KR101743284B1 - Method for enhancing direct reprogramming efficiency from somatic cells to hepatocyte-like cells using small molecules - Google Patents

Method for enhancing direct reprogramming efficiency from somatic cells to hepatocyte-like cells using small molecules Download PDF

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KR101743284B1
KR101743284B1 KR1020150147402A KR20150147402A KR101743284B1 KR 101743284 B1 KR101743284 B1 KR 101743284B1 KR 1020150147402 A KR1020150147402 A KR 1020150147402A KR 20150147402 A KR20150147402 A KR 20150147402A KR 101743284 B1 KR101743284 B1 KR 101743284B1
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한동욱
임경태
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건국대학교 글로컬산학협력단
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Abstract

(A) introducing a transcription gene comprising FOXA3 , HNF1A and HNF4A into a somatic cell and culturing the same; And (b) culturing the somatic cells transfected with the transcription gene in a culture medium containing a small molecule compound to directly reprogram the somatic cells into hepatocytes (direct reprogramming). to provide.
According to the method of enhancing direct crossing differentiation efficiency from human somatic cells to hepatocytes according to the present invention, somatic cells treated with the combination of FOXA3 , HNF1A and HNF4A are treated with Tgfβ signaling (A-83-01), which blocks and suppresses signaling, and Wnt signaling-activating small molecule compound (CHIR99021), which induces cell proliferation, to direct cross-differentiation efficiency from somatic cells to induced hepatocytes to 5 More than double.

Figure R1020150147402

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for enhancing direct crossing differentiation efficiency from a human somatic cell to a hepatocyte using a small molecule compound,

The present invention relates to a method for improving the direct crossing differentiation efficiency from human somatic cells to hepatocytes using a small molecule compound.

Converted to the pluripotent stem cell status (induced pluripotent stem cells, iPSCs) is Oct4, Sox2, Klf4, cMy c of four kinds of foreign capable omnipotence ES cells by introducing a gene in normal somatic cell differentiation (differentiation) into the body all cells of Refers to cells that have been de-differentiated.

However, although inducible pluripotent stem cells have the advantage of having the same pluripotency as embryonic stem cells, there is a possibility that a small amount of undifferentiated cells that are not sufficiently differentiated under specific culture conditions to differentiate into specific cells Because of the possibility of forming tumors (teratoma) during transplantation, it has become a serious obstacle to the application of clinical research to humans.

Recently, in the field of stem cell research, introduction of a minimal transcription factor combination into somatic cells in which the differentiation has completely progressed, and the conversion into somatic or adult stem cells having completely different characteristics, that is, direct reprogramming, This is a situation where research has been published around the world. Here, 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. Cells (cell type B).

In 2014, Chinese researchers selected human hepatocyte-specific 8 transcription factors using human-derived somatic cells and then finally cross- fertilized human somatic cells directly with hepatocytes using at least three transcription factors: FOXA3 , HNF1A and HNF4A And has announced success.

In the same year, another Chinese research team also used human somatic cells with genetic modifications to treat p53 siRNA and C-MYC to produce up to six additional hepatocyte-specific transcription factors ( HNF1A, HNF4A, HNF6, CEBPA, ≪ / RTI > and ATF5 ) to direct cross-differentiation into human hepatocytes.

Nevertheless, the efficiency of direct cross-differentiation of human somatic cells to hepatocytes is significantly lower than the previously reported human somatic cells. Non-Patent Document 1 reports that about 10 to 20% of ALB / AAT-positive cells were formed by forming an epithelial-like colony after about 12 days of transcription factor introduction, and non-patent document 2 Reported that 91% of ALB-positive cells and 99% of AAT-positive cells were present within 30 days, and both of the researchers showed low direct cross-over efficiency to hepatocytes There is a need for research on ways to supplement this.

Korean Patent Laid-Open No. 10-2015-0050961 (published on May 20, 2015) Korean Patent No. 10-1158402 (Published on July 7, 2011)

 Huang et al., Cell Stem Cell 2014.  Du et al., Cell Stem Cell 2014.

DISCLOSURE Technical Problem The present invention has been conceived to solve the problems of the prior art as described above, and it is an object of the present invention to provide a method of directly crossing differentiation efficiency from human somatic cells to hepatocytes using a small molecule compound which improves the ' And to provide a technical content of the present invention.

In order to accomplish the above object, the present invention provides a method for producing a transgenic animal , comprising: (a) introducing a transgene containing FOXA3 , HNF1A and HNF4A into a somatic cell and culturing the transgenic somatic cell; and (b) The present invention provides a method for directly crossing differentiation efficiency from a human somatic cell to a hepatocyte, the method comprising directly culturing somatic cells in a culture medium containing the compound, and direct reprogramming the somatic cells with hepatocytes.

In addition, the somatic cell is a human fibroblast.

The step (a) is performed for 48 hours.

Further, the small molecule compound is characterized by containing A-83-01 and CHIR99021.

The step (b) is performed for 96 hours or more.

In addition, the present invention provides hepatocytes directly cross-differentiated in somatic cells through the above-described method.

According to the method of enhancing direct crossing differentiation efficiency from human somatic cells to hepatocytes according to the present invention, somatic cells treated with the combination of FOXA3 , HNF1A and HNF4A were transfected with Tgfβ signaling (A-83-01), which blocks and inhibits signaling, and the Wnt signaling-activating small molecule compound (CHIR99021), which induces cell proliferation, to provide direct cross-differentiation efficiency from somatic cells to induced hepatocytes 5 times or more.

Therefore, by using the method according to the present invention, the efficiency of direct cross-over differentiation into hepatocytes is improved, which can be useful for accelerating the development of a cell therapy agent using hepatocytes, verifying toxicity of a new drug substance using hepatocytes, and efficiently mass- Can be used.

FIG. 1 is a conceptual diagram schematically showing a method for enhancing direct crossing differentiation efficiency of human somatic cells according to the present invention to hepatocytes.
FIG. 2 is an actual image showing morphological changes of cells at 4 days and 6 days after the introduction of a transcription factor by the method according to Examples and Comparative Examples.
Figure 3 is the ALB or AAT antibody in the Examples and then introduced into the comparative example transcription factors and small molecule compounds by the process according to the six days elapsed time Wild type HFF and the HFF LT cells and controls Wild type HFF and HFF LT cells FIG. 2 is a graph showing the results of measuring the number of cells showing a positive response. FIG.
4 Examples and Comparative after introduction of the transcription factor and a small molecule compound by the method according to the example, 6 days have passed positive for AAT antibody in a Wild type HAF and HAF LT cells Wild type HAF and HAF LT cells in the control group at the time FIG. 2 is a graph showing the results of measurement of the number of cells exhibiting cell proliferation.
FIG. 5 is a graph showing a change in gene expression pattern upon direct cross-differentiation into hepatocytes in a somatic cell into which a transcription factor and a small molecule compound are introduced by the method according to the embodiment.

Hereinafter, the present invention will be described in detail.

FIG. 1 is a conceptual diagram schematically showing a method for directly crossing differentiation efficiency of human somatic cells according to the present invention into hepatocytes.

As shown in FIG. 1, in order to directly cross-differentiate somatic cells into hepatocytes and to improve the efficiency of crossing differentiation, the present invention provides a method for producing a transgenic plant , comprising: (a) introducing and transfecting a somatic cell with a transcription factor comprising FOXA3 , HNF1A and HNF4A ; And (b) culturing the somatic cells transfected with the transcription factor in a culture medium containing a small molecule compound to directly reprogram the somatic cells into hepatocytes (direct reprogramming), wherein the human somatic cells are directly cross- to provide.

The step (a) is a step of introducing a transcription gene including FOXA3 , HNF1A and HNF4A , which are transcription factors inducing transcription into hepatocytes into somatic cells, and then culturing.

In this step, somatic cells can be directly cross-differentiated into somatic cells by introducing the transcription genes in somatic cells through pWPI lentiviral infection (pWPI lentiviral infection) by introducing the transcription genes into somatic cells via pWPI lentivirus.

More specifically, after transferring the transcriptional gene into somatic cells via pWPI lentivirus in a gelatin-coated plate, 48 hours after the transfection, the cells were replaced with HMM (Hepatocyte Maintenance Medium) medium and cultured, And can be configured to cross differentiate.

The somatic cells are preferably cells derived from fibroblasts. The somatic cells are preferably selected from the group consisting of wild type human fetal fibroblasts (HFFs), wild type human adult fibroblasts (HAFs), SV40 large T-antigen-introduced wild-type human fetal fibroblast (HFF LT ) and SV40 large T-antigen-introduced wild-type human adult fibroblast (HAF LT ).

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 purpose, the small molecule compound may be cultured in a hepatocyte-specific culture medium for 96 hours or more of somatic cells into which the transcription gene has been introduced. The culture medium may be a hepatocyte maintaining medium (HMM).

In this step, as described above, a small molecule compound may be added to the culture medium so that the crossing differentiation efficiency can be directly increased during the culturing process in which somatic cells are directly cross-differentiated into hepatocytes.

The small molecule compound may include a small molecule compound (A-83-01) that blocks and inhibits Tgf [beta] signaling and a Wnt signaling activating small molecule compound (CHIR99021) that induces cell proliferation.

When the small molecule compound is added to the culture medium and the small molecule compound is treated with the somatic cell, direct crossing differentiation of the somatic cell into induced hepatocytes is increased, and the direct crossing differentiation efficiency can be enhanced five times or more.

Accordingly, in the present invention, hepatocytes directly cross-differentiated from somatic cells are provided through the above-described method.

Hereinafter, the present invention will be described in more detail with reference to examples.

The embodiments presented are only a concrete example of the present invention and are not intended to limit the scope of the present invention.

<Examples>

pWPI the lentivirus as a medium, the stem cell specific transcription factor combinations FOXA3, HNF1A, HNF4A (3F) was introduced into the general somatic cell of the original 70,000. The types of somatic cells used for direct crossing differentiation into hepatocytes were as shown in Table 1 below.

Figure 112015102738573-pat00001

After transfection of hepatocyte-specific transcription factors into four kinds of somatic cells as shown in Table 1, two small molecule compounds (AR) were added to a hepatocyte maintaining medium (HMM) at 48 hours, To induce cross-differentiation (3F / AR). Both small molecule compounds used small molecule compound A-83-01 (2 μM) to block and inhibit Tgf ㅯ signaling and small molecule compound CHIR99021 (3 μM) to activate Wnt signaling to induce cell proliferation .

<Comparative Example>

Two kinds of small molecule compounds (AR) for a and is pWPI lentivirus mediated by the same method as in Example, except that the non-cultured addition, the transcription factor combinations FOXA3, HNF1A, HNF4A the four types of somatic cells as shown in Table 1 (3F) and then cultured (3F).

<Experimental Example 1> Calculation of gene expression efficiency

In order to calculate the gene expression efficiency in the direct induction of crossing differentiation from somatic cells to hepatocytes, 4 days and 6 days after transcription factor introduction by the method according to Example (3F / AR) and Comparative Example (3F) The number of epithelial colonies specific for hepatocytes was visually confirmed at the time point. The morphological changes of the cells at 4 days and 6 days after the transduction factor was introduced are shown in FIG.

As shown in FIG. 1, when the test group transfected with the transcription factor was compared with the test group transfected with the transfected factor through the method according to the example, the wild type It was observed that epithelial-like cells were already formed on day 6 in HFFs and on day 4 in HFF LT (see arrow 1 and table 2 in Fig. 1).

Wild type HFF and HFF LT cells and wild type HFF and HFF LT cells (Mock) of the control group not transfected with transcription factor at 6 days after introduction of the transcription factor by the method according to Examples and Comparative Examples, respectively, The gene expression efficiency was calculated by measuring the ratio of the number of cells exhibiting positive labeling of each marker using the AAT (human A1AT) or ALB (human albumin) antibody, which is a specific marker, and the gene expression efficiency was calculated. Respectively.

As shown in FIG. 3, when wild-type HFFs and HFF LT cells were tested for the number of cells that showed a positive marker, the wild-type HFF cells were treated by the method according to the comparative example When comparing the cell groups, the number of cells showing a maximum 5-fold ALB positive response in the cell group treated by the method according to the example was observed (12.9% vs. 68.5%, see Fig. 3).

Wild type HAF and HAF LT cells and wild type HAF and HAF LT cells (Mock) of the control group not transfected with transcription factor at 6 days after introduction of the transcription factor by the method according to Examples and Comparative Examples, respectively, Using the AAT antibody as a specific marker, gene expression efficiency was calculated by FACS analysis (ratio of the number of cells that showed positive markers for each marker), and the result of cell count was shown in FIG.

As shown in FIG. 4, when comparing the cell groups treated by the method according to the comparative example, it was confirmed that the number of cells showing AAT positive reaction was higher in the cell group treated by the method according to the example.

Using the above results, the time and efficiency of direct cross-differentiation after the introduction of transcription factors for each somatic cell type were calculated. The results are shown in Table 2 below.

Figure 112015102738573-pat00002

As shown in Table 2, it can be confirmed that the reprogramming time and efficiency of the cell group treated by the method according to the embodiment is more excellent, and it can be confirmed that the direct crossing differentiation efficiency is high.

In addition, when the cell groups treated by the methods according to Examples and Comparative Examples were consistently compared with each other, it was confirmed that a larger amount of cells express hepatocyte-specific markers in the cell group of the Examples.

<Experimental Example 2> Verification of gene expression pattern

In order to confirm the expression pattern of the gene in the direct induction of differentiation from somatic cells to hepatocytes, 4 kinds of somatic cells inducing cross-differentiation by the method according to Example (3F / AR) The total RNA was extracted from the cells of each experimental group, and the cDNAs were synthesized. The quantitative polymerase chain reaction (qPCR) technique was used for the expression of specific genes.

In addition, in order to examine whether direct cross-differentiation from the somatic cell to the hepatocyte was reflected and progressed at the level of mRNA, two types of small molecule compounds (AR) Expression patterns of specific genes in cells at 8 days were analyzed, and the results of the analysis are shown in FIG.

As shown in Fig. 1, when the respective examples and comparative examples were compared, it was found that (1) expression of somatic cell specific genes was more rapidly reduced in the cell group cultured by the method according to the example, (3) the expression of mesenchymal - to - epithelial transition (MET) - related genes and the expression of hepatocyte - specific genes were significantly increased. It was confirmed that the direct crossing differentiation process is shortened and accelerated when the AR is treated together with the direct crossing differentiation into human hepatocytes.

These results indicate that when a small molecule compound is added and cultured after introduction of a combination of transcription factors necessary for inducing known known hepatocytes, the time required for direct crossing differentiation from normal somatic cells to hepatocytes is shortened, resulting in direct crossing differentiation efficiency Which is significantly improved.

As a result, the efficiency of direct cross-over differentiation into hepatocytes was improved by up to about 5 times as much as that of conventional hepatocytes, and the efficiency was evaluated. When a small molecule compound was added together with the introduction of the conventional hepatocyte-specific transcription factor, It was confirmed that the time required for direct crossing differentiation was shortened and accelerated.

Therefore, the method according to the present invention is expected to be useful for accelerating the development of a cell therapy agent using hepatocytes, constructing a toxicity test system for a new drug substance mediated by hepatocytes, and efficiently mass-producing patient-specific hepatocytes.

Claims (6)

(a) introducing a transcription factor comprising FOXA3 , HNF1A and HNF4A into a somatic cell, which is a human fibroblast, and culturing for 48 hours; And
(b) culturing the somatic cells into which the transcription factor has been introduced in a culture medium containing a small molecule compound including A-83-01 and CHIR99021 for 96 hours or more to directly direct reprogramming somatic cells with hepatocytes Direct Crossover Differentiation Efficiency from Human Somatic Cells to Hepatocytes.
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