KR20080020098A - Media compostions containing low concentrations of glucose useful for human embryonic stem cells, differentiation method of human embryonic stem cells into insulin-producing cells or cell clusters using thereof, and insulin-producing cells or cell clusters differentiated thereby - Google Patents

Abstract

A medium composition containing low concentration of glucose for culturing human embryonic stem cells is provided to reduce insulin tolerance of cells shown by long-term exposure to high concentration of glucose environment during cultivation of cells and produce insulin-producing cells having enhanced insulin production yield. A DMEM/F12(Dulbecco's modified eagle medium/Ham's F12) medium composition for inducing differentiation of human embryonic stem cells into insulin-producing cells optionally contains low concentration such as 5-10 mM of glucose, and further contains 10mM of nicotinamide. A method for producing insulin-producing cells or cell clusters differentiated thereby comprises the steps of: (1) culturing undifferentiated human embryonic stem cells in a DMEM/F12 medium containing 5-10mM of glucose, 15% of serum replacement and 2 ng/mL of FGF-2; (2) floating culturing the cultured cells in a DMEM/F12 medium containing 5-10mM of glucose and 15% of serum replacement to form embryoid body; (3) inducing differentiation from embryoid body to endodermal stem cells by using a DMEM/F12 medium containing 5-10mM of glucose, 15% of serum replacement and 50-100 ng/mL of activin A; (4) selecting the differentiated cells to the endodermal stem cells by using a DMEM/F12 medium containing 5-10mM of glucose, 0.5-2.0 mg/mL of insulin, 0.25-1.0 mg/mL of transferring and 0.25-1.0 mug/mL of sodium selenite; (5) inducing differentiation into precursor cells of insulin-producing cells and their proliferation by using a DMEM/F12 medium containing 5-10mM of glucose and 5-20ng/mL of betacellulin; (6) inducing differentiation from the precursor cells to insulin-producing cells by using a DMEM/F12 medium containing 5-10mM of glucose and 10mM of nicotinamide; and (7) inducing insulin-producing cell clusters by using a DMEM/F12 medium containing 5-10mM of glucose and 10mM of nicotinamide.

Description

A medium composition for human embryonic stem cells containing low concentration of glucose and a method for differentiating human embryonic stem cells into insulin-producing cells or cell masses using the same, and insulin-derived cells or cell masses derived therefrom human embryonic stem cells, differentiation method of human embryonic stem cells into insulin-producing cells or cell clusters using specifically, and insulin-producing cells or cell clusters differentiated thereby}

1 is a micrograph of a colony of human embryonic stem cells growing in 7.8 mM glucose medium;

2 is a graph comparing growth rates of human embryonic stem cells grown in 7.8 mM glucose medium and 17.5 mM glucose medium;

Figure 3 is a micrograph of a cystic embryoid body produced in 7.8mM glucose medium during differentiation of human embryonic stem cells into insulin producing cells according to the present invention;

4 is a micrograph of an embryoid body obtained when differentiation was induced in the same manner as in FIG. 3 using high glucose medium with a glucose concentration of 17.5 mM;

FIG. 5 is a micrograph showing the proliferation of precursors of insulin-producing cells by betacellulin used in the present invention while killing neural precursors in the network;

FIG. 6 is a micrograph in which neuronal precursors networked by fibroblast growth factor (FGF-2) grow vigorously compared to betacellulin used in the present invention; FIG.

Figure 7 is a gel electrophoresis picture of the RT-PCR product to determine the degree of differentiation of differentiation-induced cells in 7.8mM glucose medium during the differentiation of human embryonic stem cells into insulin producing cells according to the present invention;

FIG. 8 is a micrograph confirming the expression of C-peptide, a byproduct of insulin biosynthesis, by immunocytochemical analysis for insulin-producing cell masses induced by differentiation in 7.8 mM glucose medium according to the present invention; FIG.

9 is a photomicrograph confirming the expression of Pdx-1 as a transcription factor, which is one of the characteristics of insulin producing cells, against the differentiation-producing insulin cell mass induced in 7.8 mM glucose medium according to the present invention, by immunocytochemistry. ;

10 is a flow cytometry (fluorescence-activated cell sorting; FACS) to measure the ratio of cells expressing C-peptide to differentiation-induced cell mass induced in 7.8 mM glucose medium according to the present invention. The result is;

FIG. 11 is a flow chart for measuring the ratio of cells expressing C-peptide to the insulin-producing cell mass obtained when differentiation was induced by the same method as in FIG. 10 using high glucose medium having a glucose concentration of 17.5 mM. The result of performing flow cytometry; And

12 is a graph comparing the content of C-peptides by measuring the content of C-peptides for differentiation-induced insulin-producing cell masses in 7.8 mM glucose medium and 17.5 mM glucose medium, respectively.

<Technology field>

The present invention provides a medium composition for human embryonic stem cells, characterized in that the glucose content in the medium for human embryonic stem cells is selectively reduced. In particular, the present invention provides a medium composition based on containing low concentrations of glucose and designed to induce each step of differentiation in order to produce insulin producing cells or cell masses from human embryonic stem cells.

In addition, the present invention relates to a method for inducing differentiation of human embryonic stem cells from insulin embryonic stem cells into insulin producing cells or cell masses, and the insulin producing cells or cell masses induced thereby.

Conventional Technology

Diabetes Mellitus is a disease that causes various sudden and chronic complications, leading to various illnesses and disorders in individual patients, leading to premature incompetence and premature death. It is increasing the social burden.

According to the WHO's 1997 World Health Report, the number of people with diabetes will surge to about 300 million by 2025, especially in developing countries such as Asia and Africa, resulting in a diabetes epidemic In the domestic epidemiological studies, the prevalence of diabetes among Koreans aged 30 and over is estimated to be 8-9%.

Diabetes treatment is mainly dependent on insulin therapy, oral hypoglycemic agents, insulin secretagogues, insulin sensorizers, etc., and in combination with diet and exercise therapy in principle, but it is impossible to cure in modern medicine Judging and thought to be the ultimate cure, it is thought to be a transplantation of pancreatic islets, but this implies a problem with the absolute shortage of donors and the use of persistent immunosuppressants. .

Active research on embryonic stem cells has begun to raise expectations as a source of cells that can overcome various problems of cell replacement therapy based on pluripotency. In particular, the differentiation studies from these cells into insulin-producing cells with the function of pancreatic beta cells have been carried out by Lumelsky et al. (Science 292: 1389, 2001) by differentiating mouse embryonic stem cells into insulin secretory structures similar to pancreatic islet cells. Hori et al. (PNAS 99: 16105, 2002) reported that insulin-producing cells derived from mouse embryonic stem cells obtained by introducing a PI-3 kinase inhibitor in the method described above were induced by diabetes in streptozotocin-induced mice. In the model, hyperglycemia was regulated and normalized.

In 1998, Thomson et al established human embryonic stem cells from human blastocysts, and efforts have been actively made to differentiate them into human insulin-producing cells.However, human embryonic stem cells maintain their own characteristics and maintain adult characteristics. It is now known that the signaling system required to differentiate into cells is quite different from mouse embryonic stem cells. In an attempt to obtain insulin-producing cells from human embryonic stem cells, Assady et al. (Diabetes 50: 1691, 2001) described immunocytochemical and enzyme-linked immunosorbent assays during spontaneous differentiation of these cells. The production of insulin-producing cells was reported by the same group using differentiation methods such as Lumelsky (Stem Cells 22: 265, 2004). Esau's limit remained.

Moreover, rather than the intracellular biosynthesis of insulin, which has been observed by immunocytochemical staining in many papers up to now, the insulin present in the culture medium is taken up by the cells and then stimulated by other than high glucose. It is released to the medium again, in particular they have been claimed to be unhealthy cells undergoing apoptosis (Sipione et al., Diabetologia 47: 499, 2004; Hansson et al., Diabetes 53: 2603, 2004; Rajagopal et al. Science 299: 363, 2003). As these claims have been accepted by many researchers, significant modifications have to be made to the method of obtaining insulin-producing cells presented in previously published papers or patents, and research is underway to explore new differentiation methods and identification processes. . In particular, for clinical application of these cells, it is necessary to provide high-purity insulin-producing cells or cell clusters with normal functions, so the demand for efficient differentiation will continue to increase. . Based on this recent literature, the patented technology (10-2004-7008713, PCT / US2002 / 039089) owned by Xeron is technically weak. In other words, there is no evidence that intracellular biosynthesis of insulin observed by immunocytostaining in the final product obtained through this technique is demonstrated, and evidence of such function as insulin is released out of the cell by glucose stimulation outside the cell. Was not included. In addition, a number of differentiation factors that may affect differentiation are processed in the form of cocktails to induce differentiation, and analysis of the effect of specific differentiation factors is insufficient.

On the other hand, although there are patents related to the technology for differentiating insulin producing cells, such as US 7,029,915B2, most of them are studies to differentiate into insulin producing cells using adult stem cells, which uses embryonic stem cells having different developmental stages. There is a big difference between the contents of the invention and the conditions for inducing cell culture or differentiation.

Thus, in the present invention, in order to overcome the problems of the prior art, the differentiation factor and concentration and the use time of each differentiation step in detail to increase the efficiency, proved the functionality of the final product, in particular many researchers in this process The originality of the invention was secured by suggesting the importance of glucose concentration in the culture of human embryonic stem cells and differentiation medium therefrom.

Accordingly, the present invention is to provide a step-by-step medium composition for more efficiently inducing a process from culturing human embryonic stem cells to differentiation into insulin producing cells or cell mass therefrom.

Still another object of the present invention is to incubate human embryonic stem cells using the medium composition as well as a method of inducing differentiation into cells or cell masses having insulin production capacity therefrom and insulin-producing cells or cell masses induced thereby. To provide.

In order to achieve the object of the present invention, the present invention provides a medium for human embryonic stem cells, characterized in that the content of glucose is selectively reduced. The medium composition for human embryonic stem cells preferably contains 5-10 mM glucose, more preferably 7-8 mM glucose. In one preferred embodiment of the present invention, the medium composition for human embryonic stem cells is DMEM / F12 medium containing 5-10 mM, preferably 7.8 mM of glucose.

The medium composition for human embryonic stem cells according to the present invention can be used to 'culture' human embryonic stem cells in an undifferentiated state or to 'induce differentiation therefrom', in particular beta cells of the pancreas from human embryonic stem cells It may be usefully used to induce differentiation into insulin producing cells similar or identical to.

In one embodiment, the present invention further comprises a human embryo comprising 15% serum replacement (Serum Replacement, Life Techology, etc.) and 2 ng / mL FGF-2 in basal medium for human embryonic stem cells, which selectively reduced glucose content only. Provided is a medium composition effective for culturing stem cells. Preferably, the present invention is 15% serum replacement and 2ng / mL in the basal medium for human embryonic stem cells which selectively reduced only the content of glucose in the composition (DMEM / F12) with the same ratio of DMEM and F12 medium Provided is an effective medium composition for culturing human embryonic stem cells further comprising FGF-2.

In addition, in one preferred embodiment, the present invention, for example, differentiation of activin A, betacellulin, nicotine amide, etc. in each stage in a basic medium for human embryonic stem cells which selectively reduced only glucose content. Provided is a differentiation induction medium composition added with a factor.

In particular, the present invention provides a medium composition of each step from the culturing step of human embryonic stem cells to the final differentiation into cells or cell masses with insulin production capacity.

According to one preferred embodiment, the present invention specifically mixes DMEM (Dulbecco's modified Eagle's medium, Life Technologies, Inc.) and F12 (nutrition mixture medium, Life Technologies, Inc.) in the same ratio of mixed medium composition (DMEM / F12) Provided is a step-by-step medium composition comprising an effective factor for each step from culturing human embryonic stem cells to differentiation into insulin-producing cells or cell masses, using a composition in which only the amount of glucose is selectively reduced.

In a preferred embodiment, the basal medium in the media composition of the present invention selectively reduces the amount of glucose in the composition (DMEM / F12) in which DMEM and F12 medium are added in the same ratio, thereby selectively reducing the amount of glucose. Indicates that it contains 5-10 mM of glucose, preferably 7-8 mM of glucose, more preferably 7.8 mM of glucose. However, the preferred low concentration of glucose in the final suspension culture step to make the insulin-producing cells into the cell mass is 5 to 7mM, more preferably 5 to 5.5mM.

In one preferred embodiment of the present invention, specifically looking at the medium composition of each step, 1 mM L- glutamine (1% L-glutamine), 1% non-essential amino acid in the composition (DMEM / F12) in which DMEM and F12 medium is added in the same ratio (non-essential amino acid), 0.1 mM 2-mercaptoethanol, and the like, and a composition in which only the amount of glucose was selectively reduced was used as a base medium [example] DMEM / F12 + 5-10 mM glucose];

i) Human embryonic stem cell culture medium further comprising 15% serum replacement (Serum Replacement, Life Techology) and 2 ng / mL FGF-2 (e.g.) basal medium + 15% serum replacement + 2 ng / mL FGF -2];

ii) embryoid body formation differentiation inducing medium as excluding FGF-2 from the human embryonic stem cell culture medium [eg, basal medium + 15% serum substitute];

iii) endoderm differentiation inducing medium which further comprises high concentrations of activin A in the embryoid body forming medium to promote differentiation into endoderm cells [Example: basal medium + 15% serum substitute + activin A 50-100 ng / mL];

iv) Excluding serum substitutes in the embryoid formation medium, 0.5-2.0 mg / mL insulin, 0.25-1.0 mg / mL transferrin, 0.25-1.0 μg / mL sodium selenite, etc. Serum-free selection media added and used for the selection of cells initially differentiated into endoderm cells [eg basal medium + insulin + transferrin + selenium];

v) Insulin-producing cell precursor proliferation / differentiation induction medium in which betacellulin was introduced excluding serum replacement in embryoid body forming medium [eg, basal medium + betacellulin 5-20ng / mL];

vi) Insulin producing cell differentiation inducing medium including nicotine amide excluding serum replacement in embryoid body forming medium [eg, basal medium + nicotinamide 10 mM]; And,

vii) insulin-producing cell mass culture medium (eg, DMEM / F12 5-5.5mM glucose + nicotinamide 10 mM) such that the concentration of glucose in the insulin-producing cell-induced differentiation medium was further lowered to contain 5-5.5 mM glucose.

In each of the above steps, activin A is 50-100ng / mL, betacellulin is 5-20ng / mL, and nicotinamide is 10 mM.

According to the present invention, when a medium containing low concentration of glucose is used in the culture of human embryonic stem cells, human embryonic stem cells show some important characteristics. First, a faster growth characteristic is observed compared to when cultured in a medium containing high concentration of glucose, which corresponds to a growth rate of about 1.45 times compared to the population doubling time (FIG. 2). In addition, when differentiating human embryonic stem cells cultured in a low concentration of glucose medium into embryonic stages, more cystic EBs were produced compared to human embryonic stem cells cultured in a high concentration of glucose medium. By forming (see FIG. 3), it showed that differentiation is progressing faster. This demonstrates that medium containing low concentrations of glucose is beneficial for early differentiation of goblet formation from the culture of human embryonic stem cells.

In order to achieve another object of the present invention, the present invention provides a method for culturing human embryonic stem cells in undifferentiated state or inducing differentiation from the same using the medium composition for human embryonic stem cells described above. In particular, the present invention provides a method of inducing differentiation from human embryonic stem cells to insulin producing cells and cell masses using the medium composition for human embryonic stem cells described above.

In particular, the present invention provides a method of inducing differentiation from human embryonic stem cells to cells or cell masses having insulin production capacity, comprising one or more of the following steps.

1) culturing human embryonic stem cells using human embryonic stem cell culture medium (i)

2) Embryonic Stem Cells Using Embryonic Differentiation Induction Medium (ii) to Form Embryoid Body (EB)

3) differentiation into an endoderm cell from an embryoid body (EB) using an endoderm differentiation induction medium (iii) containing a high concentration of activin A

4) using only serum-free selection media (iv) to screen only the cells initially differentiated into endoderm cells or the like to survive.

5) Differentiation from endoderm cells to insulin producing cell precursor using betacellulin-induced insulin producing cell precursor proliferation / differentiation induction medium (v)

6) differentiating from insulin producing cell precursors to insulin producing cells using insulin producing cell differentiation inducing medium (vi), including nicotinamide, and

7) forming an insulin producing cell mass from the insulin producing cells by suspension culture in the insulin producing cell mass culture medium (vii) which further lowered the glucose concentration in the insulin producing cell differentiation inducing medium.

In one preferred embodiment, the present invention provides a method for inducing differentiation from human embryonic stem cells comprising all of the above steps 1) to 7) into cells or cell masses having insulin production capacity.

It is reported that activin A and betacellulin used in the present invention can differentiate pancreatic cells, which are supposed to be adult stem cells, into cells having the function of beta cells. Using stepwise from the embryonic step, embryoid cells can be differentiated into endoderm cells, and endoderm cells can be effectively differentiated into insulin producing cell precursors. In particular, beta-cellulin was used as a proliferation factor of the precursors, which showed that it can selectively induce the proliferation of endothelial cells, which are supposed to be precursors of beta cells, while blocking differentiation into neurons. This has been described in many publications (Li et al., Am J Physiol, 285: E577, 2003; Demeterco et al., J Clin Endocrinol Metab, 85: 3892, 2000) and in patents (10-2004-7008713, PCT / US2002 / 039089). Unlike the role of endoderm differentiation factor in the paper, the function of inducing selective proliferation of progenitor cells of insulin-producing cells was strongly observed in the present invention. In particular, it is believed that activin A and betacellulin may work together to achieve differentiation and proliferation. (See Figures 5 and 6)

 In the method of inducing differentiation according to the present invention, first, in the step of culturing human embryonic stem cells, the growth rate of the cultured cells is increased while maintaining inherent self-renewal and pluripotency, and then embryonic bodies In the process of forming the cystic embryoid body (cystic EB) formation rate can be relatively increased. In addition, it is possible to selectively differentiate from human embryonic stem cells into cells or cell masses having insulin production capacity, and as a result, the differentiation efficiency can be significantly improved.

Finally, the present invention relates to human embryonic stem cells and endoderm cells differentiated from human embryonic stem cells, precursors of insulin producing cells, insulin producing cells, and insulin producing cell masses cultured by the aforementioned method.

The present invention provides a embryoid body derived from the low glucose medium obtained in the differentiation step, endoderm cells induced by introducing activin A, precursor cells of insulin-producing cells induced by introducing betacellulose, and finally induced Provide insulin producing cells or cell masses.

After adapting human embryonic stem cells to a culture medium composition containing low concentration glucose, the expression of various proteins used as undifferentiated markers was examined. Embryonic trioderm (EB) formed from these cells was examined. Gene expression of proteins representing embryonic 3-germ layers) was analyzed using gene analysis (RT-PCR). As a result, even in the low glucose medium used in the present invention, it was confirmed that human embryonic stem cells have inherent self-renewal and pluripotency. In addition, cells grown in low glucose medium have a faster growth rate when compared to those in high glucose medium (which show about 1.45-fold proliferation when compared through the population doubling time), resulting in embryoid bodies. The formation rate of cystic EB was relatively high.

In addition, differentiation intermediates and final derivatives were analyzed through gene analysis (RT-PCR), insulin, a hormone that has a glycemic control function, beta cell-specific transcription factor Pdx-1, and beta cell-specific sugar transporter (glucose transporter). 2) As a result of testing the differentiation markers of pancreatic beta cells, it was confirmed that selective differentiation was induced.

In addition, pancreatic isotopes such as C-peptide, Pdx-1, glucagon, and somatostatin, which are by-products of insulin production, for final inducible products through immunocytochemistry. Expression of endocrine hormone (hormone) and major transcription factors secreted by islet) was tested in many cells, strong expression of C-peptide, Pdx-1 and somatostatin in some cells, but not glucagon Did.

In addition, as a result of testing the ratio of cells stained with the antibody to the C-peptide through flow cytometry, it was confirmed that it reached about 57.9%, and compared with the insulin production through the enzyme immunoassay method Insulin-producing cell masses induced by differentiation in glucose medium contained about 6.4-fold C-peptide compared to high glucose products.

Differentiation from human embryonic stem cells into insulin producing cells with higher insulin production capacity compared to previous techniques when using stepwise media compositions based on low concentration glucose culture media or using the differentiation method according to the invention according to the present invention Can be derived. These insulin producing cells could be used as a new source of diabetes treatment as a cell source to replace pancreatic beta cells. In addition, all cells in the embryological stage, including end-stage insulin producing cells or cell masses, as well as endoderm cells obtained in the middle of differentiation and precursors of insulin producing cells, may be useful for screening the safety and efficacy of the drug. Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, and the scope of the invention is not limited by these examples.

Example 1 Culture of Human Embryonic Stem Cells in Low Glucose Medium

SNUhES3 (Oh et al., Stem Cells, 23: 211, 2005), a human embryonic stem cell line, was mixed with DMEM (Dulbecco's modified Eagle's medium, Life Technologies) and F12 (nutrition mixture medium, Life Technologies). / F12) glucose concentration to 7.8mM, 15% serum replacement (Life Technologies), 2ng / mL FGF-2, 1mM L-glutamine, 1% non-essential amino acid, 0.1mM 2-mercaptoethanol Culture was added using the added medium as a basal medium. STO (ATCC # 1503) was used as a feeder cell.

 1 is a photograph of a colony of human embryonic stem cells growing in 7.8 mM glucose medium. As shown in FIG. 1, these cells express appropriate cell surface markers, such as SSEA-4, TRA-1-60, TRA-1-81, alkaline phosphatase, while maintaining an intrinsic morphology of embryonic stem cells. Was confirmed by immunocytochemical staining.

Figure 2 is a graph comparing the growth rate of human embryonic stem cells growing in 7.8mM glucose medium and 17.5mM glucose medium. Incubation at low concentrations allowed the cells to grow faster than at other concentrations, corresponding to a growth rate of about 1.45 fold in terms of population doubling time.

In addition, in order to confirm whether human embryonic stem cells cultured in low glucose medium have characteristics of pluripotency, a medium containing serum substitutes in culture medium (i) of human embryonic stem cells and excluding FGF-2 is used. By forming a embryoid body, and cultured for 21 days, it was confirmed by gene expression whether the embryonic 3-germ layer marker is expressed among the naturally differentiated cells. (Schuldiner et al., PNAS 97: 11307, 2000; Reubinoff et al., Nature Biotechnol, 18: 399, 2000; Bhattachary et al., Blood 103: 2956, 2004)

Figure 3 is a picture of the cyst embryoid body (cystic embryoid body) generated in 7.8mM glucose medium during the differentiation of human embryonic stem cells into insulin producing cells according to the present invention, Figure 4 is to increase the glucose concentration to 17.5mM It is a micrograph of the embryoid body obtained when inducing differentiation by the same method as FIG. 3 using high glucose medium. The use of the low glucose medium of the present invention showed more differentiation by forming more cystic type embryoid bodies (cystic EBs) compared to other concentration groups.

Example 2 Differentiation from Human Embryonic Stem Cells to Insulin Producing Cells or Cell Masses

As shown in Example 1, human embryonic stem cell cultures were cultured using human embryonic stem cell culture medium (i) (step 1), and embryonic bodies through suspension culture using embryoid body forming medium (ii) therefrom. (Step 2), the early differentiation of constituent cells of the embryoid body into endoderm cells was induced by using endoderm-induced differentiation medium (iii) containing a high concentration of activin A of 70 ng / mL (step). 3). It is attached to the culture dish to induce monolayer culture, in which 1.0 mg / mL insulin, 0.5 mg / mL transferrin, 0.5 μg / mL sodium selenite Serum-free selection media containing this (serum-free selection media) (iv) and incubated for 4 days to ensure that only the initial differentiated cells such as endoderm cells (step 4), then beta-cellulose (10ng / mL) Using the insulin-producing cell precursor proliferation / differentiation medium (v), the proliferation and differentiation of insulin-producing cell progenitor cells were simultaneously induced from cells of an initial differentiation state including endoderm cells (step 5, FIG. 5). And 6). Again, planar culture was continued in the insulin producing cell induced differentiation medium (vi) containing nicotinamide (10 mM) for about 2 days to allow differentiation into insulin producing cells (step 6), followed by glucose concentration in the composition of the medium. Suspension culture was continued for 5 days in an insulin-producing cell mass culture medium (vii) adjusted to 5 mM, insulin-producing cells in the form of clusters, ie, insulin clusters. The formation of was induced (step 7).

Experimental Example 1: Marker gene expression experiment in insulin producing cells

RT-PCR analysis was performed to confirm whether the insulin producing cells of Example 2 induced differentiation according to the differentiation method of the present invention express genes characteristic of the beta cells of the pancreas at the mRNA level. RNA was isolated using Qiagen's RNeasy kit, cDNA was prepared therefrom, and PCR was performed using respective primers and Taq polymerase shown in Table 1. The total reaction was done 35-40 cycles. As a result, it was confirmed that the main genes such as insulin, Pdx-1, glucose transporter-2 (Glut-2, glucose transporter-2) is expressed, and as the differentiation progresses, the label of undifferentiated state such as Oct-3 / 4 The factor was confirmed that the expression is reduced. Figure 7 is an electrophoresis picture of the RT-PCR product showing the expression of beta cell related factors for each differentiation stage. (1: human embryonic stem cell-culture day 4, 2: embryonic body-culture day 4, 3: embryonic body-cultured without activin A treatment day 11, 4: embryonic body-cultured with activin A treatment Day 11, 5: Screening process after adherence culture—Day 4, 6: Concentration of precursor cells—Day 4 Day 7: Suspension culture-5 mM glucose-culture 5)

name primer  ( primer ) Insulin (179 bp) forward  5'-TCA CAC CTG GTG GAA GCT C-3 ' backward  5'-ACA ATG CCA CGC TTC TGC-3 ' Oct3 / 4 (219 bp) forward  5'-GAG AAC AAT GAG AAC CTT CAG GAG A-3 ' backward  5'-TTC TGG CGC CGG TTA CAG AAC CA-3 ' Pdx-1 (218 bp) forward  5'-GGA TGA AGT CTA CCA AAG CTC ACG C-3 ' backward  5'-CCA GAT CTT GAT GTG TCT CTC GGT C-3 ' Glut-2 (909 bp) forward  5'-GCA GCT GCT CAA CTA ATC AC-3 ' backward  5'-TCA GCA GCA CAA GTC CCA CT-3 ' β-actin (396 bp) forward  5'-TGG CAC CAC ACC TTC TAC AAT GAG C-3 ' backward  5'-GCA CAG CTT CTC CTT AAT GTC ACG C-3 '

Experimental Example 2: Immunocytochemical Study of Insulin Producing Cells

In order to examine whether the insulin-producing cells obtained in the present invention produce normal hormones or related transcription factors at the protein level, Example 2 fixed with 4% paraformaldehyde The final product was treated with a primary antibody specific to each protein and washed, and then treated with a secondary antibody to which the fluorescent substance was attached. As a result, the final differentiated insulin producing cells produced C-peptide, a by-product of insulin biosynthesis (FIG. 8), and confirmed that Pdx-1, a transcription factor specific for beta cells of the pancreas, was expressed in the nucleus (FIG. 9).

Experimental Example 3: Analysis of the proportion of insulin producing cells by flow cytometry

In order to compare the effect of the low glucose medium used in the present invention with the existing high glucose medium, two conditions (same as the conditions of Example 2 vs. Example 2, except that the concentration of glucose in all media was adjusted to 17.5 mM) The number of insulin-producing cells expressing C-peptide among the products of the final stage differentiated in the conditions) was compared. This experiment was performed using Becton-Dickinson's FACSCalibur, and analyzed using CELLQuest software. As a result, in the cell group induced differentiation in the low glucose medium of Example 2, the ratio of expressing C-peptide was about 57.9% (FIG. 10), and the ratio obtained in the medium of high glucose (17.5 mM) was about 10.4% (FIG. 11). ), The yield was about 5.6 times higher in low glucose medium.

Experimental Example 4: Insulin production comparison experiment of insulin producing cells

Example 2 To compare the amount of insulin biosynthesized in the differentiation-induced insulin producing cell masses in each of the low glucose medium and the high glucose (17.5 mM) medium, enzyme immunoassay (ELISA) Measured through. As a result, the insulin-producing cell mass induced by differentiation in low glucose medium contained about 6.4 times C-peptide compared to the product of high glucose. (See Figure 12. 598.30 ± 61.88 and 93.22 ± 7.53ng of C-peptide per milligram protein)

Experimental Example  5: Insulin production capacity test of insulin producing cells

In order to measure glucose responsiveness of the resulting insulin-producing cell mass, the insulin-producing cell mass is placed in a buffer containing glucose of various concentrations and released into the buffer in response to the concentration of glucose in the extracellular fluid. The amount of released C-peptide was measured by enzyme-linked immunosorbent assay (ELISA), resulting in a 13.4% increase in the amount of insulin secreted in response to stimulation of the high glucose environment (35 mM) compared to the low glucose environment (5 mM). Could confirm. In addition, when insulinX, an agonist known to control insulin secretion, is treated, insulin secretion increases, and consequently, insulin secretion is decreased by nifedipine, an antagonist that inhibits insulin secretion. By confirming the trend, it was confirmed that the obtained insulin-producing cells have a characteristic of reacting normally with accelerators and antagonists.

As described above, the insulin having higher insulin production capacity from human embryonic stem cells compared to the conventional technique when using the stepwise medium composition based on the low concentration glucose culture medium according to the present invention or using the differentiation method according to the present invention. Differentiation into production cells can be induced. These insulin producing cells could be used as a new source of diabetes treatment as a cell source to replace pancreatic beta cells. In addition, all cells in the embryological stage, including end-stage insulin producing cells or cell masses, as well as endoderm cells obtained in the middle of differentiation and precursors of insulin producing cells, may be useful for screening the safety and efficacy of the drug. Can be.

<110> DONG-A PHARM.CO., LTD.          MOON, SHIN YONG          OH, SUN KYUNG <120> Media compostions containing low concentrations of glucose useful          for human embryonic stem cells, differentiation method of human          embryonic stem cells into insulin-producing cells or cell          clusters using according, and insulin-producing cells or cell          clusters differentiated thereby <130> 06p-207 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> insulin forward primer <400> 1 tcacacctgg tggaagctc 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> insulin backward primer <400> 2 acaatgccac gcttctgc 18 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oct3 / 4 forward primer <400> 3 gagaacaatg agaaccttca ggaga 25 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Oct 3/4 backward primer <400> 4 ttctggcgcc ggttacagaa cca 23 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Pdx-1 forward primer <400> 5 ggatgaagtc taccaaagct cacgc 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Pdx-1 backward primer <400> 6 ccagatcttg atgtgtctct cggtc 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Glut-2 forward primer <400> 7 gcagctgctc aactaatcac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Glut-2 backward primer <400> 8 tcagcagcac aagtcccact 20 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 9 tggcaccaca ccttctacaa tgagc 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> beta-actin backward primer <400> 10 gcacagcttc tccttaatgt cacgc 25  

Claims (28)

In the medium for human embryonic stem cells, the medium composition for human embryonic stem cells, characterized in that the content of glucose is selectively reduced The medium composition for human embryonic stem cells according to claim 1, which contains 5-10 mM glucose. The medium composition for human embryonic stem cells according to claim 2, which contains 7-8 mM glucose. The medium composition of claim 1, wherein the medium composition is DMEM / F12 medium containing 5-10 mM glucose. The medium composition for human embryonic stem cells according to claim 1, wherein the medium composition is used to culture human embryonic stem cells in undifferentiated state or to induce differentiation therefrom. [Claim 6] The medium composition for human embryonic stem cells according to claim 5, wherein the medium composition is used to induce differentiation from human embryonic stem cells to insulin producing cells. It contains glucose, which has been selectively reduced to 5-10 mM, further contains 15% serum replacement and 2 ng / mL FGF-2, and is used to culture undifferentiated human embryonic stem cells. Human embryonic stem cell culture medium composition Human embryonic stem cell differentiation induction medium characterized by containing glucose which has been selectively reduced to 5-10 mM, further comprising 15% serum replacement and used to form embryoid bodies from human embryonic stem cells. Composition Contains glucose selectively reduced to 5-10 mM, further contains 15% serum replacement and 50-100 ng / mL activin A and differentiated from human embryonic stem cells Human embryonic stem cell differentiation induction medium composition, characterized in that it is used to induce differentiation from embryoid body to endoderm Contains glucose with a reduced content of 5-10 mM, 0.5-2.0 mg / mL insulin, 0.25-1.0 mg / mL transferrin, 0.25-1.0 μg / mL sodium selenite A serum-free medium further comprising a human embryonic stem cell differentiation induction medium composition, characterized in that it is used for the selection of early differentiated cells from human embryonic stem cells to endoderm cells From human embryonic stem cells, early differentiated endoderm cells to precursor cells of insulin-bearing cells, containing glucose, optionally reduced to 5-10 mM, further containing 5-20 ng / mL betacellulin Human embryonic stem cell differentiation inducing medium composition, characterized in that it is used to induce differentiation and proliferation It contains glucose which has been selectively reduced to 5-10 mM, further contains 10 mM nicotinamide, and is used to induce differentiation from insulin-producing cells differentiated from human embryonic stem cells from precursor cells to insulin producing cells. Human embryonic stem cell differentiation induction medium composition Characterized in that it is used to induce differentiation from insulin producing cells differentiated from insulin producing cells in human embryonic stem cells containing glucose which has been selectively reduced to 5-5.5 mM and further comprising 10 mM nicotinamide. Human embryonic stem cell differentiation induction medium composition A method for culturing human embryonic stem cells in an undifferentiated state or inducing differentiation therefrom using the medium composition for human embryonic stem cells according to any one of claims 1 to 5. A method of inducing differentiation from human embryonic stem cells to insulin producing cells and cell masses using the medium composition for human embryonic stem cells according to any one of claims 1 to 6. Method for culturing and maintaining undifferentiated human embryonic stem cells using the medium composition according to claim 7 A method for inducing differentiation from undifferentiated human embryonic stem cells into embryoid bodies using the medium composition according to claim 8 A method of inducing differentiation from embryonic bodies differentiated from human embryonic stem cells to endoderm cells using the medium composition according to claim 9 A method for selecting early differentiated cells from human embryonic stem cells into endoderm cells using the medium composition according to claim 10 A method for inducing differentiation and proliferation from early embryonic endoderm cells from human embryonic stem cells into precursors of insulin producing cells using the medium composition according to claim 11 A method of inducing differentiation from precursors of insulin producing cells differentiated from human embryonic stem cells to insulin producing cells using the medium composition according to claim 12 A method of inducing the formation of insulin producing cell mass from insulin producing cells differentiated from human embryonic stem cells using the medium composition according to claim 13 and using a suspension culture method. 1) Undifferentiated state using human embryonic stem cell culture medium composition containing glucose which has been selectively reduced to 5-10 mM and containing 15% serum replacement and 2ng / mL FGF-2. Culturing human embryonic stem cells; 2) the human embryonic stem through the method of suspension culture, using a human embryonic stem cell differentiation medium composition containing glucose, which has been selectively reduced to 5-10 mM, and containing 15% of serum replacement. Forming embryoid bodies from cells; 3) Human embryonic stem cell differentiation medium composition containing glucose, which has been selectively reduced to 5-10 mM, and containing 50% to 100% of serum replacement and activin A (activin A). Using to induce differentiation from the goblet cells to endoderm cells; 4) 0.5-2.0 mg / mL insulin, 0.25-1.0 mg / mL transferrin, 0.25-1.0 μg / mL sodium selenide, containing glucose, optionally reduced to 5-10 mM selecting initially differentiated cells into the endoderm cells using a serum-free human embryonic stem cell differentiation medium composition containing selenite) 5) Initially differentiated cells into the endoderm cells using a human embryonic stem cell differentiation medium composition containing glucose, the content of which is selectively reduced to 5-10 mM, and containing 5-20 ng / mL of betacellulin. Inducing differentiation and proliferation of insulin producing cells into progenitor cells 6) Differentiation of the insulin-producing cells from the precursor cells to the insulin-producing cells using a human embryonic stem cell differentiation medium composition containing glucose, which has been selectively reduced to 5-10 mM, and containing 10 mM nicotinamide Inducing; And 7) Induce the formation of insulin producing cell mass from insulin producing cells differentiated from human embryonic stem cells using a medium composition containing glucose selectively reduced to 5-5.5 mM and containing 10 mM nicotinamide Steps to Method for inducing differentiation from human embryonic stem cells to cells or cell masses having insulin producing ability, comprising Human embryonic stem cells cultured by the method of claim 23 Endoderm cells differentiated by the method of claim 23 Insulin-producing cell precursor cells differentiated by the method of claim 23 Insulin-producing cells differentiated by the method of claim 23 Insulin producing cell mass formed by the method of claim 23

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