KR20220050656A - A method of embryonic stem cell differentiation into cardiac/nerve like tissue using Wnt agonist - Google Patents

Abstract

The present invention relates to a method for differentiating embryonic stem cells into cardiac-like or nerve-like tissues using a Wnt agonist, and to cells prepared therefrom. By using the method according to the present invention, it is possible to efficiently differentiate embryonic stem cells into nerve cells or cardiac cells, so that the present invention can be effectively used in drug development and research fields associated therewith.

Description

A method of embryonic stem cell differentiation into cardiac/nerve like tissue using Wnt agonist

The present invention relates to a method for differentiating embryonic stem cells into cardiac/nerve-like tissue using a Wnt agonist, and to a cell prepared therefrom.

Stem cell research focuses on mechanisms for maintaining uniqueness and regulating self-renewal ability, and development of methods for inducing differentiation into specific target tissues. Studies have been reported that Wnt signaling is involved in the process of determining the fate of cells, and in particular, the role of Wnt signaling in regulating the differentiation of stem cells has been elucidated. Pluripotent embryonic stem cells can be differentiated into any cell according to the induction method, and there is a report that Wnt/β-catenin signaling maintains the pluripotency and self-renewal ability of human and mouse embryonic stem cells, promoting the differentiation of stem cells appears to be involved in Wnt signaling.

It is important that exogenous bioactive molecules such as Wnt, which are required for inducing specific cell differentiation from stem cells, be added to the culture system in timely manner, and research on this is also continuously required.

Republic of Korea Patent Publication No. 10-0986149

The present inventors have completed the present invention by confirming a method for differentiating embryonic stem cells into cardiac or nerve cells by treating embryoid body, which is a Wnt agonist, CHIR99021, generated using a 3D culture system for embryonic stem cells.

Accordingly, an object of the present invention is to provide a method for inducing differentiation of embryonic stem cells.

Another object of the present invention is to provide a cell differentiated by the above method.

In order to achieve the object of the present invention, the present invention comprises the steps of: a) forming an embryoid body from embryonic stem cells;

b) differentiating the embryoid body formed in step a) into an ectoderm to form an organoid; and

c) provides a method for inducing differentiation of embryonic stem cells, comprising the step of treating the organoid formed in step b) with a Wnt agonist.

In one embodiment of the present invention, the differentiation into the ectoderm of step b) is treated with BMP4 (Bone morphogenetic protein 4) and TGF-βi (Transforming growth factor beta inhibitor), and then FGF2 (fibroblast growth factor) and BMP4i (Bone) morphogenetic protein 4 inhibitor).

In one embodiment of the present invention, the ectoderm differentiation of step b) is to treat BMP4 and TGF-βi on the second day of differentiation, and treat FGF2 and BMP4i on the third day of differentiation, and the Wnt agonist of step c) is the 7th day of differentiation may be processing.

In one embodiment of the present invention, the organoid in step b) may be a beating organoid.

In one embodiment of the present invention, the Wnt agonist in step c) may be one represented by the following formula (1).

[Formula 1]

In one embodiment of the present invention, the cells differentiated through step c) may be cardiac cells or nerve cells.

In addition, the present invention provides a cell having the following characteristics 1) or 2) differentiated by the above method.

1) Tuj1 (Neuron-specific class III beta-tubulin) expression

2) Expression of Ctp1 (Cardiac Troponin 1)

The present invention relates to a method for inducing differentiation of embryonic stem cells into cardiac/nerve-like tissue, and since the method enables efficient differentiation from embryonic stem cells into nerve cells or cardiac cells, it is useful in related drug development and research fields, etc. can be used

1 schematically shows the differentiation method of the present invention.
2 is a result of observing the morphological characteristics of EBs according to the presence or absence of a core under a microscope.
3 and 4 confirm the formation of beating organoids in Wnt-activated EBs (video corresponding to the figure can be found at https://youtu.be/3JJH--qOwy0 or as a file attached to this application).
5 is a result confirming using a confocal microscope that Wnt-activated organoids generate nerve cells (A) and cardiac cells (B) after 30 days of cell culture.

The present inventors have completed the present invention by confirming a method for differentiating embryonic stem cells into cardiac or nerve cells by treating embryoid body, which is a Wnt agonist, CHIR99021, generated using a 3D culture system for embryonic stem cells.

Accordingly, the present invention comprises the steps of: a) forming an embryoid body from embryonic stem cells;

b) differentiating the embryoid body formed in step a) into an ectoderm to form an organoid; and

c) provides a method for inducing differentiation of embryonic stem cells, comprising the step of treating the organoid formed in step b) with a Wnt agonist.

In the present invention, the embryonic stem cell (embryonic stem cell) refers to an undifferentiated cell that has the ability to differentiate into cells of all tissues, but is not yet differentiated, as a cell extracted during embryonic development.

In the present invention, the term "embryoid body" refers to the formation of an aggregate that is a round ball-shaped cell mass by aggregating stem cells at the initial stage of differentiation of stem cells.

In the present invention, the term "ectoderm" refers to a cell layer that covers the outermost layer of the embryo among the three germ layers formed through gastrulation at an early stage of development.

In the present invention, organoid refers to self-renewal and self-organization from adult stem cells (ASC), embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). It means a three-dimensional cell aggregate formed through

In the present invention, Wnt agonist activates signaling in the Wnt signaling system that regulates various processes such as cell fate determination, structural reorganization, polarity, morphology, adhesion and growth, and maintenance and proliferation of undifferentiated cells during embryonic development. means a substance that

In the present invention, the differentiation into the ectoderm of step b) is treated with BMP4 (Bone morphogenetic protein 4) and TGF-βi (Transforming growth factor beta inhibitor), and then FGF2 (fibroblast growth factor 2) and BMP4i (Bone morphogenetic protein 4 inhibitor) ) may be processed.

Also, in the present invention, the ectoderm differentiation in step b) is to treat BMP4 and TGF-βi on the second day of differentiation, and treat FGF2 and BMP4i on the third day of differentiation, and the Wnt agonist in step c) may be treated on the 7th day of differentiation there is.

In the present invention, the organoid in step b) may be a beating organoid.

In the present invention, the beating organoid refers to the differentiation of embryoid bodies derived from embryonic stem cells into organoids characterized by morphological, genetic and immunological profiles similar to those of an embryonic heart and pulsatile movements.

In the present invention, the Wnt agonist in step c) may be represented by the following formula (1).

[Formula 1]

The compound, also called CHIR99021, is an amino pyrimidine derivative that is a very potent glycogen synthase kinase (GSK) 3 inhibitor that inhibits both GSK3β and GSK3α. GSK3 acts as a Wnt activator, as it is a serine/threonine kinase, a major inhibitor of the Wnt pathway.

In the present invention, the cells differentiated through step c) may be cardiac cells or nerve cells.

In another aspect, the present invention provides a cell having the following characteristics 1) or 2) differentiated by the above method.

1) Tuj1 (Neuron-specific class III beta-tubulin) expression

2) Expression of Ctp1 (Cardiac Troponin 1)

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1: Materials and Differentiation Method>

Example 1-1. Cells and cell culture

Undifferentiated mouse ES-J1 (ATCC, Manassas, VA, USA) was used and cultured on gelatin-coated plates without feeder cells and 15% (v/v) heat-activated fetal bovine serum (FBS; ATCC, Manassas, VA, USA). ), 0.1 mM β-mercaptoethanol (Gibco, Invitrogen, Carlsbad, CA, USA), 0.1 mM Glutamax (Gibco), 0.1 mM ES qualified non-essential amino acid (NEAA; Welgene, Daegu, Korea), 1% penicillin-streptomycin (PS; ATCC), high glucose supplemented with 1000 U/mL leukemia inhibitory factor (Millipore, Merck, Burlington, MA, USA), 0.033% CHIR99021 (Tocris Bioscience, Bristol, UK), and 0.125% PD035901 (Tocris Bioscience) 5% CO ₂ , 37 ° C incubator in which humidity is maintained using ESC medium composed of high-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) was maintained in

Example 1-2. embryoid body formation

Embryoid body (EB) was created using a hanging drop technique. Briefly, after embryonic stem cells (ESCs) were dissociated with trypsin/EDTA (TE), 30 μL drops containing 4 x 10 ⁵ cells/mL in maintenance medium were inoculated into the lid of the culture dish and inoculated with 5% CO ₂ . Incubate for 48 h at 37 °C in a humidified incubator containing The resulting EBs were harvested, washed with phosphate buffered saline (PBS), plated in 24-well plates, and cultured in ectoderm differentiation medium.

Examples 1-3. erupt into organoids

ESCs differentiated into inner ear-like organoids as described by Koehler and Hoshino. Ectodermal differentiation was performed with 1.5% knockout serum replacement (Gibco), 0.1 mM β-mercaptoethanol, 1 mM sodium pyruvate (Stem Cell Technologies, Vancouver, BC, CAN), 0.1 mM NEAA, and 1% PS and 2% Matrigel (Corning, New York). York, United States) supplemented with Glasgow minimal essential medium (Gibco) was used.

On the second day of differentiation, 10 ng/mL recombinant BMP4 (Stemgent, Beltsville, MD, USA) and 1 μM SB431542 (Stemgent), which is TGF-βi, were added to the cultured cells to induce non-neural ectoderm.

On the third day of differentiation, 25 ng/mL FGF-2 (Peprotech, Rocky Hill, NJ, USA) and 1 μM LDN-193189 (Stemgent), which is BMP4i, were added to induce preplacodal ectoderm (PPE) cells. was cultured for 2 more days.

On day 7 of differentiation, the differentiation medium was removed and the EBs were washed with PBS and then supplemented with 1% N ₂ supplement (Gibco), 1% Glutamax, 0.1% Normocin (InvivoGen, San Diego, CA, USA) and 1% Matrigel. Resuspended in maturation medium consisting of DMEM/F12 (Sigma-Aldrich). On the same day, EBs were supplemented with a single dose of 0.033% CHIR99021. Half of the medium was replaced with maturation medium without Matrigel every other day until day 30.

Examples 1-4. Morphological analysis of EBs

The size and morphological characteristics of EBs formed using different culture techniques were imaged using bright-field microscopy (CKX53, Olympus, Tokyo, Japan) and the average organoid formation rate, number of vesicles formed per EB, and other characteristics were measured at 15 days. It was recorded from day 30.

Examples 1-5. Epifluorescence analysis

Frozen sections were prepared in advance for immunofluorescence analysis of differentiated EBs. Briefly, EBs were immersed in 4% paraformaldehyde solution, fixed at 4°C for 16-18 hours, and then washed 3 times with cold PBS for 10 minutes each. Cells were immersed in 10%, 20%, and 30% (wt/vol) sucrose solution for 30 min each to prevent crystallization of the sample, transferred to a cyromold, and then an OCT compound (optical cutting temperature compound, Tissue-Tek, Torrence , CA, USA).

The samples were flash frozen on dry ice to make blocks and cut into sections with a thickness of 5 μm using a cryosectioning machine (Leica, Wetzlar, Germany). The cut samples were mounted on slides and stained for imaging. Using the prepared slides, permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) for 10 minutes at room temperature (RT), 10% normal goat serum (NGS; Vector Laboratories, Burlington) and 0.1% Triton X -100 was used to prevent non-specific binding at room temperature.

Sectioned EBs were reacted for 16-18 hours at 4 °C in PBS containing 3% NGS and 0.1% Triton X-100 containing the smooth muscle cell marker cardiac troponin primary antibody (Ctp1, 1:100, Abcam, UK). made it It was washed 3 times with PBS for 5 minutes and then reacted with the corresponding secondary antibody for 1 hour. Samples were washed 3 times for 10 min each, then washed and fixed with FITC-conjugated phalloidin to identify F-actin. After fixing the sample with a mounting solution containing DAPI (4',6-diamidino-2-phenylindole), it was observed.

<Example 2: Confirmation of morphological differences according to the presence or absence of cores of EBs>

As a result of morphological observation of EBs through bright field microscopy, when Wnt was activated using CHIR99021 in the maturation stage, as shown in FIG. It was observed that the inner and outer layers of the EB were subjected to inverted extraction, and as shown in FIG. And as shown in (C) and (D) of Figure 2, it was confirmed that the self-organization of the excised EBs showed a very different form after the cell culture was further progressed.

<Example 3: Confirmation of beating of Wnt-activated organoids>

It was confirmed that during the maturation stage, Wnt-activated EBs produced beating organoids that could be easily observed using light microscopy (see Figs. 3 and 4). All the beating organoids had the same shape but different sizes, and the rhythm of the beats were also different (https://youtu.be/3JJH--qOwy0 or the attached video file to confirm the beating of the organoid of the present invention) .

On the other hand, not all treated EBs formed beating organoids, but 33.3% formed beating organoids. And it was confirmed that only one beating organoid was formed per EB.

<Example 4: Confirmation of differentiation of Wnt-activated organoids into nerve or cardiac-like tissue>

It was confirmed using confocal microscopy that Wnt-activated organoids produced nerve cells and cardiac cells after 30 days of cell culture. EBs with an intact core showed positive staining for the neuronal marker tubullinJ1 (Neuron-specific class III beta-tubulin, Tuj1), and a fine neurite structure was confirmed through a higher magnification photograph (FIG. 5A). In the case of EBs from which the nucleus has been removed, it was confirmed that the organoids exhibited filamentous actin formation and were positive for the smooth muscle cell marker Cardiac Troponin 1 (Ctp1) ( FIG. 5B ).

These results indicate that the differentiation of EBs into neurons or cardiac cells is promoted by the Wnt agonist.

Claims (7)

a) forming an embryoid body from embryonic stem cells;
b) differentiating the embryoid body formed in step a) into an ectoderm to form an organoid; and
c) Inducing differentiation of embryonic stem cells comprising the step of treating the organoid formed in step b) with a Wnt agonist.
According to claim 1, wherein the differentiation into the ectoderm of step b) is treated with BMP4 (Bone morphogenetic protein 4) and TGF-βi (Transforming growth factor beta inhibitor), then FGF2 (fibroblast growth factor 2) and BMP4i (Bone morphogenetic protein) A method of inducing differentiation of embryonic stem cells by treatment with protein 4 inhibitor).
The method according to claim 1, wherein the ectoderm differentiation in step b) is to treat embryoid bodies with BMP4 and TGF-βi on the second day of differentiation and FGF2 and BMP4i for embryoid bodies on the third day of differentiation, and the Wnt agonist of step c) is to be treated on the 7th day of differentiation, a method for inducing differentiation of embryonic stem cells.
The method of claim 1, wherein the organoid in step b) is a beating organoid.
The method of claim 1, wherein the Wnt agonist in step c) is represented by the following formula (1).
[Formula 1]

The method of claim 1, wherein the cells differentiated through step c) are cardiac cells or nerve cells.
A cell differentiated by the method of any one of claims 1 to 6, having the following 1) or 2) characteristics:
1) Tuj1 (Neuron-specific class III beta-tubulin) expression
2) Expression of Ctp1 (Cardiac Troponin 1)

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