KR20180025817A - Composition for promoting direct conversion comprising decellularized extracellular matrix and use thereof - Google Patents

Abstract

The present invention relates to a composition for promoting direct crossing conversion comprising a decellularized extracellular matrix and to a method for direct crossing conversion into neurons or hepatocytes using the same. The composition for promoting direct crossing conversion according to the present invention comprises a tissue-derived extracellular matrix separated from a living body, and as the culture surface or hydrogel based on the extracellular matrix effectively provides a tissue-specific microenvironment, the direct crossing conversion efficiency can be remarkably improved, so that the present invention can be applied not only to various tissue engineering application technologies such as a culture platform for producing a cell therapy agent or a biomaterial for a transplant, but also to a toxicity evaluation and a drug screening field.

Description

Compositions for Promoting Direct Cross-Differentiation Containing Depleted Outer Extracellular Substrates and Uses Thereof < RTI ID = 0.0 >

The present invention relates to a composition for accelerating direct crossing differentiation comprising a deteriorated extracellular matrix and a method for direct crossing differentiation into neurons or hepatocytes using the same.

As the population ages and the incidence of intractable diseases increases, the demand for regenerative medicine tissue regeneration and cell therapy is rapidly increasing, and thus there is a great need for the development of a stem cell therapeutic agent. In particular, in the treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, cerebral infarction, cerebral hemorrhage and spinal cord injury, various therapeutic methods using adult stem cells, embryonic stem cells, and induced pluripotent stem cells have been developed . However, adult stem cells have a limited ability to differentiate and have low therapeutic efficacy. In the case of embryonic stem cells, these stem cells are difficult to develop as autologous cell therapy due to ethical issues, safety issues and low efficiency of differentiation . In order to produce a patient-customized cell therapy agent, research on inducing induced pluripotent stem cells from somatic cells and differentiating them into tissue-specific cells has been recently proposed by a reprogramming technique. However, There is a problem to be solved such as efficiency and possibility of teratoma formation.

On the other hand, direct cross-differentiation techniques that induce the conversion of specific somatic cells to other tissue-specific cells can be avoided in terms of immune rejection, teratoma formation, and ethical problems. However, researchers use different transcription factor genes to induce direct cross-over differentiation, and when a transcription factor gene that can induce direct cross-over differentiation into neurons is introduced into somatic cells, However, it is difficult to expect the efficacy as a practical cell therapy agent because the conversion efficiency and the differentiation ability are still very low and the function as nerve cell is limited by existing culture technology alone.

Under these circumstances, various attempts have been made to improve the efficiency of direct crossing differentiation (Korean Patent Laid-open Publication No. 10-2015-0015294), but there is a need for a technique of direct crossing differentiation which can be effectively used in vivo for actual cell therapy Development has not been done yet.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and the present inventors have made intensive efforts to develop a technique capable of enhancing the efficiency of direct crossing differentiation. As a result, , The direct crossing differentiation efficiency of nerve cells or liver cells is remarkably improved, and based on this, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for promoting direct cross-differentiation, which comprises an extracellular matrix (ECM) derived from a tissue isolated from a living body.

Another object of the present invention is to provide a culture container for direct cross-differentiation containing the composition or a three-dimensional support for direct cross-differentiation.

It is still another object of the present invention to provide a method for producing a nerve cell, which comprises culturing a somatic cell into which a transcription factor has been introduced, in a culture container for direct crossing differentiation or a three-dimensional support comprising an extracellular matrix derived from a tissue isolated from a living body Or a method of direct crossing differentiation into hepatocytes.

It is still another object of the present invention to provide a cell therapy agent containing the directly cross-differentiated neural or hepatocytes by the above method.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a composition for promoting direct cross-differentiation, which comprises an extracellular matrix (ECM) derived from a tissue isolated from a living body.

In one embodiment of the present invention, the tissue may be brain or liver tissue.

In another embodiment of the present invention, the extracellular matrix can be obtained by decellularizing a tissue isolated from the living body.

In another embodiment of the present invention, the extracellular matrix may be included in the composition in the form of a lysate.

The present invention also provides a culture container for direct cross-differentiation, the surface of which is coated with the composition.

In one embodiment of the present invention, the surface of the culture container for direct cross-differentiation may contain the extracellular matrix at a concentration of 0.005 to 0.03 mg / ml.

The present invention also provides a three dimensional support for direct crossing differentiation comprising the composition and the hydrogel.

In one embodiment of the present invention, the hydrogel may be selected from the group consisting of collagen, hyaluronic acid, alginate, gelatin, polyethylene glycol, chitosan, matrigel, cordyitin sulfate, and cellulose.

In another embodiment of the present invention, the direct crossover differentiation three-dimensional support may contain extracellular matrix at a concentration of 4 to 16 mg / ml.

Also, there is provided a method for directly crossing differentiation into neurons or hepatocytes, comprising culturing a somatic cell transduced with a transcription factor in a culture container for direct crossing differentiation or a three-dimensional support containing an extracellular matrix derived from a tissue isolated from a living body .

In an embodiment of the present invention, the extracellular matrix comprises (a) lyophilizing the extracellular matrix obtained by decellularizing the tissue isolated from a living body, and then pulverizing the extracellular matrix; And (b) dissolving the pulverized material in a solvent to prepare a melt.

In another embodiment of the present invention, the somatic cell may be a fibroblast.

In another embodiment of the present invention, the step of culturing in the direct crossing differentiation three-dimensional support is cultivated in a microfluidic device, the microfluidic device comprising: (a) A culture channel in which a somatic cell into which a 3D support and a transcription factor are introduced is loaded; And (b) a reservoir connected to the culture channel via a micro-tube capable of fluid movement.

In addition, the present invention provides a cell therapy agent comprising the directly cross-differentiated neural or hepatocytes by the above method.

The composition for promoting direct cross-differentiation according to the present invention comprises a tissue-derived extracellular matrix separated from a living body, and the culture surface or the hydrogel based on the extracellular matrix effectively provides a tissue-specific microenvironment, The present invention can be applied not only to various tissue engineering application technologies such as a culture platform for producing a cell therapy agent or a biomaterial for a transplantation but also to a toxicity evaluation and a drug screening field It will be possible.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram schematically illustrating a process for producing a brain tissue-derived extracellular matrix lysate according to the present invention and its application. FIG.
FIG. 2 is a graph showing the results of (a) visual observation of cell component removal by deaeration treatment, (b) cell nucleus change by H & E staining, and (c) d) the results of comparing the contents of glycosaminoglycan (GAG).
Fig. 3 shows the results of confirming distribution of (a) protein and (b) lipid components present in brain tissue after de-saturation treatment.
FIG. 4 shows the results of (a) surface element analysis and (b) water contact angle measurement according to the surface coating of the extracellular matrix component in the culture container for direct cross-differentiation according to the present invention.
Fig. 5 shows the result of (a) laminin immunostaining and (b) scanning electron microscope observation of the substrate surface in the culture container for direct cross-differentiation according to the present invention.
FIG. 6 shows the result of (a) observation of the internal structure of the three-dimensional support for direct crossing differentiation according to the present invention by a scanning electron microscope, and (b) measurement of the elastic modulus and the viscosity coefficient using a rotary flow meter.
FIG. 7 is a diagram schematically showing a direct crossing differentiation process to a neuron using a culture container for direct crossing differentiation according to the present invention. FIG.
8 shows the expression of Sox2, Nestin, and Tuj1 according to the concentration of the extracellular matrix (0.01 0.05, 0.1 mg / ml) on the 7-10 day after the BAM-encoding plasmid was transferred to the fibroblasts by qRT-PCR (B) confirmed the formation of neurosphere-like clusters.
FIG. 9 shows the results of (a) immunostaining of Tuj1 + cells according to the concentration of the extracellular matrix (0.005, 0.01 mg / ml), and (b) This is the result of quantification.
Fig. 10 shows the expression of Nestin, Tuj1, and MAP2 according to the extracellular matrix concentration (0.005, 0.01 mg / ml) on the 14th day after the addition of the neural cell inducing medium by immunohistochemistry. B) -PCR. ≪ / RTI >
Fig. 11 shows the results of comparing the average length of neurites according to the concentration of the extracellular matrix (0.005, 0.01 mg / ml) on the 14th day after the addition of the neuron-inducing medium.
Figure 12 shows the PLL-LN group containing a single component of the PLL-BEM (0.01) extracellular matrix according to the present invention, the PLL-LN group containing the extracellular matrix complex component (B) Expression of Nestin, Tuj1, and MAP2 by qRT-PCR in immunostaining of synapsin I + and Tuj1 + cells of FN group.
FIG. 13 is a graph showing the results of RNA-sequencing analysis of gene expression profiles of directly cross-differentiated inducible neurons (iN-BEM) in a culture container for direct cross-differentiation according to the present invention. ontology (GO) category results.
Figure 14 shows the electrophysiological function evaluation of direct crossed differentiated guinea neural cells (iN-BEM) in a culture vessel for direct cross-differentiation according to the present invention. (A) Rest membrane potential, (b) (D) post-synaptic current and current changes due to PiTx and CNQX treatment, and (e) reactivity to glutamate neurotransmitter. Results.
FIG. 15 is a schematic diagram illustrating an experimental procedure for confirming the therapeutic effect on the stroke animal model of the induction neuron according to the present invention. FIG.
FIG. 16 shows the distribution of DiI + BrdU + cells and (c) DiI + Tuj1 + cells by immunohistochemical staining of (a) DiI fluorescence distribution on the seventh day after transplantation of inducible neurons .
FIG. 17 shows the neurobehavioral characteristics of the mouse model of the transplantation of the inducible neurons. (A) Rotarod test, (b) Ladder walking test, and (c) Grip strength test.
FIG. 18 shows the results of immunostaining of Tuj1 expression of inducible neurons in a culture container for direct cross-differentiation according to the present invention having a substrate surface having a nanopattern structure.
FIG. 19 shows the result of direct cross-differentiation from adult rat-derived tail fibroblasts to neurons using the culture container for direct cross-differentiation according to the present invention, and then the expression of Tuj1 and NeuN was confirmed by immunostaining.
(A) direct crossing differentiation into neurons, (b) difference in culture conditions between the comparative group and the present invention, (c) microfluid Fig.
FIG. 21 shows quantitative results of (a) Tuj1 and MAP expression of (a) inducible neurons in response to changes in microenvironmental conditions. (B) Tuj1 and MAP expression were examined by immunohistochemical staining. H3K4me3 or H3K27Ac protein by Chromatin immunoprecipitation (ChIP) assay.
FIG. 22 shows (a) quantitative comparison of YAP expression in nuclear (a) cytoplasmic YAP expression by immunohistochemistry, and (b) nuclear YAP expression according to changes in microenvironmental conditions.
23 shows the results of (a) quantitative comparison of YAP expression in nuclear (a) cytoplasmic YAP expression by Y-27632 or blebbistatin treatment, (b) (D) treatment of blebbistatin (5, 15 μM). The results of the comparison of Tuj1 expression with the treatment concentration of -27632 (5, 10 uM) were compared.
FIG. 24 shows (a) the degree of H3K4me3 histone modification of inducing neurons according to changes in microenvironmental conditions through immuno-staining, and (b) comparison results thereof.
Fig. 25 shows the results of immunohistochemical staining of expression of NeuN, Tuj1, and MAP2 at 7-9 days after culturing in a microfluidic device.
Fig. 26 shows the results of qRT-PCR for the expression of Nestin, Tuj1, and MAP2 at 7-9 days after culturing in a microfluidic device.
FIG. 27 shows the results of (a) average length of neurites, (b) number of Tuj1 + cells, and (c) reactivity to glutamate neurotransmitter at 7-9 days after culturing in a microfluidic device.
FIG. 28 shows the result of confirming the adhesion of the induced nerve cells and the nerve processes in the three-dimensional support for direct crossing differentiation according to the present invention.
FIG. 29 shows the therapeutic effects of the induction neuron according to the present invention using an animal model of stroke. (A) The distribution of the transplanted cells through the DiI fluorescence distribution was confirmed. (B) Rotarod test results.
FIG. 30 shows the results of (a) expression of Tuj1 and MAP2 in inducible neurons induced by direct crossing differentiation using hyaluronic acid. (B) Expression of Tuj1, MAP2 and Nestin by qRT- PCR.
31 is a diagram schematically illustrating a process of direct crossing differentiation into hepatocytes using the culture container for direct crossing differentiation according to the present invention.
FIG. 32 shows the result of (a) observation of the morphology of the cells by light microscopy and (b) expression of albumin and E-cadherin by immunostaining.
FIG. 33 shows the results of confirming the presence of (a) albumin and (b) glycogen in induced hepatocytes according to the extracellular matrix concentration (0.02 mg / ml, 0.1 mg / ml).
Figure 34 shows the expression of hepatic specific markers (HNF4A, AFP, ALB, CYP7A1), (b) albumin per cell, (c) This is the result of comparing the amount of urea secreted.
FIG. 35 shows the result of immunostaining of the expression of albumin by Bi-directional flow or co-culture with HUVEC in direct crossing differentiation into hepatocytes using a three-dimensional support for direct crossing differentiation according to the present invention.
Figure 36 shows the results of (a) urea secretion per cell, (b) albumin secretion per cell, and (c) activity of CYP3A4A according to Bi-directional flow or co-culture with HUVEC.
Figure 37 shows the results of comparing the activity of CYP3A4A with (a) Rifampicin or (b) Verapamil treatment.
Figure 38 shows the results of comparing (a) F-actin and vinculin, (b) E-cadherin, and (c) Caspase-3 expression according to Bi-directional flow or HUVEC co-culture.
39 is a graph showing the relationship between the conditions in the microfluidic device and the difference in the hydrogels albumin were immunostained.
FIG. 40 shows liver toxicity of drugs using induction hepatocytes and HUVEC cell clusters derived from a direct crossing differentiation three-dimensional support according to the present invention. (A) Acetaminophen and (b) Alcohol treatment And the degree of apoptosis of the cells was determined.
FIG. 41 shows liver transplantation using a cell mass composed of inducible hepatocytes and HUVEC cells derived from a direct crossing differentiation three-dimensional support according to the present invention, and the distribution of transplanted cells through DiI fluorescence distribution was confirmed .

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for promoting direct cross-differentiation, which comprises an extracellular matrix (ECM) derived from a tissue isolated from a living body.

As used herein, the term " direct lineage reprogramming (Direct conversion, transdifferentiation) "is a technique for inducing the conversion of adult cells having completely different cell types, Which is different from the conventional technique in that it induces the conversion into a cell that is a cell line. Currently, direct cross-differentiation technology has been recognized in the field of disease modeling, drug discovery, gene therapy, and regenerative medicine, and research on the treatment of neurological diseases using this technique has been actively carried out. However, It is difficult to apply it. In addition, application of cross-differentiation in vivo through direct cross-differentiation has a significant clinical significance, but there is a problem in commercialization due to lack of proper induction method. In contrast to the direct cross-differentiation technique, in the context of the differentiation of stem cells, it has been conventionally used a surface coating using a chemical (Poly L-lysine, Poly L-ornithine) or a part of the extracellular matrix (fibronectin, collagen) Techniques, or hydrogels based on collagen or metry gel. However, they also have difficulties in improving the survival rate, differentiation ability, and functionality of the cells. Thus, the present invention aims to improve the efficiency of direct crossing differentiation by simulating tissue-specific microenvironment using tissue-derived extracellular matrix separated from living body.

As used herein, the term "extracellular matrix" is a collection of biopolymers that fill tissue or extracellular spaces and include fibrous proteins such as collagen and elastin, complex proteins such as proteoglycan and clicosaminoglycan, Cell adhesion glycoproteins such as fibronectin, laminin, and vitronectin. Meanwhile, in the present invention, the extracellular matrix derived from the tissue can be obtained through a de-saturation technique widely known in the art such as a de-saturated solution and the like, and the above-described tissue can be applied to all living tissues containing an extracellular matrix component However, for purposes of the present invention, the tissue may be brain or liver tissue.

The composition according to the present invention may also include extracellular matrix in the form of a melt for application to a culture container or a three-dimensional support having a substrate surface for direct cross-differentiation. In one embodiment, the depolymerized extracellular matrix may be lyophilized, and the lysate may be prepared using a pepsin solution or the like, but is not limited thereto.

Meanwhile, the extracellular matrix contained in the composition according to the present invention includes not only extracellular matrix protein components such as collagen and fibronectin, but also various lipid components. When the extracellular matrix is contained in excess , The hydrophilicity of the whole composition is lowered, and accordingly, the adhesion of the cells is decreased, which may directly cause a factor of lowering the crossing differentiation efficiency. Therefore, it is necessary to use an extracellular matrix having a proper concentration capable of minimizing the reduction of the direct crossing differentiation efficiency due to the lipid component, while providing a living tissue simulation environmental condition, which is the main object of the present invention, , 0.005 to 0.03 mg / ml, more preferably 0.007 to 0.02 mg / ml in the direct cross-differentiation culture surface, and 4 to 16 mg / ml in the direct cross-differentiation three-dimensional scaffold, But is not limited to.

In another aspect of the present invention, the present invention provides a culture container for direct cross-differentiation coated with the composition; And a three-dimensional support for direct cross-differentiation comprising the composition and the hydrogel.

The culture container for direct cross-differentiation according to the present invention provides a surface to which somatic cells to which transcription factors have been introduced are attached and can be directly cross-differentiated. In particular, the surface is coated with extracellular matrix components, It is possible to improve the efficiency of crossing differentiation directly in a living tissue simulation environment condition.

The three-dimensional support for direct crossing differentiation according to the present invention provides a space in which somatic cells to which transcription factors have been introduced are supported and can be directly cultured and cross-differentiated. As described above, the extracellular matrix components in the hydrogel are directly cross- And can contribute greatly to improve the efficiency. The hydrogel may be composed of various biocompatible polymers such as collagen. However, it is possible to use a gel composed of hyaluronic acid, which is known to be present in large amounts in brain tissues, in direct crossing differentiation into neurons.

On the other hand, as another embodiment of the present invention, there is provided a method for producing a nerve cell, comprising culturing a somatic cell into which a transcription factor has been introduced, in a culturing container for direct gentle germination differentiation comprising a cell- Or direct crossing differentiation into hepatocytes.

As used herein, the term "somatic cell" refers to a cell that has undergone complete differentiation or loss of pluripotency, which refers to the ability to differentiate into various types of cells, and fibroblasts and other nerves Or may be all somatic cells except hepatocytes.

As used herein, the term "nerve cell" means dopamine neuron, gawa neuron, glutam neuron, spinal nerve cell, and all kinds of neuron, May be included without limitation.

The term hepatocyte used in the present invention is a cell that constitutes the hepatic lobule and has a function of decomposing harmful substances and producing serum proteins and bilirubin of various species such as albumin, Such cells may be included without limitation if they are transplanted into a tissue and these functions can be improved.

As used herein, the term "transcription factor" is a factor that induces direct cross-differentiation. Factors that induce direct cross-differentiation into neurons are preferably Lmx1a, Nurr1, Lhx3, Hb9, Isl1, Ngn2, Sox2, Foxa1, Zic1, Olig2, Foxa2, Brn2, Ascl1, and Myt1l, and the factors that induce direct cross-differentiation into hepatocytes are HNF1a, HNF4a, Foxa3, Gata4, Foxa1, and Foxa2 But it is not limited thereto. Such transcription factors may also be introduced into somatic cells by methods well known in the art, such as viruses or nonviral delivery vehicles, and include, by way of example, electroporation or non-viral delivery of PBAE polymer-based nanoparticle delivery vehicles C32-122 poly (beta-amino) esters), but this is not so limited.

In addition, the direct crossing differentiation method according to the present invention can be applied in various forms. In one embodiment, the surface of the culture vessel for direct cross-differentiation of the present invention may include the surface of the nanopattern structure coated with the direct cross-differentiation composition (see Figure 18), the microfluidic device (see Figure 20c) Can be used to provide conditions that are more similar to biological tissue. In one embodiment, the microfluidic device comprises a culture channel in which the direct cross-differentiation three-dimensional support and somatic cells into which the transcription factor is introduced are loaded; And a reservoir containing a medium for inducing neural or hepatocyte differentiation, etc., which can be connected to a micro-tube capable of fluid interaction to provide microenvironmental conditions to the cells.

In yet another embodiment, the method comprises, in conjunction with the composition described above, a substance that modulates the activation of a particular signaling pathway by a change in mechanical properties, such as cell adhesion and contractility, mechanotransduction), it is possible to further improve the direct crossing differentiation efficiency. The material that modulates the mechanical dynamics signaling may be, but is not limited to, a material that preferably reduces the mechanical properties of the culture conditions, such as Y27632 or blebbistatin.

Therefore, the direct crossing differentiation method of the present invention can be further combined with the above-described additional technical elements to further enhance the direct crossing differentiation efficiency.

On the other hand, as another embodiment of the present invention, there is provided a cell therapeutic agent comprising the neuronal or hepatic cell directly cross-differentiated by the above method.

The term "treatment" used in the present invention means all the actions of improving or alleviating symptoms of cerebral or liver diseases by administration of the cell therapy agent according to the present invention.

The term "cell therapeutic agent" used in the present invention is a drug used for the purpose of treatment, diagnosis and prevention with cells and tissues prepared by isolation, culture, and special mangement from a human, Refers to a medicament that is used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as living, homologous, or xenogeneic cell propagation, screening, or otherwise altering the biological characteristics of the cell.

In another aspect of the present invention, there is provided a method for treating a brain or liver disease comprising administering the composition or the cell treatment agent to a subject.

In the present invention, the term "individual" refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, Of mammals.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of a composition containing an extracellular matrix by de-saturating treatment

In this Example, a composition containing a de-fatified brain tissue-derived substrate was prepared as a component for direct cross-differentiation promotion according to the procedure shown in Fig. Specifically, depleted solution of brain tissue was sequentially treated to remove cellular components in brain tissue. 1 mM sucrose [Sigma, St. Louis, MO) was added to the culture medium for 2 hours. After the brain tissue was rinsed with distilled water, the remaining tissue was washed with distilled water (Sigma) for 2 hours, and then with phosphate buffered saline (Sigma) The solution was removed, sterilized with penicillin / streptomycin, lyophilized under reduced pressure, and stored until use. The extracellular matrix derived from the brain tissue was lysed in the form of particles, and a hydrochloric acid solution containing pepsin was added to the pulverized product to obtain a brain tissue-derived extracellular matrix lysate (concentration of extracellular matrix: 40 mg / ml, Pepsin concentration: 4 mg / ml, concentration of hydrochloric acid: 0.01 M). The extracellular matrix lysate derived from hepatocyte was dissolved in 1% (v / v) Triton-X 100 (Wako Pure Chemical Industries, Osaka, Japan) and 0.1% (v / v) Was prepared in the same manner as described above.

In the meantime, changes in brain tissues following de-saturation treatment were compared through visual inspection, H & E (Hematoxylin & eosin) staining, and DNA content change measurement. In addition, Q-TOF mass spectrometry was performed to compare the distribution of proteins present in brain tissue after de-saturation treatment through the unused protein score (sum of peptide distribution values).

As a result, as shown in Fig. 2, it was confirmed that after the de-saturating treatment, the cellular components were removed and became transparent (see Fig. 2a). In addition, the cell nucleus and DNA content in the brain tissue were significantly reduced (see FIGS. 2B and 2C), while the content of glycosaminoglycan (GAG) did not show a large difference, Most cellular components were removed, but extracellular matrix components were still preserved (see FIG. 2d). As shown in FIG. 3, a large amount of collagen, fibronectin, etc., known as proteins constituting the extracellular matrix, was detected (see FIG. 3A), and glycerophosphocholines (PC), glycerophosphoethanolamines (PE), glycerophosphoglycerols , And cholesterol (Cho) were present in a large amount (see FIG. 3B).

Example 2. Two-dimensional Surface Coating and Characterization Based on Depleted Saturated Subcellular Substrate

To provide a substrate surface capable of promoting direct cross-differentiation, the lysate of Example 1 was diluted with acetic acid (AcOH, 0.02M) to a concentration of 0.005-0.1 mg / ml of the extracellular matrix. Thereafter, the diluted solution was treated to coat the plate, and the plate was washed with PBS solution to prepare a plate for direct crossing differentiation (hereinafter, referred to as direct crossing differentiation culture container) containing a degassed extracellular matrix (BEM coating). Then, X- ray photoelectron spectroscopy (XPS) was performed in order to confirm the atomic chemical change of the extracellular matrix component according to the surface coating in the culture vessel for direct cross-differentiation prepared by the above method. A drop of water was dropped on the coated surface, and the surface hydrophilic surface hydrophilicity of the culture container was evaluated by measuring the water contact angle thereof. Laminin, which is known to play an important role in adhesion of neurons, was detected by immunohistochemistry and the surface of the substrate was observed by Scanning Electron Microscope (SEM) to confirm the presence of adherent material. No coating was used as a control, and PLL coating, a poly L-lysine coating, which is widely used for culturing neurons, was used as a comparative group.

As a result, as shown in FIG. 4, the culture container for direct crossing differentiation of the present invention showed a similar tendency to the comparative group widely used for culturing neurons, in which C1s and N1s peaks were observed unlike the control group in which the coating was not performed (See FIG. 4A), and the water contact angle was 56.9, indicating that the hydrophilic properties of the surface did not show any significant change (see FIG. 4B). In addition, as shown in FIG. 5, the surface of the laminin, which was hardly detected in the control group and the comparison group, was uniformly present (see FIG. 5A), confirming the presence of the adhering substance.

From these results, the substrate surface of the present invention coated with the extracellular matrix component maintains the hydrophilic property of the surface of the conventional substrate, so that the present invention can also be applied to a direct crossing differentiation technique including a process of contacting the substrate surface And it was found.

Example 3. Hydrogel and characterization based on degassed saturated extracellular matrix

In order to provide a three-dimensional culture condition capable of promoting direct crossing differentiation as well as the two-dimensional culture of Example 2, the lysate of Example 1 was mixed with PBS diluted with distilled water and collagen I, (Hereinafter, referred to as a three-dimensional support for direct crossing differentiation) containing an outer substrate (BEM). Specifically, the lysate of Example 1 and the collagen I solution were mixed with distilled water, 10X PBS, and finally the extracellular matrix in the mixture; 8mg / ml, PBS; 1X, Collagen I; 2 mg / ml. The pH in the mixture was adjusted to pH 7.4 and then incubated at 37 ° C for 30 minutes. On the other hand, in the case of extracellular matrix-based hydrogels derived from liver tissue, it was prepared only from extracellular matrix solutes without mixing with collagen I.

Thereafter, the internal structure of the direct crossing differentiation three-dimensional support produced by the above method was observed through a scanning electron microscope (SEM), and the elastic modulus in a frequency range of 0.1-1 Hz and The viscous modulus was measured by a rotating flow meter to confirm the mechanical properties of the direct three dimensional support for crossing differentiation. On the other hand, a hydrogel (Col) consisting solely of collagen I was used as a comparative group.

As a result, as shown in FIG. 6, the three-dimensional support for direct crossing differentiation of the present invention was composed of a collagen nanofiber structure. However, unlike the comparative group, the nanofiber phase, (See Fig. 6A). As a result of checking the mechanical properties, the elastic modulus showing the solid phase was higher in all the frequency regions than the viscosity coefficient showing the liquid phase, and the collagen component and the extracellular matrix Component was polymerized to form a stable hydrogel (see FIG. 6B).

From these results, the hydrogel of the present invention containing the extracellular matrix component not only can be manufactured in a homogeneous composition, but also can provide a biologic tissue simulation environment more efficiently, and the present invention can provide a tissue- It can be applied to direct crossing differentiation technology which has a great influence on environment.

Example 4. Direct crossing differentiation into neurons using a culture vessel for direct cross-differentiation

In this example, a direct cross-differentiation culture vessel (PLL-BEM) coated with poly-L-lysine (PLL) solution containing extracellular matrix derived from brain tissue was used to directly Cross-differentiation was induced. Specifically, as shown in Fig. 7, the BAM-encoding plasmid was delivered to the fibroblasts cultured in PMEF medium (DMEM, 10% FBS, 1% P / S) through electroporation, The PMEF medium was replaced with a neural cell inducing medium (DMEM / F12, N2 supplement, 10ng / ml bFGF, 10ng / ml EGF).

4-1. Direct crossover differentiation efficiency into neuron and confirmation of maturation enhancement of induced neuron

The expression of early differentiation-related genes (Sox2, Nestin, Tuj1) by the extracellular matrix concentration (0.01, 0.05, 0.1 mg / ml) was analyzed by qRT-PCR at 10-12 days after the BAM- . Similar to the above, the formation of neurosphere-like clusters and the number of Tuj1 + cells as neuronal specific markers were compared. As a comparative group, PLL (PLL) coated with PLL solution and PLL-LN coated with Laminin were used.

On the other hand, to evaluate the maturation of inducible neurons, changes in expression of Nestin, Tuj1, and MAP2 by extracellular matrix concentration (0.005, 0.01 mg / ml) on the 14th day after culturing in neural cell- Immunohistochemistry and qRT-PCR were performed to compare the mean length of the neurites. Similarly, the PLL group and the PLL-LN group were used as the comparative group. On the 30th day after culturing in the culture medium for inducing neurons, mature (PLL-LN + FN) group (fibronectin was added to the PLL-LN solution) and PLL-BEM The distribution of neuronal markers Synapsin I +, Tuj1 + cells was compared by immunohistochemistry and the expression of Nestin, Tuj1, and MAP2 was compared by qRT-PCR.

As a result, as shown in Figs. 8 and 9, in the group coated with PLL-BEM (0.01) containing 0.01 mg / ml brain tissue-derived extracellular matrix as compared with the PLL-LN group, The expression of Nestin and Tuj1 genes was significantly increased (see FIG. 8A), and neurosphere-like cluster formation was confirmed (see FIG. 8B). In addition, the number of Tuj1 + cells was calculated by immunohistochemistry. As a result, the largest number of Tuj1 + cells were observed in the group coated with PLL-BEM (0.01) , A smaller number of Tujl + cells were observed than in the PLL-LN group (see Fig. 9). That is, since the degassed brain tissue-derived extracellular matrix of the present invention contains a lipid component together with proteins such as collagen and fibronectin, when the excessive concentration is contained, (0.01 mg / ml) may be insufficient because it may be insufficient to provide environment conditions for biotissue simulation when an extremely small amount of the concentration is contained. On the other hand, Was found to play an important role in this technique.

10 and 11, in the group coated with PLL-BEM (0.01), the highest expression of Tuj1 and MAP2 was observed (see Fig. 10), and the neurite length was also measured to be the longest 11). In addition, the group coated with PLL-BEM (0.01) showed higher levels of synapsin I + and Tuj1 + cells than the PLL-LN group containing a single component of extracellular matrix as well as the PLL-LN + FN group containing extracellular matrix components The early neuronal marker Nestin expression was the least and the later neuronal markers Tuj1 and MAP2 expression were the highest (see FIG. 12). The extracellular matrix component derived from brain tissue according to the present invention was found to be a single or extracellular matrix And that the degree of differentiation and maturation of the inducible neurons could be further increased as compared with that of the compound.

4-2. Expression of transcripts and electrophysiological functions of inducible neurons

(IN-BEM) directly cross-differentiating directly from fibroblasts (PMEF), inducing neurons (iN-LN) cross-differentiating directly from the surface of the laminin coating, and culture vessels for direct cross- We analyze the characteristics of these by analyzing them. The gene expression profiles of these cells were compared by RNA-sequencing and the expression patterns of iN-BEM and iN-LN-specific neuronal specific genes were compared based on gene ontology (GO) category analysis.

In addition, the electrophysiological function of inducing neurons directly cross-differentiated over a long period of time (30 days) in the BEM (0.01) group, which showed the best effect in the above Example 3-1, was confirmed by patch clamp analysis.

As a result, direct cross-differentiated inducible neurons (iN-LN, iN-BEM) showed similar expression profiles, whereas they showed distinctly different patterns from fibroblasts (PMEF) . In addition, the expression of neural cell-specific markers such as synapse part, synapse, neuron projection, and neuron differentiation greatly increased in the induction neuron (iN-LN, iN-BEM) iN-BEM (Fig. 13B).

Further, as shown in FIG. 14, the membrane membrane maturity level was gradually improved as the rest membrane potential (RMP) value was lowered according to the culture period (14 days, 21 days, 30 days) - In the clamped or current-clamped state, the generation of current by the action potential and the voltage-openable sodium channel was confirmed, and the action potential and the sodium channel-induced current disappearing by the treatment with TTX (Tetrodotoxin) (See Figs. 14B and 14C). In addition, current reduction by post-synaptic current and PiTx treatment was observed, and current was completely lost when PiTx and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) And inhibitory population were present (see FIG. 14d), and the reactivity to glutamate neurotransmitter was confirmed by calcium imaging (see FIG. 14e).

4-3 Identification of the therapeutic effects of guinea neurons using an animal model of stroke

BEM (0.01) group for 30 days to directly cross-differentiate the inducible neurons into the striatum and cortex areas of the mouse model of brain ischemic injury. 15). Prior to transplantation into the disease-induced mouse model, cells were treated with DiI fluorescent staining to allow for the tracking of transplanted guinea pig neurons.

Specifically, the distribution of the transplanted cells through the DiI fluorescence distribution was confirmed on day 7 after transplantation of the inducible neurons, and the characteristics of the transplanted cells were confirmed through the distribution of DiI + BrdU + cells and DiI + Tuj1 + cells. In addition, in order to examine the therapeutic effect of stroke induced by induction of neuronal cell transplantation, the neurons were observed through the Rotarod test (14 days, 28 days and 56 days), Ladder walking test (56 days), and Grip strength test Behavioral analysis was performed. On the other hand, in the neurobehavioral analysis, PBS group (PBS) was used as a comparative group.

As a result, as shown in FIG. 16, it was confirmed that most of the cells were present at the transplanted site even after 7 days of transplantation of the inducible neurons (see FIG. 16A), and DiI + BrdU + cells and DiI + When Tuj1 + cells were observed, some of the transplanted cells proliferated and they were found to be involved in the neurogenesis of lesion sites (see FIGS. 16B and 16C).

On the other hand, as shown in Fig. 17, the neurobehavioral characteristics of the transplanted mice showed that the time taken from the treadmill to the bottom for four weeks after transplantation was significantly longer (Rotarod test; see Fig. 17A). Similar to the above results, it was confirmed that the ratio of the slip to the ladder and the increase of the grip strength were increased as compared with the comparison group. By transplanting the directly cross-differentiated guinea neural cells according to the present invention, (Ladder walking test; Fig. 17B, Grip strength test; Fig. 17C).

4-4. Direct cross-fertilization using a culture vessel for direct cross-differentiation containing nanopattern structures

As another embodiment of the culture container for direct crossing differentiation according to the present invention, a culture container for direct cross-differentiation comprising a substrate surface of a 300-300 nm groove / ridge nanopattern structure is prepared in the same manner as in Example 2, Coated with an outer matrix component. Then, the fibroblasts were directly cross-differentiated to produce inducible neurons, and the expression of Tuj1, a neuron-specific marker, was confirmed. On the other hand, as a comparative group, a group using a culture vessel for direct cross-differentiation including a general substrate surface not including a nanopattern structure was used.

As a result, as shown in FIG. 18, when the BEM coating is applied to the surface of a substrate made of a nanopattern structure (300-300 nm nanopattern), a brain tissue-specific microenvironment is provided along with topographical stimulation, Direct crossing differentiation efficiency was improved.

4-5. Direct cross-differentiation from Tail-tip fibroblast cells to inducing neurons

Using the culture container for direct cross-differentiation according to the present invention, it was confirmed whether direct cross-differentiation from adult rat-derived tail-type fibroblast (TTFs) to inducing neurons was possible in addition to fetal fibroblast (MEF) . Direct crossing differentiation was induced by the same method as shown in FIG. 7, and then changes in expression of Tuj1 and NeuN, which are neuron-specific markers, were measured and evaluated through immunostaining.

As a result, as shown in Fig. 19, it was confirmed that the expression of Tuj1 and NeuN was significantly increased even when the method of the present invention was applied to adult mouse-derived tailed fibroblasts in which the pluripotency was completely lost. From the above results, it can be seen that the direct crossing differentiation technique according to the present invention can be applied to various kinds of skin cells irrespective of the remaining ability of the differentiation.

Example 5. Direct crossing differentiation into neurons using a three-dimensional support for direct crossing differentiation

In this Example, the effect of the fibroblasts of the mouse of the Example 3 on neuronal cells was examined using a hydrogel (three-dimensional support for direct cross-differentiation) and a microfluidic device containing an extracellular matrix derived from brain tissue Direct cross-differentiation was induced. Specifically, as shown in FIG. 20A, the BAM factor gene was transfected twice using electroporation (day 0) and PBAE (poly (beta amino ester)) polymer nanoparticle-based gene delivery vehicle (day 2) After 4-5 days, the cells were harvested and the cells were encapsulated in a three-dimensional support (BEM gel) of the present invention. Then, the encapsulated cells were filled in the channel of the microfluidic device, and then the neuron-inducing medium was supplied to the cells through a chamber connected to the channel, followed by three-dimensional culture under microenvironmental conditions.

5-1. Comparison of direct crossing differentiation efficiency according to change of micro environmental condition

The direct crossover differentiation system according to the present invention has a technical feature in that it efficiently simulates the microenvironment of actual brain tissue. In order to confirm this effect specifically, various comparative groups are set up to determine the influence on direct gonadal differentiation . Specifically, the degree of H3K4me3 histone deformation, which is an indicator of intracellular transcription and plays an important role in direct cross-differentiation, was compared, and the amount of expression of Tuj1, a neuronal-specific marker, and the ChiP assay revealed that H3K4me3 or H3K27Ac protein The degree of binding to the promoter of the Tuj1 gene was confirmed. YAP expression is known to be influenced by the mechanical properties (strength, etc.) of the conditions under which the cells are attached and cultured. Specifically, when adhered and cultured under conditions of high mechanical properties, YAP is mainly expressed in the nucleus, which activates signals for autologous replication of the cells, whereas when adhered and cultured under conditions of low mechanical properties, , Which is known to contribute to signal activation for cell differentiation. Based on these facts, the location of YAP expression in inducible neurons in response to changes in microenvironmental conditions was confirmed by immunostaining. On the other hand, the comparison group in this example is as shown in the following Table 1 (see Fig. 20B).

As a result, as shown in FIG. 21, it was confirmed that the differentiation of inducing neurons was further promoted in the 3D BEM culture conditions as compared with the 2D BEM and 3D Col culture environments (see FIGS. 21A and 21B) The 3D BEM culture conditions of the present invention showed that the binding of Tuj1 and H3K4me3 or H3K27Ac in the inducible neurons is greatly enhanced by the 3D BEM culture environment (see FIG. 21C), and the 3D BEM condition according to the present invention promotes direct crossing differentiation into neurons It is possible to show that it is possible. In addition, as shown in FIG. 22, the expression of YAP in the nucleus was greatly elevated under the surface culture conditions coated with the 2D BEM, whereas in the 3D hydrogel culture conditions (3D Col, 3D BEM), YAP was mainly expressed in the cytoplasm (See Figs. 22A and 22B). Thus, the need for 3D culture conditions as micro-environmental conditions to enhance direct cross-differentiation of neurons was re-confirmed.

(Y-27632 (Rho-kinase inhibitor) or blebbistatin (myosin-II activity inhibitor)) and the location of YAP expression was analyzed. As a result, as shown in FIG. 23, the 3D Col and 3D BEM groups showed no significant difference after the inhibitor treatment, whereas in the 2D BEM group, the expression of YAP in the nucleus was greatly decreased by treatment with the inhibitor. These results indicate that the mechanical signaling of the culture conditions was reduced by the treatment of Y27632 or blebbistatin in the 2D BEM group, and the signal was activated to activate the signal for cell differentiation. On the other hand, when 5 μM of Y27632 or blebbistatin was treated, the expression of tuj1 mRNA was small in all groups. However, when treated with 10 μM of Y27632, the expression of tuj1 was significantly increased in the 2D BEM group, The concentration of blebbistatin significantly increased the expression of tuj1 in the 3D Col and 3D BEM groups as well as the 2D BEM group. In other words, the decrease in mechanical properties in culture conditions could lead directly to the enhancement of crossed differentiation efficiency, and this effect could be confirmed in both 2D and 3D culture conditions depending on the concentration of the treatment substance.

Therefore, the present invention shows that direct cross-differentiation efficiency can be further enhanced by controlling the epigenetic and mechanotransduction of cells involved in cell transduction according to changes in microenvironment there was.

5-2. Comparison of direct crossing differentiation efficiency according to culture in microfluidic device

Expression changes of NeuN, Tuj1, and MAP2 by extracellular matrix contents were compared by immunohistochemistry at 8-10 days after culturing in microfluidic device. Expression changes of Nestin, Tuj1, and MAP2 were analyzed by qRT-PCR (3D BEM). In addition, we compared the mean length of these liver neurites, the number of Tuj1 + cells as neuronal markers, and the reactivity to glutamate neurotransmitters. As a comparative group, the cells were enclosed with a collagen gel containing no extracellular matrix components, and the group (3DμCol) cultured in the three-dimensional manner as described above was used.

As shown in Fig. 24, when the cells were encapsulated with hydrogel and cultured under a three-dimensional environmental condition, the expression of H3K4me3 was further enhanced. When the BEM gel of the present invention containing an extracellular matrix and a microfluidic device were used together (3Dμ BEM), it was confirmed that these effects are more excellent.

Further, as shown in Fig. 25 and Fig. 26, in comparison with the 3D 占 군 l group, the group (3D 占 BEM) cultured in the three-dimensional direct support for direct crossing differentiation containing the brain tissue-derived extracellular matrix according to the present invention , The expression of NeuN, Tuj1, and MAP2 genes was significantly increased (see FIG. 25). As a result of quantifying the expression of Nestin, Tuj1, and MAP2 genes, the expression level of 3Dμ BEM group was significantly (See Fig. 26). In addition, similarly to the above results, the 3Dμ BEM group showed the longest or greatest amount of the average length of the neurites and the number of Tuj1 + cells (see FIGS. 27A and 27B), and was also excellent in reactivity to the glutamate neurotransmitter (FIG. From these results, it was found that, even in a three-dimensional culture using a microfluidic device, direct crossing differentiation into neurons is promoted by containing an extracellular matrix component.

Therefore, the direct crossing differentiation system according to the present invention not only provides a brain tissue-specific environment containing an extracellular matrix component derived from brain tissue, but also provides a micro-environment condition for neuron culturing using a microfluidic device It was found that the direct crossing differentiation efficiency was remarkably improved.

5-3. Identification of therapeutic effect of inducing neuron

First, induction neurons were collected by dissociation into single cells, reseeded on a 2-dimensional substrate coated with PLL-laminin, cultured for 1 week, and live / dead Analysis was performed to observe changes in phenotypic traits. As a comparative group, the cells were enclosed with collagen gel containing no extracellular matrix components, and the group (3DμCol) cultured as described above was used. In addition, the present inventors investigated the transplantation of the inducible neuronal cells after the transplantation into the striatum and cortex regions of the mouse model of brain ischemic injury, and the therapeutic effects thereof. Specifically, before transplantation into the disease-induced mouse model, Were treated with DiI fluorescence staining to allow for the tracking of transplanted guinea pig neurons, and a rotarod test was performed over a period of 4 weeks after transplantation. As a comparative group, 2-dimensional cultured cells (2D iN cells) were used on the surface coated with BEM.

As a result, as shown in Fig. 28, in the group cultured three-dimensionally (3D 占 BEM) in a three-dimensional support for direct crossing differentiation containing a brain tissue-derived extracellular matrix according to the present invention, And neurite extension of the neuron are maintained well. In addition, it is possible to confirm that the neuronal cell type is maintained well.

As shown in Fig. 29, it was found that a large amount of the induction neurons in the 3Dμ BEM group were present at the graft site (Fig. 29a), and the mice transplanted with the above- (FIG. 29B), it was found that the transplantation of direct-differentiated guinea neural cells according to the present invention improved the exercise capacity of the stroke animal model there was.

5-4. Confirmation of direct crossing efficiency improvement effect using hyaluronic acid

The three-dimensional support for direct cross-differentiation prepared in Example 3 was prepared by mixing extracellular matrix lysate with collagen I, whereas in the present Example, hyaluronic acid, which is known to be present in large amounts in brain tissue rather than collagen I (HA-BEM) were prepared by mixing with 10% extracellular matrix lysate (40 mg / ml). The expression of Nestin, Tuj1, and MAP2 was analyzed by immunostaining or qRT-PCR after direct cross-differentiation with the microfluidic device was induced by the same method as above. As a comparative group, hyaluronic acid gel alone group (HA) was used.

As a result, as shown in Fig. 30, the expression of Tuj1 and MAP2 or Nestin in the HA-BEM group was significantly higher than that in the comparative group. As a result, not only collagen gel but also hyaluronic acid gel- The cross - differentiation efficiency was improved.

Example 6. Direct cross-breeding of hepatocytes using a culture vessel for direct cross-differentiation

In this example, direct cross-differentiation of mouse fibroblasts into hepatocytes was induced using a culture vessel for direct cross-differentiation containing liver tissue-derived extracellular matrix. 30, electroporation (day 0) and PBAE (poly (beta amino ester)) polymer were applied to fibroblasts seeded in PMEF medium (DMEM, 10% FBS, 1% P / (HNF1a, HNF4a, Foxa3) were transferred twice using a nanoparticle-based gene delivery vehicle (day 2). On the next day, the PMEF medium was transferred to hepatic cell induction medium (10% FBS, 0.1 (DMEM / F12) supplemented with 10 mM nicotamide, HCM SingleQuot Kit (Lonza), 20 ng / mL hepatocyte growth factor (Peprotech) and 20 ng / mL epidermal growth factor (Peprotech).

Thereafter, the difference between the inducible hepatic cells (LEM) according to the present invention and the no transfection group (No transfection) was confirmed by immunostaining with an optical microscope or albumin and E-cadherin. In addition, the expression of albumin and the presence of glycogen in induced hepatocytes were confirmed by the concentration of extracellular matrix derived from liver tissue (0.02 mg / ml (LEM 0.02), 0.1 mg / ml (LEM 0.1)), (0.02 mg / ml) (Col 0.02) were used. Expression of liver specific markers (HNF4A, AFP, ALB, CYP7A1) and albumin and urea secretion levels were compared quantitatively. Primary hepatocyte was used as a comparative group.

As a result, as shown in FIG. 32 to FIG. 34, it was confirmed that transfer of the transcription factor was an essential factor in direct crossing differentiation into hepatocytes (see FIG. 32), and compared with the Col 0.02 group and the LEM 0.1 group, The albumin expression of the inducible hepatocytes of the LEM 0.02 group, which contained the tissue-derived extracellular matrix at a concentration of 0.02 mg / ml, was the most dominant (see Fig. 33a) and stained with Periodic Acid-Schiff (PAS) (See FIG. 33B). In addition, the content of albumin and urea per cell was increased as compared with the primary hepatocyte (see FIG. 34). By containing the extracellular matrix component derived from liver tissue according to the present invention, the degree of differentiation and maturation of the induced hepatocyte increased And it was found.

Example 7. Direct crossing differentiation into hepatocytes using a three dimensional support for direct crossing differentiation

7-1. Bi-directional flow and co-cultivation with HUVEC

In this example, the hydrogel containing the extracellular matrix derived from the liver tissue of Example 3, that is, the direct crossed differentiation three-dimensional support (LEM) and the microfluidic device, In the same procedure, direct cross-differentiation of mouse fibroblasts into liver cells was induced. Specifically, the fibroblasts were encapsulated in a three-dimensional support (LEM gel) of the present invention, and then cultured in a non-flow condition (No flow) in a microfluidic device, The bi-directional flow provided a flow of fluid in the microfluidic device using a rocker. Each of these cells was cultured under conditions (iHEP: HUVEC = 6: 4) or (iHep + HUVEC) without co-culture with human umbilical vein endothelial cells (HUVEC).

The expression levels of urea and albumin and the activity of CYP3A4A, which are specific for the liver, were quantitatively compared with those of CYP4A3A activity. Rifampicin, known as an inducer associated with CYP4A3A activity, When verapamil, known as the suppressor, was added to the induced hepatocytes, the change in CYP4A3A activity was measured. In addition, F-actin, vinculin, Caspase 3, and E-cadherin immunostaining were performed under the same conditions as above to compare the expression of the protein.

As a result, as shown in Fig. 35, in the No flow group, the albumin expression level was low, and when the flow was added, the expression level of albumin was high and the vasculature was formed when co-cultured with HUVEC (iHep + HUVEC) I could confirm. As shown in Figs. 36 and 37, when co-cultured with HUVEC in the presence of flow, the urea, albumin secretion amount and CYP3A4A activity of the inducible hepatic cells were the most excellent, (See Figs. 36a, 36b and 36c). When Rifampicin or verapamil was administered to the inducible liver cells, the activity of CYP3A4A was increased or decreased. (See Figs. 37A and 37B). As shown in FIG. 38, when F-actin, vinculin and E-cadherin, a protein related to intercellular interaction, were observed, when the cells were co-cultured with HUVEC in the presence of flow, (Fig. 38A and Fig. 38B). In the case of expression of Caspase-3, a cell apoptosis-related protein, expression of these genes in the no flow group was significantly increased The greatest number of cells were observed, and co-culturing with HUVEC in the presence of flow revealed that apoptosis in the direct cross-differentiation process was slowed (see FIG. 38C).

7-2. Variation of direct crossing differentiation efficiency according to conditions in microfluidic device

In this example, the microfluidic device consisting of three channels in the microfluidic device shown in Fig. 20C was used to induce direct crossing differentiation from the fibroblasts of the mouse into the liver cells. Specifically, a group (iHep) in which only the guided hepatocytes encapsulated in the LEM gel / collagen gel were channeled, a group (iHep + EC) in which the LEM gel / collagen gel- (iHep / EC) in which the HUVECs that were not encapsulated in the outer two channels were used to measure the albumin expression.

As a result, as shown in FIG. 39, when the LEM gel according to the present invention was used, the expression of albumin was higher than that in the group using the collagen gel, and the induced hepatic cells and HUVEC In the induced hepatocytes of the iHep + EC group, the expression of albumin and the formation of vasculature were the best.

Example  8. Induced stem cells  Utilization of toxicity evaluation

In this example, hepatic toxicity evaluation and liver transplantation were performed on the drug using the cell masses of the induction hepatic cells and the HUVEC cells prepared in the procedure of Example 7 above. Acetaminophen (10 mM, 20 mM) or alcohol (100 mM, 200 mM, 600 mM), which is known to have liver toxicity specifically, was added to the induced hepatocyte culture and cultured for 2 days or 3 days thereafter to perform live / dead analysis In addition, the cells are treated with DiI fluorescence staining to be able to track the transplanted induced hepatocytes before being encapsulated with the three-dimensional support (LEM gel) of the present invention, and then transplanted to the transplantation site, Respectively.

As a result, as shown in FIG. 40, it was confirmed that the number of inducing hepatocytes (red color) to die increased as the concentration of acetaminophen (FIG. 40A) or alcohol (FIG. 40B) Showed the possibility of being applied to liver toxicity evaluation. In addition, as shown in Fig. 41, it was shown that the transplanted induced hepatocytes were present and survived in a large amount at the transplantation site, and thus could be utilized as a technique for liver transplantation.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (19)

A composition for promoting direct cross-differentiation, comprising an extracellular matrix (ECM) derived from a tissue isolated from a living body.
The method according to claim 1,
The composition for promoting direct cross-differentiation, wherein the tissue is brain or liver tissue.
The method according to claim 1,
Wherein the extracellular matrix is obtained by decellularizing a tissue isolated from the living body.
The method according to claim 1,
Wherein the extracellular matrix is contained in the form of a lysate.
A culture vessel for direct cross-differentiation, comprising a surface coated with the composition of claim 1.
6. The method of claim 5,
Characterized in that the surface of the culture vessel for direct cross-differentiation comprises an extracellular matrix in a concentration of 0.005 to 0.03 mg / ml.
A three dimensional support for direct crossing differentiation, comprising the composition of claim 1 and a hydrogel.
8. The method of claim 7,
Characterized in that said direct crossing differentiation three-dimensional scaffold comprises extracellular matrix at a concentration of 4 to 16 mg / ml.
8. The method of claim 7,
Wherein the hydrogel is selected from the group consisting of collagen, hyaluronic acid, alginate, gelatin, polyethylene glycol, chitosan, matrigel, coderoint sulfate, and cellulose.
A method for direct crossing differentiation into a neural or hepatocyte, comprising culturing a somatic cell transfected with a transcription factor in a culture container for direct gentle differentiation comprising a surface coated with an extracellular matrix derived from a tissue isolated from a living body.
11. The method of claim 10,
Wherein the tissue is brain or liver tissue.
11. The method of claim 10,
Wherein the surface of the culture vessel for direct cross-differentiation comprises an extracellular matrix at a concentration of 0.005 to 0.03 mg / ml.
11. The method of claim 10,
RTI ID = 0.0 > 1, < / RTI > wherein the somatic cells are fibroblasts.
A method for direct differentiation into a neural or hepatocyte, comprising culturing a somatic cell into which a transcription factor has been introduced, in a three-dimensional direct support for cross-differentiation, comprising a tissue-derived extracellular matrix and a hydrogel separated from the living body.
15. The method of claim 14,
Wherein said tissue is brain or liver tissue.
15. The method of claim 14,
Wherein said direct crossing differentiation three-dimensional scaffold comprises extracellular matrix at a concentration of 4 to 16 mg / ml.
15. The method of claim 14,
RTI ID = 0.0 > 1, < / RTI > wherein the somatic cells are fibroblasts.
15. The method of claim 14,
The step of culturing is characterized in that it is cultured in a microfluidic device,
The microfluidic device comprises: (a) a culture channel in which the three-dimensional support for direct cross-differentiation and a somatic cell into which a transcription factor is introduced are loaded; And (b) a reservoir connected to the culture channel via a micro-tube capable of fluid movement.
19. A cell therapy agent comprising a neuronal or hepatocyte directly cross-differentiated by the method according to any one of claims 10 to 18.

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