KR101792865B1 - Method for inducing differentiation of pluripotent stem cell into insulin producing cells using co-culture with mature islet cells - Google Patents

Abstract

INDUSTRIAL APPLICABILITY The composition for inducing differentiation and the method for co-culturing pancreatic islets according to the present invention can effectively induce the differentiation of pluripotent stem cells into insulin-secreting cells, facilitating the production of insulin-secreting cells, It can be used to solve the shortage of islet cell supply and effectively treat diabetes.

Description

[TECHNICAL FIELD] The present invention relates to a method for inducing differentiation of pluripotent stem cells into insulin-producing cells using an islet cell co-

The present invention relates to a composition for inducing differentiation of pluripotent stem cells into insulin-secreting cells, a method for inducing differentiation of pluripotent stem cells into insulin-secreting cells by co-culturing with an islet cell, And a cell therapy agent for treating diabetes comprising insulin secreting cells.

Diabetes mellitus is a disease characterized by chronic hyperglycemia as the most important feature. It prolongs wound healing caused by microvascular injury, neurological disease, renal failure, heart disease, retinal disease, and so on, which increases social and economic burden. There is no cure to cure diabetes until now, and insulin-enriched therapy which repeated insulin insufficiently in diabetic patients has been used in clinical practice for a long time, but it is inconvenient to be repeated several times a day, It is a reality that diabetes complication can not be prevented.

Diabetes mellitus is divided into two major types (type 1 and type 2) depending on the cause. In the case of type 1 diabetes, it occurs because the immune cells destroy insulin-producing cells and diabetes mellitus can not regulate blood glucose due to lack of insulin-producing cells. The type 2 diabetes is caused by the fact that the organs / tissues / cells in the body have resistance to various insulin due to various causes. The insulin-producing cells due to hyperglycemia are inferior in function and disappearance, Resulting in diabetes mellitus with hyperglycemia and subsequent complications.

Islet transplantation has been recognized as a successful strategy for the treatment of diabetes. However, due to the lack of pancreatic islets, islet transplantation for the treatment of diabetes has been limited in its use. To overcome this, research is underway to find candidates that can differentiate into insulin-producing cells (IPCs). Since pluripotent stem cells (PSCs) have self-renewal ability and differentiation potential into various cells, they can become a cell therapy agent capable of differentiating into IPC. Recent studies have shown that PSC can be differentiated into IPC in a manner similar to the differentiation stages of embryonic developmental stages. However, differentiated IPCs have many problems due to low insulin secretion, differentiated cell diversity, and teratoma forming ability. Therefore, a method for inducing differentiation into an efficient insulin-secreting cell has been actively studied.

Unlike stem cells or progenitor cells, pancreatic beta cells have limited proliferative capacity under normal conditions. However, it has been reported that beta cells increase in certain conditions such as pregnancy, obesity, and pancreatectomy, and external stimuli such as hormones and growth factors seem to be necessary for the growth and differentiation of existing beta cells.

Therefore, the present inventors have made efforts to develop a new cell therapy agent for treating diabetes. As a result, they have been able to effectively induce the differentiation of pluripotent stem cells into insulin secretory cells by co-culturing with mature islet cells, And thus the present invention has been completed.

It is an object of the present invention to provide a composition for inducing differentiation from pluripotent stem cells into insulin secreting cells, which comprises a culture medium of islet cells.

It is still another object of the present invention to provide a medium composition and a kit for inducing differentiation of pluripotent stem cells into insulin-secreting cells, which comprises an islet cell culture.

Another object of the present invention is to provide a method for inducing differentiation of pluripotent stem cells into insulin-secreting cells by co-culturing with an islet cell.

Still another object of the present invention is to provide an insulin secretory cell produced by the above method, a cell therapy agent for treating diabetes comprising the same, and a method for producing the same.

In order to achieve the above object, the present invention provides a composition for inducing differentiation of pluripotent stem cells into insulin-secreting cells, which comprises a culture medium of islet cells.

The present invention also provides a kit and a culture medium for inducing differentiation of pluripotent stem cells into insulin-secreting cells, which comprises an islet cell culture.

In addition, the present invention provides a method for inducing differentiation of pluripotent stem cells into insulin-secreting cells by co-culturing with an islet cell.

The present invention also provides an insulin-secreting cell produced by the above method, a cell therapy agent for treating diabetes comprising the same, and a method for producing the same.

INDUSTRIAL APPLICABILITY The composition for inducing differentiation and the method for co-culturing pancreatic islets according to the present invention can effectively induce the differentiation of pluripotent stem cells into insulin-secreting cells, facilitating the production of insulin-secreting cells, It can be used to solve the shortage of islet cell supply and effectively treat diabetes.

FIG. 1 is a diagram showing gene and protein expression analysis of definitive endoderm and pancreatic progenitor cells induced differentiation from mouse embryonic stem cells.
FIG. 2 shows that cells induced to differentiate from pancreatic endoderm cells by co-culturing with mature islet cells express beta cell factor.
FIG. 3 shows that cells induced to differentiation by mature islet cells and co-culture were induced into functional insulin-secreting cells.
FIG. 4 is a graph showing the expression patterns of Newport Green and proinsulin of insulin-secreting cells differentiated from mouse embryonic stem cells using a flow cytometer.
FIG. 5 is a graph showing that beta cell factor expression was improved from human de-differentiating pluripotent stem cells differentiated by co-culturing with mature islet cells.
FIG. 6 is a graph showing that cells transfected with mature islet cells and differentiated by co-culture were differentiated into cells expressing insulin and reacting with glucose when they were transplanted into diabetic mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for inducing the differentiation of pluripotent stem cells into insulin-secreting cells, comprising a culture medium of islet cells.

In the present invention, "islet cell culture fluid" is obtained by culturing mature islet cells.

As used herein, the term "stem cell" refers to a cell that has the ability to self-replicate and has the ability to differentiate into two or more different types of cells.

In the present invention, "pluripotent stem cell" refers to a pluripotent stem cell capable of differentiating into three germ layers constituting the living body, that is, endoderm, mesoderm and ectoderm . In the present invention, the pluripotent stem cells include embryonic stem cells and dedifferentiated pluripotent stem cells.

The term "embryonic stem cell" in the present invention refers to a cell cultured separately from the inner cell mass of a blastocyst, which is an early stage of development, and has pluripotency. In the present invention, "dedifferentiated pluripotent stem cell" refers to a cell that has induced pluripotency by artificially performing a reprogramming process (reprogramming) on adult cells that have already undergone differentiation.

In the present invention, "differentiation" refers to a phenomenon in which a structure or function is specialized with each other while a cell is growing and growing, that is, a cell or tissue of an organism is changed in shape or function to perform a task given to each.

"Progenitor cells" in the present invention refers to cells that have been differentiated into specific types of cells or are allowed to form certain types of tissues.

The present invention also provides a medium composition for inducing differentiation of pluripotent stem cells into insulin-secreting cells, which comprises a culture medium of islet cells.

In addition, the present invention provides a kit for inducing differentiation from pluripotent stem cells into insulin secreting cells, comprising the above-mentioned culture medium composition.

 The term "culture medium" in the present invention means a medium which enables the induction and survival of differentiation into insulin producing cells in vitro, and includes all conventional culture media used in the art suitable for cell culture . Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culture is preferably a cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source and a trace element component. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, αMEM Glasgow's Minimal Essential Medium, and Iscove's Modified Dulbecco's Medium. The medium may include antibiotics such as penicillin, streptomycin, gentamicin and the like.

The medium may further include at least one insulin secretory cell differentiation inducing substance known in the art, in addition to the islet cell culture fluid.

Also, the present invention provides a method for producing a pluripotent stem cell, comprising co-culturing an islet cell and an allogenic stem cell;

And a method for inducing differentiation from pluripotent stem cells into insulin-secreting cells.

Such culture includes suspension culture, adhesion culture and the like, and is not limited to the culture system.

In addition, the present invention provides a cell therapy agent for treating diabetes comprising insulin-producing cells produced by the above method.

The present invention also provides a method for producing a cell therapy agent for treating diabetes, comprising the step of producing an insulin producing cell by the above method.

The diabetes includes type 1 diabetes or type 2 diabetes.

The term "cell therapeutic agent" in the present invention is a pharmaceutical (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues prepared by isolation, cultivation and specific works from animals, Refers to a drug used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as living, homologous, or xenogeneic cells that multiply, select, or otherwise alter the biological characteristics of a cell.

"Administration" in the present invention means providing the subject invention with a composition of the invention in any suitable manner.

The preferred dosage of the cell therapy agent of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several doses, and the dosage is not limited in any way to the scope of the present invention.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

Experimental Example 1. Culture and Differentiation Conditions of Mouse Embryonic Stem Cells

Mouse embryonic stem cells (mES) were cultured on mouse embryonic fibroblasts treated with mitomycin. At this time, the medium was supplemented with 15% FBS, 1% penicillin-streptomycin, 100 μM β-mercaptoethanol, 100 μM nonessential amino acid, 1000 units / ml leukemia inhibition DMEM containing factor (LIF, leukemia inhibitory factor) was used. Mouse embryonic stem cells were subcultured at a ratio of 1: 5 to 1: 10 every 2 to 3 days. To remove mouse embryonic fibroblasts, the cells were cultured in 0.1% gelatin before the differentiation experiment, And transferred to induce differentiation. In step 1, the cells were cultured for 3 days in an X-vivo 10 medium containing 0.1% BSA, 50 ng / ml activin A and 1 uM LY294002 for 3 days. In step 2, 0.5% BSA, 1% insulin- The cells were cultured for 2 to 3 days in a differentiation medium, F12 / IMDM medium containing transferrin-selenium (ITS, insulin-transferrin-selenium) and 2 uM retinoic acid (RA). To this, 300 nM indolactam V (ILV, indolactam V) and 50 ng / ml FGF10 were added, and further differentiation was induced for 2-3 days. In the third step, 0.5% BSA. The cells were cultured in DMEM medium containing 1% ITS, 10 ng / ml bFGF and 20 ng / ml EGF for 3 days. In the final step, DMEM / F12 medium containing 10 ng / ml bFGF, 10 mM nicotinamide and exendin- Lt; / RTI > for 3-5 days. Differentiation by co-cultivation with mature islet cells was performed using cells that had been differentiated in three stages. The islet cell co-culture groups were as follows: 1) an experimental group that induced differentiation in differentiation medium (DMEM / F12 containing bFGF, nicotinamide, and exendin-4), 2) (3) Islet cells (Islet cells were separated from the differentiated cells by placing them in culture membrane insert wells) were cultured for 3-4 days Were cultured for induction of differentiation. In order to remove insulin from the medium of each experimental group, the cells were further cultured for 2 days in DM (except for pancreatic islets).

EXPERIMENTAL EXAMPLE 2 Human degenerative pluripotent stem cell culture and differentiation conditions

Human induced pluripotent stem cells (hiPS) were treated with definitive endoderm (DE, stage 1), primitive gut tube (PG, stage 2), posterior foregut (PF, stage 3), pancreatic endoderm and endocrine precursors (PE, stage 4), and insulin-producing cells (IPC, stage 5). In the first-stage restricted endoderm cells, undifferentiated hiPS was applied to a plate coated with a matrigel (1:40 dilution), and induction of differentiation was carried out in a cold state of 70 to 80%. Differentiation was induced in RPMI 1640 medium containing 100 ng / ml Activin A and 25 ng / ml Wnt3a for 1 day and induced to differentiate into RPMI 1640 containing 0.2% FBS and 100 ng / ml Activin A for the next 2 days Respectively. Two-stage primitive intestinal cells were differentiated in RPMI 1640 medium supplemented with 50 ng / ml FGF10, 250 nM KAAD-Cyclopamine, 2 uM RA, and 2% FBS for 3 days. After the third step, differentiation of the full-length cells was induced for 3-4 days in DMEM medium supplemented with 100 ng / ml noggin, 250 nM KAAD-Cyclopamine, 2 uM RA, and 1% B27. Pancreatic endoderm and endoderm progenitor cells were cultured for 3 days in DMEM medium supplemented with 1% B27 and exendin-4. The final stage insulin secreting cells were 50 ng / ml IGF1, 50 ng / ml HGF , 50 ng / ml exendin-4, 10 mM nicotinamide, and 1% B27 in DMEM / F12 medium for 3 days or more. Experiments were carried out in the same conditions as those for mouse embryonic stem cells.

Experimental Example 3. Islet Cell Isolation

Mouse islet cells were isolated by injecting 0.8 mg / kg collagenase P into total bile ducts, and only the islet cells were purified using a Ficoll density gradient. Pancreatic islet cells were cultured in RPMI 1640 containing 10% FBS and 1% penicillin / streptomycin, and only the islet cells were pipetted using a dissecting microscope. As shown in Figure 2A, in order to isolate islet cells from the differentiated cells in the co-culture experiment, the islet cells were cultured in 12 well plates (100 islets / well) with culture membrane inserts (Millipore, MA, USA) and cultured in the differentiation medium (DM) used in the final stage. Islet cells were cultured in DMEM / F12 medium (differentiation factor To examine the induction of differentiation by cell-cell contact, isolated islet cells were suspended in an enzyme-free dissociation buffer (enzyme-free dissociation buffer) buffer) and then fixed with Cyto / Perm and stored in a liquid nitrogen tank until use.

Experimental Example 4. Immunostaining

Differentiation-induced cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed 3 times with 1xPBS, and then blocked with 5% normal serum containing 0.2% Triton X-100 for 1 hour. Cells were then incubated with primary antibody overnight at 4 ° C. The primary antibodies used were goat anti-SOX17, goat anti-FOXA2, rabbit anti-PDX-1 or goat anti-PDX-1, rabbit anti-HNF- -6, rabbit anti-SOX9, goat anti-NKX6.1, rabbit anti-PAX6, guinea pig anti-insulin, rabbit anti-glucagon, and rabbit anti-C-peptide. The next day, cells were washed three times with PBS and incubated with secondary antibody for 1 hour at room temperature (Alexa-488-conjugated goat anti-rabbit or rabbit anti-goat, and Alexa-546 or -568-conjugated donkey or rabbit anti-goat). Cell nuclei were stained with DAPI.

After the end of the experiment, the renal transplanted kidneys in the kidney subcutaneous membrane were harvested, fixed in 4% paraformaldehyde, washed with PBS, and frozen in 30% sucrose. Tissues were cut at 10 μm to perform tissue staining.

Experimental Example 5. DTZ staining

DTZ (dithizone) was dissolved in DMSO to a storage concentration of 10 mg / ml. DTZ was diluted in HBSS at a ratio of 1: 2 and stained at 37 ° C for 30 minutes.

Experimental Example 6. Dyeing on Newport Green (NG)

Newport Green is a fluorescent dye used to detect zinc rich in beta cells. Finally, the differentiated cells were washed with 1xPBS, treated with 1 uM NG and 1 ul / ml pluronic F127, and cultured in a 37 ° C incubator for 30 minutes. The stained cells were washed with 1xPBS supplemented with 5% FBS, followed by flow cytometry (FACS ananalysis) and sorting.

Experimental Example 7. Flow cytometric analysis (FACS ananalysis) and cell sorting

The final differentiated cells were separated into trypsin / EDTA for flow cytometry analysis and FACS analysis was performed. Cells ⁽⁵ × 10 5) were fixed and transmitted in Cytofix / Cytoperm solution kit. After staining for anti-proinsulin and isotype controls, data were obtained and analyzed using CellQuestPro software. NG-stained cells were separated into NG-positive cells and negative cells using FACS AriaIII, and cells were collected for RNA analysis.

Experimental Example 8. RT-PCR and Real-Time Quantitative PCR Analysis

RNA was extracted from mouse embryonic stem cells, human de-differentiating pluripotent stem cells, and staged-induced differentiation cells using TRIzol reagent. 2 ug of RNA was reverse transcribed with cDNA using SuperScrip II reverse transcriptase. RT-PCR amplified cDNA using AccuPower PCR premix. Q-PCR was performed with SYBR Premix EX Taq.

Experimental Example 9. Measurement of insulin secretion by sugar stimulation

In order to determine whether the final differentiation-induced cells had insulin secretion ability for the sugar stimulation, the stimulation was given at a concentration of 60 mg / dL and 300 mg / dL. Cells were washed with PBS and incubated with Krebs-Ringer bicarbonate buffer for 90 minutes at 37 ° C. Stimulated with 60 mg / dL glucose for 1 hour, then with 300 mg / dL glucose Stimulation was given for 1 hour. Insulin levels were measured at each concentration using the Rat / Mouse Insulin ELISA Kit and the C-peptide ELISA Kit. Insulin and C-peptide concentrations were calibrated to the protein of each experimental group.

Experimental Example 10. Glucose-tolerance test under the renal capsule.

To prepare a diabetic animal model before cell transplantation, 180 mg / kg of streptozotocin (STZ) was injected intraperitoneally into BALB / c nude mice at 10-12 weeks of age and mice having blood glucose level of 20 mmol / Lt; / RTI > Differentiation-induced cells were collected in an E-tube, transferred to a PE50 tube using a Hamiton syringe, and then transplanted under a mouse kidney capsule. The diabetic animal model was divided into two groups, and the differentiation-inducing cells (2x10 ⁶ ) and the differentiation-induced cells (2x10 ⁶ ) were co-cultured with the islet cells in the normal differentiation medium. Blood glucose was measured twice a week, and on the 90th day, human C-peptide and glucose tolerance test were performed in mouse serum. In the glucose tolerance test, the mice were fasted for 20 hours, and 1 g / kg of glucose was injected into the abdominal cavity and blood glucose was measured with time (0, 15, 30, 45, 60, 90, 120 minutes).

Experimental Example 11. Statistical Analysis

Data are expressed as mean ± standard error of measurement. The differences between the groups were analyzed using the unpaired t-test and ANOVA, and considered as statistically significant at p-value <0.05.

Example 1. Optimal Differentiation Protocol Design Using Activin A, PI3K Inhibitor, Indolactam V, and FGF10

A co-culture system with mature islet cells was designed to improve the differentiation of mouse embryonic stem cells into insulin-secreting cells, and the results are shown in Fig.

As shown in FIG. 1A, differentiation was induced using differentiation inducing factors for each differentiation stage, and cells at each differentiation stage were identified using expression markers of the differentiation stage cells. Activin A and LY294002 were used to induce efficient endoderm in the first step. In Fig. 1B, SOX17 and FOXA2, which are endogenous endogenous expression markers, exhibited the highest expression at 50 ng / ml of actin A concentration compared to the other concentrations (10, 25 and 100 ng / ml) It was confirmed that the expression of beta cell expression factors (NeuroD, NGN3, PDX-1, NKX2.2, and insulin 1 and 2) was enhanced at ng / ml of actin A concentration. In step 2, pancreatic progenitor cells induced differentiation in the same manner as in Experimental Example 1. As shown in FIG. 1C, pancreatic progenitor cell expression markers HNF-1β and HNF-4α were highly expressed, and SOX17 was continuously expressed in HNF-1β-expressing cells. This suggests that the pancreatic progenitor cells are the endogenous origin of the endomembrane. In addition, most PDX-1 was expressed with other pancreatic progenitor cell expression markers (HNF-1β, HNF-4α, FOXA2, SOX9) (FIG. In the conventional expression protocol, PDX-1 was expressed in about 10% of cells, but after addition of indolactam V (ILV) and FGF10, 30 to 50% or more of PDX-1 positive cells could be observed. RT-PCR was performed to determine if ILV-induced cells were efficiently induced into more mature beta-cell differentiation at the differentiation stage. Cells treated with ILV and FGF10 expressed the beta cell expression markers (insulin 1, 2, NKX6.1, PDX-1, Glut2) more strongly than untreated cells. These results suggest that ILV and FGF10 promote the differentiation of pancreatic progenitor cells into beta cells.

Example 2. Enhancement of differentiation into insulin-secreting cells by co-culturing with mature islet cells

To evaluate the effect of mature islet cells on the induction of beta cell differentiation, adult pancreatic islet cells were isolated from mice to establish a co-culture system. Pancreatic endoderm cells (PE) express NGN3, NKX6.1, and NKX2.2, which are expression markers of PE, but not mature beta cell markers insulin 1,2, IAPP, Glut2, and glucagon After the confirmation, the effect of inducing the differentiation of pancreatic endoderm cells (PE) into insulin-secreting cells of the pancreatic islets was confirmed. The results are shown in Fig.

As shown in FIG. 2A, pancreatic endoderm cells (PE) induced differentiation from islet cells using an insert well. Pancreatic endoderm cells (PE) were divided into three groups and were divided into three groups: basic differentiation medium (DM) containing no mature islet cells (medium containing bFGF, exendin-4, nicotinamide) And cultured in differentiation medium (co-culture with pancreatic islet cells) and conditioned medium (CM) from pancreatic islets for 3-4 days. Fluorescence immuno staining was used to investigate the effect of differentiation by co-culture, and the expression pattern of C-peptide, insulin, and beta-cell important factors was measured. After 3 to 4 days of culture, additional cultivation was performed for 2 days in DM to remove insulin from the medium. In the immunostaining of Fig. 2A, it was confirmed that C-peptide and insulin were stained in the cytoplasm and PDX-1 was stained in the nucleus. These cells were also stained with DTZ. (Fig. 2B and C), PDX-1 and NKX6, which are important transcription factors of beta cells, were more frequently observed than those of DM cells (Fig. 2D). In addition, PDX-1 was co-expressed with C-peptide, insulin, and NKX6.1. These results suggest that the enhancement of the differentiation-induced cell differentiation is due to the factors secreted from the pancreatic islet cells, and that these factors can induce differentiation into beta cells.

Example 3. Differentiation into functional insulin-secreting cells by co-culturing with an islet cell

RT-PCR was performed to investigate the expression pattern of transcription factors in differentiation-inducing cells by co-culturing with an islet cell. The results are shown in Fig.

As shown in FIG. 3A, islet co-cultured cells and CM-differentiated cells were differentiated from DM-induced cells by insulin 1 and 2, PDX-1, IAPP, Glut2, Isl-1, glucagon, NKX6.1, NKX2 .2 was more strongly expressed. However, there was no change in the expression pattern of mRNA of somatostatin, pancreatic polypeptide, and ghrelin. In addition, as shown in FIG. 3B, Q-PCR showed similar results, and insulin 1, PDX-1, and IAPP expression were shown to increase 10-fold over the cells cultured in DM.

In addition, to examine whether the cells induced by different differentiation conditions regulate insulin secretion by the sugar, the differentiated cells were stimulated with glucose, and the insulin secretion ability was examined. As shown in FIG. 3C, cells differentiated by islet co-culture and cells differentiated from CM were better than those cultured in DM, and stimulation index was high in both low and high concentration of glucose stimulation. Cells differentiated by Edo-pancreatic co-culture and cells differentiated from CM showed similar results to those of mature islet cells (Fig. 3D). These results suggest that co - culture with islet cells can effectively improve the differentiation of insulin - secreting cells.

Example 4. Increased beta cell marker expression in Newport green sorting cells

We used Newport Green (NG) to separate pure beta cells from differentiation-induced cells. NG is a fluorescent dye that can be detected by staining zinc-rich features in beta cells. The differentiated cells were stained with NG and stained heterogeneously in the cytoplasm. NG-positive cells were significantly more detected in differentiation-induced cells (25.5 ± 3.2 vs. 45 ± 3%, P <0.05) in co-culture than in cells induced to differentiation in DM (FIGS. 4A and B). NG-positive cells were analyzed by RT-PCR using NGC-positive and negative cells by flow cytometry. As a result, insulin 1 and 2, PDX -1, NKX6.1, NKX2.2, and Glut2 were more highly expressed (Fig. 4C). In addition, flow cytometry using mouse proinsulin-specific proinsulin monoclonal antibody, which does not recognize mouse or human insulin, showed that proinsulin-positive cells were more proliferated in DM- (48.1 ± 4.2 vs. 23.7 ± 2.3%, P <0.01) (Fig. 4D and E), respectively, compared with islet cells co-cultured with pancreatic islets.

Example 5. Improvement of differentiation ability from human de-differentiating pluripotent stem cells into insulin-secreting cells through co-cultivation with mature islet cells

It was confirmed that differentiation from mouse embryonic stem cells into insulin - secreting cells was improved by co - culturing with an islet cell, and whether this co - culture differentiation method was also possible in human depleted pluripotent stem cells (hiPS). First, it was confirmed whether hiPS was induced to cells of differentiation stage. hiPS showed the highest expression of SOX17 and FOXA2 on day 3 (step 1), and SOX17 and OCT4 were observed to decrease rapidly over time. Through fluorescence immunohistochemistry, it was confirmed that SOX17 and FOXA2 were coexpressed in 60% or more of cells. In stage 2 and 3, HNF1β and HNF4α were the most highly expressed, and 60-80% of the cells were positive for HNF1β. Pancreatic progenitor cells expressing PDX-1, HLXB9, SOX9 and HNF6 were detected by Q-PCR and fluorescent immunostaining (stage 3), and cells in stage 4 were found to express NKX6.1 and PAX6 . At the final stage, PTF1a, NKX2.2, GCK and insulin were expressed. However, ALB (albumin) was rarely expressed in the differentiation process.

As shown in FIG. 5A, the efficiency of hiPS differentiation was confirmed by co-culturing with an islet cell in the same manner as that for mouse embryonic stem cells. As a result of Q-PCR, PDX-1, NKX6.1, NKX2.2, GCK, and insulin were 7.8, 2.3, 3.4, 2.6, and 4.5 times higher than the control (DM differentiated cells) (Fig. 5B). Further, on the electron microscope, more granules were observed in co-cultured cells (Fig. 5C). The percentage of NG-positive cells in co-cultured cells using flow cytometry showed that more NG-positive cells were detected in co-cultured cells than in the control (14.11 ± 1.6% vs. 24.64 ± 1.82%, P < 0.01) (Figures 5D and E).

Example 6. Effect of cell transplantation differentiated by islet cell co-culture in an animal model of diabetes

We examined whether cells co-cultured with islet cells express insulin and function as cells responsive to the stimulation with glycogen. Cells differentiated by DM and cells differentiated by islet cell co - culture were transplanted under the renal capsule of diabetic nude mice and insulin was injected during the experiment to prevent death due to severe hyperglycemia. In the islet cell co-cultured cell transplantation group, 4 out of 6 mice maintained normal blood glucose level, whereas in the DM transplantation group, 6 out of 7 mice showed severe weight loss and hyperglycemia (FIG. 6A). One of them had a teratoma, and the surviving one maintained hyperglycemia. At 90 days after transplantation, the kidneys of mice maintaining normal blood glucose were removed and blood glucose levels were elevated again after renal elimination (FIG. 6A). In the glucose tolerance test, the islet cell co-cultured cell transplantation group showed improved glucose control ability as compared to the DM transplantation cell group (Fig. 6B). The human C-peptide was measured in the serum of the mouse to confirm whether the transplanted islet cell co-cultured cells showed diabetic therapeutic effect by differentiation into insulin-secreting cells. A much higher C-peptide was detected in the islet cell co-cultured cell transplantation group than in the DM differentiated cell transplantation group (Fig. 6C). In order to confirm the presence of C-peptide or insulin-positive cells in the kidney tissue to which the cells were transplanted, fluorescent immunostaining was performed. As a result, insulin-positive cells were present in the fixed transplantation site and co-stained with C-peptide. Insulin-positive cells were also detected in the tissues of hyperglycemic mice, but the numbers of insulin-positive cells were much smaller than in tissues of normal blood glucose mice (Fig. 6D). These results suggest that cells induced to differentiate into islet cells and co-cultured cells function as insulin-secreting cells in vivo.

Claims (10)

Insulin secretion from pancreatic progenitor cells differentiated from transcription factor-uninvolved pluripotent stem cells, which contains the conditioned medium in which islet cells were cultured, Indolactam V and FGF10 as active ingredients, A composition for inducing differentiation into a cell. The composition of claim 1, wherein the conditioned medium in which the islet cells are cultured is obtained by culturing mature islet cells. The composition according to claim 1, wherein the pluripotent stem cells are embryonic stem cells or dedifferentiated pluripotent stem cells. Differentiation from pancreatic progenitor cells differentiated from transcription factor-uninvolved pluripotent stem cells into insulin-secreting cells, which contains the conditioned medium in which islet cells were cultured, Indolactam Ⅴ, and FGF10 as active ingredients Gt; A kit for inducing differentiation of pancreatic progenitor cells into insulin secreting cells differentiated from pluripotent stem cells into which the transcription factor is not introduced, comprising the culture medium composition of claim 4. delete delete delete delete delete

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