KR20150115304A - Method for preparation of insulin secreting cells differentiated from mesenchymal stem cells speroid and use thereof - Google Patents

Abstract

The present invention relates to a producing method for inducing differentiation to insulin secreting cells after culturing adult stem cells in a state of speroid in a cell incubator using a well plate consisting of microwells having an inverse pyramid form and, more specifically, to a cell therapy agent for treating diabetes which cultures mesenchymal stem cells from umbilical cord blood in a speroid form. The insulin secreting cells differentiated from mesenchymal stem cell speroid according to the present invention can be helpfully used as a cell therapy agent for transplant having improved efficacy for treating type I diabetes due to being produced at the most effective size for transplant therapy.

Description

TECHNICAL FIELD The present invention relates to a method for preparing insulin secreting cells differentiated from mesenchymal stem cell spoloids,

The present invention relates to a method for inducing differentiation of human adult stem cells into insulin-secreting cells after culturing in a spleen state in a well plate cell culture dish composed of microwells in the form of a pyramidal horn, and more particularly, Derived mesenchymal stem cells are cultured in the form of a spleod.

Currently, diabetes is a disease that afflicts about 200 million patients, and the number is expected to rise every year, and the number of patients will be expected to reach about 300 million by 2025. Diabetes is classified as type 2 diabetes due to the problem of pancreatic beta cells that secrete insulin and type 2 diabetes due to decreased insulin sensitivity but normal secretion of insulin. Type 1 diabetes is an insulin-dependent diabetes mellitus (autoimmune disease) in which the beta cells of the pancreas that secrete insulin are destroyed by autoimmune reactions.

Approximately 60-80% of endocrine tissue cells, which account for about 1% of pancreatic tissue, are insulin-secreting beta cells. Insulin produced here maintains the homeostasis of glucose in the blood of our body. If insulin is not produced, the concentration of glucose in the blood will be high and it can lead to life-threatening complications as well as symptoms that impair the functions of various organs such as the kidneys.

In the treatment of type 1 diabetes, there are methods such as continuously injecting insulin or transplanting another person's pancreas. However, in the former case, it can not be a fundamental treatment method, It is not. Also, in the latter case, the number of patients who are willing to undergo transplantation of other pancreas is too much compared to the number of donors, and the number of patients who can benefit from considering immunologic tissue compatibility is extremely limited. Therefore, it is very urgent to develop a new treatment method that can overcome these limitations.

Recently, many studies have focused on the development of treatment methods for diabetes mellitus using stem cells. Most of the results reported so far include embryonic stem cells or adult stem cells derived from bone marrow.

Examples of human embryonic stem cells that differentiate into insulin-secreting cells include Soria et al., Diabetes 49: 157-162,2000; Assady et al ., Diabetes 50: 1691-1707, 2001; Fujikawa et al ., Am J Pathol 166: 1781-1791, 2005. In this way, transplantation of cells differentiated in vitro into an animal model that has induced diabetes restores normal blood glucose (Soria et al ., Diabetes 49: 157-162,2000; Fujikawa et al ., Am J Pathol 166: 1781-1791, 2005). However, embryonic stem cells have the problem that they are differentiated into ethical problems, cancer development problems and unwanted tissues. Adult stem cells, on the other hand, are the most appropriate cell therapy because they do not cause cancer and are not prone to ethical problems.

The use of human mesenchymal stem cells as candidates for cell therapy for the treatment of type 1 diabetes has been extensively studied. Bone marrow mesenchymal stem cells, abdominal fat stem cells (Hwang et al, TERM 8 ( 5):. 482-488, 2011), umbilical cord blood stem cells (Tasi et al ., J Biomed Sci 19:47, 2012), and it has been reported that the hyperglycemia-induced rats show a blood glucose lowering effect.

However, the effect is limited and requires a large number of cells, and in the case of rats, when a single type of stem cell is injected, the transplanted single cells may be lost by the immune system or the circulatory system (Tasi et al al ., J Biomed Sci 19:47, 2012). In addition, the size of pancreatic islets that can be supplied with oxygen and nutrients during human pancreas transplantation is 50-150 μm in diameter, and the size of the pancreatic islets is a condition for success in the success rate of transplantation (Lehmann et < RTI ID = 0.0 > al ., Diabetes 56 (3): 594-603, 2007).

Accordingly, the present inventors have found that the umbilical cord blood-derived mesenchymal stem cells induce differentiation into a uniformly sized cell spoloid form, and that the differentiated insulin secreting cells are excellent in stem cell function and differentiation efficiency, thereby completing the present invention.

It is an object of the present invention to provide a method for producing insulin-secreting cells in the form of cell spheroids having a size of 50 to 150 mu m from adult stem cells.

It is another object of the present invention to provide a cell therapy agent for treating diabetes comprising the above-described method, wherein the cell is a steroid-type insulin-secreting cell.

In order to achieve the above object, the present invention provides a method for producing a cell spoiled type insulin secreting cell having a size of 50 to 150 mu m from adult stem cells comprising the following steps.

(a) culturing adult stem cells in DMEM / F12 medium containing B27 additive, N2 additive, activin A and glucagon-like peptide (GLP-1) to differentiate into insulin secreting cells; And

(b) incubating the differentiated cells of the first step with a B27 additive, N2 additive, activin A, nicotinamide, hepatocyte growth factor (HGF), glucagon-like peptide (GLP-1) In DMEM / F12 medium to differentiate into insulin-secreting cells.

The present invention also provides a cell therapy agent for treating diabetes comprising the above-described method, wherein the cell is a steroid-insulin-secreting cell as an active ingredient.

The insulin-secreting cells differentiated from the adult stem cell spleod according to the present invention can be produced in the most efficient size for the transplantation procedure, and thus can be effectively used as a cell therapy agent for transplantation for the treatment of type 1 diabetes, .

FIG. 1 (A) is a comparison of the spheroid sizes of umbilical cord blood stem cells differentiated from the Ultra low attachment dish and AggreWell ^™ (scale bar = 200 μm). (B) shows pancreas-related gene expression during spoloid differentiation (DF-A: plate culture differentiation, DF-S: speroid differentiation)
FIG. 2A is a cord blood stem cell (a: 125 cells, b: 250 cells, c: 500 cells, scale bar = 200 μm) cultured in a microwell with a width of 400 μm of AggreWell ^™ . (B) represents the expression ratio of pancreatic-related transcription factors according to the number of spleen cells. (C) represents the amount of insulin secretion according to the number of spleen cells.
Figure 3 (A) shows the appearance of 4,700 microwells in each round well and the cell spheroids. (B) shows a microwell cross section having a 400 μm wide horn or a 150 μm wide rounded dome.
Figure 4 shows the expression of pancreatic differentiation-related genes upon induction of differentiation of 250 spoloids (con: control group, DF: differentiation group).
FIG. 5 is a graph showing the cell viability and differentiation ability of the umbilical cord blood stem cells differentiated from the AggreWell ^{™ according} to the size of the spheroids. (A) 3 days culture (1: 100 cells, 2: 300 cells, 3: 600 cells). (B) Culture for 4 days.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In order to solve the problems of the conventional stem cell treatment agent, the present invention provides a method of producing a cell spoloid form by three-dimensional culture instead of the conventional method of transplanting a two-dimensional cell culture result, Insulin-secreting cells and a method for producing the same. Conventional spore culture methods include shaking culture or ultra low attachment dish culture, but they are not suitable for making uniform and small sized spheroids. In the method of the present invention, The wells can be prepared by using a well plate cell incubator composed of the wells. Preferably, a microwell with a 400 占 퐉 wide horn pyramidal shape or a 150 占 퐉 wide round dome shape can be used. Spheroids having a uniform size ranging from 50 to 150 mu m in diameter can be produced, and spoloids having a size of preferably 70 to 120 mu m and more preferably 90 to 110 mu m can be produced. In addition, the microwells having a pyramidal or rounded dome shape may have a width of 50 to 600 mu m, preferably 100 to 500 mu m in width, and most preferably 150 to 400 mu m in width.

Despite the transplantation through muscle, peripheral blood vessels or portal vein, existing cell therapy agents composed of single cells have a problem of rapidly filtering through the blood vessels in the lungs, resulting in very low incidence of the desired tissue. In addition, some cell treatments differentiated into the tertiary structure have various sizes, so that large cell clumps can block blood vessels.

The cell therapy agent of the present invention is developed in a uniform size based on the size of the pancreas, has a high engrafting rate in transplantation through the portal vein, and does not interfere with blood flow even when flowing through the blood vessel. In addition, when transplanted into muscle, unlike single cells, the rate of absorption of blood vessels is low.

(A) adult stem cells are cultured in DMEM / F12 medium containing a B27 additive, an N2 additive, activin A and a glucagon-like peptide (GLP-1) to differentiate into insulin secreting cells ; And (b) culturing the differentiated cells of the first stage in a medium containing B27, N2, activinA, nicotinamide, hepatocyte growth factor (HGF), glucagon-like peptide (GLP-1) And a second step of culturing the cells in DMEM / F12 medium containing insulin-secreting cells and inserting them into insulin-secreting cells. The present invention also relates to a method for producing insulin-secreting cells of 50 to 150 mu m in size from adult stem cells.

The spheroids of the present invention are preferably formed by inoculating 100 to 300 adult stem cells into microwells having a pyramidal or circular dome shape having a width of 150 to 400 m, but are not limited thereto.

It is preferable that the above-mentioned spheroids of the present invention are cultured in a well plate cell culture apparatus composed of microwells having a pyramidal or circular dome shape having a width of 50 to 600 μm, more preferably 100 to 500 μm Size, and most preferably in a microwell plate having a size of 150 to 400 mu m in width, but is not limited thereto.

The adult stem cells of the present invention are preferably, but not limited to, umbilical cord blood-derived stem cells.

In the present invention, the first step is a step of culturing DMEM / F12 medium (DMEM / F12 medium) containing 1 to 2% B27, 1 to 2% N2, 10 to 50 ng / ml actin A and 10 to 100 nM glucagon- Preferably 5 to 10 days and most preferably DMEM / DMEM containing 1% B27 additive, 1% N2 additive, 50 ng / ml actin A and 100 nM glucagon analog peptide (GLP-1) F12 medium for 7 days, but the present invention is not limited thereto. The second step may include 1 to 2% B27, 1 to 2% N2, 10 to 50 ng / ml of activin A, 10 mM of nicotinamide, 10 to 50 ng / ml of hepatocyte growth factor (HGF) It is preferable to culture for 5 days to 10 days in DMEM / F12 medium containing 10 nM to 100 nM glucagon analog peptide (GLP-1) and 1 to 10 mM valproic acid or 100 to 200 nM tricostatin A, and most preferably B27 , 1% N2 additive, 50 ng / ml actinA, 10 mM nicotinamide, 50 ng / ml hepatocyte growth factor (HGF), 10 nM glucagon-like peptide (GLP-1) and HDAC inhibitor 10 mM valproic acid Or other DMAC / F12 medium containing HDAC inhibitor 100 nM tricostatin A for 7 days, but the present invention is not limited thereto. The present invention is characterized by a two-step culture, wherein each step is preferably a serum-free medium.

The spleen-type insulin-secreting cell of the present invention is characterized by expressing at least one pancreatic differentiation-related gene selected from the group consisting of neuroD, pdx1, nkx6.1 and insulin, and is selected from the group consisting of Oct4, Sox2 and Nanog Or more of the stem cell function marker gene.

In another aspect, the present invention relates to a cell therapy agent for treating diabetes, which comprises, as an active ingredient, a steroid-type insulin-secreting cell produced by the above method.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1: Differentiation of cord blood-derived stem cells into insulin-secreting cells

The umbilical cord blood-derived mesenchymal stem cells were separated by the method of Bieback et al., Stem Cells 22: 625-634, 2004 and a two-step differentiation method was used to induce differentiation into insulin-secreting cells.

In this example, DMEM / F12 was used as the differentiation medium as a basic differentiation medium, and differentiation induction into insulin-secreting cells was induced by sequential induction of differentiation step by step.

Specifically, in the case of the first step, 1% B27 (Invitrogen, USA), 1% N2 additive (Invitrogen, USA) was used as a basic medium with DMEM / F12 (Dulbeco's Modified Eagle's Medium / Ham's F12 medium, Gibco, USA) ), 50 ng / ml actin A (Peprotech, USA) and 100 nM glucagon-like peptide (GLP-1) for 7 days. Thereafter, 1% B27 additive (Invitrogen, USA), 1% N2 additive (Invitrogen, USA), 50 ng / ml actin A (Peprotech, USA), 10 mM nicotinamide (Peprotech, USA), 10 nM glucagon analog peptide (GLP-1) and 10 mM valproic acid (VAP, Sigma, USA) or 100 nM tricostatin A (TSA, Sigma, USA) Differentiation was induced. Each step of the first to second steps exchanges the medium itself, and the medium of the liver stage is a serum-free medium to which no serum is added.

Example  2: cells into insulin-secreting cells Speroid  Induction of differentiation into form

When the umbilical cord blood-derived mesenchymal stem cells of Example 1 were differentiated into insulin-secreting cells using the differentiation method of Example 1, they were prepared in the form of cell spheroids of uniform size.

In this example, cells of uniform size were prepared using AggreWell ^™ 400Ex (Stem Cell Tech., Inc., Canada). Spheroids were prepared by placing about 125, 250, and 500 cells in microwells of 4,700 square pyramids each having a width of 400 m (FIG. 2).

As a result, it was possible to produce spoiloids having a size much smaller than that of the conventional cell spoloids (Fig. 1A), and it was confirmed that the efficiency of differentiation into insulin secretory cells was also excellent (Fig. 1B).

2B) and insulin secretion amount (FIG. 2C) according to the number of spleen cells. As a result, it was found that the efficiency of spleen prepared from 250 cells per microwell as the insulin-secreting cell I could confirm.

Example  3: Cell Speroid  Depending on size Stem cell ability

The umbilical cord blood-derived mesenchymal stem cells of Example 1 were prepared in the form of cell spheroids by the method of Example 2, and the stem cell function according to the size of the spheroids was analyzed.

In this embodiment, about 100, 300, and 600 cells are inserted into each microwell of a histangular pyramid having a width of 400 μm using AggreWell ^™ 400Ex (Stem Cell Tech., Inc., Canada) Respectively.

As a result, 100 and 300 cells of cord blood stem cell spoloids cultured for 3 days showed the best stem cell performance (Fig. 5A). In addition, the expression of NeuroD and Pdx1, which are the most important transcription factors in insulin differentiation, was increased in 250 cell spoloids cultured for 4 days (Fig. 5B).

Cells with a cell number of 100 cells were cultured for 24 hours at a diameter of about 70 μm, and cell spheroids having a cell number of 300 cells were about 100 μm in diameter. When cultured for 4 days, the cell spheroids, which were made of 100 cells, had a diameter of about 60 μm, and the cell spheroids made of 300 cells had a diameter of about 80 μm.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

CLAIMS 1. A method of producing a cell spheroid-shaped insulin-secreting cell having a size of 50 to 150 mu m from adult stem cells comprising the steps of:
(a) culturing adult stem cells in DMEM / F12 medium containing B27 additive, N2 additive, activin A and glucagon-like peptide (GLP-1) to differentiate into insulin secreting cells; And
(b) incubating the differentiated cells of the first step with a B27 additive, N2 additive, activin A, nicotinamide, hepatocyte growth factor (HGF), glucagon-like peptide (GLP-1) In DMEM / F12 medium to differentiate into insulin-secreting cells.
2. The method according to claim 1, wherein the spoloid is formed by inoculating 100 to 300 adult stem cells.
2. The method of claim 1, wherein the sphere is cultured in a well plate cell culture dish consisting of a micromill in the form of a historical pyramid or a round dome.
The method according to claim 1, wherein the adult stem cells are cord blood-derived stem cells.
2. The method according to claim 1, wherein the medium of the first step and the second step are each serum-free medium.
The method of claim 1, wherein the first step comprises adding 1 to 2% B27 additive, 1 to 2% N2 additive, 10 to 50 ng / ml of activin A and 10 to 100 nM of glucagon-like peptide (GLP-1) Lt; RTI ID = 0.0 > DMEM / F12 < / RTI > medium for 5 to 10 days.
2. The method of claim 1, wherein said second step comprises valproic acid or tricostatin A as an HDAC inhibitor.
8. The method of claim 7, wherein the second step comprises adding 1-2% B27, 1-2% N2, 10-50 ng / ml activin A, Wherein the cells are cultured in DMEM / F12 medium containing growth factor (HGF), 10 nM to 100 nM glucagon-like peptide (GLP-1) and 1 to 10 mM valproic acid or 100 to 200 nM tricostatin A for 5 days to 10 days Way.
2. The method of claim 1, wherein said spleen-shaped insulin-secreting cells express one or more genes selected from the group consisting of neuroD, pdx1, nkx6.1, and insulin.
2. The method according to claim 1, wherein said spleen-type insulin-secreting cells express at least one gene selected from the group consisting of Oct4, Sox2 and Nanog.
A cell therapy agent for treating diabetes comprising an effective ingredient of a sphere type insulin secretory cell produced by the method of any one of claims 1 to 10.

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