKSM, pcOKSM or mcOKSM were activated after one week. This level was normalized to the level of non-transgenic NSCs control, respectively, and was expressed as mean ± SD, n = 3.

Similar trends have been observed in lentiviral mcBKSM-transduced MEFs and as a result of lentiviral mcBKSM transduced MEFs iNSCs were also EYFP-. Thus, mcBKSM can activate NSC markers at MEF faster and at a higher level than pcBKSM or OKSM, but not up-regulate endogenous versatile markers such as Oct4.

Example  5: Oct4  And Of Brn4  Structural dissection Brn4 Versatility  Indicates unable to induce

Immediately after the invention of iPSC technology, Oct4 appeared as the only reprogramming factor for reprogramming cocktails that could not be replaced by other members of the same transcription factor family. Conversely, the introduction of poly- sistronic Brn4 into fibroblasts showed that endogenous Oct4 could be activated and chimeric grade iPSC could be produced. Both Oct4 (Pou5f1) and Brn4 (Pou3f4) belong to the POU transcription factor group (IV and III, respectively), and N- and C-terminal transactivation regions (NTD and CTD). The POU domain itself consists of a POU unique (POUsp) and a POU home domain (POUhd) connected by the non-persistent linker.

The two POU factors consist of five domains: the N-terminal transactivation domain (NTD), the POU specific domain (POUsp), the linker, the POU homeodomain (POUhd) and the C-terminal transactivation domain (CTD). O or B indicates that the domain is Oct4 or Brn4, respectively.

As shown in Figure 3c, a series of reciprocal Oct4-Brn4 chimeras were generated by exchanging their domains to determine which portion of Oct4 and Brn4 was responsible for reprogramming somatic cells multipotentially.

Specifically, the Oct4-Brn4 chimeric library was generated using an overlap extension assembly of two or three PCR fragments with overlapping ends of about 20 bp in length as previously described. The final chimeric PCR product was inserted into the pMX backbone using EcoRI and XhoI restriction sites and sequenced.

pHAGE2-tetO-OKSM (STEMCCA) and pHAGE-tetO-BKSM lentiviral vectors were prepared. All other lentivirus expression cassettes used in this study were cloned into the same backbone. The last two residues constituting the self-cleaved Pro-Gly site were mutated to Ala-Ala in order to completely disable the self-cleaving ability of the F2A peptide. The following P2A self-cleaving peptide and poly-glycine linker sequences were used: GSGATNFSLLKQAGDVEENPGP and GSGGGSGGGSGGGGSGGGGSG.

The chimera then tested its ability to generate iPSCs with SKM using Oct4-GFP reporter MEFs and individual retroviruses. Specifically, Oct4-GFP MEFs were reprogrammed into iPSCs using Oct4, Brn4 or Oct4-Brn4 chimeric proteins with SKM. The efficiency was measured by counting the number of GFP + colonies after 2 weeks and 3 weeks. The data are expressed as means ± SD, n = 3.

Oct4-GFP (OG2 MEFs or heterozygous ROSA26-rtTA / GOF18) MEFs were used after 3-4 passages for iPSC generation. MEFs were plated on 12-well plates at a density of 2.5-3x10 ⁴ per well in 1 ml of fresh MEF medium. On the same day, virus supernatants were added to the cells on the same day with 60 ul of non-enriched tetO-OKSM and 30 ul of rtTA (for OG2 MEFs) plus 6 ug / ml protamine sulfate (Sigma-Aldrich). The volume of the remaining structures was adjusted according to Q-PCR titration. After 48 hours, the medium was replaced with embryonic stem cell medium (high glucose DMEM, 15% KSR, LIF) supplemented with dox (1 ug / ml). The numbers of GFP + colonies were counted 1 and 2 weeks after induction. Dox was removed after induction 12 days (two days before the second calculation).

Lentiviral stocks were quantified using Q-PCR for cDNA samples WPRE in MEFs 24 h after dox induction.

As can be seen in Figure 3D, unlike Oct4, wild-type Brn4 could not produce GFP + iPSC colonies. Interestingly, Oct4-Brn4 chimeras containing single domain substitutions from Brn4 (NTD, POUsp, linker, POUhd or CTD) still maintained their reprogramming function despite decreased efficiency for POUsp, POUhd and CTD. Notably, the POUsp domain appeared to be the most important for the reprogramming function of Oct4, as the replacement with the Brn4 counterpart (O-BOO-O chimera) dramatically (30-fold) reduces the reprogramming ability of the chimera. However, since a single domain of Oct4 alone could not provide reprogramming to Brn4, it implies that multi-domain cooperation of Oct4 is required for reprogramming functions.

As can be seen in FIG. 3E, FACS analysis of the bulk cell population three weeks after transduction indicated that wild-type Brn4 could not produce iPSC.

As can be seen in Figure 3f, the difference in reprogramming efficiency is not due to an imbalanced transgene expression as confirmed by the qPCR titration of the retroviral vector.

Taken together, the results show that Brn4 does not induce pluripotency due to functional incompatibility for the purposes of the three domains, of which the POUsp domain is the most important.

Unlike mcBKSM, pcBKSM activated endogenous Oct4 (R26-IL-EYFP MEF) that produced EYFP + iNSC from Oct4-CreER, and to determine if pcBKSM could generate fully reprogrammed iPSCs, Oct4-GFP MEF And the cells were cultured in ESC medium. Reprogramming efficiency was measured by counting GFP + colonies 1 and 2 weeks after induction.

As can be seen in Figures 3g and 3h, GFP + colonies were observed for both pcOKSM and pcBKSM 8 days after dox treatment, with remarkably similar reprogramming efficiencies (scale bar, 250 [mu] m). Other POU factors cloned in the same polytronic cassette, such as Oct1 and Oct6, did not produce iPSC (mean ± SD, n = 3), except for Brn2, which could lead to very low GFP + colonies.

As shown in FIG. 3i, although the polyistronic vector can induce fibroblasts to a pluripotent state, the individually expressed factors directly differentiate into iNSCs without inducing MEFs into a pluripotent state.

Example 6: Multipotency induction in reprogramming of Brn4-Klf4 fusion protein

A polycistronic reprogramming cassette (also known as STEMCCA) used in Bar-Nur et al. The present invention has transcription factors that are separated into two autolysis 2A peptides and an internal ribosome entry site (IRES) (POU-F2A-KIf4-IRES-Sox2-E2A-cMyc). The sequence of the reprogramming factor of the polysytronic cassette determines the stoichiometry and the other 2A peptides are known to have a pronounced self-cleavage efficiency. Indeed, the F2 peptide F2A used to link Brn4 and Klf4 of the original STEMCCA cassette and pcBKSM cassette exhibits the lowest autolysis efficiency. This suggests that Brn4-F2A-Klf4 can produce untreated high molecular weight polyproteins that can alter cell responses and alter reprogramming results.

As can be seen in FIG. 4A, the F2A peptide was replaced with P2A (the most efficient self-cleaving peptide) or the non-cleavable mutant F2A (F2Am) by replacing two residues of the self-cleavage site (GP-> AA) A series of POU-KSM vectors were generated. A synthetic polyglycine (GL) linker was also used to eliminate the effect specific to the F2A sequence.

As can be seen in FIG. 4B, Western blot analysis confirmed the complete cleavage of POU and Klf4 linked by P2A, partial cleavage by F2A and cleavage by F2Am.

We then tested the function of the construct to reprogram Oct4-GFP MEFs to iPSCs. As we expected, only Oct4 in the P2A construct could generate GFP + colonies if it were not another POU factor. Surprisingly, the noncoding ligands F2Am and GL were able to reprogram not only Brn4 but also Oct6 and Brn2. There are two nerve-specific POU3 factors reported previously that can not generate an iPSC or even that closure chromatin is unavailable.

As can be seen in Figure 4c, fibroblasts. Brn2-, Oct6-, and Brn4-F2Am-KSM-generated iPSC lines can generate a passable clone cell line (scale bar, 250 um).

As can be seen in Figure 4d, genomic integration of each transgene was confirmed by PCR genotyping. The resulting iPSCs were able to differentiate into pluripotent markers Nanog and SSEA-1 and to differentiate into all three bacterial layers in teratoma analysis and could contribute to the development of the germline of chimeric mice.

In particular, Brn4-F2Am-KSM and Brn4-GL-KSM are worse than native F2A constructs suggesting that a mixture of Brn4-Klf4 that is not cleaved and cleaved is advantageous for efficient reprogramming. Therefore, we decided to investigate the functions of POU and Klf4 in fusion proteins. The ZHBTc4 ESC line was used to perform a pluripotency rescuing assay. Oct4 can be conditionally removed by dox treatment. Unexpectedly, the Oct4-deficient ESCs, which were rescued by P2A-, F2A-, F2Am- or GL-linked Oct4-Klf4 constructs, showed no difference in their morphology, survival or proliferative capacity, and that POU TF function was similar to POU-Klf4 polyprotein . We next used a pure ESC transformation assay primed to test the function of Klf4 in the fusion protein using Goff18 mouse epithelial stem cells. Both F2Am and GL structures were significantly impaired in the conversion of Gof18 + stem cells to Gof18 + stem cells compared to vectors containing cleavable peptides determined by FACS analysis.

Therefore, the reprogramming experiment was repeated using the POU-Klf4 bissistron structure with KSM to compensate for functionally damaged Klf4. Reprogramming efficiency was measured by counting GFP + colonies 1 and 2 weeks after induction. The data are expressed as means ± SD, n = 3.

As can be seen in Figure 4E, replacing F2A with P2A, which cleaves more efficiently, prevented reprogramming to iPSC with POU factors other than Oct4, but the non-cleavable peptide relieved reprogramming efficiency and was originally partially cleaved Lt; RTI ID = 0.0 > F2A. ≪ / RTI > This means that Brn4 obtains a reprogramming function in the STEMCCA cassette by forming a polyprotein with Klf4.

As can be seen in Figure 4f, to further confirm the reprogramming function of the Brn4-Klf4 polyprotein, the reprogramming ability of the fully cleavable Brn4-P2A-KSM cassette with addition of the non-cleavable Brn4-Klf4 construct was similar to that of Oct4 delivery To-the-bottom level.

To understand how the Brn4-Klf4 fusion protein gained reprogramming activity, chromatin immunoprecipitation (ChIP) -qPCR was performed on the POU and Klf4 targets. The N-terminus of Brn4 or Oct4 was labeled with 3xFLAG and ChIP was performed on a population of large reprogrammed cells after 4 days of dox induction.

Specifically, 2 × 10 ⁷ cells were cross-linked with formaldehyde to a final concentration of 1% at 4 ° C. for 20 minutes and quenched with glycine (final concentration 0.1375 M). The cross-linked cells were sonicated with Bioruptor (Diagenode) for 45 cycles of 30 sec pulse / 30 sec rest period. 25 ug of ultrasound chromatin in each sample was immunoprecipitated with anti-FLAG antibody (Sigma, F1804). Primers were designed based on known ChIP-Seq data. Immunoprecipitated DNA is expressed as fold enrichment compared to the Oct4 sample. Data are presented as means ± SD of the biological replicates.

As shown in FIG. 4g, the fused Brn4-Klf4 polyprotein was significantly increased in pluripotency-related Sox-Oct motif-containing loci (Pou5f1, Nanog and Sox2), while POUR3 factor specific MORE- , Lingo1, and Foxo1).

Similar to the epiblast-to-naive ESC conversion results, the significant Klf4 target was not affected by the damaged cleavage. Jerabek et al. In this study, the mutation of Oct6 reduced homodimerization to the MORE motif that converted Oct6 to a multipotent inducer. Methionine 151, the residue responsible for homodimerization of the MORE motif, POU3, is located at the end of POUhd. Fusion of relatively short CTDs with relatively large Klf4 and POU3 factors can potentially interfere with homodimerization of POU TF without affecting heterodimerization with Sox2, which is dependent on the region of the POU TF region.

Taken together, the unexpected iPSC generation capability of pcBKSM is caused by incomplete cleavage of BrN4-F2A-Klf4. This function allows reprogramming of multiple differentiability with other neural-related POU factors.

Claims (4)

A universal state of crossed differentiation cell comprising the steps of:
(Cre-recombinase) gene coding sequence operably linked to the Oct4 promoter, a loxP coding nucleic acid sequence removed by the CRE gene activation, and a somatic cell containing a fluorescent protein coding nucleic acid sequence expressed upon loxP removal Preparation phase;
A transfection step of introducing into said somatic cell each vector that individually codes a nucleic acid sequence encoding Brn4, Klf4, Sox2 and cMyc; And
And determining that the crossed differentiated cells are generated via the pluripotent state when the somatic cells are cultured and fluorescence is expressed. The method of claim 1, wherein the fluorescent protein is EYFP (Enhanced yellow fluorescent protein). The method according to claim 1, wherein the somatic cell is a fibroblast. 2. The method of claim 1, wherein the cross-differentiating cells are inducible neural stem cells.

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