KR20190041304A - Composition for direct conversion of somatic cell into hepatocyte using non-viral vector and method of direct conversion using the same - Google Patents

Abstract

The present invention relates to a method of direct conversion and a composition for direct conversion of somatic cells into hepatocytes using a non-viral vector. More particularly, the present invention provides highly efficient directly converted induced hepatocytes (iHep) by using a non-viral vector, and to a method of direct conversion from somatic cells into hepatocytes in a more rapid, accurate and economical way using the same.

Description

[Technical Field] The present invention relates to a composition for directly crossing differentiation of somatic cells into a hepatocyte using a non-viral vector, and a direct crossing differentiation method.

The present invention relates to a composition for direct crossing differentiation of somatic cells into hepatocytes using a non-viral vector and a direct cross-differentiation method. More specifically, the present invention provides highly efficient direct crossed differentiated hepatocytes (iHep) using non-viral vectors, which can be used to produce faster, more accurate, and economical somatic cells To a method for directly crossing differentiation of hepatocytes into high efficiency.

Induced pluripotent stem cells (iPSCs) are four types of foreign genes, Oct4, Sox2, Klf4, and cMyc, which are introduced into general somatic cells to produce differentiated pluripotent stem cells. Refers to cells that have been de-differentiated.

In 2006, Professor Yamanaka succeeded in creating inducible pluripotent stem cells by introducing four transcription factors into mouse fibroblasts. In the following year, he succeeded in producing human induced pluripotent stem cells, which are induced by the same four transcription factors in human dermal fibroblasts Respectively. In the same year, James Thompson, a professor at the University of Wisconsin-Madison, USA, succeeded in introducing genes (Oct4, Sox2, Nanog and Lin28) as retroviruses to produce induced pluripotent stem cells from human skin cells.

However, although inducible pluripotent stem cells have the advantage of pluripotency as embryonic stem cells, there is a possibility that a small amount of 'undifferentiated cells' that do not sufficiently differentiate under specific culture conditions to differentiate into specific cells may remain Because of the potential for tumor formation (teratoma) during transplantation, it is considered to be a major impediment to the application of clinical research to humans.

In order to overcome and overcome the disadvantages of inducible pluripotent stem cells, a technique of direct direct conversion has been spotlighted. 'Direct cross-differentiation' is a process that does not re-differentiate a given somatic cell (cell type A) into inducible pluripotent stem cells capable of differentiating into all cells, It is a technology to switch directly. Direct conversion, which induces various tissue-specific cell differentiation, has a problem of low efficiency.

The background of the present invention is Korean Patent Laid-Open No. 10-2015-0052228 (May 05, 2015), which discloses a method of reprogramming non-hepatocytes using reprogramming factors such as FOXA3, HNF1A, and HNF4A A method for use in generating induced hepatocytes is disclosed.

The present invention relates to the establishment of a cross-differentiation stem cell line based on a non-viral system based on a virus system, and to a somatic cell of a pig, wherein codon optimization and GC content optimization (HNF1A (A-83-01), and an episomal vector inserted with hHN4A and hFOX3 were efficiently introduced into the cells to enable stable cell differentiation. And a suitable culture method were selected to construct a proliferative cross-over stem cell line.

It is an object of the present invention to provide a composition for direct cross-differentiation of somatic cells into hepatocytes using the non-viral vector.

Another object of the present invention is to provide a highly efficient Directed Differentiated Hepatocyte (iHep) using the above composition.

It is still another object of the present invention to provide a method for directly crossing differentiation from somatic cells to hepatocytes with high efficiency using the above composition in a fast, accurate and economical manner.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a non-viral vector comprising the HNF1A gene fragment of SEQ ID NO: 1; And a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2, for direct cross-differentiation of somatic cells into hepatocytes.

According to one embodiment of the present invention, there is provided a composition for direct cross-differentiation of somatic cells into hepatocytes, wherein said non-viral vector is an episomal vector.

According to one embodiment of the present invention, the non-viral vector is a vector for direct crossing differentiation of somatic cells into hepatocytes, which is a vector into which a transcription factor inserted with codon optimization and GC content optimization is inserted / RTI >

According to one embodiment of the present invention, there is provided a composition for promoting direct crossing differentiation of somatic cells further comprising a small molecule compound A83-01 into hepatocytes.

According to one embodiment of the present invention, there is provided a composition for direct cross-differentiation of somatic cells into hepatocytes, wherein the somatic cells are fibroblasts of pigs.

According to another aspect of the present invention, there is provided a directly cross-differentiated stem cell line using the composition.

(A) a non-viral vector comprising the HNF1A gene fragment of SEQ ID NO: 1 and a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2, Introducing factor into somatic cells and culturing; And (b) culturing the somatic cells into which the transcription factor has been introduced in a culture medium containing a small molecule compound to directly reprogram the somatic cells to hepatocytes (direct reprogramming), thereby providing a method of directly crossing differentiation into hepatocytes .

According to one embodiment of the present invention, the somatic cell is provided with a method of directly crossing differentiation from somatic cells, which are fibroblasts derived from pigs, into hepatocytes.

According to an embodiment of the present invention, the non-viral vector is provided with a method of directly crossing differentiation from a somatic cell, which is an episomal vector, into a hepatocyte.

According to one embodiment of the present invention, there is provided a method for directly cross-dividing somatic cells into hepatocytes, wherein the small molecule compound is A83-01.

According to one embodiment of the present invention, there is provided a vector comprising a vector inserted with a transcription factor that is optimized for codon optimization and GC content optimization (GC content optimized), and which can be used for direct crossing differentiation of somatic cells into hepatocytes Vector. ≪ / RTI > The use of the non-viral vector generally has the advantage that it does not induce an immune response, has low toxicity, and is easy to mass-produce.

According to one embodiment of the present invention, it is possible to provide a composition for direct cross-differentiation of somatic cells into hepatocytes that can be used for direct crossing differentiation of somatic cells into hepatocytes.

According to an embodiment of the present invention, the non-viral vector may be used to directly provide a cross-differentiated stem cell line.

According to one embodiment of the present invention, a method of direct crossing differentiation from a somatic cell to a hepatocyte in a more efficient and economical manner is provided, thereby expanding supply of heterologous organs and helping to solve insufficient organ transplantation problems .

According to the present invention, it is possible to develop a cell line for toxicity testing for the development of a new drug, and can be utilized in the development of a safe cell therapy agent customized for liver failure patients.

1 is a schematic diagram of a non-viral vector comprising a HNF1A gene fragment according to an embodiment of the present invention.
2 is a schematic diagram of a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment according to an embodiment of the present invention.
Figures 3A and 3B are graphs showing codon optimization and GC content optimization according to an embodiment of the present invention.
FIG. 4 is a photograph showing morphological changes of cells subjected to second passage passage and third passage passage of direct cross-differentiated hepatocytes using a composition for direct crossing differentiation of somatic cells according to an embodiment of the present invention to be.
FIG. 5 is a graph showing the gene expression characteristics of piHep according to the addition period of the small molecule compound (A83-01) for directly cross-differentiated hepatocytes according to an embodiment of the present invention.
FIG. 6 is a photograph showing immunohistochemistry (IHC) analysis of the physiological characteristics of direct cross-differentiated hepatocytes according to an embodiment of the present invention.
FIG. 7 is a graph showing physiological characteristics of direct crossed differentiated hepatocytes according to Integrated Optical Density (IOD) according to an embodiment of the present invention.
FIG. 8 is a graph showing the expression pattern of CYP enzyme after direct inducible treatment of direct cross-differentiated hepatocytes according to an embodiment of the present invention.
9A and 9B are photographs and graphs showing the maturation of the directly cross-differentiated hepatocytes through lipid differentiation according to an embodiment of the present invention.
FIGS. 10A and 10B are photographs and graphs showing the maturation of direct cross-differentiated hepatocytes through glycogen accumulation according to an embodiment of the present invention. FIG.

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context clearly indicates otherwise, the singular form of the term also includes plural forms thereof, and plural forms of the term should be understood as including its singular form.

Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, there is provided a non-viral vector comprising the HNF1A gene fragment of SEQ ID NO: 1; And a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2, for direct cross-differentiation of somatic cells into hepatocytes.

Direct cross-differentiation refers to a process in which a cell type A is de-differentiated into an inducible pluripotent stem cell capable of differentiating into all cells, and then re-differentiation is performed without re-differentiation. (Cell type B).

The viral vector system which has been used in the gene therapy method has a problem of biological stability such as immunogenicity in the clinical application after the foreign gene is inserted into the host gene. On the other hand, the non-viral vector system has a problem of low transfer efficiency. The present inventors have developed a highly safe nonviral vector system capable of efficiently transferring an interstitial factor gene to a target cell and capable of expressing the gene continuously and for a long time in order to solve the above problems simultaneously.

That is, the present invention is characterized by using a non-viral vector comprising the HNF1A gene fragment and a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment, and is thus a non-viral vector system The transmission efficiency can be increased.

According to one embodiment of the present invention, there is provided a composition for direct cross-differentiation of somatic cells capable of producing a high-efficiency transformed cell line, wherein the non-viral vector is an episomal vector, into hepatocytes.

An " episomal vector " is an integration-free vector, which has the advantage of being able to replicate and propagate in chromosomally infected cells, without genetic insertions.

According to one embodiment of the present invention, the non-viral vector is a vector directly inserted into a hepatocyte of a somatic cell, in which a transcription factor inserted with codon optimization and GC content optimization is optimized for expression optimization A composition for cross-differentiation is provided. For optimization of expression in a species, it is preferable to optimize the expression by adjusting the GC content to 80% or more but not to 30% or less.

In the case of the present invention, the average GC content of the HNF1A gene was 65% and the average GC content of the Hnf4a-F2A-Foxa3 gene was 64% using a gene optimization program in accordance with the codon of the pig gene. (See Figs. 3A and 3B). In the optimization process, the following cis-acting sequence motifs were avoided.

- internal TATA-boxes, chi-sites and ribosomal entry sites

- AT-rich or GC-rich sequence stretches

- RNA instability motifs

- repeat sequences and RNA secondary structures

- (cryptic) splice donor and acceptor sites in higher eucaryotes

In the present invention, " Codon Optimization " refers to a method of enhancing the production of proteins by highlighting a prefered codon that is biased in the amino acid codon at a site coding for the protein and modifying the rare codom .

In the present invention, the codon-optimized nucleotide sequence represents the HNF1A gene fragment of SEQ ID NO: 1 and the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2, and although this is a non-viral vector system by the codon optimization, .

In the present invention, the somatic cell is a fibroblast of porcine.

The present invention utilizes porcine fibroblasts which are used as donors for organ transplantation in xenotransplantation.

"Pigs" are suitable for xenotransplantation, and for now pigs are considered to be the best candidates. Pigs are highly available in supply, and these organs are similar in size to humans. Moreover, even though it is not medicines, it has been regarded as relatively easy to handle because it has been raised by humans for centuries.

According to one embodiment of the present invention, there is provided a composition for direct cross-differentiation of somatic cells into hepatocytes further comprising the small molecule compound A83-01.

The small molecule compound A83-01 has an effect of blocking and inhibiting Tgfβ signaling in somatic cells treated with combination 1 (hHNF1A) and combination 2 (hHF4A and hFOX3), respectively. Thus, direct crossing differentiation efficiency of somatic cells into hepatocytes .

The composition of the present invention may further include a known small molecule compound. But are not limited to, for example, a Wnt signaling activating small molecule compound (CHIR99021) that induces cell proliferation.

According to another aspect of the present invention, there is provided a directly cross-differentiated stem cell line using a composition for direct cross-differentiation of somatic cells into hepatocytes.

The direct-differentiated stem cell line according to the present invention has higher relative gene expression of CYP, which is a representative enzyme of pig, than wild-type strain, and the degree of accumulation of adipocytes and glycogen, which can confirm liver function of iHep, Respectively.

According to another aspect of the present invention there is provided a kit comprising: (a) a non-viral vector comprising the HNF1A gene fragment of SEQ ID NO: 1; And a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2; And (b) culturing the somatic cells into which the transcription factor has been introduced in a culture medium containing a small molecule compound to directly reprogram the somatic cells to hepatocytes (direct reprogramming), thereby providing a method of directly crossing differentiation into hepatocytes .

The step (a) is a step of introducing HNF1A, HF4A, and FOX3, which are transcription factors inducing transcription into hepatocytes into somatic cells, into the combination 1 (hHNF1A) and combination 2 (hHF4A and hFOX3) and then culturing.

The somatic cells are preferably cells derived from fibroblasts, and fibroblasts from pigs are preferred.

In the step (b), the somatic cells are cultured in a culture medium containing a small molecule compound to directly cross-differentiate the somatic cells into which the transcription genes and the transcription genes are directly induced, thereby directly cross- So as to directly improve the crossing differentiation efficiency.

For this, the small molecule compound can be constructed to cultivate the somatic cells into which the transcription gene has been introduced for 48 hours in a hepatocyte-specific culture medium, and the composition of the culture solution is shown in Table 2.

According to one embodiment of the present invention, the somatic cell is provided with a method of directly crossing differentiation from somatic cells, which are fibroblasts derived from pigs, into hepatocytes.

According to one embodiment of the present invention, the non-viral vector is provided as a direct cross-differentiating method from a somatic cell, which is an episomal vector, to a hepatocyte.

According to one embodiment of the present invention, there is provided a method for directly cross-dividing somatic cells into hepatocytes, wherein the small molecule compound is A83-01. The small molecule compound A83-01 has an effect of blocking and inhibiting Tgf [beta] signaling in somatic cells treated with combination 1 (hHNF1A) and combination 2 (hHF4A and hFOX3), respectively.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1: codon optimization ( codon  optimization) and GC  Content optimization GC  content optimization) of three transcription factors into two episomal vectors.

Three of the human liver transcription factors (hHNF1A, hHNF4A, hFOX3) were selected and considered as the combination of 1 (hHNF1A) and combination 2 (hHF4A and hFOX3) Two codon optimization and GC content optimization vectors consisting of HNF1A (1914 bp) and HNF4A-2A-hFOX3FH (2577 bp) were constructed to allow synthesis (see Figures 1 and 2) .

Example  2: Episomal  vector( Episomal  Optimization of inserting vectors

⁵ × 10 ⁵ cells of pork fibroblasts were placed in a collagen-coated 35 mm dish, and 1 μM 5 azacitidine was added to the cells to induce dimethylation for 1 day. Twenty-four hours later, two vectors were added using Lipofectamine 3000 and cultured for 48 hours in 2 ml of serum and opti-MEM without antibiotics. On day 4, the medium was replaced with liver differentiation medium.

Expand Induced Hepatocytes (Expand iHep) and the composition of the culture medium were constructed as shown in Table 1 below.

Example  3: Expand  Induction Hepatocyte (Expand iHep ) Production

After 2 weeks of culture, the cells were divided into 1: 4 and subcultured. At this time, the whole cell was subcultured to passage 2 until passage 2, and single cells (single cell) were formed using trypsin EDTA only after the formed colonies from passage 3-4, followed by seeding in a 35 mm dish. The cell line was subcultured and stored until passage 13 (see FIG. 4).

Subsequently, all the cells are subcultured to increase the number of colonies from 0 to 1, followed by collecting only colonies in the second to third stages of subculturing to increase the frequency and purity, and continuously cultivate the colonies 4, it is possible to induce a typical hepatocyte-like cell with a polygonal and polynuclear chain.

In addition, the small molecule compound A83-01 blocked TGF-β-induced epithelial-to-mesenchymal transitions and promoted the formation of iHep.

The efficiency of piHep according to the period of addition of the small molecule compound (A83-01) is shown in Fig. Using the small molecule compound a83-01, we were able to determine how much they were able to induce expression of transcription factors. We compared the number of cells treated with 1 week to 4 weeks (+) and those without treatment (-) according to the number of days. The three genes injected from the outside were confirmed to be silent over time (FIG. 5B), but it was confirmed that the genes were increased in the same week compared to the untreated group.

It was confirmed that the introduction of the external transcription factor could induce the activity of the internal electronic element (FIG. 5C), and it was confirmed that the addition of the drug significantly improved the expression of the internal electronic element.

And it was confirmed that continuous treatment of drug contributes to increase gene expression rate. (+ → - VS. + → +). And albumin and trans-perine.

Experimental Example 1: Immunofluorescence

To examine the physiological functions and characteristics of the direct crossed differentiated hepatocytes according to the present invention, tissue immunostaining was performed as follows.

Cells cultured on a 4-well culture dish covered with 3.7% formaldehyde were fixed at room temperature for 20 minutes, and washed three times with PBS containing 1% BSA. The cells were then incubated at room temperature for 1 hour with PBS containing 0.1% Triton X-100 (Sigma) and 1% BSA (except for E-cadherin, cell surface factor). After 1 hour, the primary anti-body was added and the reaction was induced at 4 ° C for over night . Details of the antibodies used for the experiments are shown below.

Thereafter, the cells were washed three times with PBS containing 1% BSA, and a secondary anti-body with fluorescence was added thereto, followed by incubation at room temperature for 1 hour. After incubation, the cells were rinsed three times with PBS containing 1% BSA, stained with DAPI Solution (1 mg / mL) (Thermo Scientific ™) for comparison, and observed under fluorescence microscope.

FIG. 6 is a photograph showing immunohistochemistry (IHC) analysis of the physiological characteristics of direct cross-differentiated hepatocytes according to an embodiment of the present invention. As shown in FIG. 6, the expression of CYP genes involved in typical drug toxicity degradation and the expression of related proteins in the synapses between proteins such as albumin and hepatocytes expressed in hepatocytes were confirmed.

FIG. 7 is a graph showing physiological characteristics of direct crossed differentiated hepatocytes according to Integrated Optical Density (IOD) according to an embodiment of the present invention. As shown in FIG. 7, when the fluorescence expression level was confirmed, it was confirmed that the group using the codon optimization vector according to the present invention was more efficient.

Experimental Example  2: piHep's  Drug Metabolic activity

Cross - differentiation cells using episomal vectors between passages 7 and 10 were observed to proliferate well, and the expression of liver - specific genes was observed. Especially, sleeping pig transcription factors were activated. In particular, the cell lines using the codon optimization and GC content optimization vectors showed relatively higher iHep incidence than the wild type and showed excellent results in CYP expression and immunoprotein staining (see FIG. 8).

FIG. 8 is a graph showing the expression pattern of CYP enzyme after direct inducible treatment of direct cross-differentiated hepatocytes according to an embodiment of the present invention. As shown in FIG. 8, the expression of typical CYP enzymes in the pigs was confirmed, and the reaction force according to the vector before and after the use of the drug was confirmed in the cell line reached to the subculture 10, and the codon optimization and the GC content optimization (GC content optimized) group was highly expressed.

Cyp450 expression in the generated iHep was confirmed after treatment with inducers, and it was confirmed that codon optimization and GC content optimization vectors were superior (see FIG. 8).

The type of Cyp enzyme, the kind of prototypical inducer and the induction time are shown in Table 2 below.

For the Cyp1A2 enzyme, the inducer was 3-methylcholanthrene (3-MC) and the induction rime was 72 hours.

For the Cyp1A4 enzyme, the inducer was Rifampicin (RIF) and the induction time was 72 hours.

Experimental Example  3: The induced hepatic cells ( iHep ) Maturity check

To confirm the maturation of cross-differentiated hepatocytes, lipid differentiation was confirmed.

9A and 9B are photographs and graphs showing the maturation of the directly cross-differentiated hepatocytes through lipid differentiation according to an embodiment of the present invention. As shown in FIGS. 9A and 9B, both codon optimization and GC content optimization (Opti-) group showed relatively high mRNA expression and Oil Red O staining reaction Respectively.

The mature hepatocytes showed the accumulation of glycogen, which was confirmed by staining.

FIGS. 10A and 10B are photographs and graphs showing the maturation of direct cross-differentiated hepatocytes through glycogen accumulation according to an embodiment of the present invention. FIG. As shown in FIGS. 9A and 9B, codon optimization and GC content optimization group (Opti-) showed relatively high mRNA expression and PAS staining reaction regardless of progress of subculture .

Therefore, in the cross-differentiation stem cell line of the present invention, both early passage P1-2 and late passage P9-10 cells exhibited relatively high mRNA expression and Oil red O staining reaction , And it can be seen that even in the case of early cultured liver stem cells, it has high maturity.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Composition for direct conversion of somatic cell into hepatocyte          using non-viral vector and method of direct conversion using the          same &Lt; 130 > NPF-31254 <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 1914 <212> DNA <213> Sus scrofa <400> 1 aagcttgcca ccatggtttc caagctgagc cagctgcaga ccgaactgct ggctgctctg 60 ctggaaagcg gcctgtctaa agaggccctg attcaggccc tgggagagcc tggaccttat 120 ctgcttgctg gcgagggccc actggataag ggcgaatctt gtggcggagg aagaggcgaa 180 ctggccgagc tgcctaatgg actgggcgag acaagaggca gcgaggacga gacagatgac 240 gacggcgagg atttcacccc tcctatcctg aaagagctgg aaaatctgag ccccgaggaa 300 gccgctcacc agaaagctgt ggtggaaacc ctgctgcaag aggacccttg gagagtggcc 360 aagatggtca agagctacct gcagcagcac aacatccctc agcgcgaggt ggtggatacc 420 acaggcctga accagagcca cctgtctcag cacctgaaca agggcacccc tatgaagacc 480 cagaagcggg ctgccctgta cacttggtac gtgcggaagc agagagaggt ggcccagcag 540 tttacacacg ctggacaagg cggactgatc gaggaaccta caggcgacga gctgcccacc 600 aagaaaggca gacggaaccg gtttaagtgg ggccctgcct ctcagcagat cctgtttcag 660 gcctacgagc ggcagaagaa ccccagcaaa gaggaaagag agacactggt cgaggaatgc 720 aaccgggccg agtgtatcca gaggggcgtt agtccttctc aggcccaagg cctgggcagc 780 aatctggtta cagaagtgcg ggtctacaat tggttcgcca accggcggaa agaggaagcc 840 ttcagacaca agctggccat ggacacctac agcggacctc cacctggacc aggacctgga 900 cctgcacttc ctgctcattc tagccctgga ctgcctcctc ctgctctgtc tccatctaag 960 gtgcacggcg tcagatatgg ccagcctgcc acatctgaga cagccgaggt tccatctagc 1020 tctggcggac ctctggtcac cgtgtctaca cctctgcatc aggtgtcccc aaccggcctg 1080 gaaccttctc acagcctgct gagcacagag gccaaactgg tgtcagctgc aggcggacca 1140 ctgcctccag tctctacact gacagccctg cacagcctgg aacagacaag ccccggactg 1200 aatcagcagc cccagaacct gattatggcc tctctgcccg gcgtgatgac aatcggacct 1260 ggcgaacctg cttctctggg ccccaccttt acaaacaccg gcgccagcac actggtcatc 1320 ggactggctt ctacacaggc ccagagcgtg cccgtgatca atagcatggg cagcagcctg 1380 accacactgc agcctgtgca gtttagccag cctctgcacc cctcttacca gcagcctctg 1440 atgccaccag tgcagagcca cgtgacacag agccctttca tggccacaat ggcccagctg 1500 caaagccctc acgctctgta ctctcacaag cccgaagtgg cccagtacac ccacacagga 1560 ctgctgcctc agaccatgct gatcaccgac accaccaatc tgagcgccct ggctagcctg 1620 acacctacca aacaggtgtt caccagcgac accgaggcca gctctgaatc tggactgcac 1680 acaccagcca gccaggccac aacactgcat gtgccatctc aggatccagc cggcatccag 1740 catctgcagc cagctcatag actgagcgcc tctccaacag tgtccagctc tagcctggtg 1800 ctgtaccaga gcagcgacag cagcaatggc cagagccatc tgctgcctag caaccacagc 1860 gtgatcgaga cattcatcag cacccagatg gccagcagca gccagtaggg atcc 1914 <210> 2 <211> 2577 <212> DNA <213> Sus scrofa <400> 2 aagcttgcca ccatgcggct gagcaagacc ctggtggata tggacatggc cgactacagc 60 gccgctctgg atcctgccta caccacactg gaattcgaga acgtgcaggt cctgaccatg 120 ggcaacgaca catctccaag cgagggcacc aacctgaacg cccctaatag cctgggagtg 180 tctgccctgt gtgccatctg tggcgataga gccacaggca agcactacgg cgccagctct 240 tgtgatggct gcaagggctt ctttcggcgg agcgtgcgga agaaccacat gtacagctgc 300 cggttcagcc ggcagtgcgt ggtggacaag gacaagagaa accagtgccg gtactgcaga 360 ctgaagaagt gcttcagagc cggcatgaag aaagaggccg tccagaacga gcgggacaga 420 atcagcacca gaagaagcag ctacgaggac agcagcctgc ctagcatcaa tgccctgctg 480 caggccgagg ttctgagcag acagatcaca agccctgtgt ccggcatcaa cggcgacatc 540 agagccaaga agatcgccag cattgccgac gtgtgcgaga gcatgaagga acagctgctg 600 gtgctggtgg aatgggccaa gtacatcccc gccttttgcg agctgcccct ggatgatcaa 660 gtggccctgc tgagagcaca cgccggcgaa catttgctgc tgggagccac caagcggagc 720 atggtgttca aggatgtgct gctgctcggc aacgattaca tcgtgcccag acactgtcct 780 gagctggccg agatgagccg ggtgtccatc agaatcctgg acgaactggt gctgcccttc 840 caagagctgc agatcgacga caacgagtac gcctacctga aggccatcat ctttttcgac 900 cccgacgcca agggcctgag cgatcctgga aagatcaagc ggctgcggag ccaggtgcaa 960 gtgtccctgg aagattacat caacgaccgg cagtacgaca gccggggcag atttggagaa 1020 ctgctgctcc tgctgccaac actgcagagc atcacctggc agatgatcga gcagatccag 1080 ttcatcaagc tgttcggcat ggccaagatc gacaacctgc tgcaagagat gctgcttggc 1140 ggcagccctt ctgatgcccc tcatgctcat caccctctgc accctcacct gatgcaagaa 1200 cacatgggca ccaatgtgat cgtggccaac acaatgccca cacacctgag caacggccag 1260 atgtgcgaat ggcctagacc tagaggacag gccgccacac ctgaaacacc tcagccttct 1320 ccacctggcg gatctggcag cgagccttac aaacttctgc ctggcgctgt ggccaccatc 1380 gtgaaacctc tgtctgccat tcctcagcct accatcacca agcaagaagt gatccggaag 1440 cggagagccc ctgtgaagca gaccctgaac ttcgacctgc tgaaactggc cggcgacgtg 1500 gaaagcaacc ctggacctat gctgggcagc gtgaagatgg aagcccatga tctggccgag 1560 tggtcttact atcctgaggc cggcgaggtg tacagccctg tgacacctgt tcctacaatg 1620 gcccctctga acagctatat gccctgaat cctctgagca gcccctatcc tcctggtgga 1680 cttcctgcta gccctctgcc ttctggacct cttgctcctc ctgctccagc tgctcctctg 1740 ggccctacat ttcctggact gggagtttct ggcggctcta gcagctctgg atatggcgct 1800 cctggaccag gactggtgca cggcaaagag atgcccaagg gctacagaag gcctctggct 1860 ccgccaagc ctccatacag ctacatcagc ctgatcacca tggccatcca gcaggcccct 1920 ggcaagatgc tgacactgag cgagatctac cagtggatca tggatctgtt cccttactac 1980 cgcgagaacc agcagcggtg gcagaacagc attagacaca gcctgagctt caacgactgc 2040 ttcgtgaagg tggccagatc tcccgacaag cctggcaagg gctcttactg ggctctgcat 2100 cctagcagcg gcaacatgtt cgagaatggc tgctacctgc ggcggcagaa gcggttcaag 2160 ctggaagaga aagtgaagaa aggcggctcc ggcgctgcca ccacaaccag aaatggaaca 2220 ggatctgccg ccagcaccac aacaccagcc gctacagtta caagccctcc tcagccacct 2280 cctccagcac ctgaacctga agctcaaggc ggagaagatg tgggcgccct ggattgtgga 2340 agccctgcca gctctacccc ttacttcacc ggacttgagc tgcccggcga gctgaaactc 2400 gacgcccctt acaacttcaa tcaccccttc agcatcaaca acctgatgtc cgagcagacc 2460 cctgctcctc caaagctgga tgttggcttt ggcggctatg gcgctgaagg cggcgaacct 2520 ggtgtctact atcagggcct gtacagcaga agcctgctga acgcctcttg aggatcc 2577

Claims (10)

A non-viral vector comprising the HNF1A gene fragment of SEQ ID NO: 1; And
A composition for direct cross-differentiation of somatic cells into hepatocytes, comprising a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2.
The method according to claim 1,
Wherein said non-viral vector is an episomal vector, for direct cross-differentiation of somatic cells into hepatocytes.
The method according to claim 1,
Wherein the non-viral vector is a vector into which a transcription factor inserted with codon optimization and GC content optimization is inserted, wherein the non-viral vector is a vector for direct crossing differentiation of somatic cells into hepatocytes.
The method according to claim 1,
A composition for direct cross-differentiation of somatic cells into hepatocytes further comprising the small molecule compound A83-01.
The method according to claim 1,
Wherein said somatic cell is a fibroblast of porcine.
A stem cell line which is directly cross-differentiated using the composition for direct cross-differentiation of somatic cells according to any one of claims 1 to 5 into hepatocytes.
(a) a non-viral vector comprising the HNF1A gene fragment of SEQ ID NO: 1; And a non-viral vector comprising the Hnf4a-F2A-Foxa3 gene fragment of SEQ ID NO: 2, wherein the transcription factor is introduced into somatic cells and cultured; And
(b) culturing somatic cells transfected with the transcription factor in a culture medium containing a small molecule compound to directly reprogram the somatic cells to hepatocytes (direct reprogramming).
8. The method of claim 7,
Wherein the somatic cell is a fibroblast derived from a swine, directly crossing differentiation from somatic cells into hepatocytes.
8. The method of claim 7,
Wherein said non-viral vector is an episomal vector, from a somatic cell to a hepatocyte.
8. The method of claim 7,
Wherein said small molecule compound is A83-01, said method comprising direct crossing differentiation from somatic cells to hepatocytes.

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