KR20120095257A - A METHOD FOR PRODUCING PANCREAS β LIKE CELLS FROM ADULT STEM CELLS - Google Patents

Abstract

PURPOSE: A manufacturing method of pancreas beta similar cells from adult stem cells is provided to maximize efficiencies of differentiation by consecutively applying a step by step differentiating method. CONSTITUTION: A manufacturing method of pancreas beta similar cells from adult stem cells comprises the following steps: differentiating the adult stem cell by cultivating the adult stem cells in the culture medium including the activin -A; and differentiating into a pancreas beta similar cell by culturing the differentiated cell in a culture medium including KGF(Keratinocyte Growth Factor) and let differentiate to the pancreas beta similar cell. The culture mediums of the first and the second steps are the invitrogen mediums which do not include the respective blood serum. The first differentiation step comprises the following: cultivating in a medium including activin -A and sodium butyrate; and cultivating in the culture medium which includes the activin -A and bovine serum albumin.

Description

A method for producing pancreas β like cells from adult stem cells}

The present invention is a method for producing pancreatic beta-like cells by inducing differentiation of pancreatic beta-like cells from adult stem cells, the composition for inducing differentiation, the pancreatic beta-like cells and pancreatic beta-like cells prepared by the method as an active ingredient. It relates to a cell therapy for treating diabetes. The present invention also relates to a method for producing pancreatic precursor cells by inducing differentiation of pancreatic precursor cells from adult stem cells, the composition for inducing differentiation, and a cell therapeutic agent for treating diabetes comprising the pancreatic precursor cells as an active ingredient. . The method for producing pancreatic beta-like cells of the present invention was maximized the efficiency of differentiation by sequentially applying stepwise differentiation as a two-step differentiation induction method, and established a differentiation method from various adult stem cells to diabetic disease. To provide new treatment alternatives.

Diabetes is a disease that more than 200 million patients are suffering from, and it is a problem caused by high incidence, mortality rate and complications from diabetes in countries around the world, including Korea, where the number of patients is expected to double by 2025. . Diabetes is classified as type 1 diabetes due to the problem of pancreatic beta cells secreting insulin and type 2 diabetes due to its normal secretion but reduced sensitivity to insulin. Among these, insulin-dependent type 1 diabetes is a disease that is destroyed by the autoimmune response of the beta-cells in the pancreas. Patients with this disease must be continuously injected with insulin and complications such as retinal diseases, heart and kidney diseases, and neurological disorders. You will suffer extreme pain. In case of type 1 diabetes, pancreatic beta cells are transplanted because normal insulin secretion can be treated. However, pancreatic beta cell transplantation does not have many donors. Problems remain due to tissue rejection with patients who will receive cells. Due to these problems, type 1 diabetes is difficult to treat. In order to solve these therapeutic problems, research on diabetes treatment using stem cells as a cell therapy is receiving new attention.

Most of the studies reported so far use embryonic stem cells or bone marrow-derived stem cells among adult stem cells. According to the conventional report, the differentiation using embryonic stem cells naturally adopts the method of differentiating embryonic stem cells into pancreatic beta cells, so the number of differentiated pancreatic cells is very small, and the ethical problem of using embryonic stem cells. And there is a problem that cancer is caused and differentiation into unwanted tissue. Among adult stem cells, bone marrow-derived stem cells have a problem in that they need to acquire cells in an invasive manner and there are not many donors to receive bone marrow. In addition, the differentiation of pancreatic beta cells using adult stem cells has been followed by the differentiation of embryonic stem cells. In addition, direct transplantation of pancreatic islets is known to be effective in diabetes. However, the lack of donors and the difficulty of directly extracting and amplifying cells from Langerhans is difficult as cell therapy.

As a means to solve this problem, a lot of research has been focused on how to use stem cells having infinite proliferation. Stem cells are cells that have the ability to replicate themselves, that is, to self-replicate, and to differentiate into two or more germ-derived cells. Stem cells can be divided into adult stem cells and embryonic stem cells, depending on the source from which the cells are obtained. These stem cells are being used as cell therapeutics in many diseases in that differentiation ability, that is, the specific ability of stem cells itself, that can differentiate into various cells and can be specialized.

Interest in stem cell research has been raised due to the ability of such stem cells, and research is being conducted on refractory diseases that have been incurable or difficult until now. The possibility of treatment by stem cells for diseases such as Parkinson's disease, brain injury disease, spinal cord injury disease, cancer-related disease, diabetes and Burger's disease has been suggested.

Among the stem cells, embryonic stem cells have excellent differentiation ability. In particular, because of the differentiation ability (pluripotency) was considered as a source that can be used as a cell therapy, many studies have been conducted. However, in the case of embryonic stem cells, there are many limitations, such as the problem pointed out above in that the stem cell acquisition itself must use the embryo. In other words, the use of embryos to obtain embryonic stem cells is limited in securing the number of cells to treat the disease. Above all, the use of embryonic stem cells cannot avoid ethical problems. In addition, as demonstrated in several animal experiments, there is a problem that it is difficult to overcome the risk of cancer when using embryonic stem cells.

Adult stem cells are more limited than embryonic stem cells in that their differentiation capacity is multipotency compared to embryonic stem cells. However, ethical problems faced by embryonic stem cells can be overcome and there is no risk of cancer from developing embryonic stem cells in animals. In particular, adult stem cells have various advantages in their sources. Of these, placental-derived epithelial stem cells are adult stem cells, and various studies have recently been actively conducted. Placenta-derived epithelial stem cells are extracted from the placenta during delivery. To date, studies have shown that a large amount of stem cells can be extracted, and since the placenta itself is classified as a waste, it is used as a source of adult stem cells. Suffice. In other words, the placenta-derived epithelial stem cells, unlike other adult stem cells, utilize easily discarded resources and are easily accessible and have excellent utility. For this reason, many studies using adult stem cells are being conducted. However, no established differentiation method of pancreatic beta cells using placental-derived epithelial stem cells has been reported.

Against this background, the present inventors focus on adult stem cells as a source of stem cells capable of overcoming ethical and cancer development problems, and to develop an active method for differentiating into efficient and simple pancreatic beta cells. As a result of our efforts, we established a two-step method of differentiating adult stem cells into pancreatic beta-like cells by sequentially treating activin-A, sodium butyrate and KGF, and differentiated pancreatic beta-like cells secrete insulin and c-peptide. After confirming that the present invention was completed.

In accordance with the present invention, adult stem cells are sequentially treated with activin-A, sodium butyrate and KGF based on EGM-2 medium to induce differentiation into pancreatic progenitor cells and pancreatic beta-like cells. An object of the present invention is to provide a method for producing pancreatic progenitor cells and pancreatic beta-like cells. In addition, to provide a composition for inducing differentiation used in the production method, pancreatic progenitor cells and pancreas-like cells prepared by the production method, and the pancreatic progenitor cells and pancreas-like cells as an active ingredient for treating diabetes mellitus For the purpose of

The present invention comprises the steps of: (a) culturing adult stem cells in a medium containing activin-A to differentiate into pancreatic progenitor cells; And (b) a second step of culturing the differentiated cells of the first step in a medium containing KGF (Keratinocyte Growth Factor) to differentiate into pancreatic beta-like cells, preparing pancreatic beta-like cells from adult stem cells. Provide a way to.

In addition, the present invention provides a method for producing pancreatic progenitor cells by culturing adult stem cells in a medium containing activin-A and sodium butyrate, and in the medium containing activin-A and BSA to differentiate into pancreatic progenitor cells. do.

Furthermore, the present invention provides a kit for inducing pancreatic progenitor differentiation from adult stem cells, comprising activin-A and BSA in EGM-2 medium.

The present invention also provides a composition for inducing pancreatic beta-like cell differentiation from pancreatic progenitor cells, comprising KGF and BSA in EGM-2 medium.

The present invention provides pancreatic beta-like cells prepared by the method for producing pancreatic beta-like cells; And it provides a cell therapy for the treatment of diabetes comprising the pancreatic beta-like cells as an active ingredient.

The present invention is a pancreatic progenitor cell prepared by the method for producing pancreatic progenitor cells; And it provides a cell therapy for treating diabetes comprising the pancreatic progenitor cells as an active ingredient.

The present invention provides a method for treating diabetes comprising administering the cell therapy agent for treating diabetes to a subject suffering from diabetes.

In one embodiment, the present invention comprises the steps of (a) culturing adult stem cells in a medium containing activin-A to differentiate into pancreatic progenitor cells; And (b) culturing the cells of the first step in a medium containing keratinocyte growth factor (KGF) to differentiate into pancreatic beta-like cells, the method of producing pancreatic beta-like cells from adult stem cells. To provide.

In the present invention, the term "adult stem cells" refers to stem cells that appear during the development or development of each organ of the embryo or adult stage. Adult stem cells are generally limited to cells that make up a specific tissue. These adult stem cells remain in most organs after adulthood and play a role in supplementing the loss of normal or pathological cells. This means that the stem cells existing in the tissues are cultured in vitro and have a pluripotent (multipotency) cell. For the purpose of the present invention, adult stem cells to which the two-step differentiation induction method of the present invention is applied are examples thereof. Although not limited, there are bone marrow stem cells, retinal stem cells, intraretinal Müller cells, neural stem cells, placental-derived epithelial stem cells, Watson Jelly-derived stem cells or umbilical cord blood-derived stem cells, preferably placental-derived epithelial stem cells. It may be a Watson jelly-derived stem cells or umbilical cord blood-derived stem cells. In a preferred embodiment of the present invention, the inventors confirmed that placental-derived epithelial stem cells, Watson Jelly-derived stem cells, or cord blood-derived stem cells differentiate into pancreatic beta-like cells by the two-step differentiation method of the present invention (FIG. 3). To 9).

Animals in the present invention include not only humans and primates, but also domestic animals such as cattle, pigs, sheep, horses, dogs, mice, rats, and cats, and are preferably humans.

As used herein, the term "pancreatic progenitor cell" refers to a cell having a multipotency capable of expressing a gene involved in pancreas development and differentiating into all cells of the pancreas, which are various kinds constituting the pancreas. Because of its pluripotency to differentiate into cells, it can be used for the treatment of various pancreatic diseases requiring transplantation of pancreatic cells, tissues or organs. Preferably, the pancreas may be used for the treatment of type 1 diabetes caused by problems with insulin secretion of the pancreas.

In the present invention, the term "pancreatic beta-like cell" or "pancreatic beta cell" may be used interchangeably, which is one of the cells constituting the islet of pancreatic islet, which is a cell that produces and secretes insulin. Problems with pancreatic beta cells can lead to insulin production, which can lead to type 1 diabetes. Therefore, such cells can be used for the treatment of type 1 diabetes caused by pancreatic insulin secretion.

According to the present invention, the method for producing pancreatic beta-like cells by inducing differentiation of adult stem cells into pancreatic beta-like cells is induced through differentiation induction process in two steps. Such two differentiation induction steps are briefly shown in FIG. 1.

In a preferred embodiment, (a) the first step of culturing adult stem cells in a medium comprising activin-A to differentiate into pancreatic progenitor cells further comprises (i) activin-A and sodium butyrate. Culturing in a medium containing; And (ii) culturing in a medium comprising activin-A and bovine serum albumin (BSA). Activin-A and sodium butyrate promote differentiation into endoderm and BSA promotes cell growth. That is, after inducing adult stem cells into endoderm, especially pancreatic endoderm, BSA was added to promote cell growth so that a large amount of cells could be differentiated, thereby preventing induction of differentiation into other lines.

In the first step, adult stem cells may be cultured without restriction for an appropriate period of time to differentiate into pancreatic progenitor cells, but preferably 18 hours to 84 hours, more preferably 3 days. Step (i) is preferably 18 hours to 30 hours, more preferably 1 day. Step (ii) is preferably 42 hours to 54 hours, more preferably 2 days.

The concentration of activin-A in the first step may be used without limitation as long as it is a concentration suitable for differentiating adult stem cells into pancreatic progenitor cells, preferably 2 nM to 6 nM, more preferably 4 nM. have. The concentration of activin-A in step (i) is preferably 2 nM to 6 nM, more preferably 4 nM, the concentration of sodium butyrate is preferably 0.5 mM to 1.5 mM, more preferably Preferably 1 mM. The concentration of activin-A in the step (ii) is preferably 2 nM to 6 nM, more preferably 4 nM, the concentration of BSA is preferably 0.05wt% to 0.15wt%, more Preferably 0.1 wt%.

In a preferred embodiment, in the second step of culturing the cells of the first step in a medium containing KGF (Keratinocyte Growth Factor) to differentiate into pancreatic beta-like cells, BSA may be further included.

In the second step, the pancreatic progenitor cells may be cultured without restriction for an appropriate period of time to differentiate into pancreatic beta-like cells, but preferably 66 hours to 78 hours, more preferably 3 days.

As described above, the method for producing pancreatic beta-like cells by inducing differentiation of the present invention can efficiently produce pancreatic beta-like cells from adult stem cells by simple two-step culture and at least 6 days of culture. It was first developed by the inventors.

The concentration of KGF in the second stage can be used without limitation as long as it is a concentration suitable for differentiating pancreatic progenitor cells into pancreatic beta-like cells, preferably 25 ng / ml to 75 ng / ml, more preferably 50 ng. / Ml. The concentration of BSA is preferably 0.5wt% to 1.5wt%, more preferably 1.0wt%.

The basal medium used in the first step and the second step may be used without limitation, a medium for stem cell culture that is commonly used as a medium for culturing stem cells known in the art, for example DMEM, DMEM / F12, α-MEM, EGM-2, preferably EGM-2. Also preferably, the medium used in the first and second steps may be a serum-free medium containing no serum.

As used herein, the term "serum-free medium" refers to any cell culture medium that does not include foreign or homologous serum that may contaminate the cultured cell or cause transplant rejection, and may further include a serum substitute. Examples of serum-free media include, but are not limited to, commercially available serum substitutes such as knockout serum replacement. The use of such serum-free media has clinical applications due to the possibility of containing contaminants, including mad cow disease virus due to proprietary materials such as fetal bovine serum (FBS) or bovine serum (FCS) present in serum medium. This can eliminate the risks that can arise. In addition, when serum-free medium is used, the pancreatic progenitor cells or pancreatic beta produced by the differentiation method of the present invention can be eliminated because the protein in serum can induce the formation of antibodies and thereby cause the rejection of transplanted cells. Like cells can be applied as a cell therapy for pancreatic related diseases, ie diabetes, without clinical risk. In addition, since the serum-free medium of the present invention does not have various growth factors and the like present in the serum, the present invention enables more efficient induction of differentiation into the desired pancreatic progenitor cells or pancreatic beta-like cells.

In a preferred embodiment of the present invention, the inventors of the present invention are human adult stem cells, placenta-derived epithelial stem cells, Watson Jelly-derived stem cells, and umbilical cord blood-derived stem cells, respectively, using serum-free EGM-2 medium as a base medium, 4 nM Differentiation of pancreatic progenitor cells by treatment of activin-A and 1 mM sodium butyrate for 1 day, followed by differentiation medium containing 4 nM of activin-A and 0.1% BSA in EGM-2 basal medium for 2 days. After the first step of inducing the two-step differentiation method comprising a second step of differentiating into pancreatic beta-like cells by treating the EGM-2 basal medium with 50 ng / ㎖ KGF for 3 days. As a result of confirming the expression of specific genes of pancreatic beta-like cells by each step by RT-PCR, it was confirmed that Nkk6.1, a pancreatic progenitor cell-related gene, was expressed in the first stage, and CX36, known as a mature pancreatic beta cell marker, Confirming that INS, GCG, PPY, etc. are expressed in the second step (Fig. 3), it was confirmed that the differentiation induction method of the present invention can be prepared in a short time pancreatic beta-like cells and pancreatic progenitor cells. In addition, as a result of confirming protein expression but not mRNA level, it was confirmed that the cells differentiated from c-peptide and insulin by the second step were expressed (FIGS. 4 to 6), and secreting insulin and c-peptide into the medium. It was confirmed (FIGS. 7-9). In addition, by confirming that insulin responds to glucose (FIGS. 7 to 9), it was confirmed that pancreatic beta-like cells having normal functions in the human body can be prepared from various adult stem cells by the differentiation method of the present invention.

As another aspect, the present invention provides a method for producing pancreatic progenitor cells comprising culturing adult stem cells in a medium comprising activin-A to differentiate into pancreatic progenitor cells.

The method preferably comprises the steps of: (i) culturing in a medium comprising activin-A and sodium butyrate; And (ii) culturing in a medium comprising activin-A and bovine serum albumin (BSA).

The concentration of activin-A, sodium butyrate, BSA, incubation period and the like are as described above.

As another aspect, the present invention provides a kit for inducing pancreatic progenitor differentiation from adult stem cells, comprising activin-A in EGM-2 medium.

The medium contained in the kit for inducing differentiation according to the present invention can maximize the differentiation efficiency by excellent cell viability compared to other media. In one preferred embodiment of the present invention, the present inventors compared the differentiation efficiency of the EGM-2 medium and EBM-2 medium, low glucose DMEM medium, high glucose DMEM medium of the present invention, when using EGM-2 medium By confirming that the differentiation efficiency was maximized (FIG. 10), it was confirmed that the medium of the present invention is the optimal medium.

 The kit for inducing differentiation of the present invention may include, without limitation, a substance known in the art as required for the culture of stem cells in addition to the medium for inducing differentiation.

The differentiation induction kit is preferably separated into EGM-2 medium containing activin-A and sodium butyrate and EGM-2 medium containing activin-A and BSA, respectively, which can be used sequentially.

More preferably, EGM-2 medium containing 4 nM activin-A and 1 mM sodium butyrate was treated for 1 day, and EGM-2 medium containing 4 nM activin-A and 0.1% BSA was treated for 2 days. Can induce differentiation of pancreatic progenitor cells.

In another aspect, the present invention provides a composition for inducing pancreatic beta-like cell differentiation from pancreatic progenitor cells, comprising KGF and BSA in EGM-2 medium.

The differentiation inducing composition of the present invention can maximize the differentiation efficiency by excellent survival rate compared to other media.

KGF can be used without limitation so long as it is a concentration suitable for differentiating pancreatic progenitor cells into pancreatic beta-like cells, preferably 25 ng / ml to 75 ng / ml, more preferably 50 ng / ml. The concentration of BSA is preferably 0.05% to 0.15%, more preferably 0.1%.

As another aspect, the present invention provides a pancreatic beta-like cell which can be prepared by a two-step differentiation method for producing pancreatic beta-like cells from the adult stem cells.

The pancreatic beta-like cells produced by the present invention have the following characteristics:

(a) expresses one or more genes selected from the group consisting of Nkk6.1, NGN3, CX36, INS, GCG and PPY; (b) secrete insulin; And (c) secrete c-peptide.

The pancreatic beta-like cells of the present invention express Nkk6.1, NGN3, CX36, INS, GCG or PPY, markers of pancreatic progenitors and differentiated pancreatic beta-like cells (FIG. 3), and immunostaining and ELISA result c-peptides. (FIGS. 4-9) and insulin (FIGS. 4-9) are detected. These results support the successful differentiation of cells produced by the methods of the invention into insulin producing cells. c-peptide is a useful indicator of the insulin secretion of pancreatic beta-like cells. The differentiation may also be performed using techniques such as flow cytometry or immunocytochemistry to measure cell surface labeling (e.g., staining cells with tissue-specific or cell-labeling specific antibodies) and morphological changes, Or by examining the morphology of the cells using confocal microscopy, or by measuring changes in gene expression patterns using techniques well known in the art such as PCR and gene-expression profiles.

As another aspect, the present invention provides a pancreatic progenitor cell that can be prepared by the first step of producing pancreatic progenitor cells from the adult stem cells.

The pancreatic progenitor cells produced by the present invention have the following characteristics.

(a) Expresses Nkx6.1 and NGN3 genes.

The pancreatic progenitor cells of the present invention express Nkk6.1, NGN3, markers of pancreatic progenitor cells (FIG. 3). The differentiation may be performed using techniques such as flow cytometry or immunocytochemistry to measure cell surface labels (e.g., staining cells with tissue-specific or cell-label specific antibodies) and changes in morphology, with light microscopy or co-operation. This can be confirmed by examining the morphology of the cells using a focal microscope or by measuring changes in gene expression using techniques well known in the art such as PCR and gene-expression profiles.

Pancreatic progenitor cells according to the methods of the present invention can be used for testing the efficacy and safety of drugs associated with pancreatic progenitor cells.

As another aspect, the present invention provides a cell therapy agent for treating diabetes, comprising the pancreatic precursor cells or pancreatic beta-like cells.

As used herein, the term "cell therapeutic agent" refers to a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared from isolation, culture, and special chewing from animals including humans. Refers to a drug used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as proliferating or screening living autologous, allogeneic, or heterologous cells in vitro or altering the biological characteristics of cells in order to restore function do. Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.

Cell therapy for the treatment of diabetes mellitus of the present invention induces the differentiation of pancreatic progenitor cells from adult stem cells derived from humans or animals in vitro, thereby proliferating and activating pancreatic progenitor cells to be administered to diabetic patients to grow into pancreatic beta cells or It refers to cell therapies that can be used to treat diabetes by forming cell aggregates such as Langerhans Island. Or it refers to a cell therapy made in vitro to induce the differentiation of adult stem cells derived from human or animal from pancreatic beta-like cells to proliferate and activate pancreatic beta-like cells to be administered to diabetic patients for use in the treatment of diabetes.

Cell therapy for the treatment of diabetes of the present invention may be prepared in the form of a formulation common in the art, for example, injectable, and may be surgically implanted directly into the pancreatic region, or may be intravenously administered and then moved to the pancreatic region. The dose of the cell therapy of the present invention may vary depending upon the degree of jungham degree, the route of administration the patient, the age and sex of the patient, and the disease, and preferably an average for an adult, 1 × 10 ⁷ to 10 ¹¹ cells To administer.

In another aspect, the present invention provides a method for treating diabetes comprising administering the cell therapy agent for treating diabetes to a subject suffering from diabetes.

Administration of the cell therapy of the present invention is applicable to any animal, and animals include humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, mice, rats, and cats. As used herein, the term "administration" means introducing the cell therapy of the invention into a patient in any suitable manner and includes the transplantation of differentiated cells. The route of administration of the cell therapy of the present invention may be administered through various routes as long as it can reach the target tissue, and preferably, may be administered to the tissue in the island of Langerhans.

The method for producing pancreatic beta-like cells by inducing differentiation from adult stem cells to pancreatic beta-like cells of the present invention is advantageous in practical clinical practice because adult stem cells are free from cancer and ethical problems. In addition, the differentiation induction method of the present invention is a simple and efficient method of two steps, the pancreatic beta-like cells produced by differentiation secrete insulin, and pancreatic beta-like cells in which insulin increases in response to glucose, which is used to treat diabetes. It can be used as a possible cell therapy. In addition, the cells obtained in the intermediate stage of the differentiation stage can be used for pancreatic progenitor cells to test the efficacy and safety of the drug associated with them.

Figure 1 is a schematic diagram showing the step-by-step differentiation method of adult stem cells from the pancreatic beta-like cells of the present invention. The differentiation process applied to each differentiation step used in the present invention is shown from the first stage (stage 1) to the second stage (stage 2). It also shows the differentiation application time for each differentiation step.
Figure 2 is a micrograph of the cells when the differentiation of adult stem cells from the stage of differentiation into pancreatic beta-like cells step by step. Micrographs of the respective undifferentiated, first and second stages for umbilical cord blood-derived stem cells (UCB-MSC), Watson Jelly-derived stem cells (WJ-MSC) and placental-derived epithelial stem cell-derived stem cells (AESC). . Scale bars each represent 50 μm.
Figure 3 is a diagram showing the expression of pancreatic beta-like cell-related gene expression by RT-PCR by extracting RNA from the stage of differentiation to differentiation into pancreatic beta-like cells using the differentiation method of the present invention. RT-PCR of each gene in the differentiation stage, the first stage and the second stage for umbilical cord blood-derived stem cells (UCB-MSC), Watson Jelly-derived stem cells (WJ-MSC) and placental-derived epithelial stem cells (AESC) It is a diagram showing the expression pattern through.
Figures 4, 5 and 6 shows the differentiation of cord blood-derived stem cells (Fig. 4), Watson Jelly-derived stem cells (Fig. 5) and placental-derived epithelial stem cells (Fig. 6) and differentiation into pancreatic beta-like cells. It is a diagram showing the expression pattern of pancreatic beta cell-related protein using the staining method.
7, 8 and 9 are the ELISA technique when applied to differentiation stages of the cord blood-derived stem cells (Fig. 7), Watson Jelly-derived stem cells (Fig. 8) and placental-derived epithelial stem cells (Fig. 9) and pancreatic beta-like cells, respectively. In the insulin and c-peptide secretion and glucose stimulation test (glucose stimulation test) is a graph confirming the degree of insulin response.
FIG. 10 shows a step-by-step step of differentiation into pancreatic beta-like cells from the undifferentiated phase after performing the two-stage differentiation method of the present invention using EBM-2 medium, low glucose DMEM medium, high glucose DMEM medium, and EGM-2 medium. Is a diagram showing the expression of pancreatic beta-like cell-related genes by RT-PCR technique.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1: Staged Differentiation Method for Differentiation into Pancreatic Beta-like Cells

The present inventors have described placental epithelial stem cells (AESC), which are adult stem cells derived from humans, from Toshio Miki et al. Watson Jelly-derived stem cells (WJ-MSCs) were prepared by Giampiero La Rocca et al. (Current Protocols in Stem Cell Biology, 1E.3.1-1e.3.9, 2007). (Histochemical Cell Biology, 131: 267-282, 2009) by using the method of umbilical cord blood-derived stem cells (UCB-MSC) Hyung-Sik Kim et al. (PLOS ONE 2010 Oct 22; 5 (10): e15369), respectively, were isolated and two differentiation methods were established to differentiate into pancreatic beta-like cells. In the present invention, EGM-2 medium was used as a differentiation medium, and differentiation was induced into pancreatic beta-like cells by sequential differentiation induction method for each step.

Specifically, in the case of the first stage of differentiation used in the present invention, EGM-2 medium (Endothelial Cell) 4 nM with Basal Medium-2 (EBM-2) (catalog # CC-4176, Lonza, Walkersville, MD, USA) plus growth factors EGEM-2 SingleQuots (catalog # CC-4176 (Lonza)) Activin-A (activin-A), 1 mM sodium butyrate (sodium butyrate) was treated for 1 day. Thereafter, differentiation was induced by replacing the differentiation medium with 4 nM of activin-A and 0.1 wt% of BSA (bovine serum albumin) added to the basal medium. In the second step, KGF was treated at a concentration of 50 ng per ml of medium and replaced with differentiation medium to which 1 wt% of BSA was added to induce differentiation for 3 days. This differentiation induction method of the present invention is shown in FIG. Each step of the first to second step is to replace the medium itself, the medium of each step is a serum-free medium without the addition of serum.

Example  2: Pancreatic beta-like cells confirmed the change in cell shape of differentiation-inducing cells at each step

Olympus differentiation status and shape of cells during differentiation of each step of Example 1 presented in the present invention by separating human-derived adult stem cells, placental-derived epithelial stem cells, Watson jelly-derived stem cells, and umbilical cord blood-derived stem cells CKX41, and observed under a microscope 200 times magnification.

As a result, when the general medium was used, the basic shape of human adult stem cells was maintained, but the change in morphology was observed as the differentiation was induced step by step. Specifically, the umbilical cord blood and Watson Jelly-derived stem cells show no significant change in shape even after the differentiation stage, but in placental-derived epithelial stem cells, the cells become slightly larger and flatter than the undifferentiated cells in the first stage of differentiation. However, in the second stage of differentiation, the cells were slightly thicker than the first stage (FIG. 2).

Example  3: Identification of specific gene expression patterns in each stage of induction of differentiation into pancreatic beta-like cells

Treatment of human-derived adult stem cells with stepwise differentiation medium in sequence, and identification of specific gene expression patterns of pancreatic beta-like cells in each step of differentiation of pancreatic beta-like cells differentiated in Example 1 by the step-wise differentiation induction method For this purpose, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. The expression patterns of the genes were compared with the undifferentiated state of human adult stem cells, placental-derived epithelial stem cells, Watson Jelly-derived stem cells, and cord blood-derived stem cells. TRIzol Reagent (Invitrogen, Carlsbad, CA, USA.) Was used to extract the RNA of the cells in each differentiation step. After lysing the cells with TRIzol Reagent, pure RNA was isolated by gradual treatment with chloroform (Invitrogen, Carlsbad, CA, USA.) And isopropanol (Invitrogen, Carlsbad, CA, USA.). The extracted RNA was synthesized into cDNA (complementary DNA) through reverse transcription. The synthesized cDNA was amplified by PCR according to the conditions shown in Table 1 below. Table 1 below shows the names and related information of pancreatic beta cell-related specific gene primers for confirming their differentiation into pancreatic beta cells using reverse transcriptase-polymerase chain reaction.

Gene name primer order
number Temperature
(℃) cycle size
(bp) Nestin
Forward: AAC AGC GAC GGA GGT CTC TA One 57 33 220 Reverse: TTC TCT TGT CCC GCA GAC TT 2 Glucagon
(Glucagon) Forward: AGG CAG ACC CAC TCA GTG A 3 65 33 308 Reverse: AAC AAT GGC GAC CTC TTC TG 4 NGN3
Forward: CGA ATG CAC AAC CTC AAC TC 5 54 33 126 Reverse: AGT CAG CGC CCA GAT GTA GT 6 Nkx6.1
Forward: ACA CGA GAC CCA CTT TTT CCG 7 57 33 361 Reverse: TGC TGG ACT TGT GCT TCT TCA AC 8 Connectin 36
(Connexin 36, cx36) Forward: AAC GCC GCT ACT CTA CAG TC 9 57 33 426 Reverse: TTG GCC GGG ACA CAT AAC AT 10 insulin
Forward: TTC TTC TAC ACA CCC AAG AC 11 57 33 192 Reverse: CTA GTT GCA GTA GTT CTC CA 12 Pdx1
Forward: CTC CTA CAG CAC TCC ACC TT 13 57 33 153 Reverse: CCG AGT AAG AAT GGC TTT AT 14 PPY
Forward: CTG CTG CTC CTG TCC ACC TG 15 65 33 206 Reverse: CTC CGA GAA GGC CAG CGT GT 16 Nkx6.1
Forward: ACA CGA GAC CCA CTT TTT CCG 17 57 33 361 Reverse: TGC TGG ACT TGT GCT TCT TCA AC 18 Sox17
Forward: AGA TTG AAA AAA CAC CCA GG 19 57 33 153 Reverse: TTG TTC AGA GAT TTG TTT CC 20 Beta-actin
Forward: CGG CAT CGT CAC CAA CTG GGA 21 62 20 281 Reverse: CGT AGA TGG GCA CAG TGT GGG 22

As a result, the expression patterns of pancreatic beta progenitor cell-related genes Nkx6.1 and NGN3 were confirmed as differentiation was induced at each stage. In addition, expression patterns of Cx36, INS, GCG, PPY, etc., known as mature pancreatic beta cell markers, were also confirmed (FIG. 3). These results suggest that the cells differentiated by the differentiation method of the present invention are pancreatic beta cells.

Example  4: Identification of specific protein expression patterns at each stage when inducing differentiation into pancreatic beta-like cells

Treatment of human-derived adult stem cells with a staged differentiation medium was performed sequentially, and pancreatic beta-like cells related to pancreatic beta-like cells differentiated in Example 1 by the differentiation-induced differentiation in each step were expressed. Confirmed. To identify pancreatic related specific proteins, immunocytoscopy (ICC, immunocytochemistry) was used. The cells of each differentiation stage were fixed with PFA (paraformaldehyde, 4%), and the primary protein of insulin (# ab8302, Abcam, Cambridge, MA, USA) and c-peptide (4020-01, Linco), the specific proteins to be identified Antibodies were treated. Subsequently, the secondary antigen was treated with an antibody expressing fluorescence, and Hoechst staining was performed to observe the nuclei of the cells. Expression of c-peptide and insulin, which are specific proteins to be identified according to each differentiation step, was confirmed using a confocal laser microscope system (CLSM).

The expression of insulin and c-peptide by immunofluorescence staining was compared between the undifferentiated state and the second stage state of umbilical cord blood-derived stem cells in human-derived adult stem cells. After the differentiation induction was confirmed to increase strongly (Fig. 4). Comparative analysis of insulin and c-peptide by immunofluorescence staining of the undifferentiated state and the second-stage state of Watson jelly-derived stem cells showed that the expression of insulin and c-peptide increased strongly after induction of differentiation compared to the undifferentiated state. It was confirmed (FIG. 5). As a result of comparative analysis of the undifferentiated state and the second stage state of placental-derived epithelial stem cells by immunofluorescence staining, it was confirmed that the expression of insulin and c-peptide increased strongly after induction of differentiation compared to the undifferentiated state (FIG. 6). .

Example  5: Induce differentiation into pancreatic beta-like cells at each stage ELISA  Insulin and c- Peptide  Confirmation of expression

Human pancreatic beta-like cells differentiated in Example 1 by sequentially processing differentiation medium to adult stem cells and inducing differentiation in each step, insulin and c, which are one of the characteristics of pancreatic beta cells during differentiation process Peptide secretion was confirmed by ELISA technique. In addition, a certain amount of glucose (15mM) was processed in the differentiation step and each step of differentiation process to test the response of insulin (glucose stimulation test) was conducted.

As a result of comparing the undifferentiated state of umbilical cord blood-derived stem cells and the expression level of insulin and c-peptide in each stage of differentiation, the secretion of insulin and peptide increased as the stage of differentiation progressed. In addition, insulin increased response to glucose as the differentiation step progressed (FIG. 7). As a result of comparing the undifferentiated state of the Watson Jelly-derived stem cells and the expression patterns of insulin and c-peptide in each differentiation stage, the secretion of insulin and c-peptide increased as the differentiation stage progressed. In addition, insulin increased response to glucose as the differentiation step progressed (FIG. 8). As a result of comparing the undifferentiated state of the placental-derived epithelial stem cells and the expression patterns of insulin and c-peptide in each differentiation stage, the secretion of insulin and c-peptide increased as the differentiation stage progressed. In addition, insulin increased response to glucose as the differentiation step progressed (FIG. 9). These results suggest that the differentiation method of the present invention can be used to differentiate pancreatic beta-like cells capable of secreting insulin and c-peptides from various kinds of adult stem cells. Pancreatic beta-like cells, which increase insulin in response, suggest that pancreatic beta cells function normally in the human body. The pancreatic beta-like cells having such a function can be used as a therapeutic agent for type 1 diabetes having a problem in insulin secretion. The differentiation method of the present invention and the pancreatic beta-like cells prepared by the present invention can be usefully used as a therapeutic agent for diabetes. It can be supported.

Example  6: Comparison of Differentiation Efficiency of Each Differentiation Medium

As in Example 1, adult stem cells were differentiated into pancreatic beta-like cells by a two-step differentiation method, and in contrast groups, EBM-2 medium, low glucose DMEM medium, and high glucose DMEM medium were used instead of EGM-2 medium. Differentiation was used. Expression of insulin, Pdx1, SOX17, and β-actin gene was confirmed in the same manner as in Example 3 with respect to the cells of each step cultured in the EGM-2 medium and the comparative group medium (FIG. 10).

As a result, when using the EGM-2 medium of the present invention than the comparison group, it was confirmed that the expression of insulin, which is a marker of mature pancreatic beta-like cells, appeared to maximize the differentiation efficiency.

<110> SNU R & DB FOUNDATION <120> A method for producing pancreas beta like cells from adult stem          cells <130> PA100843 / KR <160> 22 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for nestin <400> 1 aacagcgacg gaggtctcta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for nestin <400> 2 ttctcttgtc ccgcagactt 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Glucagon <400> 3 aggcagaccc actcagtga 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Glucagon <400> 4 aacaatggcg acctcttctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NGN3 <400> 5 cgaatgcaca acctcaactc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for NGN3 <400> 6 agtcagcgcc cagatgtagt 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Nkx6.1 <400> 7 acacgagacc cactttttcc g 21 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Nkx6.1 <400> 8 tgctggactt gtgcttcttc aac 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Connexin 36 <400> 9 aacgccgcta ctctacagtc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Connexin 36 <400> 10 ttggccggga cacataacat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer fod insulin <400> 11 ttcttctaca cacccaagac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for insulin <400> 12 ctagttgcag tagttctcca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Pdx1 <400> 13 ctcctacagc actccacctt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Pdx1 <400> 14 ccgagtaaga atggctttat 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PPY <400> 15 ctgctgctcc tgtccacctg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PPY <400> 16 ctccgagaag gccagcgtgt 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Nkx6.1 <400> 17 acacgagacc cactttttcc g 21 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Nkx6.1 <400> 18 tgctggactt gtgcttcttc aac 23 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Sox17 <400> 19 agattgaaaa aacacccagg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Sox17 <400> 20 ttgttcagag atttgtttcc 20 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for beta-actin <400> 21 cggcatcgtc accaactggg a 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for beta-actin <400> 22 cgtagatggg cacagtgtgg g 21

Claims (21)

(a) a first step of culturing adult stem cells in a medium containing activin-A to differentiate into pancreatic progenitor cells; And
(b) preparing pancreatic beta-like cells from adult stem cells, comprising the second step of culturing the differentiated cells of the first step in a medium containing KGF (Keratinocyte Growth Factor) to differentiate into pancreatic beta-like cells. Way.
The method of claim 1, wherein the medium of the first and second stages is a serum-free medium, each of which does not contain serum.
The method of claim 1, wherein the first step
(i) culturing in a medium comprising activin-A and sodium butyrate; And
(ii) culturing in a medium comprising activin-A and bovine serum albumin (BSA).
The method of claim 1, wherein the second step is culturing in a medium containing KGF and BSA.
The method of claim 1, wherein the first step is 18 hours to 84 hours and the second step is 66 hours to 78 hours.
The method of claim 3, wherein step (i) is 18 to 30 hours and step (ii) is 42 to 54 hours.
The method of claim 1, wherein the concentration of activin-A is 2 nM to 6 nM and the concentration of KGF is 25 ng / ml to 75 ng / ml.
The method of claim 3, wherein the concentration of sodium butyrate is 0.5 mM to 1.5 mM and the concentration of BSA is 0.05 wt% to 0.15 wt%.
The method of claim 4, wherein the concentration of BSA is 0.5 wt% to 1.5 wt%.
The method of claim 1, wherein the medium of the first and second stages is independently Endothelial Growth Medium-2 (EGM-2) medium.
The method of claim 1, wherein the stem cells are derived from humans.
The method of claim 1, wherein the adult stem cells are placental-derived epithelial stem cells, Watson Jelly-derived stem cells or umbilical cord blood-derived stem cells.
A method for producing pancreatic progenitor cells from adult stem cells, comprising culturing the adult stem cells in a medium containing activin-A to differentiate into pancreatic progenitor cells.
The method of claim 13, wherein the method is
(i) culturing in a medium comprising activin-A and sodium butyrate; And
(ii) culturing in a medium comprising activin-A and BSA to differentiate adult stem cells into pancreatic progenitor cells.
Kit for inducing pancreatic progenitor differentiation from adult stem cells, comprising activin-A in EGM-2 medium.
The kit of claim 15 comprising EGM-2 medium comprising activin-A and sodium butyrate, and EGM-2 medium comprising activin-A and BSA.
A composition for inducing pancreatic beta-like cell differentiation from pancreatic progenitor cells, comprising KGF and BSA in EGM-2 medium.
A pancreatic beta-like cell produced by the method of any one of claims 1 to 12 with the following characteristics:
(a) expresses one or more genes selected from the group consisting of Nkk6.1, NGN3, CX36, INS, GCG and PPY;
(b) secrete insulin; And
(c) secretes c-peptides.
Pancreatic progenitor cells produced by the method of claim 13 or claim 14 having the following characteristics:
(a) Expresses Nkx6.1 and NGN3 genes.
Cell therapy for diabetes treatment comprising pancreatic beta-like cells of claim 18 as an active ingredient.
Cell therapy for diabetes treatment comprising the pancreatic progenitor cells of claim 19 as an active ingredient.

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