KR20200049669A - Composition for inducing dedifferentiation for insulin producing cells from human induced pluripotent stem cells and method for inducing insulin producing cells using the same - Google Patents

Abstract

The present invention relates to a composition for inducing differentiation for insulin producing cells derived from human induced pluripotent stem cells and a method for inducing insulin producing cells using the same. The composition for inducing differentiation for insulin producing cells derived from human induced pluripotent stem cells of the present invention shortens a differentiation process compared to a conventional technique and increases differentiation efficiency to the insulin producing cells. Also, the composition is composed of a low molecular material, not a protein formulation, such that stability of the composition is improved. Accordingly, the composition can be used for a process for mass producing a cell treatment agent and insulin for treating diabetes.

Description

Composition for inducing dedifferentiation for insulin producing cells from human induced pluripotent stem cells and method for inducing insulin producing cells using the same}

The present invention relates to a composition for inducing human-derived pluripotent stem cell-derived insulin-producing cell differentiation and a method for inducing insulin-producing cell differentiation using the same, and more specifically, a composition for inducing differentiation into insulin-producing cells containing a low molecular substance is a differentiation process. Since it can increase the efficiency of differentiation into insulin-producing cells while shortening, it can be used as a cell therapy for the treatment of diabetes.

Diabetes mellitus is a disease characterized by chronic hyperglycemia as the most important disease, and when prolonged, it causes complications such as delayed wound healing due to microvascular damage, neurological disease, renal failure, heart disease, and retinal disease, thereby increasing the social and economic burden. Until now, there is no treatment to cure diabetes, and insulin-enhancing therapy that repeatedly administers insulin that is insufficient in diabetic patients has been used in clinical practice for a long time, but there is a discomfort that must be repeatedly administered several times a day. The reality is that diabetes complications cannot be prevented.

Diabetes is largely divided into two types (type 1 and type 2) depending on the cause. In the case of type 1 diabetes, it occurs because one's own immune cells destroy insulin-producing cells, and the insulin-producing cells are insufficient to control blood sugar. In the case of type 2 diabetes, due to various reasons, organs / tissues / cells in the body have resistance to insulin, and blood sugar cannot be controlled smoothly due to dysfunction and loss of insulin-producing cells due to hyperglycemia. As a result, the symptoms of diabetes with high blood sugar and complications are caused.

Islet cell transplantation has been recognized as a successful strategy for the treatment of diabetes. However, due to the lack of pancreatic sharers, pancreatic islet transplantation to treat diabetes is limiting its use. Many recent studies have focused on the development of cell therapy products using stem cells, and representative stem cells include embryonic stem cells and adult stem cells. Embryonic stem cells can differentiate into cells that secrete insulin outside the body, and studies have shown that these cells can be used to treat hyperglycemia when transplanted into diabetic rats (Fujikawa T. et al. , Am. J. Pathol . , 166: 1781-1791, 2005; D'Amour KA et al. , Nat. Biotechnol . , 24: 1392-1401, 2006). However, embryonic stem cells have a great disadvantage that cancer can occur when transplanted in clinical applications.

Previous studies have reported that it is possible to differentiate from ectodermal-derived progenitor cells to insulin-expressing cells (Hori Y. et al. , PLOS Med ., 2: 103, 2005) and pancreatic ductal cells. There have been reports that epithelial cells were able to differentiate into pancreatic islet-like structures (Bonner-Weir S. et al. , Prod. Natl. Acad. Sci. , 97: 7999-8004, 2000), but in the former case, neurospheres Differentiation was induced using a cell line (Neurosphere cell line), and it was difficult to demonstrate its efficiency because bovine insulin was added to the culture medium. In the latter case, the amount of stem cells obtained from the ductal tissue was extremely limited. There are disadvantages. In particular, in the case of embryonic stem cells, when inducing differentiation in the culture medium containing insulin, it is reported that the secreted insulin absorbs and releases the insulin in the culture medium. It was demonstrated as a result of not secreting the truncated C-peptide (Rajagopal J. et al. , Science , 299: 363, 2003; Hansson M. et al. , Diabetes , 53: 2603-2609, 2004).

Therefore, many studies have recently attempted to induce differentiation without adding insulin to the culture medium, but embryonic stem cells are difficult to use for the purpose of treatment because ethical problems and cancer may occur as mentioned above. , Inducing pluripotent stem cells using fibroblasts derived from the skin, or attempting differentiation using mesenchymal stem cells, but there is a problem in that insulin and C-peptide secretions are remarkably low. To overcome this problem, research is underway to find candidates capable of differentiating into insulin-producing cells (IPC).

Induced pluripotent stem cells (iPSCs) are self-proliferative and capable of differentiating into various cells, and thus can be differentiated cell therapies to IPC. According to a recent study, iPSC showed that it is possible to differentiate into insulin-secreting cells in a manner similar to the stage of differentiation during embryonic formation. However, differentiated insulin-secreting cells have many problems due to low insulin secretion, diversity of differentiated cells, and teratoma-forming ability. Therefore, research has been actively conducted on a method for inducing differentiation into efficient insulin secretory cells. Unlike stem cells or progenitor cells, islet beta cells have limited proliferative capacity under normal conditions. However, it has been reported that beta cells are increased under certain conditions such as pregnancy, obesity, and pancreatic resection, and external stimuli such as hormones and growth factors appear to be necessary for the growth and differentiation of existing beta cells.

Accordingly, the present inventors have tried to develop a composition for inducing differentiation with high efficiency of differentiation into insulin producing cells, and invented a composition for inducing differentiation of insulin producing cells derived from human induced pluripotent stem cells containing small molecules. , The composition for inducing differentiation of insulin-producing cells of the present invention has been completed by confirming that the differentiation step and differentiation period are shortened and the differentiation efficiency is increased as compared with the prior art.

An object of the present invention is to provide a composition for inducing differentiation of insulin-producing cells derived from human induced pluripotent stem cells.

Another object of the present invention is to provide a method for inducing differentiation of insulin-producing cells derived from human induced pluripotent stem cells using the composition.

Another object of the present invention is a pharmaceutical composition for treating diabetes comprising the insulin-producing pancreatic endocrine cell and the insulin-producing pancreatic endocrine cell prepared as a method for inducing differentiation of the human-derived pluripotent stem cell-derived insulin producing cells. To provide.

Another object of the present invention is to provide a method for mass production of insulin derived from the insulin-producing pancreatic endocrine cell.

In order to achieve the above object,

The present invention,

(Iv) a composition for inducing differentiation from human induced pluripotent stem cells (iPSCs) including activin A, CHIR99021, B27 and LY294002 to definitive endoderm (DE);

(Ii) a composition for inducing differentiation from endoderm cells including activin A and B27 to a primitive gut tube (PGT);

(Iii) a composition for inducing differentiation of pancreatic endoderm (PE) from the primitive intestinal tract including B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1; And

(Iii) B27, a composition for inducing differentiation of pancreatic endoderm, including B27, forskolin, dexamethasone, and nicotinamide to insulin-producing pancreatic endocrine cells (endocrine pancrease; EP). Provided is a composition for inducing differentiation of insulin-producing cells derived from human induced pluripotent stem cells (iPSC).

In a preferred embodiment of the present invention, the composition for inducing differentiation in step (iii) is 50 to 150 ng / mL activin A, 1 to 5 μM CHIR99021, 1 to 5% 1X B27, and 15 to 25 μM LY294002 The composition for inducing differentiation in step (ii) may include 50 to 150 ng / mL activin A and 1 to 5% 1X B27, and the composition for inducing differentiation in step (iii) 1 to 5% 1X B27, 1 to 5 μM dolsomorphine, 1 to 5 μM retinoic acid, 5 to 15 μM SB431542, 1 to 5 μM FR180204 and 0.1 to 0.5 μM SANT-1, and may include (i) The composition for inducing differentiation of the step may include 1 to 5% 1X B27, 5 to 15 μM forskolin, 5 to 15 μM dexamethasone, and 5 to 15 μM nicotinamide.

In a preferred embodiment of the present invention, the human induced pluripotent stem cells are derived from fibroblasts, and Oct4, Sox2, Klf4, c-Myc and SSEA4 can be expressed on the cell surface.

The present invention also provides a human induced pluripotent stem cell derived insulin producing cell differentiation induction medium comprising the composition for inducing human induced pluripotent stem cell derived insulin producing cell differentiation as an active ingredient.

In one embodiment of the invention, the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), Improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI 1640 (Advanced RPMI1640), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's) Medium) may include a medium selected from the group consisting of.

The present invention also

(a) inducing differentiation into endoderm by culturing human induced pluripotent stem cells in a medium containing activin A, CHIR99021, B27 and LY294002;

(b) inducing differentiation into the primitive intestine by culturing the differentiation-derived endoderm in the medium containing activin A and B27 in step (a);

(c) The differentiation-induced primitive intestinal tract in step (b) is cultured in a medium containing B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1 to induce differentiation into pancreatic endoderm. To do; And

(d) Inducing differentiation into pancreatic endocrine cells capable of producing insulin by culturing the differentiation-induced pancreatic endoderm in the medium containing B27, forskolin, dexamethasone, and nicotinamide in step (c). It provides a method for inducing differentiation of human-derived pluripotent stem cell-derived insulin-producing cells.

In one embodiment of the present invention, the step (a) is human induced pluripotent stem cells 50 ~ 150 ng / mL activin A, 1 ~ 5 μM CHIR99021, 1 ~ 5% 1X B27, and 15 ~ 25 μM LY294002 Incubation in the medium containing this induces differentiation into the endoderm, and step (b) incubates the endoderm in a medium containing 50 to 150 ng / mL activin A and 1 to 5% 1X B27 to differentiate into the native intestinal tract. Induction, the step (c) is 1 ~ 5% 1X B27, 1 ~ 5 μM dolsomorphine, 1 ~ 5 μM retinoic acid, 5 ~ 15 μM SB431542, 1 ~ 5 μM FR180204 and 0.1 ~ 0.5 μM SANT Cultured in a medium containing -1 to induce differentiation into pancreatic endoderm, step (d) comprises 1 to 5% 1X B27, 5 to 15 μM forskolin, 5 to 15 μM dexamethasone and 5 to 15 μM in step (d). Incubation in a medium containing nicotinamide can induce differentiation into pancreatic endocrine cells capable of producing insulin.

The present invention also provides an insulin-producing pancreatic endocrine cell or an endocrine aggregate produced by the method for inducing differentiation of the human-derived pluripotent stem cell-derived insulin producing cells.

The present invention also provides a pharmaceutical composition for the treatment of diabetes comprising the insulin-producing pancreatic endocrine cells or endocrine aggregates as an active ingredient.

The present invention also, (a) culturing the insulin-producing pancreatic endocrine cells or endocrine aggregates; And

(b) comprising the step of separating insulin from the culture by the culture of step (a), provides a method for mass production of insulin.

The composition for inducing human-derived pluripotent stem cell-derived insulin-producing cell differentiation of the present invention can increase the efficiency of differentiation into insulin-producing cells while shortening the differentiation process compared to the prior art, and is composed of a low-molecular substance rather than a protein preparation. Since it has the advantage of improving stability, it can be used in cell therapeutics for the treatment of diabetes and processes for mass production of insulin.

1 is a schematic diagram showing the mechanism of a low-molecular substance used in an insulin-producing cell differentiation-inducing composition.
Figure 2 is data confirming the identification and differentiation of the major gene expression of human induced pluripotent stem cells (A: microscopic observation of the formation pattern of human induced pluripotent stem cells, B ~ E: Oct4 (B) in induced pluripotent stem cells , Nanog (C), Sox2 (D), SSEA4 (E) expression, F: Implantation of induced pluripotent stem cells subcutaneously in immunodeficient mice resulted in teratoma formation, and histological cross-sectional histological changes were examined. Confirm that exocrine, endocrine, and mesoderm were formed).
Figure 3 shows the degree of expression of endoderm markers (Sox17, FoxA2, CXCR4) in order to confirm the differentiation efficiency according to the composition of the differentiation-inducing composition from human induced pluripotent stem cells to endoderm, immunocytochemical staining (A) and PCR analysis (B). It is the data confirmed through.
Figure 4 is data confirming the differentiation efficiency according to the composition of the composition for inducing differentiation from the primitive intestinal tract to pancreatic endoderm (PE) (A: microscopic observation of cell differentiation, B: insulin production on the 5th day of final differentiation induction culture) Checking the degree, C: Checking the degree of insulin production on the 8th day of the final differentiation induction culture, D: Checking the degree of insulin production on the 12th day of the final differentiation induction culture, E: Checking the glucose stimulated insulin secretion (GSIS) after the final differentiation step is completed) .
FIG. 5 is data confirming insulin, glucagon, and Pdx-1 produced by differentiated insulin-producing cells using the insulin-producing cell differentiation-inducing composition of the present invention through immunocytochemical staining.
6 is FACS analysis of insulin (ginsulin) and glucagon (glucagon) secretion level and expression level of Ngn3 transcription factor of insulin-producing cells differentiated using the insulin-inducing cell differentiation-inducing composition of the present invention.
7 is a data analyzed by PCR of the expression level of pancreatic-related electron factors of differentiated insulin-producing cells using the composition for inducing differentiation of insulin-producing cells of the present invention.
FIG. 8 is data obtained by culturing differentiated insulin-producing cells in a spheroid form after the final differentiation step (A: observation of a spheroid form formed under a microscope, B: insulin and glucagon secreted from the spheroid form) Observed through chemical staining, C: The amount of C-peptide secretion according to the culture medium composition and period was confirmed by ELISA method).
9 is a schematic diagram showing a process for inducing differentiation and differentiation induction of insulin producing cells derived from human induced pluripotent stem cells of the present invention.
FIG. 10 shows data obtained by immunocytochemical staining of insulin and glucagon produced in differentiated insulin producing cells after the final differentiation step when cultured according to each differentiation protocol.
FIG. 11 shows data obtained by immunocytochemical staining of insulin and glucagon generated in differentiated insulin producing cells after the final differentiation step when cultured according to each differentiation protocol.
12 is a data analyzed by FACS differentiation rate of differentiated insulin-producing cells and glucagon-producing cells after the final differentiation step when cultured according to each differentiation protocol.

Hereinafter, the present invention will be described in detail.

As described above, studies have been conducted to induce differentiation into insulin producing cells using induced pluripotent stem cells or adult stem cells, but there are still problems in that insulin and C-peptide secretions are remarkably low.

Accordingly, the present inventors tried to develop a composition for inducing differentiation with high efficiency of differentiation into insulin producing cells. A composition for inducing human-derived pluripotent stem cell-derived insulin-producing cell differentiation containing various small molecule substances was prepared, and as a result of inducing differentiation from inducing pluripotent stem cell to insulin-producing cells, compared to a conventional technique It was confirmed that the differentiation step and differentiation period were shortened and the differentiation efficiency increased.

Thus, the present invention, (i) activin A (activin A), CHIR99021, B27 and LY294002 human induced pluripotent stem cells (iPSC) from the endoderm (definitive endoderm; DE) differentiation Composition for induction;

(Ii) a composition for inducing differentiation from endoderm cells including activin A and B27 to a primitive gut tube (PGT);

(Iii) a composition for inducing differentiation of pancreatic endoderm (PE) from the primitive intestinal tract including B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1; And

(Iii) B27, a composition for inducing differentiation of pancreatic endoderm, including B27, forskolin, dexamethasone, and nicotinamide to insulin-producing pancreatic endocrine cells (endocrine pancrease; EP). Provided is a composition for inducing differentiation of insulin-producing cells derived from human induced pluripotent stem cells (iPSC).

"Pluripotent stem cells" in the present invention refers to stem cells having pluripotency capable of differentiating into all three germ layers constituting the living body, that is, endoderm, mesoderm, and ectoderm. . In the present invention, induced pluripotent stem cells (iPSC) were used, and induced pluripotent stem cells are artificially made pluripotent stem cells that artificially express specific genes to induce adult cells, which are non-potent cells, and embryonic stem cells. In many ways, it is known to be similar to natural pluripotent stem cells such as cells.

The term "differentiation" used in the present invention is a phenomenon in which structures or functions of each other are specialized during cell division and growth, that is, the form or function of cells or tissues of an organism to perform a task given to each. It means changing.

Specifically,

The composition for inducing differentiation in step (iii) includes 50 to 150 ng / mL activin A, 1 to 5 μM CHIR99021, 1 to 5% 1X B27, and 15 to 25 μM LY294002, in step (ii). The composition for inducing differentiation so that 50 to 150 ng / mL activin A and 1 to 5% 1X B27 are included, the composition for inducing differentiation in step (i) is 1 to 5% 1X B27, 1 to 5 μM dolsomorphine, The composition for inducing differentiation in step (iii) is 1-5% 1X B27, 5 ~, so that 1-5 μM retinoic acid, 5-15 μM SB431542, 1-5 μM FR180204 and 0.1-0.5 μM SANT-1 are included. It can be added to the base culture medium to include 15 μM forskolin, 5 to 15 μM dexamethasone and 5 to 15 μM nicotinamide.

The basic culture medium is Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Improved MEM (MEM), Basal Medium Eagle (BME), Roswell Park Memorial Institute medium 1640 (RPMI 1640), Advanced RPMI 1640 (Advanced RPMI1640), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) The medium may include, but is not limited to, a medium capable of supporting differentiation induction and survival of pluripotent stem cells into insulin-producing cells can be used without limitation.

1 is a schematic diagram showing the mechanism of a low-molecular substance used in an insulin-producing cell differentiation-inducing composition, and in the present invention, the composition, concentration, and treatment duration of known low-molecular substances are adjusted to increase the differentiation rate into insulin-producing cells. Did.

When the composition for inducing differentiation is a protein, there is a problem in that it is costly in industrialization and has problems in stability of the composition in clinical applications, but in the case of a low-molecular substance, there is a small economic cost and an advantage in ensuring stability of the composition There is this.

As shown in FIG. 1, the components CHIR99021 and LY294002 serve to promote differentiation from induced pluripotent stem cells to endoderm. In the process of differentiation from endoderm to pancreatic progenitor cells, dolsomorphine inhibits the BMP / AMPK pathway, SB431542 suppresses the ALK / NODAL signal, FR180204 suppresses the ERK signal, and SANT-1 suppresses the hedgehog signal to suppress pancreatic progenitors. Cells increase the efficiency of differentiation. In addition, FGF-2, FGF-10 and retinoic acid serve to promote differentiation into pancreatic progenitor cells.

In the present invention, the human induced pluripotent stem cells (iPSC) are derived from fibroblasts, and may be characterized in that Oct4, Nanog, Sox2 and SSEA4 are expressed.

In a specific embodiment of the present invention, as a result of confirming the identification and differentiation ability of major gene expression of human induced pluripotent stem cells, it was confirmed that specific markers of Oct4, Nanog and Sox2 are expressed in the iPSC nucleus, and representative cell surface of iPSC It was confirmed that SSEA4, one of the markers, was expressed. In addition, it was confirmed that Klf4 and c-Myc were also expressed.

As a result of performing teratoma analysis, iPSCs transplanted into mice grew into solid masses (teratomas) of tissues for 40 days, and the surface showed various types of tissue clusters. In addition, as a result of examining histological variations of the formed teratoma cross section, representative tissues of endoderm, ectoderm and mesoderm were identified. Based on this, it was confirmed that the iPSC used in the present invention has polydifferentiation ability, including the ability to differentiate into insulin producing cells.

In a specific embodiment of the present invention, to determine the differentiation efficiency according to the composition of the differentiation-inducing composition from human induced pluripotent stem cells to endoderm. As shown in FIG. 3, as a result of confirming the differentiation efficiency to definitive endoderm, an early stage of differentiation, by combining activin A, Ly294002, and CHIR99021, activin A, Ly294002, and CHIR99021 were compared to the group treated with low-molecular substances alone. It was confirmed that the expression level of the endoderm markers Sox17, FoxA2 and CXCR4) was highest in the group treated with.

Therefore, in the present invention, in the process of inducing differentiation from induced pluripotent stem cells to endoderm, all of activin A, Ly294002 and CHIR99021 among low molecular substances are treated, and in the process of inducing differentiation from endoderm to primitive intestinal tract, only activin A is to be treated alone. Did.

In another specific embodiment of the present invention, to determine the differentiation efficiency according to the composition of the composition for inducing differentiation from the primitive intestinal tract to pancreatic endoderm (PE). As shown in FIG. 4, induction of differentiation from the primitive intestinal tract to pancreatic endoderm was performed for a total of 6 days, and in addition to basic differentiation conditions (dolsomorphine, retinoic acid, and SB431542), FGF-2, FR180204, and SANT-1 were combined to induce differentiation. Was performed. As a result of inducing differentiation into pancreatic endocrine cells and confirming the differentiation efficiency according to the above conditions, the insulin concentration in the culture medium of the group additionally treated with SANT-1 and FGF-2, FR180204 and SANT-1 was the highest. It was confirmed that it was high.

As a result of confirming the expression level of insulin, glucagon, and Pdx-1 produced in differentiated insulin-producing cells using groups additionally treated with FGF-2, FR180204, and SANT-1, as shown in FIG. 5. Similarly, on day 11 of induction of differentiation into pancreatic endocrine cells, cells expressing insulin / glucagon were identified by immunostaining, and cells expressing insulin / Pdx-1 were also confirmed. As a result of FACS analysis of the level of insulin and glucagon secretion and the expression level of Ngn3 transcription factor in all differentiated cells, as shown in FIG. 6, insulin secreted cells 38.1%, glucagon expressing cells 23.0% , Ngn3 expressing cells were confirmed to be 66.7%.

In another specific embodiment of the present invention, flotation culture was performed to make the pancreatic endocrine cells finally induced in differentiation by the above method in a spheroid form. As shown in FIG. 8A, a small spheroid form was formed from 1 day after flotation culture, and a spheroid form with a larger size was observed after 5 days of culture. In addition, as shown in Figure 8B, as a result of immunostaining the formed spheroid form, it was confirmed that there is a large amount of cells expressing insulin / glucagon. In addition, as shown in Figure 8C, when cultured in a spheroid form, as a result of confirming the amount of C-peptide secretion according to the composition and duration of the culture medium, when continuously cultured in a spheroid form, islet cells rather than the existing differentiation 4-step culture solution When cultured with the culture medium, CMRL 1644, the secretion of C-peptide appeared low, but it was confirmed that it remained stable until 7 days of culture.

In the present invention, according to the above results, as shown in Figure 9, was established a composition for inducing pluripotent stem cell-derived insulin-producing cell differentiation.

In another specific embodiment of the present invention, it was intended to confirm the differentiation efficiency from induced pluripotent stem cells to insulin producing cells according to the initial differentiation condition (Protocol A) and the differentiation induction condition (Prococol B) of the present invention.

As shown in FIG. 10, after confirming the amount of C-peptide secretion generated in the culture medium after the final differentiation step, 6.5 ng / ml was detected in the case of Protocol A, while 37.5 ng / ml was detected in the case of enhanced Procotol B. It was confirmed that the conditions established in the present invention (protocol B) increased about 5.7 times C-peptide secretion.

As shown in FIG. 11, as a result of confirming the expression level of insulin and glucagon produced in differentiated insulin-producing cells after the final differentiation step by immunofluorescence staining, insulin and glucagon expressing cells rarely exist in conventional protocol A. Although found, it was observed that insulin and glucagon expressing cells form a significant number of colonies under the conditions established in the present invention (protocol B).

In addition, as shown in FIG. 12, after the final differentiation step, after the differentiation, the overall differentiation rate of insulin and glucagon cells was analyzed by FACS, and protocol A showed almost no insulin-expressing cells, and only glucagon-expressing cells differentiated by 29.2%. On the other hand, under the conditions established in the present invention (Protocol B), it was confirmed that insulin-expressing cells differentiated to 38.1% and glucagon-expressing cells to 23.1%.

That is, the composition for inducing human-derived pluripotent stem cell-derived insulin-producing cell differentiation established in the present invention is a result of adjusting the composition, concentration, and treatment period of various low-molecular substances, resulting in shorter differentiation and differentiation periods compared to conventional techniques. It was confirmed that the efficiency increased. In addition, the composition for inducing differentiation of insulin-producing cells of the present invention is composed of a low-molecular substance rather than a protein preparation, and thus has an advantage of improving the stability of the composition, and thus can be used in a cell therapeutic agent for diabetes treatment and a process for mass production of insulin. .

The present invention also provides a human induced pluripotent stem cell derived insulin producing cell differentiation induction medium comprising the composition for inducing human induced pluripotent stem cell derived insulin producing cell differentiation as an active ingredient.

In the present invention, the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), Improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI 1640 (FRP), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) It may include a medium selected from the group, but is not limited thereto, and a medium capable of supporting differentiation induction and survival of pluripotent stem cells into insulin-producing cells can be used without limitation.

The present invention also, (a) inducing differentiation into endoderm by culturing human induced pluripotent stem cells in a medium containing activin A, CHIR99021, B27 and LY294002;

(b) inducing differentiation into the primitive intestine by culturing the differentiation-derived endoderm in the medium containing activin A and B27 in step (a);

(c) The differentiation-induced primitive intestinal tract in step (b) is cultured in a medium containing B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1 to induce differentiation into pancreatic endoderm. To do; And

(d) Inducing differentiation into pancreatic endocrine cells capable of producing insulin by culturing the differentiation-induced pancreatic endoderm in the medium containing B27, forskolin, dexamethasone, and nicotinamide in step (c). It provides a method for inducing differentiation of human induced pluripotent stem cell-derived insulin-producing cells comprising the step of treating the composition for inducing human induced pluripotent stem cell-derived insulin-producing cell differentiation to human induced pluripotent stem cells.

Specifically, in step (a), human induced pluripotent stem cells are cultured in RPMI1640 medium containing activin A, CHIR99021, B27 and LY294002 to induce differentiation into endoderm, and step (b) is endoderm And incubated in RPMI1640 medium containing activin A and B27 to induce differentiation into the primordial intestine, step (c) comprises primordial intestine B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1 containing incubated in MEM (improved MEM) containing medium to induce differentiation into pancreatic endoderm, step (d) is pancreatic endoderm B27, forskolin (forskolin), dexamethasone (dexamethasone) ) And nicotinamide, and cultured in an impregnated MEM (MEM) medium containing nicotinamide to induce differentiation into pancreatic endocrine cells capable of producing insulin.

More specifically, in step (a), human induced pluripotent stem cells contain 50 to 150 ng / mL activin A, 1 to 5 μM CHIR99021, 1 to 5% 1X B27, and 15 to 25 μM LY294002 containing RPMI1640 medium. Incubation to induce differentiation into endoderm, step (b) induces differentiation into primitive intestinal tract by culturing endoderm in RPMI1640 medium containing 50 to 150 ng / mL activin A and 1 to 5% 1X B27, In step (c), the primary intestine is 1-5% 1X B27, 1-5 μM dolsomorphine, 1-5 μM retinoic acid, 5-15 μM SB431542, 1-5 μM FR180204 and 0.1-0.5 μM SANT-1. Cultured in an embedded MEM (improved MEM) medium to induce differentiation into pancreatic endoderm, step (d) is 1 ~ 5% 1X B27, 5 ~ 15 μM forskolin, 5 ~ 15 μM dexamethasone And incubation in 5 to 15 μM nicotinamide containing impregnated MEM (MEM) medium to induce differentiation into pancreatic endocrine cells capable of producing insulin. can do.

The method for inducing differentiation of human-derived pluripotent stem cell-derived insulin-producing cells of the present invention can be preferably performed according to the conditions of FIGS. 9 and 9, and the details are the same as described above.

The present invention also provides an insulin-producing pancreatic endocrine cell prepared by the method for inducing differentiation of insulin-producing cells derived from the human induced pluripotent stem cells.

The present invention also provides an endocrine aggregate of insulin-producing pancreatic endocrine cells prepared by the method for inducing differentiation of insulin-producing cells derived from the human induced pluripotent stem cells.

The pancreatic endocrine cells or endocrine aggregates are preferably reacted with glucose in vitro to increase the secretion amount of the insulin precursor C-peptide, but is not limited thereto. The pancreatic endocrine cells or endocrine aggregates are preferably 1 × 10 ⁴ to 1 × 10 ⁵ individuals in the process of increasing cell proliferation, but are not limited thereto.

Furthermore, the present invention provides a pharmaceutical composition for treating diabetes comprising the insulin-producing pancreatic endocrine cells or endocrine aggregates as an active ingredient.

The present invention also, (a) culturing the insulin-producing pancreatic endocrine cells or endocrine aggregates; And

(b) comprising the step of separating insulin from the culture by the culture of step (a), provides a method for mass production of insulin.

Insulin separated by the method for mass production of insulin according to the present invention can be usefully used for the prevention or treatment of diabetes.

The diabetes is preferably type 2 diabetes, which is insulin-independent diabetes, which is relatively insufficient in insulin, but is not limited thereto.

The therapeutically effective amount of the composition of the present invention can vary depending on several factors, such as the method of administration, the target site, the patient's condition, and the like. Therefore, when used in the human body, the dosage should be determined in an appropriate amount in consideration of safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments.

The compositions of the present invention may also include carriers, diluents, excipients, or combinations of two or more of those commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one of these components. It is possible to mix and use more than the components, and if necessary, other conventional additives such as antioxidants, buffers, bacteriostatic agents can be added.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main-use formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the art.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally, depending on the desired method, and the dosage may include the patient's weight, age, sex, health status, The range varies depending on the diet, administration time, administration method, excretion rate and disease severity. The daily dosage of the composition according to the present invention is 00001 to 10 mg / ml, preferably 00001 to 5 mg / ml, and more preferably divided and administered once to several times a day.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Human-derived induced pluripotent stem cell culture and characterization

1-1: Human-derived induced pluripotent stem cell culture

The human induced pluripotent stem cells (iPSC) used in the present invention were provided by the stem cell research center of the Asan Research Institute, and specifically, after the fibroblasts were separated and cultured from the skin fibrous tissue, CytoTune ^TM -iPS 2.0 Sendai Reprogramming Kit (ThermoFisher) was used. The Klf4, Oct3 / 4, Sox2, and cMyc genes were introduced. The first colonies were identified 12 days after virus treatment, and colonies composed of 20-30 cells were transferred to a new culture dish and propagated.

The prepared iPSC was cultured in a culture dish coated with vitronectin using 8TM basic medium (Gibco, USA) containing essential 8TM supplement (Gibco, USA) and antibiotic (Gibco, USA). . Induced pluripotent stem cells were passaged 1: 5 to 1: 6 using StemPro Accutase medium (Gibco, USA), and cultured at 37 ° C and 5% CO ₂ conditions.

1-2: Confirmation of human-derived induced pluripotent stem cell characteristics

In order to confirm that the human-derived induced pluripotent stem cells have the ability to differentiate into insulin producing cells, it was confirmed that specific markers of Oct4, Nanog, Sox2 and SSEA4 are expressed in the iPSC nucleus.

First, the cells were treated with 4% paraformaldehyde (PFA) on the cells, fixed at room temperature for 30 minutes, and then washed 3 times with PBS. The fixed cells were prepared by infiltration with 0.1% Triton X-100 for 5 minutes, followed by washing with PBS. For blocking, cells were treated with a solution containing normal horse serum (1:30) for 30 minutes, and then anti-Oct4 antibody (1:50; abcam, USA) , Anti-Nanog antibody (mouse anti-Nanog antibody, 1: 200; abcam), anti-Sox2 antibody (rpabbit anti-Sox2 antibody, 1: 1000; abcam) and anti-SSEA4 antibody (mouse anti-SSEA4 antibody, 1: 200; abcam) were each treated and reacted overnight at 4 ° C. Then, after washing with PBS, a secondary antibody (donkey antibody or goat antibody) against mouse IgG or rabbit IgG conjugated with Alexa Fluor 488 or Alexa Fluor 594 is 1: Diluted to 250 and then reacted for 1 hour at room temperature. Nuclei were stained using Hoechst 33342 (Thermo Scientific, Germany) and observed using a fluorescence microscope.

As a result, as shown in FIG. 2, it was observed that the human induced pluripotent stem cells prepared in the present invention grow by forming a cluster (FIG. 2A), and that specific markers of Oct4, Nanog, and Sox2 are expressed in the iPSC nucleus. It was confirmed that SSEA4, one of the representative cell surface markers of iPSC, was expressed (FIGS. 2B to 2E).

1-3: Observation of the pluripotency of human-derived induced pluripotent stem cells

In the present invention, teratoma analysis was performed to observe the pluripotency of human-derived induced pluripotent stem cells.

On the day of transplantation, iPSC was separated from the culture dish using Accutase, and then suspended as a single cell. A total of 1 x 10 ⁶ cells were administered subcutaneously directly to 8-week-old immune-deficient SCID mice, and then non-specific pathogen-free (SFP) in a laboratory animal facility at Asan Hospital. It was kept in a state. Tissues of 1 cm ³ or more were generated in mice, or tissues were observed after 40 days of cell administration. The tissue containing the tumor was prepared by using a 4% paraformaldehyde (PFA) paraffin block, and then cut into 4 micron cross sections. The tissue section slides were stained with hematoxylin and eosin, and then histological analysis was performed using a microscope.

As a result of the observation, as shown in FIG. 2F, iPSC implanted in the mouse grew into a solid mass of tissue for 40 days, the surface showed various types of tissue clusters, and the tissue of the solid mass showed histological variation. Representative tissues of endoderm, ectoderm and mesoderm were identified, and it was confirmed that the iPSC used in the present invention has polydifferentiation ability, including the ability to differentiate into insulin producing cells.

Establishment of composition to induce differentiation from human induced pluripotent stem cells to endoderm

2-1: Identification of differentiation efficiency according to the composition of the differentiation-inducing composition from human induced pluripotent stem cells to endoderm

In the present invention, to determine the differentiation efficiency according to the composition of the differentiation inducing composition from human induced pluripotent stem cells to endoderm.

Induced pluripotent stem cells were prepared by culturing as in Example 1-1, and cultured in RPMI 1640 medium containing 1% B27 (Invitrogen, USA) for induction of differentiation into endoderm, 100 ng / as shown in Table 1. Differentiation into endoderm by incubation for 24 hours in a medium containing ml activin A (PeproTech, USA), 3 μM CHIR99021 (Sigma-Aldrich, USA), or 5 μM LY294002 (Sigma-Aldrich, USA), respectively or all Induced.

The next day, after washing with PBS, 100ng / mL Activin A was added to RPMI 1640 medium, and cultured with the same composition was treated on the third day to complete differentiation into endoderm cells. When the differentiation was completed with the endoderm, it was confirmed that the cells were densely packed and the induced pluripotent stem cells, which formed colonies, opened in the colony and had a small polygonal shape.

Composition composition for inducing endoderm differentiation One 2 3 100 ng / ml Activin A + + + 5 μM LY294002 +  3 μM CHIR99021 + +

To confirm the degree of differentiation into endoderm, mRNA expression levels of Sox17, FoxA2 and CXCR4, known as endoderm markers, were confirmed using real-time PCR.

Total RNA was isolated according to the product manual using TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using a reverse transcription master premix (Reverse transcription master premix (ELPiS, Korea)). Real-time PCR was performed using Cyber Green Supermix (SsoAdvancedTM Universal SYBR Green Supermix; BIO-RAD, USA), and amplified each mRNA using the primers shown below: PCR was 95 ° C for 15 seconds, 58 ° C. The cycle was repeated 30 cycles at 30 ° C and 30 seconds at 72 ° C., and the relative level of mRNA expression of each gene for GAPDH mRNA was analyzed by 2 ^-ΔΔCT method.

Sox17-forward: 5`-CGCTTTCATGGTGTGGGCTAAGGACG-3` (SEQ ID NO: 1)

Sox17-reverse: 5`-TAGTTGGGGTGGTCCTGCATGTGCTG-3` (SEQ ID NO: 2)

FoxA2-forward: 5`- ACTGGAGCAGCTACTTAGCAGAGC-3` (SEQ ID NO: 3)

FoxA2-reverse: 5`-TCATGGAGTTCATGTTGGCGTAG-3` (SEQ ID NO: 4)

CXCR4-forward: 5`-GGTGGTCTATGTTGGCGTCT-3` (SEQ ID NO: 5)

CXCR4-reverse: 5`-TGGAGTGTGACAGCTTGGAG-3 (SEQ ID NO: 6)

GAPDH-forward: 5`-AAGGGCTCATGACCACAGTCCAT-3` (SEQ ID NO: 7)

GAPDH-reverse: 5`-CAGGAAATGAGCTTCACAAAGTT-3` (SEQ ID NO: 8)

Comparison of expression of endoderm markers qPCR Sox17 ActivinA ActivinA
+ CHIR99021 ActivinA
+ CHIR99021
+ LY294002 No1 One 3.0951 3.6808 No2 One 2.7132 5.1694 No3 One 3.0738 7.1602 Mean One 2.9607 5.3368 Std. Dev 0 0.2146 1.7458 FoxA2 ActivinA ActivinA
+ CHIR99021 ActivinA
+ CHIR99021
+ LY294002 No1 One 2.8679 2.4284 No2 One 2.6027 2.6759 No3 One 5.9381 4.2575 Mean One 3.8029 3.1206 Std. Dev 0 1.8539 0.9923 CXCR4 ActivinA ActivinA
+ CHIR99021 ActivinA
+ CHIR99021
+ LY294002 No1 One 5.579 5.3147 No2 One 5.2054 5.6178 No3 One 5.3517 7.0616 Mean One 5.3787 5.998 Std. Dev 0 0.1883 0.9335

As shown in Figure 3 and Table 2, as a result of confirming the differentiation efficiency to definitive endoderm, an early stage of differentiation, by combining activin A, Ly294002, and CHIR99021 in differentiation stage 1, it was found that the low-molecular substance was treated alone. It was confirmed that the expression level of endoderm markers Sox17, FoxA2 and CXCR4) was highest in the group treated with both activin A, Ly294002 and CHIR99021.

Accordingly, in the present invention, in the process of inducing differentiation from induced pluripotent stem cells to endoderm, both activin A, Ly294002 and CHIR99021 among low molecular substances are treated, and RPMI 1640 containing 1% B27 in the process of inducing differentiation from endoderm to primitive intestine. The medium was treated with 100 ng / ml activin A alone to incubate for 12 hours.

Establishment of composition for inducing differentiation from primitive intestinal tract to pancreatic endoderm

3-1: Establishment of composition for inducing differentiation from primitive intestinal tract to pancreatic endoderm

In the present invention, to determine the differentiation efficiency according to the composition of the composition for inducing differentiation from the intestinal tract to pancreatic endoderm (PE).

Specific expression genes of the pancreatic endoderm include Pdx1, Nkx6.1, Ngn3, etc. In the present invention, cells expressing Pdx1 and Nkx6.1 at the same time have a high possibility of differentiating into insulin producing cells, so Pdx1, Nkx6.1 are used. It was intended to specify the stage of expression. Therefore, rather than the gene expression level of the endoderm of the pancreas, it was intended to establish a condition in which the differentiation of insulin-producing cells is finally higher.

Induction of differentiation from the primitive intestinal tract to pancreatic endoderm was performed for a total of 6 days, and induction of differentiation was performed by combining FGF-2, FR180204 and SANT-1 in addition to basic differentiation conditions (dolsomorphine, retinoic acid, and SB431542).

Specifically, MEM medium containing 1% B27, 1 μM dolsomorphin (Dorsomorphin; Sigma-Aldrich, USA), 4 μM retinoic acid (Retinoic acid, Tocris Bioscience, UK) and 10 μM SB431542 (Tocris Bioscience, UK) ( Improved MEM Zinc Option culture medium, Invitrogen, USA), 50 ng / ml FGF2 (PeproTech, USA), 3 μM FR180204 (Sigma-Aldrich, USA) and 0.25 μM SANT-1 (Sigma-Aldrich, USA) as shown in Table 3 below. ) Was cultured for 6 days in a medium containing each or both, and the medium was changed daily.

Composition composition for inducing pancreatic endoderm differentiation #One #2 # 3 #4 # 5 # 6 Dolsomorphine, retinoic acid and SB431542 + + + + + + 50 ng / ml FGF2 + + + 3 μM FR180204 + + + 0.25 μM SANT-1 + +

To induce differentiation of pancreatic endoderm under the same conditions as above, and then induce differentiation into pancreatic endocrine cells, which are insulin-producing cells, 10 μM forskolin (forskolin; Sigma-Aldrich, USA), 10 μM dexamethasone (dexamethasone; Sigma-) Aldrich, USA) and 10 μM nicotinamide (Sigma-Aldrich, USA) were cultured for 10 days, and the medium was changed daily.

3-2: Identification of differentiation efficiency according to the composition of the composition for inducing differentiation from the primitive intestinal tract to the pancreatic endoderm-insulin concentration analysis

When finally differentiated into insulin-producing cells, in order to confirm the degree of insulin production according to the composition component for inducing differentiation from the primitive intestinal tract to the pancreatic endoderm, insulin in the culture medium 6, 8, and 12 days after induction of differentiation into pancreatic endocrine cells Concentrations were analyzed. Insulin secreted in differentiation-inducing medium was measured according to the product manual using an ELISA kit (ALPCO, USA).

Analysis of insulin concentration in culture medium 5 days after differentiation into pancreatic endocrine cells 5 days #One #2 # 3 #4 # 5 # 6 No1 5.9493 4.2476 3.7195 5.6969 3.2101 4.2255 No2 4.3837 4.2348 3.0205 5.0443 3.1624 3.9021 No3 5.0339 5.17 4.3151 6.8623 4.1569 4.2743 Mean 5.1223 4.5508 3.685 5.8678 3.5098 4.134 Std. Dev 0.7865 0.5363 0.648 0.921 0.5609 0.2023

Analysis of insulin concentration in culture medium 8 days after differentiation into pancreatic endocrine cells 8 days #One #2 # 3 #4 # 5 # 6 No1 75.2997 77.0488 76.4624 80.0546 76.8867 80.0546 No2 80.0546 75.0676 80.0546 80.0546 74.4612 80.0546 No3 75.9781 68.6577 80.0546 80.0546 80.0546 80.0546 Mean 77.1108 73.5913 78.8572 80.0546 77.1342 80.0546 Std. Dev 2.5719 4.386 2.074 0 2.8049 0

Analysis of insulin concentration in culture medium 12 days after differentiation into pancreatic endocrine cells 12 days (forskolin removed) #One #2 # 3 #4 # 5 # 6 No1 25.804 30.4482 37.3353 50 37.1488 50 No2 37.8704 34.6849 31.5237 50 32.1674 50 No3 30.4112 23.9494 38.4222 50 36.3467 50 Mean 31.3619 29.6942 35.7604 50 35.221 50 Std. Dev 6.0891 5.4073 3.7091 0 2.6747 0

As a result, as shown in Tables 4 to 6 and 4, it was confirmed that the insulin concentration in the culture medium of the group additionally treated with only SANT-1 and FGF2, FR180204 and SANT-1 was the highest.

In addition, in order to confirm the insulin secretion ability by glucose stimulation, cells differentiated into insulin producing cells are glucose-free Krebs-Ringer bicarbonate HEPES buffer, Krebs buffer; 115 mM Nacl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, After treating for 2 hours at 37 ° C with 1.2mM KH2PO4, 1.2mM MgSO4, 25mM HEPES and 0.5% BSA), the reaction was sequentially performed for 1 hour using 2.8mM glucose and 30mM glucose Krebs buffer. Then, insulin secreted in Krebs buffer was measured according to the product manual using an ELISA kit (ALPCO, USA).

Confirmation of glucose stimulated insulin secretion (GSIS) after completion of the final differentiation step GSIS # 1 (2.8mM) # 1 (30mM) # 4 (2.8mM) # 4 (30mM) # 5 (2.8mM) # 5 (30mM) No1 0.9649 1.1859 2.2166 7.5033 1.3775 5.7036 No2 0.5217 0.9164 1.4694 3.9295 0.8436 4.5796 No3 0.4808 0.9483 1.0326 4.2807 0.8142 4.4685 Mean 0.6558 1.0168 1.5729 5.2378 1.0117 4.9172 Std. Dev 0.2685 0.1472 0.5988 1.9698 0.3171 0.6833

As a result, as shown in Table 7 and FIG. 4E, it was confirmed that the insulin secretion ability by glucose stimulation was the highest in the culture medium of only the group additionally treated with SANT-1 and the group additionally treated with FGF2, FR180204 and SANT-1. .

Confirmation of insulin-producing cell differentiation using established insulin-producing cell differentiation-inducing composition

4-1: Identification of differentiation efficiency through immunocytochemical staining

In the present invention, when the induced pluripotent stem cells were cultured under the conditions established in Examples 2 and 3, it was intended to confirm the differentiation efficiency of insulin producing cells.

After the cells differentiated into the insulin-producing cells were fixed in the same manner as in Example 1-2, anti-insulin antibody (mouse anti-insulin antibody, 1: 200; Santa Cruz), anti-glucagon antibody (rabbit anti- The primary antibodies against Glucagon antibody, 1: 200; Santa Cruz) and anti-Pdx1 antibody (goat anti-Pdx1 antibody, 1: 10000; abcam) were each treated and reacted overnight at 4 ° C. Then, after washing with PBS, a secondary antibody (donkey antibody or goat antibody) against mouse IgG or rabbit IgG conjugated with Alexa Fluor 488 or Alexa Fluor 594 is 1: Diluted to 250 and then reacted for 1 hour at room temperature. Nuclei were stained with Hoechst 33342 (Thermo Scientific, Germany) and observed using a fluorescence microscope.

As a result, as shown in FIG. 5, cells expressing insulin / glucagon were identified by immunostaining on day 11 of induction of differentiation into pancreatic endocrine cells, and cells expressing insulin / Pdx-1 were also confirmed.

4-2: Check differentiation efficiency through flow cytometry

Differentiated cells were separated from the culture dish using Accutase, and then treated with 4% paraformaldehyde (PFA) to fix the cells. Cells were treated with a solution containing normal horse serum (1:30) for 30 minutes, followed by mouse anti-insulin antibody and anti-glucagon antibody. And treated with anti-Ngn3 antibody (rabbit anti-Ngn3 antibody) for 30 minutes at room temperature. Then, a second antibody against mouse or rabbit IgG conjugated with Alexa Fluor 488 or Alexa Fluor 594 was treated for 30 minutes at room temperature, and then reacted for 30 minutes at room temperature. It was analyzed using an analyzer (FACSAria II; Becton Dickinson).

As a result of FACS analysis of the level of insulin and glucagon secretion and the expression level of Ngn3 transcription factor in all differentiated cells, as shown in FIG. 6, insulin secreted cells 38.1%, glucagon expressing cells 23.0% , Ngn3 expressing cells were confirmed to be 66.7%.

4-3: Identification of gene expression related to differentiation through polymerase chain reaction (PCR)

It was confirmed that the differentiated cells expressed pancreatic-related transcription factors including insulin. Specifically, after extracting total RNA in the same manner as in Example 2-1, PCR was performed, and Pdx1, Pdx6, NeuroD, Ngn3, Sox9, and cytokeratin 19, which are the main transcription factors of cells that secrete insulin through PCR, 19 (Cytokeratin 19), insulin (insulin), somatostatin (Somatostatin) and expression of 18s RNA was confirmed. Each mRNA was amplified using the primers shown below:

Pdx1-forward: 5`-GCATCCCAGGTCTGTCTTCT-3` (SEQ ID NO: 9)

Pdx1-reverse: 5`-ATCCCACTGCCAGAAAGGTT-3` (SEQ ID NO: 10)

Pax6-forward: 5`-GAGGCCCTGGAGAAAGAGTT-3` (SEQ ID NO: 11)

Pax6-reverse: 5`-CTTCTCCATTTGGCCCTTCG-3` (SEQ ID NO: 12)

NeuroD-forward: 5`-GCCGACGGAGATTAGGAGAA-3` (SEQ ID NO: 13)

NeuroD-reverse: 5`-TCTTGTCCTGACACTGGCAT-3` (SEQ ID NO: 14)

Ngn3-forward: 5`-CTTGCTGCTCAGGAAATCCC-3` (SEQ ID NO: 15)

Ngn3-reverse: 5`-CTTCTGGTCGCCAAGTTCAG-3` (SEQ ID NO: 16)

Sox9-forward: 5`-TACGACTACACCGACCACCA-3` (SEQ ID NO: 17)

Sox9-reverse: 5`-TCAAGGTCGAGTGAGCTGTG-3` (SEQ ID NO: 18)

Cytokeratin 19-forward: 5`-GGACCTGCGGGACAAGATTCTTGGTG-3` (SEQ ID NO: 19)

Cytokeratin 19-reverse: 5`-CGAGATCGGTGCCCGGAGCGGAATCC-3` (SEQ ID NO: 20)

insulin-forward: 5`-ATCCTGGATCTCAGCTCCCT-3` (SEQ ID NO: 21)

insulin-reverse: 5`-CTCACAGCCCTTCAGAGACA-3` (SEQ ID NO: 22)

Somatostatin-forward: 5`-AACCCAACCAGACGGAGAAT-3` (SEQ ID NO: 23)

Somatostatin-reverse: 5`-TAGCCGGGTTTGAGTTAGCA-3` (SEQ ID NO: 24)

18s RNA-forward: 5`-GAGCCTGCGGCTTAATTTGA-3` (SEQ ID NO: 25)

18s RNA-reverse: 5`-AACTAAGAACGGCCATGCAC-3` (SEQ ID NO: 26)

As shown in FIG. 7, it was confirmed that Pdx1, NeuroD, and Ngn3 are expressed in three different differentiated cells, and that insulin and somatostatin are also strongly expressed.

Spheroid type induction using pancreatic endocrine cells

In the present invention, flotation culture was performed to make the pancreatic endocrine cells, which were finally differentiated and induced into the spheroid form, under the conditions established in Examples 2 and 3 above.

After the four-step differentiation was completed, the cells were made into single cells using trypsin-ID, and cultured in a culture dish without cell attachment. After 1 day of cultivation, it can be confirmed that the cells adhere to each other in a small bundle of spheroid form. After 5 days of cultivation, the spheroid form was enlarged.

As shown in FIG. 8A, a small spheroid form was formed from 1 day after flotation culture, and a spheroid form with a larger size was observed after 5 days of culture. In addition, as shown in Figure 8B, as a result of immunostaining the formed spheroid form, it was confirmed that there is a large amount of cells expressing insulin / glucagon.

As shown in Figure 8C, when cultured in a spheroid form, as a result of confirming the amount of C-peptide secretion according to the culture medium composition and duration, when continuously cultured in a spheroid form, the culture of pancreatic islet cells rather than the existing 4-step differentiation culture It was confirmed that it was better to grow with CMRL 1644 culture.

Forskolin is added to the STEP 4 medium, so even if the same insulin producing cells are secreted, more insulin can be detected in the medium. Therefore, after removing the forskolin from the STEP 4 medium, the culture was compared with the amount of insulin secretion when cultured in CMRL 1644, which is a medium for culturing human pancreatic islet cells. Even if only the forskolin is removed from the STEP 4 medium, each culture dish While the expression of insulin was uneven per CMRL 1644 medium, the expression of insulin evenly in each culture dish was confirmed. Therefore, in order to secrete relatively stable insulin, it was determined that CMRL 1644 medium that does not stimulate insulin producing cells would be better.

Establishment of composition conditions for inducing differentiation of insulin producing cells

As shown in FIG. 9, the present invention finally established a composition for inducing differentiation of insulin-producing cells derived from human induced pluripotent stem cells based on the contents of Examples 2 to 5.

The following table shows the initial differentiation induction conditions (Table 8) and the differentiation induction conditions established in the present invention (Table 9).

Initial differentiation induction conditions (Protocol A) iPSC-> DE DE-> PGT PGT-> PE PE-> EP Culture period 3-4 days 3 days 5 days 10 days Basic badge DMEM RPMI1640 DMEM DMEM / F12 Additive 1XB27 100ng / mL Activin A
3uM CHIR99021 1XB27
1uM Dorsomorphin
50ng / mL FGF10 1XB27
1uM Dorsomorphin
50ng / mL FGF10
2uM Retinoic acid
3uM FR180204 1XB27
10uM Forskolin
10uM Nicotinamide
40ng / mL EGF
10uM Betacelluin

 Differentiation-inducing conditions established in the invention (Protocol B) iPSC-> DE DE-> PGT PGT-> PE PE-> EP Culture period 3-4 days 3 days 5 days 10 days Basic badge RPMI1640 RPMI1640 improved MEM (ZINC option) improved MEM (ZINC option) Additive 1XB27 100ng / mL Activin A
3uM CHIR99021
20uM LY294002 1XB27
100ng / mL Activin A 1XB27
1uM Dorsomorphin
2uM Retinoic acid
3uM FR180204
0.25uM SANT-1 1XB27
10uM Forskolin
10uM Dexamethasone
10uM Nicotinamide

In Tables 8 and 9, iPSC is induced pluripotent stem cells, DE is definitive endoderm, PGT is primitive gut tube, PE is pancreatic endoderm, EP is insulin It refers to the resulting pancreatic endocrine cell (endocrine pancrease).

Identification of differentiation induction efficiency according to differentiation induction conditions

7-1: Confirmation of the amount of C-peptide secretion

In order to confirm the differentiation efficiency from induced pluripotent stem cells to insulin producing cells according to the initial differentiation condition (protocol A) and the differentiation induction condition (prococol B) of the present invention. With the composition of Tables 8 and 9, differentiation medium was prepared for each step, and differentiation was induced by exchanging the medium every day.

After the final differentiation step, the amount of C-peptide secreted in the culture medium was confirmed, and the c-peptide secreted in the differentiation-inducing medium was measured according to the product manual using a human c-peptide ELISA kit (Alpha Diagnostic, USA). .

As a result, as shown in FIG. 10, as a result of confirming the amount of C-peptide secretion generated in the culture medium after the final differentiation step, 6.5 ng / ml was detected for Protocol A, whereas 37.5 ng / for enhanced Procotol B It was confirmed that ml was detected, and the conditions established in the present invention (protocol B) increased by about 5.7-fold C-peptide secretion.

7-2: Identification of differentiation efficiency through immunocytochemical staining

After the final differentiation step, the expression level of insulin and glucagon generated in differentiated insulin producing cells was confirmed through immunocytochemistry.

Specifically, after the final differentiation, the cells were washed several times with PBS and then fixed with 4% paraformaldehyde for 1 hour, then washed again with PBS and treated with 0.5 triton X 100 (trition X-100). The permeability of the cell membrane was made, and after PBS washing, 1% normal horse serum (normal horse serum) was blocked for 1 hour. Thereafter, the insulin antibody diluted 1: 100 and the glucagon antibody diluted 1: 100 were reacted at 4 ° C overnight. Thereafter, the cells were washed several times with PBS and treated with fluorescently fluorescent secondary antibody to each cell to observe cells expressed with a fluorescent microscope.

As a result, as shown in FIG. 11, as a result of confirming the expression level of insulin and glucagon generated in differentiated insulin-producing cells after the final differentiation step by immunofluorescence staining, the existing protocol A expresses insulin and glucagon. Although cells were rarely found, it was observed that insulin and glucagon expressing cells formed a large number of colonies under the conditions established in the present invention (Protocol B).

7-3: Identification of differentiation efficiency through flow cytometry

 After the final differentiation step, the overall differentiation rate of insulin and glucagon cells after differentiation was analyzed by FACS.

Specifically, after the differentiation was completed, the cells were treated with trypsin-IDA, separated into single cells, washed several times with PBS, and fixed with 4% paraformaldehyde for 20 minutes. After washing again with PBS several times, 0.5 triton X-100 was treated to make cell membrane permeability, and insulin antibody diluted 1: 100 and glucagon antibody diluted 1: 100 were reacted for 30 minutes at room temperature. Ordered. Subsequently, after washing with PBS, FACS analysis was performed by treating the fluorescently fluorescent secondary antibody to each group.

As a result, as shown in FIG. 12, after the final differentiation step, the overall differentiation rate of insulin and glucagon cells after differentiation was analyzed by FACS, and protocol A showed almost no insulin-expressing cells, and only glucagon-expressing cells differentiated by 29.2%. On the other hand, under the conditions established in the present invention (Protocol B), it was confirmed that insulin-expressing cells differentiated to 38.1% and glucagon-expressing cells to 23.1%.

<110> UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION          THE ASAN FOUNDATION <120> Composition for inducing dedifferentiation for insulin producing          cells from human induced pluripotent stem cells and method for          inducing insulin producing cells using the same <130> 1066489 <150> KR 10-2018-0131257 <151> 2018-10-30 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sox17-forward <400> 1 cgctttcatg gtgtgggcta aggacg 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sox17-reverse <400> 2 tagttggggt ggtcctgcat gtgctg 26 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FoxA2-forward <400> 3 actggagcag ctacttagca gagc 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FoxA2-reverse <400> 4 tcatggagtt catgttggcg tag 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4-forward <400> 5 ggtggtctat gttggcgtct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4-reverse <400> 6 tggagtgtga cagcttggag 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-forward <400> 7 aagggctcat gaccacagtc cat 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-reverse <400> 8 caggaaatga gcttcacaaa gtt 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pdx1-forward <400> 9 gcatcccagg tctgtcttct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pdx1-reverse <400> 10 atcccactgc cagaaaggtt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pax6-forward <400> 11 gaggccctgg agaaagagtt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pax6-reverse <400> 12 cttctccatt tggcccttcg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NeuroD-forward <400> 13 gccgacggag attaggagaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NeuroD-reverse <400> 14 tcttgtcctg acactggcat 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ngn3-forward <400> 15 cttgctgctc aggaaatccc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ngn3-reverse <400> 16 cttctggtcg ccaagttcag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox9-forward <400> 17 tacgactaca ccgaccacca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox9-reverse <400> 18 tcaaggtcga gtgagctgtg 20 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cytokeratin 19-forward <400> 19 ggacctgcgg gacaagattc ttggtg 26 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cytokeratin 19-reverse <400> 20 cgagatcggt gcccggagcg gaatcc 26 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> insulin-forward <400> 21 atcctggatc tcagctccct 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> insulin-reverse <400> 22 ctcacagccc ttcagagaca 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Somatostatin-forward <400> 23 aacccaacca gacggagaat 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Somatostatin-reverse <400> 24 tagccgggtt tgagttagca 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s RNA-forward <400> 25 gagcctgcgg cttaatttga 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s RNA-reverse <400> 26 aactaagaac ggccatgcac 20

Claims (11)

(Iv) a composition for inducing differentiation from human induced pluripotent stem cells (iPSCs) including activin A, CHIR99021, B27 and LY294002 to definitive endoderm (DE);
(Ii) a composition for inducing differentiation from endoderm cells including activin A and B27 to a primitive gut tube (PGT);
(Iii) a composition for inducing differentiation of pancreatic endoderm (PE) from the primitive intestinal tract including B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1; And
(Iii) B27, a composition for inducing differentiation of pancreatic endoderm, including B27, forskolin, dexamethasone, and nicotinamide to insulin-producing pancreatic endocrine cells (endocrine pancrease; EP). A composition for inducing differentiation of insulin-producing cells derived from human induced pluripotent stem cells (iPSC).
According to claim 1, wherein the composition for inducing differentiation in step (i) comprises 50 to 150 ng / mL activin A, 1 to 5 μM CHIR99021, 1 to 5% 1X B27, and 15 to 25 μM LY294002,
The composition for inducing differentiation in step (ii) includes 50 to 150 ng / mL activin A and 1 to 5% 1X B27,
The composition for inducing differentiation in step (i) is 1-5% 1X B27, 1-5 μM dolsomorphine, 1-5 μM retinoic acid, 5-15 μM SB431542, 1-5 μM FR180204 and 0.1-0.5 μM SANT- Contains 1,
The composition for inducing differentiation in the step (i) is 1 to 5% 1X B27, 5 to 15 μM forskolin, 5 to 15 μM dexamethasone, and 5 to 15 μM nicotinamide, human induced pluripotent stem cells A composition for inducing differentiation of derived insulin producing cells.
According to claim 1, The human induced pluripotent stem cells are fibroblast (fibroblast) derived, characterized in that expressing Oct4, Sox2, Klf4, c-Myc and SSEA4, human induced pluripotent stem cell derived insulin producing cell differentiation Composition for induction.
A medium for inducing human-derived pluripotent stem cell-derived insulin-producing cell differentiation, comprising the composition for inducing human-derived pluripotent stem cell-derived insulin-producing cell differentiation according to any one of claims 1 to 3 as an active ingredient.
The medium of claim 4, wherein the medium is Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Improved MEM (MEM), Basal Medium Eagle (BME), Roswell Park Memorial Institute medium 1640 (RPM 1640). , Advanced RPMI 1640 (Advanced RPMI1640), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) Insulin producing cell differentiation induction medium derived from human induced pluripotent stem cells, characterized in that it is selected from the group consisting of.
(a) inducing differentiation into endoderm by culturing human induced pluripotent stem cells in a medium containing activin A, CHIR99021, B27 and LY294002;
(b) inducing differentiation into the primitive intestine by culturing the differentiation-derived endoderm in the medium containing activin A and B27 in step (a);
(c) The differentiation-induced primitive intestinal tract in step (b) is cultured in a medium containing B27, dorsomorphin, retinoic acid, SB431542, FR180204 and SANT-1 to induce differentiation into pancreatic endoderm. To do; And
(d) Inducing differentiation into pancreatic endocrine cells capable of producing insulin by culturing the differentiation-induced pancreatic endoderm in the medium containing B27, forskolin, dexamethasone, and nicotinamide in step (c). Including; comprising, human induced pluripotent stem cell-derived insulin producing cells differentiation induction method.
The method of claim 6, wherein step (a) comprises human induced pluripotent stem cells containing 50 to 150 ng / mL activin A, 1 to 5 μM CHIR99021, 1 to 5% 1X B27, and 15 to 25 μM LY294002. Cultured in the medium to induce differentiation into the endoderm,
In step (b), the endoderm is cultured in a medium containing 50 to 150 ng / mL activin A and 1 to 5% 1X B27 to induce differentiation into the primitive intestine,
In step (c), the primary intestine is 1-5% 1X B27, 1-5 μM dolsomorphine, 1-5 μM retinoic acid, 5-15 μM SB431542, 1-5 μM FR180204 and 0.1-0.5 μM SANT-1. Incubation in the included medium induces differentiation into pancreatic endoderm,
In step (d), pancreatic endoderm is cultured in a medium containing 1 to 5% 1X B27, 5 to 15 μM forskolin, 5 to 15 μM dexamethasone, and 5 to 15 μM nicotinamide to differentiate into insulin-producing pancreatic endocrine cells. Characterized in that, inducing differentiation of human-derived pluripotent stem cell-derived insulin producing cells.
The human induced pluripotent stem cell of claim 6, wherein the human induced pluripotent stem cell of step (a) is derived from fibroblasts and expresses Oct4, Sox2, Klf4, c-Myc and SSEA4. Method for inducing differentiation of derived insulin producing cells.
An insulin-producing pancreatic endocrine cell prepared by the method of claim 6.
A pharmaceutical composition for treating diabetes comprising the insulin-producing pancreatic endocrine cell of claim 9 as an active ingredient.
A method for mass production of insulin comprising the following steps:
(A) culturing the insulin-producing pancreatic endocrine cells of claim 9; And
(B) separating the insulin from the culture by the culture of step (a).

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