SC-β cells and compositions, and methods for producing the same.

Abstract

To provide SC-β cells and compositions, as well as methods for producing them. [Solution] To provide stem cell-derived β-cells (SC-β) that exhibit an in vitro glucose-stimulated insulin secretion (GSIS) response. A method for generating SC-β cells from insulin-positive endocrine cells includes contacting a cell aggregate containing insulin-positive endocrine cells with at least two β-cell maturation factors, including a) a transforming growth factor β (TGF-β) signaling inhibitor and b) a thyroid hormone signaling pathway activator, under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least one insulin-positive endocrine cell in the aggregate into an SC-β cell.

Description

[Technical Field]

[0001] The present invention relates to SC-β cells, compositions, and methods for producing them.

[0002] Cross-reference of related applications This application is based on U.S. Provisional Application No. 61 / 833,89, filed on June 11, 2013. No. 8 and U.S. Provisional Application No. 61 / 972,212 filed on March 28, 2014 To assert an interest. Its content is incorporated by reference. [Background technology]

[0003] To date, research has focused on the response to continuously changing glucose levels. Insulin-expressing cells that do not secrete sufficient amounts of insulin, or cells that only function abnormally, Pancreatic progenitor cells can only mature into functional insulin-expressing cells three months after transplantation into a Uss host. This is being generated (Cheng et. al., 2012; D'Amour et. al., 2005;D'Amour et al., 2006;Kroon et al. al., 2008;Nostro et al., 2011;Rezania et al. al., 2012;Schulz et al., 2012;Xie et al. ., 2013). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cheng, X., Ying, L., Lu, L., Galvao, AM, Mills, JA, Lin, HC, Kotton, DN, Shen, SS, Nostro, MC, et al. (2012). Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell Stem Cell 10, 371-384. [Overview of the project] [Problems that the invention aims to solve]

[0005] High levels of glucose in glucose-stimulated insulin secretion (GSIS) assays In response to insulin, high levels of insulin are released, and this can be repeated, correct In contrast to normal pancreatic islets or scattered adult β-cells, hPSCs generated by existing methods. Insulin-expressing cells respond appropriately to the addition of glucose at various concentrations. Insulin cannot be secreted. Therefore, the phenotype of normal pancreatic islets or mature adult β-cells is not present. There is a need for methods to obtain hPSC-derived cells that demonstrate this need. [Means for solving the problem]

[0006] In some embodiments, the present disclosure provides stem cell-derived β-cells (SC-β).

[0007] In some embodiments, the cells are mature. In some embodiments, the cells are This shows the glucose-stimulated insulin secretion (GSIS) response in vitro. In the application morphology, the cells exhibit an in vivo GSIS response. In some embodiments, The aforementioned cells exhibit glucose-stimulated insulin secretion in vitro and in vivo. (GSIS) responses are shown. In some embodiments, the cells have a GSIS response to at least one glucose challenge. In some embodiments, the cells have a GSIS response to at least two consecutive glucose challenges. In some embodiments, the cells have a GSIS response to at least three consecutive glucose challenges. In some embodiments, the GSIS response is observed immediately after transplanting the cells into a human or an animal. In some embodiments, the GSIS response is observed within about 24 hours after transplanting the cells into a human or an animal. In some embodiments, the GSIS response is observed within about two weeks after transplanting the cells into a human or an animal. In some embodiments, the stimulation index of the cells, characterized by the ratio of insulin secreted in response to a high glucose concentration compared to a low glucose concentration, is similar to the stimulation index of endogenous mature pancreatic β cells. In some embodiments, the stimulation index is 1 or more or 1. 1 or more or 1.3 or more or 2 or more or 2.3 or more or 2.6 or more. In some embodiments, the cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, the cytokines are interleukin-1β (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α) and combinations thereof selected from the group consisting of. In some embodiments, insulin secretion from the cells is enhanced in response to anti-diabetic agents. In some embodiments, the anti-diabetic agents are incretin mimetics, sulfonylureas, meglitinides and combinations thereof . In some embodiments, the insulin secretion from the cells is enhanced in response to anti-diabetic agents. In some embodiments, the anti-diabetic agents are incretin mimetics, sulfonylureas, meglitinides and combinations thereof It contains a secretion-promoting substance selected from the group consisting of the following. In some embodiments, the cells are mono It is hormonal. In some embodiments, the cells take the form of endogenous mature pancreatic β-cells. They exhibit similar morphology. In some embodiments, the cells are intrinsic mature pancreatic β-cells. The image shows inclusion crystal insulin granules, similar to insulin granules, under electron microscopy. In one embodiment, the cells exhibit slow replication. In some embodiments, the cells are internal Glucose-stimulated Ca, similar to GSCF in resident mature pancreatic β cells 2+ Flow (GSCF) As shown, in some embodiments, the cells undergo at least one glucose challenge. The cells then exhibit a GSCF response. In some embodiments, the cells undergo at least two glucose-induced glycation tests. They exhibit a GSCF response to the challenge. In some embodiments, the cells are small Both exhibit a GSCF response to three glucose challenges. In some embodiments, The cells exhibit enhanced calcium flow. In some embodiments, the enhanced calcium Glucose flow involves either an increased amount of inflow or a low inflow ratio relative to high glucose concentration. In one embodiment, the cells contain insulin, C-peptide, PDX1, MAFA, and NK cells. X6-1, PAX6, NeuroD1, Glucokinase (GCK), SLC2A1, PCS K1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD 2. Endogenous mature pancreas selected from the group consisting of PTPRN, NKX2-2, and Pax4 It expresses at least one marker specific to β cells. In some embodiments, the details The cells consist of a)i) glucagon (GCG) and ii) somatostatin (SST). The hormones selected from, or b)i) amylase and ii) carboxypeptidase A glandular cell marker selected from the group consisting of A(CPA1); c)i) GCG, ii) Ar x, iii) α-cell markers selected from the group consisting of Irx1 and Irx2; and d) A tubular cell marker selected from the group consisting of i) CFTR and ii) Sox9. It does not express at least one marker selected from the group. In some embodiments, The aforementioned cells are insulin-positive endocrine cells or Nkx6-1-positive progenitor pancreatic cells, Pdx From the group consisting of 1-positive pancreatic progenitor cells and pluripotent stem cells, a precursor is selected, in They are differentiated in vitro. In some embodiments, the pluripotent stem cells are embryonic stems. Selected from the group consisting of cells and induced pluripotent stem cells. In some embodiments, the cells are It is human. In some embodiments, the cells are not genetically modified. In the embodiment, the cells are genetically modified. In some embodiments, the cells The insulin produced in each instance is released during a 30-minute incubation at a high glucose concentration. The amount is between 0.5 and 10 μIU per 1000 cells. In some embodiments, Insulin produced per cell is incubated at a high glucose concentration for 30 minutes. In this case, the amount is approximately 2.5 μIU per 1000 cells. In some embodiments, the above Incubation occurs ex vivo.

[0008] In some embodiments, the disclosure provides cell lines comprising SC-β cells. The cell line stably expresses insulin. In some embodiments, the cell It can withstand freezing, thawing, and other processes for at least 30 successive generations without any apparent morphological changes. It can be amplified with doubling times between approximately 24 and 44 hours.

[0009] In some aspects, the disclosure describes how insulin is used under conditions that promote cell cluster formation. A cell aggregate containing β-positive endocrine cells is identified as a) a transformation growth factor β (TGF-β) signaling pathway a) a thyroid hormone signaling pathway activator and at least two other By contacting β-cell maturation factors, at least one insulin-positive endocrine in the aggregate This includes in vitro maturation of cells into SC-β cells, and insulin-positive intracellular This invention provides a method for generating SC-β cells from secretory cells.

[0010] In some embodiments, the SC-β cells undergo at least one glucose challenge. The response to this is shown. In some embodiments, the SC-β cells undergo at least two consecutive It shows a response to a glucose challenge. In some embodiments, the SC-β cells , showing a response to at least three consecutive glucose challenges. Some embodiments Therefore, the morphology of the SC-β cells is similar to that of endogenous mature β cells. In terms of application methods, the SC-β cells are used in vitro and / or in vivo. It exhibits a lucos-stimulated insulin secretion (GSIS) response. In some embodiments, the GSIS The S response is observed immediately after transplantation of the SC-β cells. In some embodiments, The GSIS response is observed within approximately 24 hours of transplantation of the SC-β cells. In some embodiments, the GSIS response occurs approximately two weeks after transplantation of the SC-β cells. Observed within 100 nM–100 μM. In some embodiments, the cell aggregate is observed between 100 nM and 100 μM. The TGF-β signaling pathway inhibitor is brought into contact with the TGF-β signaling pathway inhibitor at the concentration of the TGF-β signaling pathway inhibitor. In some embodiments, The aforementioned cell aggregate is brought into contact with the TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway involves the TGF-β receptor type I kiner This includes ze signaling. In some embodiments, the TGF-β signaling pathway inhibitor is , including Alk5 inhibitor II. In some embodiments, the TGF-β signaling pathway is inhibited. The harmful agent includes analogs or derivatives of Alk5 inhibitor II. In some embodiments, the same applies to the... The cell aggregate was subjected to the thyroid hormone signaling pathway at a concentration between 0.1 μM and 10 μM. Bring the cell aggregate into contact with the cutiverter. In some embodiments, the cell aggregate is subjected to a concentration of 1 μM. It is brought into contact with the thyroid hormone signaling pathway activator. In some embodiments, the Thyroid hormone signaling pathway activators include triiodothyronine (T3). In some embodiments, the cell aggregate is optionally brought into contact with a protein kinase inhibitor. In some embodiments, the cell aggregate is not brought into contact with the protein kinase inhibitor. In some embodiments, the cell aggregate is brought into contact with a protein kinase inhibitor. In this embodiment, the cell aggregate is subjected to the proteinquine at a concentration between 10 nM and 1 μM. The cell aggregate is brought into contact with an enzyme inhibitor. In some embodiments, the cell aggregate is treated with a concentration of 100 nM. The protein kinase inhibitor is brought into contact with the protein kinase inhibitor. In some embodiments, the protein kinase - The enzyme inhibitor includes staurosporine. In some embodiments, the method is used to collect the cells The combination is brought into contact with at least one further β-cell maturation factor. Some embodiments Therefore, the aforementioned at least one further β-cell maturation factor controls cystic fibrosis membrane conductance. Contains a CFTR inhibitor. In some embodiments, the cell aggregate is 100 nM- The CFTR inhibitor is brought into contact with the substance at a concentration between 100 μM. In some embodiments, The cell aggregate is brought into contact with the CFTR inhibitor at a concentration between 10 nM and 10 μM. In some embodiments, the CFTR inhibitor includes Gly-H101. In some embodiments, The at least one further β-cell maturation factor comprises an O-GlcNAcase inhibitor. In some embodiments, the cell aggregate is subjected to the O at a concentration between 100 nM and 100 μM. - Contact with a GlcNAcase inhibitor. In some embodiments, the cell aggregate is 10 The O-GlcNAcase inhibitor is brought into contact with the inhibitor at a concentration of nM-10μM. In terms of form, the O-GlcNAcase inhibitor contains Thiamet G. In the embodiment, the cell aggregate is cultured in a suitable culture medium. In some embodiments, Appropriate culture media include pancreatic islet medium (CMRLS) or CMRLS supplemented with Co nought Medical Research Laboratories 10 Includes 66. In some embodiments, the CMRLS is added together with serum. In the application method, the CMRLS is added together with 10% fetal bovine serum. In some embodiments, the conditions that promote the formation of the cell clusters include suspension culture. The cell aggregate is a small number of insulin-positive endocrine cells in the cell aggregate. Suspension for at least a period sufficient to induce in vitro maturation to one SC-β cell. It is maintained in culture. In some embodiments, the period includes at least 7 days. In some embodiments, the period includes the period between 7 and 21 days. The period includes the period between 7 and 14 days. In some embodiments, the period includes the period between 10 and 14 days. This includes the period between. In some embodiments, the period includes 14 days. In some embodiments, The aforementioned β-cell maturation factor is replenished every two days. In some embodiments, the cell aggregate At least 1% of the insulin-positive endocrine cells are induced to mature into SC-β cells. In some embodiments, at least 99% of the insulin-positive endocrine cells in the collection This is induced and matures into SC-β cells. In some embodiments, the obtained fragments in the assembly are At least 30% of the cells contain SC-β cells. In some embodiments, the SC-β cells It expresses C-peptide, insulin, NKX6-1, and Pdx1, and NKX6-1 It co-expresses the insulin-positive endocrine cell and the C-peptide. In some embodiments, the insulin-positive endocrine cell The cells also express Pdx1 and NKX6-1. In some embodiments, the insulin Positive endocrine cells are pluripotent stem cells selected from a group consisting of embryonic stem cells and induced pluripotent stem cells. They are produced from a collection of cells. In some embodiments, the SC-β cells include human cells. In some embodiments, the generation of the SC-β cells in vitro is scaled up. It's possible.

[0011] In some aspects, this disclosure relates to SC- produced based on the method described in this specification. Provides an isolated collection of β-cells.

[0012] In some embodiments, the disclosure includes an isolated collection of SC-β cells encapsulated therein. To provide microcapsules.

[0013] In some aspects, this disclosure relates to SC- produced based on the method described in this specification. The present invention provides a composition containing an aggregate of β cells.

[0014] In some aspects, this disclosure relates to SC- produced based on the method described in this specification. The present invention provides an assay involving an isolated collection of β-cells.

[0015] In some embodiments, the assay involves β-cell proliferation, β-cell replication, β-cell death, and β Cell function, β-cell sensitivity to immune attack, or β-cell dedifferentiation or differentiation One or more symptoms selected from a group consisting of susceptibility, which promote or inhibit the fate of β cells. It is intended for use in identifying supplements. In some embodiments, the assay is At least one insulin-positive endocrine cell or its precursor, at least one SC -It is intended to be used to identify one or more candidate agents that promote differentiation into β cells. ru.

[0016] In some aspects, this disclosure relates to SC- produced based on the method described in this specification. The process involves administering a composition containing an isolated aggregate of β cells to a subject. The present invention provides a treatment method for the target. In some embodiments, the SC-β cells are microcapsulated They are enclosed in a container. In some embodiments, the SC-β cells are administered to the SC-β cells. It is produced from an aggregate of pluripotent stem cells obtained from the same subject as described above. In some embodiments, The SC-β cells are produced from an aggregate of iPS cells, and the iPS cells are the SC- The β cells are obtained from cells obtained from the same subject to which the β cells are administered. In some embodiments, The aforementioned subjects have diabetes or are at increased risk of developing diabetes. In this embodiment, diabetes is defined as type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and prediabetic diabetes. Selected from a group consisting of diseases. In some embodiments, the subject has a metabolic disorder or Alternatively, they have an increased risk of developing metabolic disorders.

[0017] In some embodiments, the present disclosure relates to administering to subjects who require it, as described in claim 41. Use of an isolated aggregate of SC-β cells produced by the method described in any one of item 102 Regarding use.

[0018] In some embodiments, the isolated aggregate of SC-β cells is encapsulated in microcapsules. It is then administered to the subject. In some embodiments, the subject has diabetes or Alternatively, they have an increased risk of developing diabetes. In some embodiments, the diabetes is I The group is selected from type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and prediabetes. In the embodiment of the part, the subject has a metabolic disorder or an increased condition that leads to a metabolic disorder. It carries risks.

[0019] In some aspects, this disclosure relates to a) Alk5 inhibitors, b) triiodothyronine (T3), Depending on the circumstances, c) staurosporine, and depending on the circumstances, d) CMRLS or CMRLS A culture medium containing the following components is provided.

[0020] In some aspects, this disclosure describes how insulin-positive endocrine cells can be converted into SC-β cells. The culture medium is used to induce trophic maturation, and the SC-β cells are in vit. The use includes demonstrating a GSIS response both in vivo and / or in vivo.

[0021] In some aspects, this disclosure describes how Pdx1 is used under conditions that promote cell cluster formation. a) Cell aggregates including positive pancreatic progenitor cells from the fibroblast growth factor (FGF) family a) at least one growth factor, b) at least two including a sonic hedgehog pathway inhibitor One β-cell maturation factor, and, if applicable, c) low concentrations of retinoic acid (RA) signaling. The path activator is brought into contact with at least one of the sets for a period of at least 5 days. This includes inducing the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells. Furthermore, the aforementioned NKX6-1-positive pancreatic progenitor cells express NKX6-1 and are Pdx1-positive. This invention provides a method for producing NKX6-1 positive pancreatic primordial cells from pancreatic primordial cells.

[0022] In some embodiments, the cell aggregate is expressed at a concentration between 1 ng / mL and 100 ng / mL. , to be brought into contact with at least one growth factor from the FGF family. Partial implementation Morphologically, the cell aggregate is subjected to a concentration of 50 ng / mL from the FGF family. Contact with at least one growth factor. In some embodiments, the FGF family or The aforementioned at least one growth factor includes keratinocyte growth factor (KGF). In this embodiment, the at least one growth factor from the FGF family is FGF 2. Selected from the group consisting of FGF8B, FGF10, and FGF21. Some embodiments Therefore, the aforementioned cell aggregate is not brought into contact with the RA signaling pathway activator. In this embodiment, the cell aggregate is subjected to the RA SIG at a concentration between 0.01 μM and 1.0 μM. The cell aggregate is brought into contact with the NAL signaling pathway activator. In some embodiments, the cell aggregate is brought into contact with 0.1 The RA signaling pathway activator is brought into contact with the activator at a concentration of μM. In some embodiments, The RA signaling pathway activator includes RA. In some embodiments, the The cell aggregate is brought into contact with an SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the cell aggregate is subjected to the SHH pathway inhibitor at a concentration of 0.25 μM. To bring into contact. In some embodiments, the SHH pathway inhibitor includes Sant1. The method includes exposing the cell aggregate to at least one further β-cell maturation factor. In this embodiment, the at least one further β-cell maturation factor is derived from the EGF family. It contains at least one growth factor. In some embodiments, the cell aggregate is 2 ng / m At concentrations between L and 200 ng / mL, at least one of the EGF family The cell aggregate is exposed to growth factors. In some embodiments, the cell aggregate is exposed to a concentration of 20 ng / mL. Exposed to at least one growth factor from the EGF family. Some embodiments Therefore, at least one growth factor from the EGF family is betacellin and EG Selected from the group consisting of F. In some embodiments, the cell aggregate is in a suitable culture medium. They are cultured in a suspension culture. In some embodiments, the conditions that promote the formation of the cell clusters are suspension cultures. Contains nutrients. In some embodiments, the β-cell maturation factor is replenished every two days. In this embodiment, the protein kinase C activator was added to the suspension culture for 5 days. Not done. In some embodiments, the activator of protein kinase C is used 5 days prior. It is removed from the suspension culture. In some embodiments, the protein kinase C is activated Beta includes PdbU. In some embodiments, the BMP signaling pathway inhibitor is 5 It is not added to the suspension culture for 24 hours. In some embodiments, the BMP signaling pathway is inhibited. The harmful agent is removed from the suspension culture before 5 days. In some embodiments, the BMP The Gunal signaling pathway inhibitor includes LDN193189. In some embodiments, the set At least 10% of the Pdx1-positive pancreatic progenitor cells are induced to be NKX6-1 positive pancreatic cells. Differentiates into organ progenitor cells. In some embodiments, at least 95% of the Pd in ​​the aggregate x1-positive progenitor cells are induced to differentiate into NKX6-1-positive progenitor cells. In this embodiment, the NKX6-1 positive pancreatic progenitor cells are Pdx1, NKX6-1 and Fo It expresses xA2. In some embodiments, the Pdx1-positive pancreatic progenitor cells are embryonic stem cells. It is produced from a collection of pluripotent stem cells selected from a group consisting of cells and induced pluripotent stem cells. .

[0023] In some aspects, this disclosure relates to NKX6-1 obtained by the method described in this specification. This provides an isolated collection of positive pancreatic progenitor cells.

[0024] In some embodiments, this disclosure relates to a single NKX6-1 positive pancreatic progenitor cell encapsulated therein. The invention provides microcapsules containing separated aggregates.

[0025] In some aspects, this disclosure relates to NKX produced based on the method described in this specification. The present invention provides a composition comprising an isolated aggregate of 6-1 positive pancreatic progenitor cells.

[0026] In some aspects, this disclosure relates to NKX produced based on the method described in this specification. This assay provides an isolated aggregate of 6-1 positive pancreatic progenitor cells.

[0027] In some embodiments, the assay involves at least one Pdx1-positive progenitor pancreatic cell or one or more candidate cells that promote the differentiation of the precursor into NKX6-1 positive pancreatic progenitor cells. It is used to identify the drug.

[0028] In some aspects, this disclosure relates to NKX produced based on the method described in this specification. 6-1 A composition comprising an isolated aggregate of positive pancreatic progenitor cells is administered to a subject. This provides a method for treating those who require it.

[0029] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are the NKX6-1 positive pancreatic cells. The organ progenitor cells are produced from an aggregate of pluripotent stem cells obtained from the same subject to which the cells are administered. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are encapsulated in microcapsules. In some embodiments, the subject is someone who has diabetes or is at risk of developing diabetes. It carries the risk of [having the following]. In some embodiments, the diabetes is type 1 diabetes, type 2 diabetes, Selected from the group consisting of type 1.5 diabetes and prediabetes. In some embodiments, the opposite Elephants have metabolic disorders or are at increased risk of developing metabolic disorders.

[0030] In some embodiments, the present disclosure relates to the process for differentiating into SC-β cells, as described in claims 113 to 14. Isolation of NKX6-1 positive pancreatic progenitor cells produced by the method described in any one of item 2. Regarding the use of the set.

[0031] In some embodiments, this disclosure provides for administration to subjects requiring it, as described in this specification. Use of isolated aggregates of NKX6-1-positive pancreatic progenitor cells produced by the described method. include.

[0032] In some embodiments, the isolated aggregate of NKX6-1 positive pancreatic progenitor cells is micro The substance is encapsulated in a capsule and administered to the subject. In some embodiments, the subject is sugar They have a urine disease or an increased risk of developing diabetes. In some embodiments, The aforementioned diabetes is a group consisting of type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and prediabetes. Selected from. In some embodiments, the subject has a metabolic disorder or a metabolic disorder. It carries an ever-increasing risk.

[0033] In some embodiments, this disclosure covers a) KGF, b) SANT1, and optionally c) RA The present invention provides a culture medium that contains and substantially does not contain PdbU and LDN193189. In some embodiments, the present disclosure relates to NKX6-1 positive pancreatic progenitor cells of Pdx1-positive pancreatic progenitor cells. The use of the culture medium according to claim 160 for in vitro differentiation into cells is included. nothing.

[0034] In some aspects, this disclosure describes how NKX6 can be used under conditions that promote cell cluster formation. -1-positive cell aggregates including pancreatic progenitor cells were subjected to a) TGF-β signaling pathway inhibitors and b ) At least two β-cell maturation factors, including thyroid hormone signaling pathway activators By bringing it into contact with, at least one NKX6-1 positive pancreatic progenitor cell in the aggregate This also includes inducing differentiation into another insulin-positive endocrine cell, the insulin-positive Pancreatic progenitor cells express insulin, and insulin is derived from NKX6-1 positive pancreatic progenitor cells. The present invention provides a method for producing thrin-positive endocrine cells. In some embodiments, the cell aggregate is 1 The TGF-β signaling pathway inhibitor is brought into contact with the TGF-β signaling pathway inhibitor at a concentration between 00 nM and 100 μM. In some embodiments, the cell aggregate is subjected to the TGF-β-signature at a concentration of between 10 μM. The TGF-β signaling pathway is brought into contact with a signaling pathway inhibitor. In some embodiments, the TGF-β signaling pathway is , including TGF-β receptor type I kinase signaling. In some embodiments, the TGF -β signaling pathway inhibitors include Alk5 inhibitor II. In some embodiments, the The cell aggregates at a concentration between 0.1 μM and 10 μM, and the thyroid hormone signaling pathway. Bring the cell aggregate into contact with the activator. In some embodiments, the cell aggregate is subjected to a concentration of 1 μM. The thyroid hormone signaling pathway activator is brought into contact with the thyroid hormone signaling pathway activator. In some embodiments, the The thyroid hormone signaling pathway activator contains triiodothyronine (T3). In some embodiments, the method involves forming the cell aggregate into at least one further β-cell aggregate. This includes contacting with a maturation factor. In some embodiments, the at least one further β-fiber Cell maturation factors include γ-secretase inhibitors. In some embodiments, the cell aggregate is The γ-secretase inhibitor is brought into contact with the substance at a concentration between 0.1 μM and 10 μM. In this embodiment, the cell aggregate is brought into contact with the γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor includes DAPT. At least one additional β-cell maturation factor is derived from at least one EGF family. Contains growth factors. In some embodiments, the cell aggregate is 2 ng / mL-200 ng / mL Contact with the at least one growth factor from the EGF family at a concentration between these two values. In some embodiments, the cell aggregate is given the EGF family at a concentration of 20 ng / mL. - Contact with at least one growth factor from. In some embodiments, the EGF formula The aforementioned at least one growth factor from Milly comprises betacellin. In some embodiments, The at least one growth factor from the EGF family includes EGF. In this embodiment, the at least one further β-cell maturation factor is a low concentration of retinoic acid (RA) Includes a signaling pathway activator. In some embodiments, the cell assembly is In contact with the RA signaling pathway activator at a concentration between 0.01 μM and 1.0 μM. In some embodiments, the cell aggregate is subjected to the RA signal at a concentration of 0.1 μM. It is brought into contact with the signaling pathway activator. In some embodiments, the RA signaling pathway A The cutibeta includes RA. In some embodiments, the at least one further β-cell component The maturation factor includes a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, the preceding The cell aggregate was contacted with the SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the cell aggregate is subjected to a concentration of 0.25 μM to inhibit the SHH pathway. The substance is brought into contact with the harmful agent. In some embodiments, the SHH pathway inhibitor includes Sant1. In some embodiments, the cell aggregate is optionally brought into contact with a protein kinase inhibitor. In some embodiments, the cell aggregate is brought into contact with the protein kinase inhibitor. No. In some embodiments, the cell aggregate is brought into contact with the protein kinase inhibitor. In some embodiments, the cell aggregate is subjected to the protease at a concentration between 10 nM and 1 μM. The cell aggregate is brought into contact with an in kinase inhibitor. In some embodiments, the cell aggregate is subjected to a 100 nM solution. The protein kinase inhibitor is brought into contact with the protein at a certain concentration. In some embodiments, the protein The in kinase inhibitor includes staurosporine. In some embodiments, the method is the same as above. This includes exposing the cell aggregate to glucose. In some embodiments, the cell aggregate is 1 m The cells are exposed to glucose at concentrations between M and 50 mM. In some embodiments, the cell aggregates The cells are exposed to glucose at a concentration of 25 mM. In some embodiments, the cell cluster - Conditions that promote formation include suspension culture. In some embodiments, the cell aggregate is the At least one of the NKX6-1-positive progenitor cells of the pancreatic insulin-positive endocrine cell in the aggregate They are maintained in suspension culture for a period sufficient to induce differentiation into cells. In some embodiments, The said period is at least 7 days. In some embodiments, the β-cell maturation factor is The suspension culture is replenished once every two days. In some embodiments, at least 15% of the aforementioned NKX6-1-positive pancreatic progenitor cells are induced to become insulin-positive endocrine cells. Differentiation occurs. In some embodiments, at least 99% of the aggregate is NKX6-1 positive. Pancreatic progenitor cells are induced to differentiate into insulin-positive endocrine cells. In some embodiments, The insulin-positive endocrine cells mentioned above are Pdx1, NKX6-1, NKX2-2, and Mafb. glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and / or expressing insulin. In some embodiments, the NKX6-1 positive pancreatic origin is described above. The cells are a collection of pluripotent stem cells selected from a group consisting of embryonic stem cells and induced pluripotent stem cells. It is produced from.

[0035] In some aspects, this disclosure relates to the methods used in this specification to the production of the insulators produced by the methods described herein. Provides an isolated collection of phosphorus-positive endocrine cells.

[0036] In some embodiments, the Disclosure relates to the isolation of insulin-positive endocrine cells encapsulated therein. The present invention provides microcapsules containing a collection of such collections. In some embodiments, the present invention relates to the present specification. A composition comprising an aggregate of insulin-positive endocrine cells produced according to the method described in the book. To provide.

[0037] In some aspects, this disclosure relates to the methods used in this specification to the production of the insulators produced by the methods described herein. The method involves administering a composition containing an isolated aggregate of phosphorus-positive endocrine cells to a subject. This provides a method for treating targets that require such treatment.

[0038] In some embodiments, the insulin-positive endocrine cells are the insulin-positive endocrine cells The cells are produced from a collection of pluripotent stem cells obtained from the same subject to which the cells are administered. In this application, the insulin-positive endocrine cells are encapsulated in microcapsules. In this embodiment, the subject is someone who has diabetes or is at increased risk of developing diabetes. It has. In some embodiments, the diabetes is type 1 diabetes, type 2 diabetes, type 1.5 diabetes The group is selected from the disease and prediabetes. In some embodiments, the subject is metabolic disorder. They have a constant condition or an increased risk of developing metabolic disorders.

[0039] In some aspects, the present disclosure relates to the method described in this specification for differentiating into SC-β cells. This includes the use of an isolated aggregate of insulin-positive endocrine cells produced by the method described above.

[0040] In some embodiments, this disclosure provides for administration to subjects requiring it, as described in this specification. This includes the use of an isolated aggregate of insulin-positive endocrine cells produced by the described method. .

[0041] In some embodiments, the isolated aggregate of insulin-positive endocrine cells is microc The substance is enclosed in a capsule and administered to the subject. In some embodiments, the subject is a patient with diabetes. Having or having an increased risk of developing diabetes. In some embodiments, the Diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and prediabetes. Selected. In some embodiments, the subject has a metabolic disorder or is subject to a metabolic disorder. It carries an increased risk.

[0042] In some embodiments, this disclosure relates to a) TGF-β signaling pathway inhibitors, and b) TH pathway inhibitors. Cutiverter, as well as i) XXI, ii) betacellin, iii) low concentrations of RA signaling At least one selected from the group consisting of pathway activators and iv) SHH pathway inhibitors A culture medium containing β-cell maturation factors is provided.

[0043] In some embodiments, this disclosure relates to insulin-positive endocrine cells of NKX6-1-positive pancreatic progenitor cells. This includes the use of the culture medium of claim 221 for in vitro differentiation into cells.

[0044] In some aspects, this disclosure describes how Pdx1 is used under conditions that promote cell cluster formation. Endocrine cells that are positive for NKX6-1 and insulin are i) transformed growth factor β(T GF-β) signaling pathway inhibitors, ii) thyroid hormone signaling pathway activators t, and, if applicable, iii) in contact with a protein kinase inhibitor, at least partially SC-β cells of the aforementioned Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. This includes inducing in vitro maturation of the SC-β cells, in which the SC-β cells are in vitro Methods for generating SC-β cells that exhibit a GSIS response in vivo provide.

[0045] In some embodiments, the GSIS response was obtained by (i) transplanting the SC-β cells. (ii) immediately after transplantation to the subject; or (iii) within approximately 24 hours of transplantation to the subject. Observed within a week. In some embodiments, the SC-β cells (i) at least 1 (ii) a glucose challenge; (ii) at least two consecutive glucose challenges; Or, (iii) demonstrating a response to at least three consecutive glucose challenges. In some embodiments, the morphology of the SC-β cells is similar to that of endogenous β cells. In some embodiments, the Pdx1-positive, NKX6-1-positive, and insulin-positive individuals are divided into The secretory cells were inhibited from the TGF-β signaling pathway at a concentration between 100 nM and 100 μM. The agent is brought into contact with the substance. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin Endocrine cells positive for the TGF-β signaling pathway were exposed to the TGF-β signaling pathway inhibitor at a concentration of 10 μM. To allow contact. In some embodiments, the TGF-β signaling pathway involves the TGF-β receptor type I. This includes kinase signaling. In some embodiments, the TGF-β signaling pathway is inhibited. The harmful agent includes Alk5 inhibitor II. In some embodiments, the Pdx1-positive, NKX6 -1-positive, insulin-positive endocrine cells were subjected to the following treatment at a concentration between 0.1 μM and 10 μM: It is brought into contact with the glandular hormone signaling pathway activator. In some embodiments, the P Endocrine cells that are positive for dx1, NKX6-1, and insulin were subjected to the above-mentioned treatment at a concentration of 1 μM. It is brought into contact with the thyroid hormone signaling pathway activator. In some embodiments, the Thyroid hormone signaling pathway activators include triiodothyronine (T3). In some embodiments, the endocrine cells that are Pdx1-positive, NKX6-1-positive, and insulin-positive are used. The cells are not brought into contact with the protein kinase inhibitor. In some embodiments, the Pdx Endocrine cells that are positive for 1, NKX6-1, and insulin are inhibited by the protein kinase inhibitors. In some embodiments, the Pdx1-positive, NKX6-1-positive, and insulin Phosphorus-positive endocrine cells were subjected to the protein kinase inhibition at a concentration between 10 nM and 1 μM. The agent is brought into contact with the substance. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin Endocrine cells that are positive for the protein kinase inhibitor are brought into contact with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor includes staurosporine. In some embodiments, the method involves Pdx1-positive, NKX6-1-positive, and insulin-positive individuals. Endocrine cells of the sex were exposed to a cystic fibrosis membrane conductance regulator (CFTR) inhibitor. This includes the following: In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin Endocrine cells positive for CFTR were treated with the CFTR inhibitor at a concentration between 100 nM and 100 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive, and insulin-positive individuals are brought into contact with each other. Sex endocrine cells are brought into contact with the CFTR inhibitor at concentrations of 10 nM and 10 μM. In some embodiments, the CFTR inhibitor includes Gly-H101. The above method involves endocrine cells that are Pdx1-positive, NKX6-1-positive, and insulin-positive. This includes contacting with an O-GlcNAcase inhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells were selected at 100 nM and 100 The O-GlcNAcase inhibitor is brought into contact with the patient at a concentration between μM and μM. (Some embodiments) Then, the Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells are subjected to a 10 nM injection. The O-GlcNAcase inhibitor is brought into contact with the patient at a concentration between 10 μM and 10 μM. In the application form, the O-GlcNAcase inhibitor contains Thiamet G. In this embodiment, the Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells are: The culture is performed in a suitable culture medium. In some embodiments, the suitable culture medium is pancreatic islet culture medium. (CMRLS) or Connought Medica with added CMRLS components l Includes Research Laboratories 1066. In some embodiments The CMRLS is added together with the serum. In some embodiments, the CMRLS is , added together with 10% fetal bovine serum. In some embodiments, the cell cluster form Conditions that promote growth include suspension culture. In some embodiments, the Pdx1-positive, NKX Endocrine cells that are 6-1 positive and insulin positive are at least partially Pdx1 positive and NKX6 positive. Induces in vitro maturation of -1-positive, insulin-positive endocrine cells into SC-β cells. It is maintained in suspension culture for a period sufficient to induce development. In some embodiments, the period is short It includes at least 7 days. In some embodiments, the period includes the period between 7 and 21 days. In some embodiments, the period includes the period between 7 and 14 days. The period includes 14 days. In some embodiments, the suspension culture is replenished every two days. In some embodiments, at least 30% of the generated cells include SC-β cells. In some embodiments, the SC-β cells are given C-peptide, insulin, NKX6-1, It expresses Pdx1 and co-expresses NKX6-1 and C-peptide. Morphologically, the SC-β cells include human cells. In some embodiments, in vitro The generation of SC-β cells in o can be scaled up.

[0046] In some embodiments, the insulin-positive endocrine cells promote cell cluster formation. Under these conditions, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells were subjected to i) TGF- ii) β-signaling pathway inhibitors and thyroid hormone signaling pathway activators Touching the pancreatic progenitor cells revealed that at least some Pdx1-positive and NKX6-1-positive cells were Pdx By inducing differentiation into endocrine cells that are positive for 1, NKX6-1, and insulin, The endocrine cells obtained that are Pdx1-positive, NKX6-1-positive, and insulin-positive are Pdx 1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Z It expresses nt8, SLC2A1, SLC2A3, and / or insulin.

[0047] In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are used in 100 The TGF-β signaling pathway inhibitor is brought into contact with the TGF-β signaling pathway inhibitor at a concentration between nM and 100 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are divided into 10 μM The TGF-β signaling pathway inhibitor is brought into contact with the TGF-β signaling pathway inhibitor at the concentration of the TGF-β signaling pathway inhibitor. In some embodiments, The TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In one embodiment, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are 0. At concentrations between 1 μM and 10 μM, the thyroid hormone signaling pathway activator is brought into contact with the thyroid hormone signaling pathway activator. To be touched. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are brought into contact with the pancreatic cells. The thyroid hormone signaling pathway activator is then brought into contact with the thyroid hormone signaling pathway activator at a concentration of 1 μM. In one embodiment, the thyroid hormone signaling pathway activator is triiodothi Contains ronin (T3). In some embodiments, the method is Pdx1 positive, NKX6 -1-positive pancreatic progenitor cells were treated with i) SHH pathway inhibitors, and ii) RA signaling pathway activators. iii) Beta, γ-secretase inhibitor, iv) Epidermal growth factor (EGF) family At least one of their growth factors, and optionally, a small amount of a protein kinase inhibitor. This includes contact with at least one of the Pdx1-positive and NKX6. -1-positive pancreatic progenitor cells were subjected to the SHH pathway inhibition at a concentration between 0.1 μM and 0.5 μM. The harmful agent is brought into contact with the pancreatic tissue. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic tissue is brought into contact with the harmful agent. The progenitor cells are contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, The SHH pathway inhibitor includes Sant1. In some embodiments, the Pdx1 positive NKX6-1 positive pancreatic progenitor cells were administered at a concentration between 0.01 μM and 1.0 μM, as described above. It is brought into contact with the RA signaling pathway activator. In some embodiments, the Pdx1 positive In the RA signaling pathway, NKX6-1 positive pancreatic progenitor cells were subjected to a concentration of 0.1 μM. Bring into contact with the pathway activator. In some embodiments, the RA signaling pathway activator The data includes RA. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreas The progenitor cells were brought into contact with the γ-secretase inhibitor at a concentration between 0.1 μM and 10 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are used. The γ-secretase inhibitor is brought into contact with the γ-secretase inhibitor at a concentration of μM. In some embodiments, the γ -Secretase inhibitors include XXI. In some embodiments, the γ-secretase inhibitor The harmful agent includes DAPT. In some embodiments, the Pdx1-positive, NKX6-1-positive Pancreatic progenitor cells are administered to the EGF family at concentrations between 2 ng / mL and 200 ng / mL. In some embodiments, the Pdx1 Pancreatic progenitor cells that are positive for NKX6-1 were treated with the aforementioned EGF-fat at a concentration of 20 ng / mL. Contact with at least one growth factor from Lee. In some embodiments, the EG The aforementioned at least one growth factor from the F family includes betacellin. Morphologically, the at least one growth factor from the EGF family comprises EGF. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are used in the P Avoid contact with rhodoin kinase inhibitors. In some embodiments, the Pdx1-positive, NK X6-1-positive pancreatic progenitor cells are brought into contact with the protein kinase inhibitor. In the administration method, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells were administered in a 10 nM-1 μM solution. The protein kinase inhibitor is brought into contact with the protein kinase inhibitor at a concentration between the above. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells were subjected to the aforementioned protein at a concentration of 100 nM. The protein kinase inhibitor is brought into contact with the protein kinase inhibitor. In some embodiments, the protein kinase inhibitor is , containing staurosporin. In some embodiments, the method involves glycating the cell aggregate. This includes exposure to a concentration between 1 mM and 50 mM. In some embodiments, the cell aggregate is exposed to a concentration between 1 mM and 50 mM. They are exposed to glucose at a temperature of 25 mM. In some embodiments, the cell aggregate is exposed to glucose at a concentration of 25 mM. , exposed to glucose. In some embodiments, conditions that promote the formation of the cell clusters This includes suspension culture. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreas The progenitor cells are at least some of the aforementioned Pdx1-positive, NKX6-1-positive pancreatic progenitor cells. This induces differentiation into Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. It is maintained in suspension culture for a sufficient period of time. In some embodiments, the period is less At most 7 days. In some embodiments, the suspension culture is replenished once every two days. In this embodiment, at least 15% of the Pdx1-positive, NKX6-1-positive pancreatic primordium cells are present. The cells are induced to differentiate into Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. In some embodiments, at least 99% of the Pdx1-positive, NKX6-1-positive The pancreatic progenitor cells are induced to be Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. It differentiates into cells.

[0048] In some embodiments, the Pdx1-positive, NKX6-1-positive progenitor pancreatic cells are cell clusters. Under conditions that promote raster formation, Pdx1-positive pancreatic progenitor cells are subjected to i) FGF formulation. ii) at least one growth factor from Millie, and And in some cases, iii) contact with low concentrations of RA signaling pathway activators for a period of 5 days. Therefore, at least some of the Pdx1-positive pancreatic progenitor cells, NKX6-1 Obtained by inducing differentiation into positive pancreatic progenitor cells, and being Pdx1-positive and NKX6 -1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0049] In some embodiments, the Pdx1-positive pancreatic progenitor cells are administered at a dose of 1 ng / mL-100 ng / At concentrations between mL, the at least one growth factor from the FGF family is brought into contact with the In some embodiments, the Pdx1-positive pancreatic progenitor cells are administered at a concentration of 50 ng / mL. , to be brought into contact with at least one growth factor from the FGF family. Partial implementation Morphologically, the at least one growth factor from the FGF family is used in keratinocytes. It contains growth factors (KGF). In some embodiments, the fraction of the FGF family At the very least, one growth factor consists of FGF2, FGF8B, FGF10, and FGF21. Selected from the group. In some embodiments, the Pdx1-positive pancreatic progenitor cells are 0.1 μM The at least one SHH pathway inhibitor is brought into contact with the substance at a concentration between 0.5 μM and 0.5 μM. In one embodiment, the Pdx1-positive pancreatic progenitor cells were used at a concentration of 0.25 μM. In some embodiments, the at least one SHH pathway inhibitor is brought into contact with the SHH pathway inhibitor. The SHH pathway inhibitors include Sant1. In some embodiments, the Pdx1-positive pancreas The progenitor cells were activated in the RA signaling pathway at a concentration between 0.01 μM and 1.0 μM. Bring to contact with beta. In some embodiments, the Pdx1-positive pancreatic progenitor cells are 0.1 μ The RA signaling pathway activator is brought into contact with the M concentration. In some embodiments, The RA signaling pathway activator includes RA. In some embodiments, the method The method involves the Pdx1-positive pancreatic progenitor cells being grown from at least one EGF family This includes contacting the factor. In some embodiments, the Pdx1-positive pancreatic progenitor cells are At concentrations between 2 ng / mL and 200 ng / mL, the least from the EGF family In some embodiments, the Pdx1-positive pancreatic progenitor cells are brought into contact with a single growth factor. The at least one growth factor from the EGF family at a concentration of 20 ng / mL To bring into contact with the child. In some embodiments, at least one of the EGF family The growth factor includes betacellin. In some embodiments, it is derived from the EGF family. The at least one growth factor includes EGF. In some embodiments, the Pdx1 positive The progenitor pancreatic cells are cultured in a suitable culture medium. In some embodiments, the cell cluster Conditions that promote star formation include suspension culture. In some embodiments, the suspension culture is It is replenished every two days. In some embodiments, the activator of protein kinase C is It is not added to the suspension culture for 5 days. In some embodiments, protein kinase C The activator is removed from the suspension culture before 5 days. In some embodiments, The activator of protein kinase C contains PdbU. In some embodiments, BMP The signaling pathway inhibitor is not added to the suspension culture for 5 days. In some embodiments... The BMP signaling pathway inhibitor is removed from the suspension culture before 5 days. In this embodiment, the BMP signaling pathway inhibitor includes LDN193189. In one embodiment, at least 10% of the Pdx1-positive progenitor cells in the collection are induced These cells are then guided to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells. (Some embodiments) Therefore, at least 95% of the aforementioned Pdx1-positive pancreatic progenitor cells are induced to be Pdx1-positive, They differentiate into NKX6-1 positive pancreatic progenitor cells.

[0050] In some embodiments, the disclosure a) aggregates pluripotent stem cells into Pdx1-positive pancreatic progenitor cells. to differentiate into; b) under conditions that promote cell cluster formation, the Pdx1 positive The progenitor cells of the pancreas receive i) at least one growth factor from the FGF family, and ii) a small amount of At least one SHH pathway inhibitor, and, if applicable, iii) RA signaling pathway activation Beta is brought into contact with at least a portion of the Pd in ​​the set, once every two days for a period of five days. By inducing the differentiation of x1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells, At least some of the aforementioned Pdx1-positive pancreatic progenitor cells are Pdx1-positive and NKX6-1-positive. The process involves differentiating the NKX6-1 positive primordial pancreatic cells into Pdx The differentiated cells expressing 1 and NKX6-1; c) under conditions that promote cell cluster formation In this study, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells were subjected to i) TGF-β-Sig a) TH signaling pathway inhibitors, b) TH signaling pathway activators, and, if applicable, c) small ii) One SHH pathway inhibitor, iii) RA signaling pathway activator, γ-secretase inhibitors and vi) at least one of the epidermal growth factor (EGF) family Another growth factor is brought into contact with the plant every other day for a period between days 5 and 7, at least partially. The aforementioned Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are Pdx1-positive and NKX6-1-positive. Sex, by a method of inducing differentiation into insulin-positive endocrine cells, at least some of the above Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, Pdx1-positive, NKX6-1-positive, Differentiation into insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6- Endocrine cells that are positive for 1 and insulin include Pdx1, NKX6-1, NKX2-2, and Ma fb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and The differentiation described above, which expresses and / or insulin; and d) promotes cell cluster formation. Under these conditions, the endocrine cells that are Pdx1-positive, NKX6-1-positive, and insulin-positive i) Transgenic growth factor β (TGF-β) signaling pathway inhibitors, ii) Thyroid hormone Lumont signaling pathway activators, and, if applicable, iii) protein kinase inhibitors The harmful agent is brought into contact with at least a portion of the Pd every two days for a period between 7 and 14 days. Intake of x1-positive, NKX6-1-positive, and insulin-positive endocrine cells into SC-β cells By inducing in vitro maturation, at least some of the Pdx1-positive NKX6- 1. Differentiating insulin-positive endocrine cells into SC-β cells, SC-β cells enable GSIS responses in vitro and / or in vivo. The present invention provides a method for generating SC-β cells from pluripotent cells, including the differentiation described above.

[0051] In some embodiments, the disclosure a) provides Pdx 1) Differentiation into positive pancreatic progenitor cells; b) Under conditions that promote cell cluster formation Then, the Pdx1-positive pancreatic progenitor cells are subjected to i) KGF, ii) Sant1, and, if applicable iii) Contact with a low concentration of RA once every two days for a period of 5 days, and at least one of the aggregates It also induces differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells. By this method, at least some of the Pdx1-positive pancreatic progenitor cells are Pdx1-positive, NKX The method involves differentiating the cells into NKX6-1 positive progenitor cells, which are NKX6-1 positive progenitor cells. The differentiation of cells expressing Pdx1 and NKX6-1; c) the Pdx1-positive, NK X6-1 positive progenitor cells of the pancreas are treated with i) Alk5 inhibitor II, ii) T3, and, if applicable iii) Sant1, iv) RA, v) XXI and vi) Betacerlin, every other day During the period between the 5th and 7th day, contact was observed, and at least some of the Pdx1-positive, NKX6- Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells of pancreatic progenitor cells By inducing differentiation into cells, at least some of the aforementioned Pdx1-positive and NKX6-1 positive cells can be obtained. In the case of pancreatic progenitor cells, Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells were converted to Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. Differentiation is performed, and the Pdx1-positive, NKX6-1-positive, and insulin-positive cells are differentiated. Secretory cells are Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Expressing Kir6.2, Znt8, SLC2A1, SLC2A3 and / or insulin The differentiation described above; and d) under conditions that promote cell cluster formation, the Pd x1-positive, NKX6-1-positive, insulin-positive endocrine cells, i) Alk5 inhibitor II ii) T3, and if applicable, iii) staurosporine, every other day, for 7 and 14 days. During the period between days, at least some of the Pdx1-positive, NKX6-1-positive, i By inducing the in vitro maturation of ncerin-producing endocrine cells into SC-β cells Furthermore, at least some of the Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells The process involves differentiating cells into SC-β cells, wherein the SC-β cells are in vitro Pluripotent cells, including the differentiation that exhibits a GSIS response in vivo, and / or This provides a method for generating SC-β cells.

[0052] In some aspects, this disclosure relates to S cells differentiated in vitro from pluripotent stem cells. This product provides artificial pancreatic islets containing C-β cells.

[0053] In some aspects, this disclosure relates to S cells differentiated in vitro from pluripotent stem cells. We provide an artificial pancreas containing C-β cells.

[0054] Unless otherwise specified, the implementation of this invention requires skills in cell biology, within the scope of the relevant art. Cell culture, molecular biology, genetic engineering biology, microbiology, recombinant nucleic acids (e.g., DNA) Conventional techniques in technology, immunology, and RNA interference (RNAi) will typically be used. For a non-exclusive description of these specific techniques, see the following publication: Ausubel, F., et al. al., (eds.), Current Protocols in Molecule. lar Biology, Current Protocols in Immuno logy, Current Protocols in Protein Science ce, and Current Protocols in Cell Biolog y, all John Wiley & Sons, NY, edition as of December 2008;Sambrook, Russell, a nd Sambrook, Molecular Cloning: A Laborat. ory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 20 01;Harlow, E. and Lane, D., Antibodies-A Laboratory Manual, Cold Spring Harbor L aboratory Press, Cold Spring Harbor, 198 8;Freshney, RI, “Culture of Animal Cell s, A Manual of Basic Technique”, 5th ed. See John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases is provided by Goodman and Gilman's The Pharmacological Basis of Th erapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharm. acology, McGraw-Hill / Appleton & Lange;10 See the 1st edition (2006) or the 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is provided by McKusick, V. A.:Mendelian Inheritance in Man. A Catal og of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Pres. s, 1998 (12th edition) or more recent online database S: Online Mendelian Inheritance in Man, O MIM (trademark). McKusick-Nathans Institute of G etic medicine, Johns Hopkins University y (Baltimore, MD) and National Center for Biotechnology Information, National Library ary of Medicine(Bethesda, MD), as of May 1, 2010, World Wide Web URL: http: / / www. ncbi.nlm.nih.gov / omim / and Online Mendeli an Inheritance in Animals (OMIA), a data base of genes, inherited disorders and t raits in animal species (other than huma) n and mouse), at http: / / omia.angis.org.a All of the special features mentioned in this specification can be found at u / contact.shtml. Licenses, patent applications, and other publications (e.g., scientific papers, books, websites, and databases) The entirety of this is incorporated by reference. Either this specification or the aforementioned incorporated references. If there is any inconsistency between the two, this specification (including any amendments thereto) shall apply. (This may be based on incorporated references.) However, this will be controlled. Unless otherwise specified, the standard technically accepted meaning of a term is used in this specification. Standard abbreviations for various terms are used in this specification.

[0055] This patent or application file includes at least one color drawing. Including copies of this patent or patent application publication, upon request and payment of the required fees It will likely be provided by government agencies.

[0056] In other words, the gist of this invention is, Item 1 The invention described in the specification. Regarding. [Effects of the Invention]

[0057] The present invention may provide SC-β cells and compositions, as well as methods for producing them. [Brief explanation of the drawing]

[0058] [Figure 1] Figures 1A and 1B show a comparison between the previously published control differentiation method and the novel direct differentiation method. Figure 1A shows a schematic diagram comparing the typical direct differentiation method of this disclosure for generating INS+ cells from hPSCs with the previously published control differentiation method. Figure 1B illustrates histological sections of undifferentiated HUES8 (top panel), differentiated to DE (interrupted), and differentiated to PP1 (bottom panel) stained with OCT4, SOX17, and PDX1, respectively, using the previously published control differentiation method. Scale bar = 100 μm. [Figure 2]Figures 2A, 2B, and 2C illustrate that stem cell-derived β (SC-β) cells generated in vitro secrete insulin in response to multiple consecutive high-glucose challenges, similar to primary human β cells. Figures 2A, 2B, and 2C are graphs showing ELISA measurements of human insulin secreted from SC-β (Figure 2A), primary β cells (Figure 2B), and PH cells (Figure 2C) that were consecutively challenged with 2, 20, 2, 20, 2, and 20 mM glucose. After consecutive low / high glucose challenges, the cells were depolarized with 30 mM KCl. [Figure 3] Figures 3A, 3B, and 3C illustrate further biological replicates of in vitro SC-β cells that secrete insulin, similar to primary β cells, in response to multiple consecutive high-glucose challenges. The left panel is the same as in Figure 2. SC-β cells (SC-β; Figure 3A), primary β cells (1°β; Figure 3B), and PH cells (Figure 3C) were sequentially loaded with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl, and human insulin was measured by ELISA. [Figure 4]Figures 4A, 4B, 4C, 4D, and 4E illustrate that SC-β cells, like primary β cells, migrate cytoplasmic Ca2+ in response to multiple consecutive high-glucose challenges. Figure 4A is a schematic diagram of the detection of cytoplasmic Ca2+ at the aggregate level and single-cell level using Fluo-4 AM staining. Aggregate-level measurements were performed on entire clusters (shown as large red circles in the schematic), and individual cells within intact clusters (shown as small red circles) were analyzed for single-cell analysis. Figure 4B is a graph showing aggregate measurements of dynamically normalized Fluo-4 fluorescence intensity for SC-β cells, primary β cells, and PH cells that were consecutively challenged with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl. Figure 4C shows fluorescence images of Fluo-4 AM staining used for single-cell analysis. Figure 4D shows representative images indicating the position of a single cell in response to 3 (yellow), 2 (orange), 1 (blue), and 0 (red) glucose challenges. Figure 4E shows a graphical quantification of the frequencies of SC-β cells (n=156), primary β cells (n=114), and PH cells (n=138) in response to 20 mM glucose. Scale bar = 100 μm. [Figure 5]Figures 5A, 5B, 5C, 5D, 5E, and 5F illustrate that SC-β expresses human β-cell markers at the protein and gene expression levels. Figure 5A shows immunohistochemical images of cells stained with C-peptide (green), NKX6-1 (red), and somatostatin (gray). Figure 5B shows immunohistochemical images of cells stained with C-peptide (green) and PDX1 (red). Figure 5C shows immunohistochemical images of cells stained with C-peptide (green) and glucagon (red), with corresponding DAPI staining (blue). Figure 5D shows representative flow cytometry dot plots and groupings of cells stained with C-peptide and NKX6-1. Figure 5E shows hierarchical clustering analysis based on all genes measured by microarrays of undifferentiated HUES8, PH cells, fetal β cells, and adult primary β cells isolated from INS (data from Hrvatin et al. (Hrvatin et al., 2014)), as well as SC-β cells (SC-β) isolated from INS and NKX6-1. Figure 5F shows heatmaps of the 100 genes with the highest variance across all samples. CP = C-peptide, SST = somatostatin, GCG = glucagon. Scale bar = 100 μm. [Figure 6] Figure 6 shows the histological features of SC-β cell clusters stained with DAPI (blue), insulin (green), and C-peptide (red). Scale bar = 100 μm. [Figure 7] Figures 7A, 7B, and 7C illustrate further histological staining of SC-β cells. Figure 7A shows staining with C-peptide (green) and ISL1 (red). Figure 7B shows staining with C-peptide (green) and MAFA (red). Figure 7C shows staining with C-peptide (green) and MAFB (red). Scale bar = 100 μm. [Figure 8] Figures 8A, 8B, and 8C show representative flow cytometry dot plots and aggregation ratios of SC-β cells and PH cells stained with C-peptide and SST (Figure 8A), C-peptide and GCG (Figure 8B), and SST and GCG (Figure 8C). [Figure 9]Figures 9A, 9B, and 9C illustrate the structural similarity of SC-β-cell granules to primary human β-cell granules. Figure 9A shows electron microscope images of granules with representative crystallized insulin granules (red), early insulin granules (yellow), and mixed-type endocrine granules (blue) highlighted. Scale bar = 500 nm. Figure 9B shows a higher magnification image of the granules highlighted in (Figure 9A). Scale bar = 500 nm. Figure 9C shows electron microscope images of immunogold-labeled cells showing granules containing insulin (smaller 5 nm black dots) and / or glucagon (larger 15 nm black dots). Representative immunogold particles are highlighted with red arrows (insulin) and blue arrows (glucagon). Scale bar = 100 nm. [Figure 10] Figures 10A and 10B illustrate that stem cell-derived β (SC-β) cells generated from hiPSCs in vitro secrete insulin in response to multiple consecutive high-glucose challenges, similar to primary human β cells. Figures 10A and 10B are graphs showing ELISA measurements of human insulin secreted from SC-β cells generated from non-diabetic cells (Figure 10A) and type 1 diabetic cells (Figure 10B) that were consecutively challenged with 2, 20, 2, 20, 2, and 20 mM glucose. [Figure 11] Figures 11A, 11B, 11C, 11D, 11E, and 11F show representative flow cytometry dot plots and aggregation ratios of cells stained with C-peptide and NKX6-1 from multiple hiPSC strains. Figures 11A, 11B, and 11C show representative flow cytometry dot plots and aggregation ratios of cells stained with C-peptide and NKX6-1 from non-diabetic hiPSC strains. Figures 11D, 11E, and 11F show representative flow cytometry dot plots and aggregation ratios of cells stained with C-peptide and NKX6-1 from type 1 diabetic hiPSC strains. [Figure 12]Figures 12A, 12B, 12C, and 12D illustrate the rapid functioning of transplanted SC-β cells in vivo. Figure 12A is a graph showing ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells, primary human β cells (1°β), or PH cells (cultured for 1 week in the final in vitro step). Measurements were taken before glucose injection (white bars) and 30 minutes after injection (black bars) in mice 2 weeks post-transplantation. Figure 12B shows immunohistochemical images of transplanted cells (Figure 12A) stained with C-peptide (green) and PDX1 (red) to confirm the presence of the graft. Figure 12C is a graph showing ELISA measurements of human insulin from the serum of individual mice transplanted with pancreatic progenitor cells. Measurements were taken before glucose injection (white bars) and 30 minutes after injection (black bars) in mice 2 weeks post-transplantation. Figure 12D is a graph showing ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells cultured for two weeks during the final in vitro step. Measurements were taken 30 minutes after glucose injection (black bar) in mice two weeks post-transplantation. nd = not measured. Scale bar = 100 μm. [Figure 13] Figures 13A and 13B illustrate further histological fragments of SC-β and PH cells transplanted into mice two weeks prior. Figure 13A shows low-magnification images of the grafts stained with DAPI (blue), C-peptide (green), and GCG (red). Scale bar = 200 μM. Figure 13B shows higher-magnification images of the grafts stained with C-peptide (green) and GCG (red). Scale bar = 100 μM. [Figure 14A]Figures 14A, 14B, and 14C illustrate the use of culture media in the final stage of differentiation, enabling SC-β cells to secrete more insulin in vivo. Figure 14A is a schematic diagram showing the use of various culture media at various stages of the differentiation process. Figure 14B shows that in the final stage of differentiation, the addition of further factors, e.g., Sant1, XXI, and SSP, to CMRL medium results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by the stimulation index between high-glucose and low-glucose challenges. Figure 14C shows that in the final stage of differentiation, the addition of further factors, e.g., Sant1, XXI, and SSP, to CMRL medium results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by a certain amount of released insulin. [Figure 14B-C] Figures 14A, 14B, and 14C illustrate the use of culture media in the final stage of differentiation, enabling SC-β cells to secrete more insulin in vivo. Figure 14A is a schematic diagram showing the use of various culture media at various stages of the differentiation process. Figure 14B shows that in the final stage of differentiation, the addition of further factors, e.g., Sant1, XXI, and SSP, to CMRL medium results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by the stimulation index between high-glucose and low-glucose challenges. Figure 14C shows that in the final stage of differentiation, the addition of further factors, e.g., Sant1, XXI, and SSP, to CMRL medium results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by a certain amount of released insulin. [Figure 15A-C]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can enhance the viability and quality of the generated SC-β cells. Figure 15A is a schematic description of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates the extent to which cell viability can be improved by using the Rock inhibitor in steps 3-5. Figure 15D illustrates the extent to which SOX2 can be downregulated and cell viability can be improved by using activin A together with nicotinamide. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates the extent to which a near-pure endocrine cluster can be produced by using staurosporine in step 6. Figure 15G illustrates how much higher the proportion of NKX6-1 / C-peptide+ cells is generated by using staurosporine in step 6. Figure 15I illustrates how much neuro-D+ aggregation is increased by using XXI in combination with Alk5i and T3 in steps 5-6 compared to using Alk5i and T3 alone (Figure 15H). [Figure 15D-E]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can enhance the viability and quality of the generated SC-β cells. Figure 15A is a schematic description of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates the extent to which cell viability can be improved by using the Rock inhibitor in steps 3-5. Figure 15D illustrates the extent to which SOX2 can be downregulated and cell viability can be improved by using activin A together with nicotinamide. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates the extent to which a near-pure endocrine cluster can be produced by using staurosporine in step 6. Figure 15G illustrates how much higher the proportion of NKX6-1 / C-peptide+ cells is generated by using staurosporine in step 6. Figure 15I illustrates how much neuro-D+ aggregation is increased by using XXI in combination with Alk5i and T3 in steps 5-6 compared to using Alk5i and T3 alone (Figure 15H). [Figure 15F-G]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can enhance the viability and quality of the generated SC-β cells. Figure 15A is a schematic description of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates the extent to which cell viability can be improved by using the Rock inhibitor in steps 3-5. Figure 15D illustrates the extent to which SOX2 can be downregulated and cell viability can be improved by using activin A together with nicotinamide. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates the extent to which a near-pure endocrine cluster can be produced by using staurosporine in step 6. Figure 15G illustrates how much higher the proportion of NKX6-1 / C-peptide+ cells is generated by using staurosporine in step 6. Figure 15I illustrates how much neuro-D+ aggregation is increased by using XXI in combination with Alk5i and T3 in steps 5-6 compared to using Alk5i and T3 alone (Figure 15H). [Figure 15H-I]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can enhance the viability and quality of the generated SC-β cells. Figure 15A is a schematic description of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates the extent to which cell viability can be improved by using the Rock inhibitor in steps 3-5. Figure 15D illustrates the extent to which SOX2 can be downregulated and cell viability can be improved by using activin A together with nicotinamide. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates the extent to which a near-pure endocrine cluster can be produced by using staurosporine in step 6. Figure 15G illustrates how much higher the proportion of NKX6-1 / C-peptide+ cells is generated by using staurosporine in step 6. Figure 15I illustrates how much neuro-D+ aggregation is increased by using XXI in combination with Alk5i and T3 in steps 5-6 compared to using Alk5i and T3 alone (Figure 15H). [Figure 16]Figures 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H, and 16I illustrate the clinical utility of SC-β cells as a diabetes treatment or drug discovery platform. Figure 16A is a schematic diagram of the utility of SC-β cells for the treatment of diabetes or for screening drugs that improve function or replication. Figure 16B is a table listing the diabetes drugs tested and their general therapeutic classification. Figure 16C is a graph showing ELISA measurements of human insulin secreted from seeded SC-β cells treated with the indicated drugs at 2 and 20 mM glucose. The p-values ​​shown compare insulin levels at 20 mM glucose between the drug and the control. Figure 16D is immunofluorescence imaging of dispersed and seeded SC-β cells stained with DAPI (blue), C-peptide (green), and Ki67 (red) without treatment. Figure 16E is an immunofluorescence image of SC-β cells stained with DAPI (blue), C-peptide (green), and Ki67 (red), treated with prolactin for 48 hours, dispersed, and seeded. Figure 16F shows a graphical quantification of the function of cells co-expressing C-peptide and Ki67. *p<0.05. Figure 16G is a graph illustrating the measurement of blood glucose levels in fasted Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 indicates a comparison between two cell groups on the same day. Figure 16H is a graph illustrating the measurement of blood glucose levels from progressively diabetic Akita mice transplanted with SC-β cells or PH cells. Measurements were taken before glucose injection (white bars) and 20 minutes after injection (black bars) in mice transplanted two weeks prior. Glucose levels were saturated at 550 mg / dL. *p<0.05 indicates a comparison between two cell groups at the same time after glucose injection. Figure 16I is a graph showing ELISA measurements of human insulin from serum of Akita mice 20 minutes after glucose injection. Mice were challenged with glucose for 2 weeks post-transplant. *p<0.05 indicates a comparison between the two cell groups mentioned above. Scale bar = 50 μm. [Figure 17]Figure 17 is a graph illustrating the body weight of Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 indicates a comparison between the two cell groups at 18 and 28 days. [Modes for carrying out the invention]

[0059] Aspects of this disclosure include at least one insulin-positive endocrine cell or its precursor (e.g. iPS cells, hESCs, adult endoderm cells, gastrulatum cells, Pdx1-positive pancreatic progenitor cells (Pdx1-positive, NKX6-1-positive pancreatic primordial cells, Ngn3-positive endocrine primordial cells, etc.) Furthermore, a composition for generating stem cell-derived β (SC-β) cells (e.g., mature pancreatic β cells). Methods, kits and activators, and production by their compositions, methods, kits and activators The resulting cells are used in cell therapies, assays (e.g., drug screening), and various treatment methods. Regarding SC-β cells for use.

[0060] Furthermore, aspects of this disclosure include morphological evaluation criteria and without the use of selection markers. Functional characteristics, for example, expressing insulin and responding to one or more glucose challenges In response, it secretes insulin, shows a mature GSIS response, and has an in vivo odor These tissues are organized into islets in the pancreas and are typically small, spindle-shaped structures with a diameter of approximately 9-15 μm. The present invention relates to a method for identifying SC-β cells that can be detected based on their ability to have cells.

[0061] Furthermore, aspects of this disclosure relate to methods for identifying β-cell maturation factors. Whether it can function using assays known in the art can be recognized by those skilled in the art. They would be aware of it, or it would be easily verifiable. For example, at least one instrument The ability of a beta cell maturation factor to convert a glucagon-positive endocrine cell or its precursor into an SC-beta cell can be evaluated using the assays disclosed herein. Other convenient assays include measuring the ability to activate transcription of a reporter construct that includes a beta cell marker binding site operably linked to a nucleic acid sequence encoding a detectable marker, e.g., luciferase . One assay includes determining whether a candidate beta cell maturation factor induces at least one glucagon-positive endocrine cell to become an SC-beta cell, or express markers of beta cells, or exhibit functional characteristics of mature beta cells disclosed herein . Determination of such expression of beta cell markers can be determined by any suitable method, e.g., using immunoblotting . Such assays can be readily employed to identify or confirm the activity of an agent that directly converts at least one glucagon-positive endocrine cell or its precursor into an SC-beta cell . SC-beta cells (i.e., pancreatic beta cells) matured in vitro generated according to the methods of the invention described herein have many advantages, e.g., they perform glucose-stimulated insulin secretion in vitro, are similar to human pancreatic beta cells by gene expression and ultrastructure, secrete human insulin, improve hyperglycemia when transplanted into mice, provide a new platform for cell therapy (e.g., transplantation into subjects that require additional and / or functional beta cells), drug screening (e.g., for insulin production / secretion, survival, differentiation, etc.), (e.g., between normal and diabetic beta cells)

[0062] ​​​​​​​​​​Research (determining differences in function) and (for example, as the first cell type to reconstruct pancreatic islets) This document describes providing regenerative medicine (using the aforementioned SC-β).

[0063] definition

[0064] For convenience, in this specification, the examples and the appended claims, the terms used in this specification are used. This section summarizes specific terminology. Unless otherwise specified, all technical terms used in this specification are included. Technical and scientific terms are used in the same way as they are commonly understood by those skilled in the art to which this invention belongs. It holds.

[0065] The term "differentiated cells" is defined in this specification as referring to natural cells. This refers to any primary cell that is not pluripotent in its type. In other words, the "differentiated cell" mentioned above. The term refers to cells of a cell type that is not very specific in the process of cell differentiation (for example, Cells of a more specific cell type obtained from stem cells (e.g., induced pluripotent stem cells) It tastes like it. Although not bound by theory, in a series of normal ontogeny, pluripotent stem cells The cells can first differentiate into endodermal cells capable of forming pancreatic cells and other endodermal cell types. In this case, the differentiation of endodermal cells leads to the pancreatic pathway. In this case, approximately 98% of the aforementioned cells are extradermal. They develop into urinary glands, tubules, or matrix cells, with about 2% becoming endocrine cells. The cells are pancreatic islet precursors. These precursors then produce insulin, glucagon, and somatostatin. Insulin-producing cells that secrete tin or pancreatic polypeptides (e.g., functional endocrine cells) Endoderm cells can further differentiate into other cells of endoderm origin, such as lung, liver, and intestine cells. It can also differentiate into the thymus, etc.

[0066] In use in this specification, the term "somatic cells" refers to organ cells, in contrast to germline cells. It refers to any cell that makes up the body. In mammals, it refers to germline cells (also called "germ cells"). (Publicly known) refers to sperm and egg cells that fuse during fertilization to produce cells called fertilized bodies. From the fertilized body, it develops into the entire mammalian embryo. All other cell types in the mammalian body (sperm) (Excluding the egg cell, (germ cells) and undifferentiated stem cells are formed from the aforementioned cells.) These are somatic cells. Internal organs, skin, bones, blood, and connective tissue are all composed of somatic cells. In the embodiment of the part, the somatic cell is a "non-embryonic somatic cell". The non-embryonic somatic cell is in the embryo. It does not exist, and in vitro, such cell proliferation does not occur when obtained from embryos. This refers to somatic cells. In some embodiments, the somatic cells are "adult somatic cells." Adult somatic cells are present in organs other than the embryo or in the fetus, and are obtained from the embryo, or in This refers to cells that undergo such proliferation in vitro. Unless otherwise specified. , insulin production from at least one insulin-positive endocrine cell or its precursor, gluc The conversion to cos-responsive cells is performed both in vivo and in vitro. (where at least one insulin-positive endocrine cell or its precursor is within the target) If present, in vivo is practical. Isolated cells maintained in culture If at least one insulin-positive endocrine cell or its precursor is used, in In vitro testing is practical.

[0067] When used in this specification, the term "adult cells" refers to the entire body after embryonic development. It refers to the cells that are released.

[0068] As used herein, the term "endodermal cell" means a cell derived from one of the three major germ cell layers in a very early embryo (the other two germ cell layers are the mesoderm and the ectoderm). The endoderm is the innermost of the three layers. Endodermal cells differentiate to first form the embryonic digestive tract, and then give rise to the lining of the respiratory and digestive tracts (e.g., the intestine), the liver, and the

[0069] As used herein, the term "cells of endodermal origin" means any cell that has developed or differentiated from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, digestive tract, stomach, and thyroid. Without being bound by theory, the precursors of the liver and pancreas (also referred to as pancreatic precursors) develop from endodermal cells into the embryonic foregut. Soon after that specification, the precursors of the liver and pancreas rapidly acquire quite different cell functions and regenerative capabilities. These changes are elicited by inductive signals and gene regulatory factors that are highly conserved in vertebrates. Such organ development and regeneration are stimulated by the strong demand for hepatocytes and pancreatic beta cells in the therapeutic treatment of liver failure and type I diabetes. Studies in various model organisms and humans have demonstrated an evolutionarily

[0070] As used herein, the term "mature-type endoderm" means cells that have differentiated from endodermal cells. Said cells can differentiate into SC-beta cells Germ layer cells express the marker Sox17. Other markers specific to adult endoderm cells The markers are not limited to MIXL2, GATA4, HNF3b, GSC, and FGF. 17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, Gooseco Examples include iodine proteins, C-Kit, CD99, CMKOR1, and CRIP1. In this specification, adult endodermal cells express Sox17, and in some cases Morphologically, it expresses Sox17 and HNF3B, and clearly levels of GATA4, S Adult endoderm cells do not express PARC, APF, or DAB. Not positive for Pdx1 (for example, they are Pdx1 negative). Adult type Germ layer cells have the ability to differentiate into cells including those of the liver, lungs, pancreas, thymus, intestines, stomach, and thyroid. It has. The expression of Sox17 and other markers in the adult endoderm is as known to those skilled in the art. By any method, for example, immunochemistry or quantitative RT-PCR using anti-Sox17 antibodies It can be evaluated.

[0071] The term "pancreatic endoderm" refers to a group of pancreatic cells that can differentiate into multiple pancreatic lineages, such as pancreatic β-cells. This refers to endoderm-derived cells that no longer possess the ability to differentiate into non-pancreatic lineages.

[0072] When used in this specification, the terms "gastrulatory cells" or "intestinal cells" refer to endoderm cells. This refers to cells differentiated from [another cell type]. These cells differentiate into SC-β cells (e.g., pancreatic β cells). It is possible. Gastrulatal cells may contain at least one of the following markers: HNF1-β, HNF3-β. They express HNF4-α. The gastrulatum cells include cells from the lungs, liver, pancreas, stomach, and intestines. It has the ability to differentiate into cells. The expression of HNF1-β and other markers in the gastrulation is observed in our research. It is evaluated by any method known to the public, for example, by immunochemistry using an anti-HNF1-β antibody. It is possible.

[0073] "Pancreatic precursor," "Pancreatic endocrine precursor," "Pancreatic precursor," or "Pancreatic endocrine precursor" The terms pancreatic endocrine cells, pancreatic exocrine cells, and This refers to stem cells that can form pancreatic duct cells and potentially become pancreatic hormone-expressing cells. The cells are at least one type of pancreatic cell, for example, insulin-producing beta cells; Alpha cells that produce lucagon; delta cells (or D cells) that produce somatostatin. ) and / or are left to differentiate into F cells that produce pancreatic polypeptides. These cells contain at least one of the following markers: NGN3, NKX2.2, NeuroD, IS It may express L-1, Pax4, Pax6, or ARX.

[0074] When used in this specification, the term "Pdx1-positive pancreatic precursor" means SC-β cells, e.g. For example, it refers to pancreatic endoderm (PE) cells that have the ability to differentiate into pancreatic β cells. Pdx1-positive pancreatic precursors express the marker Pdx1. Other markers It is not limited to Cdcp1 or Ptf1a or HNF6 or NRx2.2 The expression of Pdx1 can be achieved by any method known to those skilled in the art, for example, by anti-Pdx It can be evaluated by immunochemistry using one antibody or by quantitative RT-PCR.

[0075] When used in this specification, the term "pdx1-positive, NKX6-1-positive pancreatic precursor" This is the pancreatic endoderm (P) which has the ability to differentiate into insulin-producing cells, such as pancreatic β-cells. E) This refers to cells that are cells. Pancreatic precursors that are pdx1-positive and NKX6-1-positive are called MAR It expresses the carriers Pdx1 and NKX6-1. Other markers are limited to... Examples include Cdcp1, Ptf1a, HNF6, or NRx2.2. NKX6-1 expression can be performed using any method known to those skilled in the art, for example, by using an anti-NKX6-1 antibody. It can be evaluated by immunochemistry or quantitative RT-PCR.

[0076] In use in this specification, the term "Ngn3-positive endocrine precursor" refers to a transcription factor. This refers to the precursor of pancreatic endocrine cells that express neurogenin-3 (Ngn3). Progenitor cells are differentiated from pluripotent stem cells and can differentiate into a very small number of cell types. Ngn3-positive endocrine origin cells are five pancreatic endocrine cell types (α, β, δ, ε, and PP). It has the ability to differentiate into. The expression of Ngn3 can be performed by any method known to those skilled in the art, for example, It can be evaluated by immunochemistry using anti-Ngn3 antibodies or by quantitative RT-PCR.

[0077] The terms "Neuro-D" and "Neuro-D1" are used interchangeably and refer to pancreatic endocrine cells. Identify the proteins expressed in the environment and the genes that encode them.

[0078] The terms "insulin-positive β-like cells" and "insulin-positive endocrine cells" refer to pancreatic β-cells. The patient presents at least one marker indicating insulin expression, but does not have endogenous β-cells. This refers to cells that lack a cell-specific GSIS response (e.g., pancreatic endocrine cells).

[0079] In relation to insulin-positive endocrine cells, the term "their precursor" refers to the aforementioned precursor cells. When cultured under conditions suitable for differentiation into the aforementioned insulin-positive endocrine cells, Srin-positive endocrine cells, such as pluripotent stem cells, adult endoderm cells, gastrulatum cells, and pancreatic cells. This refers to any cell capable of differentiating into progenitor cells or endocrine progenitor cells, etc.

[0080] “Stem cell β cells,” “SC-β cells,” “functional β cells,” “functional pancreatic β cells,” and The term "mature SC-β cells" refers to pancreatic β cells that have at least one marker (e.g., It presents PDX-1 or NKX6-1, secretes insulin, and is directed to endogenous mature β-cells. This refers to cells that exhibit a specific GSIS response (e.g., pancreatic β-cells). Therefore, the aforementioned "SC-β cells" include mature pancreatic β cells. Any cell as a starting point (this If the invention is not intended to be limited to this method, for example, embryonic stem cells, induced pluripotent stem cells Cells, progenitor cells, partially reprogrammed somatic cells (e.g., induced pluripotent stem cells and the aforementioned Somatic cells partially reprogrammed to an intermediate state between somatic cells and somatic cells, and the resulting somatic cells ), pluripotent cells, totipotent cells, or differentiated versions of any of the aforementioned cells may be used. Using the method disclosed herein, any insulin-positive endocrine cell or its precursor can be obtained from SC- If β cells can be obtained, the SC-β cells are derived (for example, directly) from stem cells. It is important to understand that this is not necessary. In some embodiments, the SC-β cells are subjected to multiple cycles. Glucose challenge (for example, at least once, at least twice, or at least three times) The above shows the response to a series of glucose challenges. In some embodiments, the above The response is the response of endogenous pancreatic islets (e.g., human pancreatic islets) to multiple glucose challenges. Similar to the answer. In some embodiments, the morphology of the SC-β cells is the morphology of endogenous β cells. It is similar to the following. In some embodiments, the SC-β cells are the endogenous β-cell GSIS response. It exhibits a similar in vitro GSIS response. In some embodiments, the SC -β cells exhibit an in vivo GSIS response similar to the endogenous β-cell GSIS response. The answer is given. In some embodiments, the SC-β cells respond to the endogenous β-cell GSIS response. It exhibits similar GSIS responses both in vitro and in vivo. The GSIS response of C-β cells is transmitted when the SC-β cells are transferred to a host (e.g., human or animal). This can be observed within two weeks of planting. In some embodiments, the SC-β cells are secretory granulomas. Insulin is packaged within the granule. In some embodiments, the SC-β cells are inclusion granules. Crystalline insulin granules are shown. In some embodiments, the SC-β cells are stimulated to a level greater than 1. The index is shown. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. In this application, the SC-β cells, in their response to cytokines, induce cytokine induction. It exhibits conductive apoptosis. In some embodiments, insulin secretion from the SC-β cells is observed. This is enhanced in response to known antidiabetic agents (e.g., secretagogues). In some embodiments, the SC-β cells are monohormone. SC-β cells respond to other hormones, such as glucagon, somatostatin, or pancreatic polyps. They do not abnormally co-express ptide. In some embodiments, the SC-β cells are slow polymorphic The manufacturing process is shown. In some embodiments, the SC-β cells respond to glucose. , intracellular Ca 2+ To increase. In use in this specification, the term "exocrine cells" means This refers to the cells of exocrine glands, that is, glands that secrete their contents through ducts. (Specific implementation form) In this context, exocrine cells refer to pancreatic exocrine cells. These pancreatic exocrine cells are divided within the small intestine. These are pancreatic cells that produce enzymes that are secreted as food passes through the digestive tract. They help digest food. Pancreatic exocrine cells are also known as islets of Langerhans. The pancreatic islets secrete two hormones, insulin and glucagon. Secretory cells include multiple cell types: alpha-2 cells (which produce the hormone glucagon). ; or β cells (which produce the hormone insulin); and (somatos, which are regulators) It may be one of the alpha-1 cells that produce tatin. When used in this specification, non Insulin-producing exocrine cells refer to alpha-2 cells or alpha-1 cells. The term pancreatic exocrine cells refer to hormones secreted into the bloodstream (for example, those produced by beta cells). Insulin (produced by alpha-2 cells), glucagon (produced by delta cells) (produced by) somatostatin and (produced by F cells) pancreatic polypeptides. Please note that this term includes "pancreatic endocrine cells," which refer to the cells that produce pancreatic tissue.

[0081] As used in this specification, the term "insulin-producing cells" refers to pancreatic precursors or This refers to cells differentiated from precursors. These cells secrete insulin. Living cells, when the term is used in this specification, include pancreatic β-cells and constitutive cells. In an inducible manner, insulin is synthesized (i.e., the insulin gene is transcribed), Translate proinsulin mRNA, and convert the proinsulin mRNA into insulin protein (to modify it), and express it (i.e., to express the trait carried by the insulin gene), Alternatively, secreting (releasing insulin into the extracellular space) pancreatic β-like cells (i.e., insulin) Includes insulin-positive endocrine cells. For example, insulin-positive endocrine cells based on the method of the present invention. The insulin produced by differentiating secretory cells or their precursors into SC-β cells The aggregate of β-producing cells is a pancreatic β-cell or β-like cell (for example, a small number of endogenous β-cells) A cell having at least one or at least two, and endogenous adult β It exhibits a GSIS response similar to that of cells. The novelty of this composition and method is that it naturally reacts with insulin. This cannot be ruled out by the presence of cells (e.g., β-cells) in the producing aggregate. For example, in the present application The aggregate of insulin-producing cells produced by the method disclosed in the specification is mature pancreatic β-cells. Or may include SC-β cells, and non-insulin-producing cells (i.e., they This may also include cells with a β-cell-like phenotype, except that they do not produce or secrete insulin. This is also considered.

[0082] When used in this specification, the terms "endogenous β-cells," "endogenous mature pancreatic β-cells," or The term "endogenous pancreatic β-cells" refers to insulin-producing cells of the pancreas or pancreatic β-cells (β-cells). ) This refers to cells of a particular phenotype. The phenotype of the pancreatic β-cells is well known to those skilled in the art, for example, Insulin secretion, c-peptide, and Pdx1 in response to elevated glucose levels Expression of polypeptides and markers such as Glut2, and distinguishable morphological features, for example This includes organization into islets in the pancreas in vivo, typically with a diameter of approximately 9–15 mm. It has tiny, micrometer-sized spindle-like cells.

[0083] When used in this specification, the terms "SC-β cells," "pancreatic β-like cells," and "mature pancreatic β-like cells" are used. The term "insulin" refers to at least 15% of the amount of insulin expressed by endogenous pancreatic β-cells, Alternatively, at least about 20% or less of the amount of insulin secreted by endogenous pancreatic β-cells. at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 100% or more than 100%, for example, at least about 1.5 times or is at least approximately 2 times or at least approximately 2.5 times or at least approximately 3 times or less It is expressed approximately 4 times or at least approximately 5 times or more than approximately 5 times, or endogenous At least one or at least two characteristics of pancreatic β-cells, for example, without limitation, glucos Insulin secretion and β-cell markers in response to the -, e.g., c-peptide , expressing Pdx1 and glut-2, produced by the method described in this specification. This refers to cells that have been cultured. In one embodiment, the SC-β cells are immortalized cells (i.e., cultured cells). (They do not proliferate indefinitely in this state.) In one embodiment, the SC-β cells are transformed into Cells, for example, transformation properties, for example, growth or contact inhibition in soft agar are absent. These are not cells that indicate [something].

[0084] The term "β-cell marker" is not limited to proteins, peptides, nucleic acids, and proteins. and nucleic acid polymorphs, splice variants, protein or nucleic acid fragments, elements Furthermore, it refers to other specimens that are specifically expressed or present in pancreatic β-cells. As a typical β-cell marker, there are no restrictions, and pancreatic and duodenal homeobox 1 (Pd x1) Polypeptide, insulin, c-peptide, amylin, E-cadherin, HNF3 β, PCI / 3, Β2, Nkx2.2, NKX6-1, GLUT2, PC2, ZnT-8 Isll, Pax6, Pax4, NeuroD, Hnflb, Hnf-6, Hnf-3 DATA and MafA, and Zhang et al., Diabetes. 50(1 0): Examples include those described in 2231-6 (2001). In some embodiments, The β-cell marker is a nuclear 3-cell marker. In some embodiments, the β-cell marker The manufacturer is either Pdx1 or PH3.

[0085] The term "pancreatic endocrine markers" is not limited to proteins, peptides, nucleic acids, and other proteins. Polymorphisms of proteins and nucleic acids, splice variants, protein or nucleic acid fragments, elemental polymorphisms This refers to the substance expressed specifically in or present in pancreatic endocrine cells, as well as other specimens. Typical pancreatic endocrine cell markers are not limited to Ngn-3 and Neuro-D. And Islet-1 is another example.

[0086] As used in this specification, the term "non-insulin-producing cells" means constitutively or induced This allows any endoderm-derived cell that does not inherently synthesize, express, or secrete insulin. This means that, when used in this specification, the term "non-insulin-producing cells" This excludes pancreatic β-cells. Examples of non-insulin-producing cells that can be used in the method of the present invention include For example, non-β cells of the pancreas, such as amylase-producing cells, acinar cells, and cells of tubular adenocarcinoma cell lines. (For example, CD18, CD11 and Capan-I cells (Busik et al., See Schaffert et al. 1997. Non-pancreatic cells of endoderm origin, such as non-pancreatic stem cells and cells of other endocrine and exocrine organs. Cells such as liver cells, thymocytes, thyroid cells, intestinal cells, lung cells, and pituitary cells can also be used. It may be used. In some embodiments, non-insulin-producing endodermal cells are mammalian cells, or More specifically, it could be human cells. Mammalian pancreatic non-islets, pancreatic amylase-producing cells. An example of this method using pancreatic acinar cells is provided in this specification.

[0087] The term "phenotype" refers to the condition under specific environmental conditions and factors, regardless of the actual genotype. In this context, it refers to one or more collective biological characteristics that define a cell or organism. ru.

[0088] As used in this specification, the term "multi-potential" means two or more under various conditions. Differentiate into differentiated cell types, preferably into cell types specific to all three germ cell layers. It means a cell that has the ability to fertilize. Pluripotent cells are, firstly, for example, nude mouse teratodus. Using a cell formation assay, differentiate into two or more cell types, preferably all three germ layers. , characterized by its ability. Pluripotency is determined by the expression of embryonic stem (ES) cell markers. Although this can also be proven, a preferred test for pluripotency is when cells divide into any of the three germ layers. This explains the ability to transform. Simply clustering such cells is itself It should be noted that this is not described as pluripotency. Reprogrammed pluripotency Sex cells (for example, iPS cells, as defined in this specification) are primary It retains its proliferative ability relative to the parent cell and also possesses the characteristic of being able to be passed through over a long period of time. Cells generally have the ability to divide only a limited number of times in culture.

[0089] When used in this specification, the terms "iPS cells" and "induced pluripotent stem cells" are interchangeable. Used for non-pluripotent cells, typically adult somatic cells, for example, one or more genes. By inducing the forced expression of (for example, by being induced or by complete recovery) This refers to artificially obtained pluripotent stem cells.

[0090] "Progenitor" cells or "precursor" cells The term is used interchangeably in this specification and refers to cells that may be produced by differentiation. Primitive (i.e., early stages along the developmental pathway or developmental stage, rather than fully differentiated cells) This refers to cells that have a cellular phenotype (located in). Often, the progenitor cells also exhibit a prominent or very It has high proliferative capacity. The progenitor cells are formed according to the developmental pathway and the environment in which the cells develop and differentiate. This can result in multiple distinct differentiated cell types or a single differentiated cell type.

[0091] As used in this specification, the term "stem cell" means a proliferative, differentiated daughter cell. Or it has the ability to generate a large number of parent cells that can sequentially produce differentiated daughter cells, It refers to undifferentiated cells that can give rise to progenitor cells. The daughter cells themselves are induced to proliferate. It can produce offspring that differentiate sequentially into one or more mature cell types, but which possess the developmental ability of the parent. It is also possible to maintain more than one type of cell. The term "stem cell" above is used in a specific environment. , possessing the ability or potential to differentiate into more specific or differentiated phenotypes, under specific circumstances In this context, it refers to a subset of precursors that maintain the ability to proliferate without substantial differentiation. In one embodiment, the term stem cells generally refers to the progressive diversity of embryonic cells and tissues. When this occurs, in many cases the offspring (offspring) differentiate, for example, completely into individual This refers to naturally derived mother cells that acquire specific characteristics and become specialized in various directions. Cellular differentiation is a complex process that typically occurs through many cell divisions. Cells are themselves obtained from pluripotent cells, etc. While all of these cells may be considered stem cells, the range of cell types varies considerably. These can undergo various changes. Some differentiated cells also have the ability to produce cells with higher developmental potential. Such abilities may be naturally occurring or based on treatment with various factors. They can be artificially induced. In many biological cases, stem cells are also "pluripotent." This is because they can produce offspring of two or more distinct cell types. Cell type does not require "stem cell properties". Self-renewal is another classification in the definition of stem cells. It is a part and, when used in this document, is essential. Theoretically, self-replication is, It can result from one of two main mechanisms. Stem cells maintain their stem cell state. One daughter, and other daughters that exhibit several other distinguishable specific functions and phenotypes, They can divide symmetrically. Alternatively, some stem cells in the aggregate can divide symmetrically into two stem cells. Therefore, a portion of the stem cells in the set can be maintained as a whole. On the other hand, in the set Other cells only produce differentiated offspring. Formally, cells that start as stem cells, The process progresses towards a transformed phenotype, but in that case, those skilled in the art will know "dedifferentiation" or "reprogramming". The term "retrodifferentiation," often referred to as "ming" or "retrodifferentiation," describes the process of returning to the stem cell phenotype. , and the possibility of re-expressing the same phenotype. When used in this specification, "pluripotent stem cells" The term "placental stem cells" includes embryonic stem cells, induced pluripotent stem cells, and placental stem cells.

[0092] Regarding the development of cells, the adjectives "differentiated" or "in the process of differentiation" are: "Differentiated cells" are cells that have progressed further down the developmental pathway than the cells being compared. It is a relative term meaning that. For this reason, stem cells are lineage restriction precursor cells (for example) If so, it can differentiate into mesodermal stem cells. Next, the precursor cells further descend the pathway, Other types of precursor cells (e.g., cardiomyocyte precursors), and then the final differentiated cells. It can differentiate. The differentiated cells in the final stage play characteristic roles in specific tissue types. And they may maintain their ability to reproduce further, or they may not maintain that ability. be.

[0093] The term "embryonic stem cells" refers to pluripotent stem cells in the inner cell population of a blastocyst. (See U.S. Patent Nos. 5,843,780 and 6,200,806) (This refers to...) Such cells are obtained from the inner cell aggregate of blastocysts obtained from somatic cell nuclear transfer. It can be obtained in the following way (for example, U.S. Patent No. 5,945,577, and No. 5,994,6 See Specification No. 19 and No. 6,235,970.) Characteristics that distinguish embryonic stem cells. This defines the phenotype of embryonic stem cells. Therefore, one or more unique characteristics of embryonic stem cells If present, the cell will have the phenotype of an embryonic stem cell. This means that the cell will have the phenotype of other cells. It can be distinguished from other types. Typical distinguishable features of embryonic stem cells include not being limited in genes. These include expression profile, proliferation ability, differentiation ability, karyotype, and responsiveness to specific culture conditions. ru.

[0094] The term "adult stem cells" or "ASC" refers to non-embryonic tissues, such as fetal, juvenile, and adult stem cells. It is used to refer to any pluripotent stem cells obtained from tissue. Stem cells encompass a wide range of species. From adult tissues such as blood, bone, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle and cardiac muscle, They are separated. Each of these stem cells has different gene expression, factor responsiveness, and morphology in culture. They can be characterized based on the following. Typical adult stem cells include neural stem cells, neural coronal stem cells, Examples include mesodermal stem cells, hematopoietic stem cells, and pancreatic stem cells. As mentioned above, stem cells are, It has been found that they are present in virtually all tissues. Therefore, the present invention relates to stem cell clusters. Understand that the compound can be isolated from virtually any animal tissue.

[0095] The term "pancreas" refers to a glandular organ that secretes digestive enzymes and hormones. In humans, the pancreas The organ was yellowish, measuring approximately 7 inches (17.8 cm) in length and 1.5 inches (3.8 cm) in width. It is an organ. The pancreas is located below the stomach and is part of the digestive tract, extending from the lower end of the stomach (pylorus) to the opening of the anus. It is connected to the small intestine, which is a muscular, hose-like part. Most pancreatic tissue is supplied by the bile duct from the liver. Grapes are cells that produce a clear fluid (pancreatic juice) that flows through the common canal into the duodenum along with the juice. It consists of clusters similar to those shown. Bile contains proteins, carbohydrates, and fats, along with intestinal enzymes. Three digestive enzymes complete the digestion of each component: tryptase, amylase, and lipase. It includes. Among the enzyme-producing cells of the pancreas, those that are dispersed are small endocrine cells. This group is called the islets of Langerhans and contains two hormones: insulin and glucago It secretes a hormone. The islets of the pancreas are composed of multiple types of cells: they produce the hormone glucagon. β-cells; β-cells that produce the hormone insulin (in this specification, "pancreas") Also called "visceral β-cells"); and alpha-1 cells that produce the regulatory agent somatostatin. These hormones are secreted directly into the bloodstream, and they interact with each other, causing glucose in the blood. It regulates blood sugar levels. Insulin lowers blood sugar levels and reduces glycogen in the liver. It increases the amount of glycerides (storage carbohydrates). Glucagon has the opposite effect. Dysfunction of insulin-secreting cells is called diabetes or diabetes mellitus. It brings about (iabetes mellitus).

[0096] When used in this specification, the term "reprogramming" means changing the differentiation state of somatic cells. This means the process of reprogramming or returning. The cells are partially or They can ultimately differentiate into one of the following. Reprogramming is the differentiation of somatic cells. This includes the complete restoration of the pluripotent state. Such a complete restoration of differentiation induces Pluripotent (iPS) cells are produced. When used in this specification, reprogramming is performed. Partial restoration of the cell's differentiated state, for example, restoration to a pluripotent state or pluripotent or pluripotent state. Neither of the differentiation potentials, but the differentiated cells have lost one or more specific characteristics, or For example, cells that undergo direct reprogramming to differentiate into different somatic cell types. This includes the return to somatic cells. Reprogramming generally involves nucleic acid modification (e.g., At least some genetic changes such as methylation, chromatin condensation, acquired changes, and genomic imprinting. This includes a transformation of the sex pattern, for example, a reversion. The transformation is the cell differentiation that occurs when a zygote develops into an adult. It is something that occurs inside.

[0097] As used in this specification, the term “actionant” means any compound or substance, for example, It is not limited to small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. "Agent" refers to any chemical substance, entity, or part, for example, synthetic and naturally derived, without limitation. It can be a proteinaceous or non-proteinaceous entity. In some embodiments, the activator is a nucleus Acids, nucleic acid analogs, proteins, antibodies, peptides, aptamers, nucleic acid oligomers, amino acids Acids or carbohydrates, for example, proteins, oligonucleotides, ribozymes, etc. (not limited to) DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers and related This is a modification and combination of the following. In a particular embodiment, the activator is a small molecule having a chemical moiety. For example, the chemical part may be a substituted or unsubstituted alkyl, aromatic, or heterocyclyl part. This includes, for example, macrolides, leptomycin and related natural products or analogues. The compound may be known to have the desired activity and / or properties, or They can be selected from a library of various compounds.

[0098] When used in this specification, "to bring into contact" (i.e., at least one instrument) Phosphorus-positive endocrine cells or their precursors, β-cell maturation factors or combinations of β-cell maturation factors The term (bringing into contact with) refers to bringing the β-cell maturation factor and cells together in vitro. Incubating in a controlled environment (for example, adding the β-cell maturation factor to the cells during culture) It is intended to include (the act of making contact). In some embodiments, the term "making contact" means In the subject, the in viv of cells into the compounds disclosed in this specification is likely to occur in the subject. This includes exposure in (i.e., exposure that can occur as a result of natural physiological processes). It is not intended to do so. As in embodiments relating to the production of SC-β cells, at least Another insulin-positive endocrine cell or its precursor is brought into contact with β-cell maturation factors. The process can be carried out by any suitable method. For example, the cells may be cultured in adherent culture or suspended in a suspension. They can be treated in culture. In some embodiments, the cells promote cell cluster formation. The cells are processed under conditions that promote cell cluster formation. This disclosure considers any conditions that promote cell cluster formation. Examples of conditions that promote cell cluster formation include, but are not limited, low-adhesion tissue culture pre- Methods include suspension culture in a treadmill, spinner flask, or Aggrewell plate. In some embodiments, the inventors have shown that the clusters are stable in a culture medium containing 10% serum. It was observed that it remained unchanged. In some embodiments, the conditions that promote cluster formation This includes a low-serum medium.

[0099] The cells that have come into contact with the β-cell maturation factor are then subjected to another agent, such as a growth factor or other compound. A catalyst or an environment that stabilizes the cells or further differentiates the cells, It is understood that contact may occur simultaneously or sequentially.

[0100] Similarly, at least one insulin-positive endocrine cell or its precursor, It may be brought into contact with one β-cell maturation factor, and then into contact with at least another β-cell maturation factor. In some embodiments, the cells are brought into contact with at least one β-cell maturation factor. The contact is spaced out over time. In some embodiments, the cells are connected to at least one β-cell. The cells are brought into contact with the maturation factor substantially at the same time. In some embodiments, the cells are brought into contact with at least two , at least 3, at least 4, at least 5, at least 6, at least 7 , and bring into contact with at least 8, at least 9, or at least 10 β-cell maturation factors.

[0101] In this specification, the term "cell culture medium" is used. The term (also called "culture medium") refers to a medium containing nutrients that maintain cell viability and support proliferation. It is a culture medium for culturing cells. The cell culture medium is used in an appropriate combination. Note: salt, buffer, amino acids, glucose or other sugars, antibiotics, serum or It may contain serum substitutes and other components, such as peptide growth factors. The cell culture media commonly used for cytoplasm are known to those skilled in the art.

[0102] The term "cell line" refers to a single ancestral cell or a defined and / or implemented cell line of an ancestral cell. It means a collection of cells that are typically obtained from the same set, and are mainly or substantially identical. The aforementioned cell line can be maintained in culture over long periods (e.g., several months, several years, or indefinitely). The cells are maintained or it may be possible to maintain them. The cells are indefinitely cultured. The cell line may undergo simultaneous or induced transformation to confer longevity. This includes all cell lines recognized in the field. The cells undergo mutations and / or acquired changes. It will be understood that this will allow us to obtain data over time. However, some characteristics may differ from one another. In some embodiments, the cell line is as described in this application. Includes SC-β cells as described in the detailed document.

[0103] The term "exogenous" refers to a substance present in cells or organisms other than those of its natural origin. For example, the terms "exogenous nucleic acid" or "exogenous protein" are used in relation to biological systems, such as cells. Alternatively, it refers to nucleic acids or proteins that have been introduced into living organisms by human intervention. The nucleic acid or protein is not normally found in the aforementioned biological system, or is found in smaller amounts. It is discovered. The site in which the substance is introduced into the cell or into the ancestor of the cell that inherits the substance. In other words, substances can be considered to be of foreign origin. In contrast, the term "endogenous" refers to organisms. It refers to substances that are inherently present in a system.

[0104] The term "expression" refers to the production of RNA and proteins, and, if necessary, proteins. To secrete, for example, transcription, translation, folding, where applicable, without restriction. It refers to cellular processes, including modification and processing. "Expression products" include genes. Polypeptide obtained by translation of RNA transcribed from offspring and mRNA transcribed from genes. Petit Do is one example.

[0105] When used in this specification, the terms "genetically modified" or "genetically engineered" are used. The term "cell" refers to a cell into which exogenous nucleic acids have been introduced by human means (or the aforementioned This refers to the offspring of such cells that have inherited at least a portion of the nucleic acid. For example, it may include sequences that are exogenous to the cell. The nucleic acid is an innate combination The arrangement is a sequence (i.e., the arrangement naturally found in the cell), but the arrangement is of non-natural origin (for example, different arrangements). This includes coding regions linked to promoters from genes, or variants of innate sequences, etc. That is also acceptable. The method for transporting the nucleic acid to the cell can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, etc. Transduction or infection using cytoporation and viral vectors are examples. In some embodiments, polynucleotides or portions thereof are incorporated into the genome of the cell. The nucleic acid may be subsequently removed or excised from the genome. However, Such removal or excision is unmodified, but otherwise equivalent to the cells. , the condition is that it brings about a detectable change in the aforementioned cells. Genetically modified, The term includes the direct introduction of modified RNA (e.g., synthetic modified RNA) into cells. The intention is that such synthetically modified RNAs are endonucleases and exonucleases. Modifications that prevent rapid degradation by, and the cell's intrinsic immunity or injection against the RNA. This includes modifications that prevent or reduce the tarferon response. The modifications are not limited to, for example, (a) Terminal modifications, for example, 5'-end modifications (phosphorylation, dephosphorylation, conjugation, inverse bond, etc.) (b) 3' end modifications (conjugate, DNA nucleotide, inverse bond, etc.), (b) base modifications, for example, The ornamental base, stabilizing base, destabilizing base, or base pair has a partner extension repertoire. Substitution by a base or conjugate base, (c) sugar modification (e.g., at the 2' or 4' position) (d) substitution of the ) or sugar, and (d) nucleoside linkage modification, for example, phosphodi This includes modifications or substitutions of ester bonds. Such modifications interfere with translation. Furthermore, the absence of the aforementioned modification results in a 50% or greater decrease in translation—for example, in rabbit reticulocytes. Insofar as the in vitro translation assay is performed, the above modifications are as described in this specification. The method and composition described are not suitable. In some embodiments, the SC-β cells are neuro The cells are genetically modified to express genin 3. In some embodiments, the SC-β cells are modified. Cellular genetic recombination involves introducing modified mRNA for synthesis that encodes neurogenin 3. This includes the gene set of SC-β cells by synthetic modified RNA encoding neurogenin 3. The replacement is thought to increase insulin production from the aforementioned cells. Such genetic recombination in living cells is expected to increase gene production in those cells. It is predicted that this will happen.

[0106] In some embodiments, the disclosure includes detectable markers at the insulin gene locus. The present invention provides genetically modified SC-β cells. In some embodiments, the SC -β cells are detected by a detectable marker that both alleles of the insulin gene locus are located They are modified to replace. In some embodiments, the SC-β cells are modified to replace the detectable ma The maker is genetically modified to insert into the insulin gene locus. Furthermore, the detectable marker is the SC in response to the glucose challenge. -Expressed together with insulin in β-cells. In some embodiments, the SC-β cells are Instead of insulin, the detectable marker is inserted into the insulin gene locus. It is genetically modified in such a way. As a result, the detectable marker is glucose chain In response to the range, it is expressed in place of insulin in the SC-β cells. A detectable marker, such as a nucleic acid encoding a fluorescent protein (e.g., GFP). It is considered that such genes may be inserted into the insulin gene locus. Recombinant SC-β cells, for example, in response to an agonist, the detectable By assaying the markers, insulin expression and / or fractions from β cells can be detected. Those skilled in the art can use this method in various screening techniques to identify agents that stimulate secretion. If there is an example, it will be understood. For example, (for example, by GFP) in both alleles SC-β cells genetically modified to replace the thrin gene were tested with a test agent and GF. P is a candidate drug that may activate insulin gene expression in β cells. Therefore, it is possible to bring the SC-β cells into contact with the activators that cause them to fluoresce. Furthermore, the detectable markers are found in such genetically modified SC-β cells. It can be used as a surrogate marker for insulin expression.

[0107] As used in this specification, the term “identity” means two or more nucleic acids or polynucleotides This means that the sequences of the two sequences are similar to a certain extent. It represents an evaluation window between the desired sequence and the second sequence. Percent identity over a certain range, for example, over a desired sequence length, aligns the sequence, The same residues are placed opposite each other within the evaluation window to maximize identity by introducing a gap. Determine the number of residues (nucleotides or amino acids) within the desired window. By dividing by the total number of residues in the sequence or the second sequence (whichever is longer) and multiplying by 100, It can be calculated. The number of identical residues required to achieve a specific percentage of identity can be calculated. When rounding, decimals are rounded to the nearest integer. Percentage identity is relevant to the field. It can be calculated using various publicly known computer programs. For example, Programs such as BLAST2, BLASTN, BLASTP, and BLAST2 are available. T generates alignment and provides percentage identity between desired sequences. arlin and Altschul, Proc. Natl. Acad. Sc Karlin, improved in L USA 90:5873-5877, 1993. and Altschul's algorithm (Karlin and Altschul) , Proc. Natl. Acad. Sci. USA 87:22264-22 68, 1990), but Altschul et al. al., J. MoI. Biol. 215:403-410, 1990). It is incorporated into the LAST and XBLAST programs. Gap aligner for comparison purposes. To obtain the ment, the gap BLAST is Altschul et al. (Alt Schul, et al. Nucleic Acids Res. 25:3389 Used as described in -3402, 1997). BLAST and Gap BL When using AST programs, the default parameters of each program are used. It may also be used. PAM250 or BLOSUM62 matrix may be used. The software used for AST analysis is provided by the National Biotechnology Information Center (NCB). I) These programs are available to the public. For more information, see "ncbi.nlm Visit the website with the URL "nih.gov" (World Wide Web Address). Regarding illumination. In specific embodiments, the percentage identity is provided by NCBI. It is calculated using BLAST2 with the appropriate parameters.

[0108] As used in this specification, the terms “isolated” or “partially purified” are: In the case of nucleic acids or polynucleotides, if found in their natural source, The nucleic acids or polynucleotides present together with and / or secreted polypeptides In the case of [the substance], when expressed or secreted by cells, together with the nucleic acid or polynucleotide, There may be at least one other component (e.g., nucleic acid or polynucleotide) It refers to nucleic acids or polynucleotides isolated from a substance. It also refers to chemically synthesized nucleic acids or These are polynucleotides, or those synthesized using in vitro transcription / translation. It is considered to be "isolated."

[0109] As used in this specification, the term “isolated cell” refers to the organism in which it is originally found. This refers to cells extracted from or descendants of such cells. In some cases, the aforementioned details The cells are cultured in vitro, for example, in the presence of other cells. Therefore, the cells are subsequently introduced into a second organism, or (or Cells derived from [the source] are reintroduced into the isolated organism.

[0110] When used in this specification, the term “isolated aggregate” refers to an isolated aggregate of cells. This refers to a collection of cells that have been separated from a mixture or heterogeneous collection of cells. In one embodiment, the isolated aggregate is compared with the heterogeneous aggregate from which the cells are isolated or concentrated. Therefore, it is essentially a pure collection of cells.

[0111] The term "substantially pure" in relation to a specific cell collection refers to the cells that make up the total cell collection. And at least about 75%, preferably at least about 85%, more preferably at least about This means a cell collection that is 90%, and most preferably, at least about 95% pure. Regarding the collection of SC-β cells, the terms "substantially pure" or "essentially purified" This is approximately 20% or less, more preferably approximately 15%, 10%, 8%, 7% or less, and most preferably approximately 15%, 10%, 8%, 7% or less. Approximately 5%, 4%, 3%, 2%, 1% or less, or less than 1% as specified in this specification. This refers to a cell collection that includes cells that are not more defined SC-β cells. In some embodiments, The present invention relates to a proliferated collection of SC-β cells that is a substantially pure collection of SC-β cells. This includes a method for the proliferation of SC-β cells.

[0112] Similarly, "substantially pure" or "essentially purified" insulin-positive endocrine cells Regarding the set, approximately 20% or less, more preferably approximately 15%, 10%, 8%, 7% or less, most Preferably about 5%, 4%, 3%, 2%, 1% or less, or less than 1% in the present specification. This refers to a cell aggregate that includes cells other than insulin-positive endocrine cells as defined by the aforementioned terms. In some embodiments, the present invention provides a proliferated aggregate of insulin-positive endocrine cells, A collection of insulin-positive endocrine cells, which is essentially a pure collection of insulin-positive endocrine cells. It includes methods of propagation.

[0113] Similarly, "substantially pure" or "essentially purified" Ngn3-positive endocrine precursors Regarding the set, approximately 20% or less, more preferably approximately 15%, 10%, 8%, 7% or less, most Preferably about 5%, 4%, 3%, 2%, 1% or less, or less than 1% in the present specification. Cells including Ngn3-positive endocrine precursors or their descendants as defined above This refers to cell aggregation. In some embodiments, the present invention promotes the proliferation of Ngn3-positive endocrine precursors. The set is a substantially pure set of Ngn3-positive endocrine precursors, Ngn3-positive endocrine This includes methods for increasing the aggregate of precursors.

[0114] Similarly, "substantially pure" or "generally pure" pancreatic precursors that are Pdx1-positive and NKX6-1-positive. Regarding a "qualitatively refined" collection, the percentages should be approximately 20% or less, more preferably approximately 15%, 10%, and 8%. %, 7% or less, most preferably about 5%, 4%, 3%, 2%, 1% or less or less than 1%, In this specification, the terms used are defined as Pdx1-positive and NKX6-1-positive prepancreatic cells. This refers to a collection of cells that are not the progenitor or its offspring. In some embodiments, the present invention This is an aggregate of proliferated pancreatic precursors that are Pdx1-positive and NKX6-1-positive, and N A virtually pure collection of KX6-1-positive pancreatic precursors, Pdx1-positive, NKX6-1 This includes a method for proliferating a collection of positive pancreatic precursors.

[0115] Similarly, a "substantially pure" or "essentially purified" collection of Pdx1-positive pancreatic precursors. Regarding the ratio, approximately 20% or less, more preferably approximately 15%, 10%, 8%, 7% or less, and most preferably approximately 15%, 10%, 8%, 7% or less. The percentages are approximately 5%, 4%, 3%, 2%, 1% or less, or less than 1%, as specified in the present specification. Cell collections containing Pdx1-positive pancreatic precursors or non-descendants as defined by the terminology used. This means a combination. In some embodiments, the present invention relates to a proliferated aggregate of Pdx1-positive pancreatic precursors. However, a collection of Pdx1-positive pancreatic precursors is a substantially pure collection of Pdx1-positive pancreatic precursors. It includes methods for multiplying the compound.

[0116] Similarly, with respect to a “substantially pure” or “essentially purified” collection of gastrulatic cells, Approximately 20% or less, more preferably approximately 15%, 10%, 8%, 7% or less, most preferably approximately 5% %, 4%, 3%, 2%, 1% or less, or less than 1%, as defined in this specification by the terms above. This refers to a cell aggregate that includes cells that are not defined gastrulatic cells or their descendants. Morphologically, the present invention relates to a proliferated aggregate of gastrulatous cells, which is a substantially pure aggregate of gastrulatous cells. It includes a method for the proliferation of a collection of gastrullous cells.

[0117] Similarly, regarding a “substantially pure” or “essentially purified” collection of adult endoderm cells And, more preferably, about 20% or less, more preferably about 15%, 10%, 8%, 7% or less, and most preferably about 15%, 10%, 8%, 7% or less. Approximately 5%, 4%, 3%, 2%, 1% or less, or less than 1% in this specification. This refers to a cell collection that includes adult endoderm cells or cells that are not their descendants, as defined by [the relevant regulations]. In some embodiments, the present invention relates to a proliferated aggregate of adult endoderm cells, which are adult endoderm cells. This encompasses methods for the proliferation of a collection of adult endoderm cells, which are essentially a pure collection of cells.

[0118] Similarly, regarding a “substantially pure” or “essentially purified” collection of pluripotent cells, Approximately 20% or less, more preferably approximately 15%, 10%, 8%, 7% or less, most preferably approximately 5% %, 4%, 3%, 2%, 1% or less, or less than 1%, as defined in this specification by the terms above. This refers to a cell collection that includes cells that are not defined as pluripotent cells or their descendants. Morphologically, the present invention provides a proliferated collection of pluripotent cells in a substantially pure collection of pluripotent cells. It encompasses a method of proliferation for a collection of pluripotent cells.

[0119] The terms “concentrate” or “concentrated” are used interchangeably in this specification. Therefore, the yield (percentage) of one type of cell is the amount of that type of cell in the initial culture or preparation. This means that the percentage has increased by at least 10%.

[0120] The terms “replication,” “self-replication,” or “multiplication” are interchangeable in this specification. Used and divided into the same unspecified cell type over a long period and / or from months to years. By doing so, it is used to mean the ability of stem cells to replicate themselves. Proliferation refers to the proliferation of cells through repeated division of one cell into two identical daughter cells. do.

[0121] As used in this specification, the term “lineage” means cells that share a common ancestor or common Describe cells that have a developmental fate. For example, those of endoderm origin or "endodermal lineage". Regarding the cells, this means that the said cells are obtained from endoderm cells and are limited by the endoderm lineage. For example, through a pathway that gives rise to adult endoderm cells, and then these adult endoderm cells become liver cells. It can differentiate along one or more developmental pathways, which can lead to differentiation into the thymus, pancreas, lungs, and gastrointestinal tract. It means that.

[0122] As used in this specification, the term "heterogeneous" means cells obtained from different species. .

[0123] As used in this specification, “marker” describes the characteristics and / or phenotype of a cell. It is used for this purpose. Markers can be used to select cells that contain the desired characteristics. This will change depending on the specific cell. The morphological and functional characteristics of cells of a particular cell type. Regardless of biochemical (enzymatic) characteristics or molecules expressed by the aforementioned cell type, markers - is unique. Preferably, such a marker is a protein, and more preferably It has an epitope for an antibody or other binding molecule available in that field. However, the marker can be any molecule found in the cell, for example, a protein, without limitation. (Peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids may be composed of these. Examples of morphological features or characteristics, without limitation, include shape, size, and nucleus / cytoplasmic ratio. Examples of functional features or properties include, but are not limited to, the ability to adhere to a particular substrate. Ability to contain or excrete specific dyes, migratory ability under specific conditions, and differentiation ability along specific strains. These are some examples. The marker may be detected by any method available to those skilled in the art. The maker does not need to have morphological features, or it may not need to have proteins, lipids, etc. It is not necessary. Markers are a unique feature of polypeptides and other morphological features, indicating the presence or absence of these features. It could be a combination of flannel.

[0124] The term "adjust" is used in a manner consistent with its usage in that field, that is, Qualitative or quantitative changes, alterations, or modifications in a desired process, path, or phenomenon. It means to cause or facilitate. Such changes are not limited to species. Increase, decrease, or change in the relative strength or activity of each component, or process, It can be a branching of a pathway or phenomenon. The "modulating substance" is the desired process, pathway, or phenomenon. To cause qualitative or quantitative changes, alterations, or modifications in, or to easily cause It is an agent that has an effect.

[0125] As used in this specification, the term "DNA" is defined as deoxyribonucleic acid. .

[0126] In this specification, the term "polynucleotide" refers to a polymer of nucleosides. It is used interchangeably with "nucleic acid". Typically, the polynucleotides of this invention are phosphodi DNA or RNA linked by ester bonds (e.g., adenosine, thymidine, Guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyg Composed of naturally occurring nucleosides (anosine and deoxycytidine). However, the aforementioned terms apply to chemically derived nucleic acids, regardless of whether they are found in naturally occurring nucleic acids or not. or nucleosides or nucleoside-like substances containing biologically modified bases, modified skeletons, etc. It includes molecules containing bodies. Such molecules may be preferable for certain applications. In this application, when polynucleotides are referred to, both DNA and RNA, as well as single-stranded and It is provided in either a double-stranded or double-stranded form (and as complementary chains to each single-stranded molecule). This is understood. When used in this specification, "polynucleotide sequence" means polynucleotide sequence. Sequence information that biochemically characterizes the nucleic acid material itself and / or specific nucleic acids (i.e., base It can mean a sequence of characters used as an abbreviation for. Unless otherwise specified, the polynucleotide sequences are represented in the 5' to 3' direction.

[0127] As used in this specification, the term "polypeptide" means a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably in this specification. Peptides are relatively short polypeptides, typically with a length of about 2 to 60 units. It is an amino acid between . The polypeptides typically used in this specification are proteins It contains the most commonly found amino acids, for example, 20 L-amino acids. However, Other amino acids and / or amino acid analogs known in the field may be used. One or more amino acids in a lipeptide are, for example, chemical entities such as carbohydrate groups and phosphorus groups. They may be modified by adding acid groups, fatty acid groups, linkers for conjugates, or functionalization. A polypeptide having a non-polypeptide portion that is co- or non-co-associated with it. However, it can be considered a "polypeptide." Typical modifications include glycosylation and pulping. Mitoylation is one example. Polypeptides are purified from natural sources and recombinant DNA technology is used. It is produced by and can also be synthesized by chemical means, such as conventional solid-phase peptide synthesis. i. When used in this specification, the terms “polypeptide sequence” or “amino acid sequence” are: , the polypeptide material itself and / or sequence information that biochemically characterizes the polypeptide ( In other words, a sequence of letters or three-letter codes used as an abbreviation for an amino acid name. This can mean that, unless otherwise specified, the polypeptide sequences expressed in this specification are N - Represented from the terminal to the C-terminal direction.

[0128] The term "mutant" in reference to polypeptides is used, for example, in contrast to full-length polypeptides. And at least 80%, 85%, 90%, 95%, 98%, or 99% identical polypeptide The mutant may be a fragment of a full-length polypeptide. This may be a naturally occurring splice variant. The variant is a flag of the polypeptide. For the ment, at least 80%, 85%, 90%, 95%, 98%, or 99% identical. It may be a polypeptide. In this case, it may be a wild-type full-length polynucleotide or a polypeptide with the desired activity For example, SC-β cells or insulin-positive endocrine cells from which SC-β cells can be obtained. Insofar as the domain has the ability to detect the presence of the precursor, the fragment at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% It is 99%. In some embodiments, the domain is at any amino acid position in the sequence. It starts and extends toward the C-terminus, with a length of at least 100, 200, 300, or 4 It consists of 00 amino acids. In the field of removing or substantially reducing the activity of proteins. In some embodiments, known mutations are preferred to be avoided. In some embodiments, the mutants are full-length poly It lacks the N- and / or C-terminal portions of the lipeptide. For example, from either end Up to 10, 20, or 50 amino acids are missing. In some embodiments, the polyp The plutido has a sequence of mature (full-length) polypeptides. The mature polypeptide consists of one or more The upper part, for example, during normal intracellular protein degradation (for example, simultaneous translation or translation) This refers to polypeptides that have signal peptides that are removed during post-translation processing. In one embodiment, the protein is purified from cells that naturally express it. When produced externally, the protein is a chimeric polypeptide. Petit means that it contains parts derived from two or more different species. In some embodiments, The site where the aforementioned protein is produced other than by purifying it from cells that naturally express it. In addition, the protein is a derivative. The derivative is a derivative in which the protein has the same sequence as the protein. Unless it substantially reduces the biological activity of the protein, it is not related to the protein. This means it includes further sequences.

[0129] As used in this specification, the term "functional fragment" means that before it is a fragment Although smaller in size than the polypeptide, it is substantially the same as the polypeptide, which is a fragment. It is a polypeptide having a matching amino acid sequence. In this case, the functional fragment The polypeptide sequence is a fragment of the polypeptide, and at least about 5 0% or 60% or 70% or 80% or 90% or 100% or 100% Higher, for example, 1.5 times, 2 times, 3 times, 4 times, or more than 4 times, effective biological effect It is located there. The functional fragment polypeptide has reduced antigenicity, (in transcription factors and (In the process of) increased DNA binding or (in regulating RNA stability or degradation) It may also have further functions, including altered RNA-binding ability (as a functionality).

[0130] The term "vector" refers to a carrier into which a DNA sequence can be inserted for introduction into a host cell. This refers to DNA molecules. Preferred vectors enable the autonomous replication of nucleic acids bound to them and / or or it is possible to express it. The expression of genes that are manipulably bound to them can be controlled. In this specification, the vector is referred to as an "expression vector." "—" is a dedicated region containing essential regulatory regions required for the expression of desired genes in host cells. It is a vector. In some embodiments, the desired gene is another sequence in the vector. It is operably bound to the vector. The vector is either a viral vector or a non-viral vector. It is possible. A viral vector would be used. The viral vector has replication defects. It is preferable that the replication defect includes, for example, all viral nuclei encoded for replication. This can be achieved by removing acid. The replication-deficient viral vector also retains its infectivity characteristics. It will likely enter cells in a similar manner to how adenovirus vectors replicate. Furthermore, once it enters the aforementioned cells, the replication-deficient viral vector will not regenerate or multiply. The framework also includes other means for transporting liposomes, nanoparticles, and DNA molecules into cells. .

[0131] The term "operably coupled" means that in order to achieve the expression of a code sequence, the code sequence The regulatory sequence essential for the expression of the sequence is located at an appropriate position relative to the coding sequence, within the DNA molecule. This means that it is placed inside. This same definition applies to the coding sequence and in the expression vector. Suitable for the arrangement of transcription control elements (e.g., promoters, enhancers, and terminal elements). It may be used. The term “operably coupled” above means that the polyn should be expressed. The creotide sequence has an appropriate start signal (e.g., ATG) before it, and the correct reading frame is Maintain the expression of the polynucleotide sequence and the polynucleotide sequence under the control of the expression regulatory sequence. This includes enabling the production of a desired polypeptide encoded by a nucleotide sequence. .

[0132] The term "viral vector" refers to a virus acting as a carrier for nucleic acid constructs into cells. This refers to the use of a vector or virus-related vector. The construct is a non-replicating virus. Scenogene-like adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), for example, retroviruses and lenticular viruses used for cell infection or transduction. It can be incorporated into a ruth vector and packaged. The vector can be incorporated into the cell genome. It may or may not be incorporated. The structure may, if necessary, It may contain a viral sequence for sansfection. Alternatively, the construct may contain an episodic virus sequence. The vectors may be duplicated, for example, and incorporated into EPV and EBV vectors.

[0133] The terms “adjustable array” and “promoter” are used interchangeably in this specification. Nucleic acid molecules that induce or control the transcription of protein-coding sequences that are manipulably bound to them. This refers to columns, for example, start signal, enhancer, and promoter. In some examples, recombination The transcription of the recombinant gene controls the expression of the recombinant gene in the cell type in which it is intended to be expressed. The recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence). These are the same as or different from their sequences, which control the transcription of naturally occurring forms of proteins. It may be under the control of transcriptional regulatory sequences. In some cases, the promoter sequence is the synthesizer of the cell. Recognized by the structure or introduced into the synthesis mechanism, and necessary for initiating the transcription of a specific gene. It is considered essential.

[0134] As used in this specification, the term “transcription factor” refers to a DNA-binding domain. It binds to specific sites on DNA and controls the transfer (or transcription) of genetic information from DNA to RNA. It refers to a protein that is part of a system that proliferates. When used in this specification, it means "to proliferate". " and "proliferation" refer to the increase in the number of cells in an aggregate formed by cell division (proliferation). Proliferation is generally a response to the environment, for example, to growth factors and other mitogenic substances. It is understood that this leads to the coordinated activation of multiple signal transduction pathways. Cell proliferation is Intracellular or extracellular signals and It can also be promoted by being freed from the action of the mechanism.

[0135] The term "selective marker" refers to a selective phenotype when expressed, such as a cytotoxic agent. or resistance to cell proliferation inhibitors (e.g., antibiotic resistance), trophotrophicity or specific It is used as a basis for distinguishing between cells that express this protein and those that do not. This refers to a gene, RNA, or protein that confers the expression of a specific protein to a cell. Proteins whose expression can be easily detected, for example, fluorescent or luminescent proteins or An enzyme that acts on a substrate to produce a colored, fluorescent, or luminescent substance ("detectable marker") These constitute a subset of selection markers. They are typically expressed selectively or exclusively in pluripotent cells. The presence of a selection marker that binds to the expression regulatory element inherent in the gene being targeted. This makes it possible to identify and select somatic cells that have been reprogrammed into a pluripotent state. Species selection marker genes, for example, neomycin resistance gene (neo), puromycin Gamma-ion resistance gene (puro), guanine phosphoribosyltransferase (GPT), Hydrofolate reductase (DHFR), adenosine diaminase (ADA), Pyromycetin N-acetyltransferase (PAC), hygromycin resistance gene (hyg) multidrug resistance genes (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphate Suvoribosyltransferase (HPRT) and the hisD gene may be used for detection. Possible markers include green fluorescent protein (GFP), blue, sapphire, and yellow. —, red, orange, and cyan fluorescent proteins, and any variant thereof, are examples. It is possible. Luminescent proteins, for example, luciferase (for example, fireflies or sea urchins) The luciferase of this application is also useful. As will be evident to those skilled in the art, the specification of this application uses When used, the term "selection marker" refers to a gene or the expression product of said gene, for example. This could mean the encoded protein.

[0136] In some embodiments, the selection marker is either not expressed or significantly expressed. For cells that express it at a low level, proliferation occurs in cells that express it. and / or confer a survival advantage. Such a proliferation and / or survival advantage is prior This typically occurs when the cell is maintained under specific conditions, i.e., "selective conditions." To ensure effective selection, cell aggregation is performed so that cells that do not express the marker proliferate. not, and / or not to survive, and to be removed from the set, or its Under conditions and maintained for a sufficient period of time such that the number decreases to only a very small proportion of the set Obtain. Under selective conditions for removing cells that do not express the marker, almost completely or entirely. By maintaining cell aggregation in this state, a mer confers an advantage in proliferation and / or survival. The method for selecting cells expressing KAR is referred to as "positive selection" in this specification. The aforementioned marker is said to be "useful for positive selection." Negative selection and Markers useful for negative selection are also subject to the specific methods described in this specification. The expression of such markers is either absent or significantly low. It is expressing it at a certain level (or, in another possible way, expressing the marker) Cells that do not express the marker exhibit a proliferative and / or survival advantage over cells that express the marker. ) Cells that express the marker exhibit proliferation and / or This imposes a disadvantage to survival. Therefore, cells expressing the aforementioned markers are considered to have a sufficient lifespan. If maintained under interselective conditions, they are almost completely removed from the cell aggregate.

[0137] When used in this specification, the "reporter gene" is genetically introduced into a cell, and the above Includes any gene to add to the stem cell phenotype. Reporter genes disclosed in this invention The offspring include fluorescent, luminescent, enzymatic, and resistance genes, and can be easily detected by those skilled in the art. It is also intended to include the genes of the present invention. In some embodiments of the present invention, the reporter gene is It is used as a marker to identify specific stem cells, cardiovascular stem cells, and their differentiated offspring. The reporter gene is one that is monitored by measuring the expression of the reporter gene. The sequence that regulates its expression is genetically manipulated by a method that conforms to the above conditions. In some cases, the expression of the reporter gene may be measured in living cells. When reporter gene assays are used in living cells, reporter gene expression is multiple. It can be monitored at multiple points in time, for example, 2, 3, 4, 5, 6, 8, or 10 or more points. In some cases, when a reporter assay is used in living cells, the reporter gene The expression can last from at least 10 minutes to about 24 hours, for example, at 20 minutes, 1 hour, 2 hours, 3 hours. Intervals: 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours The frequency of monitoring is either a certain frequency or another frequency from any integer between approximately 10 minutes and approximately 24 hours. It can be done.

[0138] The terms "subject" and "individual" are used interchangeably in this specification, and cells are acquired. Treatment with cells as described in this specification, e.g., prophylactic treatment. It refers to an animal, such as a human, which is provided. It is specific to a particular animal, such as a human. With regard to the treatment of those specific infections, symptoms, or disease conditions, the aforementioned terms refer to their specific This refers to animals. The terms "non-human animals" and "non-human animals" are used interchangeably in this specification. "Dairy" refers to mammals, such as rats, mice, rabbits, sheep, cats, dogs, and cows. Examples include pigs and non-human primates. The term “subject” above can refer to any vertebrate, for example , without limitation, also encompassing mammals, reptiles, amphibians and fish. However, advantageously, the aforementioned subjects This includes mammals, such as humans or other mammals, such as domesticated mammals, such as dogs and cats. These include horses and other farming mammals, such as cattle, sheep, and pigs.

[0139] The terms "diabetes" and "diabetes" are used interchangeably in this specification. Health organizations state that for diabetes, a fasting plasma glucose concentration of 7.0 mmol / l (126 mg / dl) (whole blood 6.1 mmol / l or 110 mg / dl) or higher, or 2 hours A diagnostic threshold of intermittent glucose level of 11.1 mmol / L or higher (200 mg / dL or higher) is defined. Other values ​​that suggest diabetes or indicate a high risk of diabetes include elevated levels. Arterial pressure ≥ 140 / 90 mmHg; elevated plasma triglycerides (1.7 mmol / L); 150 mg / dL) and / or low HDL-cholesterol (0.9 mm for men) ol / L, less than 35 mg / dl; for women, less than 1.0 mmol / L, less than 39 mg / dl. ); Central obesity (male: waist-to-hip circumference ratio greater than 0.90; female: waist-to-hip circumference ratio greater than 0.85) to / or obesity index 30 kg / m² 2 Ultra-low albuminuria (in this case, urinary albumin Excretion rate of 20 μg / min or higher, or albumin:creatinine ratio of 30 mg / g or higher. ) are examples. The term diabetes mentioned above refers to all types of diabetes, for example, type I, type II and type 1. It includes .5 type.

[0140] When applied to isolated cells, use terms such as "to treat," "to treat," or "treatment." The word means providing the cells to any kind of process or condition, or any kind This includes performing the layering or method on the cells. When applied to the subject, the terms are: This means providing medical or surgical treatment, care, or management to an individual. Individuals are usually sick, injured, or exhibit the average characteristics of the population. The numb is at high risk of developing illness and requires such treatment, care, or management. .

[0141] When used in this specification, the terms "to treat" and "treatment" refer to the subject being a disease. A reduction in at least one sign of the disease or an improvement in the disease, for example, beneficial or desired clinical This means administering an effective amount of the composition to the target in order to obtain the desired result. The relevant, beneficial, or desired clinical outcomes are not limited to those that are detectable or not. Furthermore, improvement of one or more symptoms, a decrease in the severity of the disease, and stabilization of the disease (i.e., no worsening) i) Delay or slowing of the state, disease progression, improvement or alleviation of the aforementioned disease state, and reduction ( Examples include (whether partial or comprehensive). Treatment is necessary if treatment is not received. Compared to the measured survival, this may mean an extended survival. Therefore, the treatment may affect the aforementioned disease symptoms. Those skilled in the art will understand that while this may improve the condition, it may not completely cure the disease. When used in this specification, the term “treatment” includes prevention. Alternatively, treatment is If the progression of the aforementioned disease is reduced or stopped, it is considered "effective." "Treatment" is, It may also mean an extended survival compared to the survival predicted if no treatment is received. Those who are eligible include individuals who have already been diagnosed with cardiac symptoms, as well as those with genetic susceptibility. Other factors, such as weight, diet, and health, may potentially lead to cardiac symptoms. Those who fit this description can be mentioned.

[0142] When used in this specification, the terms "administer," "introduce," and "transplant" are used. The term "a method or method that results in at least partial localization of cells introduced into a desired site" Depending on the pathway, the placement of the cells of the present invention (e.g., SC-β cells) to the target can be interchanged. Used. The cells, for example, SC-β cells (e.g., pancreatic β cells or pancreatic β-like cells) ) can be directly implanted in the pancreas, or at a desired location in the subject. It can be administered by any suitable route that brings about delivery. In this case, at least Some of the embedded cells or components of said cells remain alive after administration to the subject. The survival period of the aforementioned cells ranges from a few hours, for example, 24 hours, to several days or even several years. It is possible. In some cases, the cells may also be administered to non-pancreatic sites, such as the liver. Or, for example, maintaining the implanted cells in the implantation location and the movement of the implanted cells In capsules that prevent movement (e.g., microcapsules), subcutaneous administration is also possible. can.

[0143] When used in this specification, the expressions "parenteral administration" and "administered parenterally" are: This refers to administration methods other than enteral and local administration, usually by injection, and is not limited to intravenous or intramuscular injection. Internal, intraarterial, subarachnoid space, ventricle, sacral, orbital, intracardiac, intradermal, intraperitoneal, transtracheal, skin Examples of injections and infusions include those below, under the epidermis, intra-articular, subcapsular, subarachnoid, intrathecal, intravertebral, and intrasternal injections. When used in this specification, the terms "systemic administration," "administered systemically," and "peripheral administration" are used. The expressions "administered" and "administered peripherally" refer to cardiovascular stem cells and / or their offspring and / or This refers to the administration of compounds and / or other materials in a manner other than direct intra-central nervous system. However, because it enters the organs of animals, it is subject to metabolic and other processes, for example, subcutaneous tissue. It is an administration.

[0144] The term "organization" refers to a specific group or layer of cells that perform a particular specialized function together. This means that the term "tissue-specific" refers to a cell source that originates from a specific tissue.

[0145] The terms "decrease," "decline," "decrease," "decrease," or "inhibit" all refer to: In this specification, it is commonly used to mean a statistically significant decrease. However, to avoid any doubt, use "decreased," "decrease," or "decrease" or "Inhibit" means a reduction of at least 10% compared to the reference level, for example, at least Also about 20% or at least about 30% or at least about 40% or at least about 50% % or at least 60% or at least about 70% or at least about 80% or less A decrease of at least 90%, or a decrease including 100% or less and 100% (i.e.) (absence level compared to the reference sample), or 10-100 compared to the reference level. This means any decrease within the percentage range.

[0146] The terms "increased," "to increase," "to improve," or "to become more active" are all... In this specification, the term is commonly used to mean an increase of a statistically significant amount. To avoid any doubt arising, the aforementioned "increased," "increasing," or " The terms "improve" or "activate" indicate an increase of at least 10% compared to the reference level. Large, for example, at least about 20%, at least about 30%, or at least about 40% or at least about 50% or at least about 60% or at least about 70% or less An increase of at least 80% or at least 90%, or less than 100% and less than 100%. Including increases, or any increase between 10-100% compared to the reference level, or less At least twice as much, or at least three times as much, or at least four times as much, or at least five times as much. Or at least a tenfold increase, or between 2 and 10 times compared to the reference level. It means any increase.

[0147] The terms "statistically significant" or "significantly" mean statistical significance, and generally refer to This refers to two standard deviations (2SD) of the marker concentration below or lower than normal. The aforementioned terms mean statistical evidence that a difference exists. The aforementioned terms mean that the null hypothesis is true. If it is actually true, it is defined as the probability of making a decision to reject the null hypothesis. This is often done using p-values.

[0148] When used in this specification, the terms "including" or "contains" are essential combinations of the present invention. Used to refer to products, methods, and their individual components, but whether essential or not, It is open to the inclusion of undefined constituent elements.

[0149] When used in this specification, the term “essentially” is used as required for the given embodiment. It refers to those components. The terms above are fundamental and novel to the present invention and its embodiments. Alternatively, it allows for the presence of further components that do not substantially affect the functional characteristics.

[0150] The term "consisting of" means the compositions, methods, and their respective components described in this specification. and is exclusive to any component not enumerated in the description of the embodiment.

[0151] When used in this specification and the attached claims, the singular forms "a", "an", and "The" refers to multiple objects unless explicitly specified otherwise. For example, a reference to "the method" means one or more methods and / or the present application. This includes the types of processes described in the specification, and this is based on reading this disclosure, etc. This will become clear to those skilled in the art.

[0152] stem cells

[0153] Stem cells possess the ability to regenerate themselves through mitotic division, and are specialized in various ranges. These are cells that can differentiate into different cell types. Two broad types of mammalian stem cells are found in blastocysts. These are adult stem cells found in embryonic stem (ES) cells and adult tissues. In developing embryos, Stem cells can differentiate into all specific embryonic tissues. In adult organisms, stem cells and primordial cells... Cells act as the body's repair system, replenishing specific cells and regenerating organs, for example, They maintain the normal turnover of blood, skin, or digestive tract tissue. Pluripotent stem cells are three types. It can differentiate into cells obtained from any of the germ layers.

[0154] In certain embodiments, SC-β cells (e.g., mature pancreatic β cells or β-like cells) or so The use of stem cells to produce precursors is mentioned below, but germline cells The cell uses a protocol similar to the exemplary protocol described in this specification, To provide at least one SC-β cell, in place of the stem cell, or the stem It may be used with cells. Suitable germline cells are, for example, about 8-1 after the last menstrual period. It can be prepared from proto-germline cells present in human fetal material collected at 1 week. For example, the method for preparing breeding cells is described in Shamblott et al., Proc. Na tl. Acad. Sci. USA 95:13726, 1998 and U.S. It is described in Specification No. 6,090,622.

[0155] ES cells, for example, possess virtually unlimited replication capacity and the potential to differentiate into most cell types. Human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs) are, in principle, clinically... Provides an unlimited supply of starting material for generating differentiated cells for bed therapy (http: / / ste mcells.nih.gov / info / scireport / 2006report (.htm, 2006). One possible use of ES cells is, firstly, hESC, for example. From this, endoderm is produced, for example, adult endoderm, and then the adult endoderm is further reduced They are then differentiated into one insulin-positive endocrine cell or its precursor, and then further, Furthermore, the at least one insulin-positive endocrine cell or its precursor is SC-β By differentiating these cells, new pancreatic β-cells for cell replacement therapy in type 1 diabetes can be generated. That is what it is.

[0156] hESC is, for example, Cowan et al. (N Engl. J. Med.) 350:1353, 2004) and Thomson et al. (Science) Described in 282:1145, 1998), it is an embryonic stem cell derived from another primate, *Aka*. Ghezakan stem cells (Thomson et al., Proc. Natl. Acad (Sci. USA 92:7844, 1995), marmoset stem cells (Tho mson et al., Biol. Reprod. 55:254, 1996) and human embryonic germline (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998) also, It may be used in the method described in the application specification. mESC is, for example, Tremml et al.(Curr Protoc Stem Cell Biol. Chapter 1: As described in Unit 1C.4, 2008). The stem cells are, for example, monocellular. It can be pluripotent, totipotent, multipotent, or pluripotent. In some cases, at least one germ layer or The present specification describes any primate-derived cell capable of producing offspring obtained from all three germ layers. It is likely to be used in the methods disclosed.

[0157] In certain cases, ES cells are used, for example, Cowan et al. (N Engl. J (Med. 350:1353, 2004) and U.S. Patent No. 5,843,780 Detailed document and Thomson et al., Proc. Natl. Acad. It can be isolated as described in Sci. USA 92:7844, 1995. For example, hESC cells are described by Thomson et al. (US. 6,200,800) Specification No. 6;Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff This is described in et al, Nature Biotech. 18:399, 2000. Using the techniques described above, it can be prepared from human blastocyst cells as an equivalent cell type to hESCs. Its pluripotent derivatives, for example, WO01 / 51610 (Bresagen), are outlined below. Examples include protoectoderm-like (EPL) cells. hESCs can also be obtained from human preimplantation embryos. This is possible. Alternatively, in vitro fertilization (IVF) embryos can be used, and A single-cell human embryo can be grown to the blastocyst stage (Bongso et al.). al., Hum Reprod 4:706, 1989). Embryos are G1, G2 and G 2.2 Cultured in medium to the blastocyst stage (Gardner et al., Fer (til. Steril. 69:84, 1998). The zona pellucida is pronase (Sig Simple exposure to ma) removes it from the developed blastocyst. The inner cell population is immune. It can be isolated by immunosurgery. In the aforementioned immunosurgery, the blastocyst is treated with a 1:50 dilution of rabbit anti-Hypermethrin. The spleen cells were exposed to antiserum for 30 minutes, then washed three times in DMEM for 5 minutes each, and 1: Exposure to 5-dilution guinea pig complement (Gibco) for 3 minutes (Solter et al.) ., Proc. Natl. Acad. Sci. USA 72:5099, 1 975). Furthermore, after washing twice in DMEM, the lysed trophectoderm cells were gently... The intact internal cell population (ICM) is removed by pipetting. These are seeded onto the mEF feeder layer. After 9 to 15 days, proliferation from the internal cell population is observed. 1 mM EDTA-containing phosphate-buffered saline, free of calcium and magnesium. Exposure to (PBS), exposure to dispase or trypsin, Alternatively, the material is separated into clumps by mechanical separation using a micropipette, and then, They can be reseeded on mEF in a culture medium. Proliferating colonies with an undifferentiated morphology are then... They can be individually selected using a pipette, mechanically separated into clumps, and reseeded. (ES) Morphologically, it is characterized as a small colony with a clearly high nucleus / cytoplasmic ratio and a prominent nucleus. It can be removed. Then, the obtained hESCs are subjected to simple trypsinization every 1-2 weeks. Exposure to Dulbecco's PBS (containing 2 mM EDTA), type IV collagenase (approximately 200 Exposure to U / mL (Gibco) or individual colonies via micropipette The process is divided into routines based on the selection of the cells. In some cases, this involves approximately 50 to 100 cells. The aggregate size is optimal. mESC cells are, for example, Conner et al. (Cur r. Prot. in Mol. Biol. Unit 23.4, 2003) It can be prepared using the techniques described.

[0158] Embryonic stem cells can be isolated from blastocysts of members of primate species (U.S. Patent No. 5,843). , 780; Thomson et al., Proc. Natl. Aca d. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells are , Thomson et al (U.S. Pat. No. 6,200,806; Sciencec e 282:1145, 1998;Curr. Top. Dev. Biol. 3 8:133 ff., 1998) and Reubinoff et al, Natur Using the technique described in e Biotech. 18:399, 2000, human embryo It can be prepared from cyst cells. Equivalent cell types to hES cells include its pluripotent derivatives, e.g. For example, the protoectoderm-like (EPL) details outlined in WO01 / 51610 (Bresagen) Examples include cysts, etc.

[0159] Alternatively, in some embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilization (IVF) embryos can be used, or one Human embryonic cells can be proliferated to the blastocyst stage (Bongso et al., (Hum Reprod 4:706, 1989). Embryos were cultured in G1.2 and G2.2 medium. The blastocyst is cultured in the above-mentioned stage (Gardner et al., Fertil). Steril. 69:84, 1998). The zona pellucida is opposed to pronase (Sigma). The inner cell population is removed from the developed blastocyst by simple exposure. They are then isolated. In the aforementioned immunosurgery, the blastocysts are treated with a 1:50 dilution of rabbit anti-human spleen cell anti- The samples were exposed to serum for 30 minutes, then washed three times in DMEM for 5 minutes each, and then diluted 1:5 in molar solution. Exposure to Mott complement (Gibco) for 3 minutes (Solter et al., Pro c. Natl. Acad. Sci. USA 72:5099, 1975). Furthermore, after washing twice in DMEM, the lysed trophectoderm cells were gently pipetated. It is removed from the intact internal cell population (ICM) by the process. The ICM is then removed by mEF. It is sown on the feeder layer.

[0160] From day 9 to day 15, proliferation from the internal cell population was observed, containing 1 mM EDTA and calcium. And exposure to magnesium-free phosphate-buffered saline (PBS) can cause disease By exposure to spase or trypsin, or by mechanical means of a micropipette. By one of the separation methods, the cells are separated into clumps, and then re-seed onto mEF in a new culture medium. The growing colonies, which have an undifferentiated morphology, are individually selected using a micropipette. They are mechanically separated into clumps and re-seeded. ES-like morphology clearly shows a high nucleus / cell ratio and prominent They are characterized as small colonies with a nucleus. Next, the obtained ES cells are 1 -Every two weeks, for example, simple trypsinization, Dulbecco's PBS (containing 2 mM EDTA) Exposure to ) and exposure to type IV collagenase (approximately 200 U / mL; Gibco) can lead to Alternatively, the colonies can be routinely divided by selecting individual colonies using a micropipette. A cluster size of approximately 50 to 100 cells is optimal.

[0161] In some embodiments, human embryonic germline (hEG) cells are obtained using the method disclosed in this specification. hEG cells are pluripotent stem cells that can be used to differentiate into primordial endoderm cells. Prepared from proto-germ cells present in human fetal material collected approximately 8-11 weeks after menstruation. It can be used. A suitable preparation method is described in Shamblott et al., Proc. N. atl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent This is described in Patent No. 6,090,622. The entire document is referred to in this application. This will be incorporated by the specification.

[0162] In short, the genital ridges are treated to form deaggregated cells. The EG growth medium is DMEM DMEM is 4500 mg / L D-glucose, 2200 mg / L mM N aHCO3; 15% ES-grade fetal bovine serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U / mL human recombinant white Blood disease suppressor factors (LIF, Genzyme); 1-2 ng / mL Human recombinant bFGF(G enzyme; and 10 μM forskolin (in 10% DMSO). LI F, bFGF, or forcholine-free modified EG growth medium was cultured for 3 days, and 50 Feeder cells (e.g., STO cells, ATC) inactivated by 00 rad gamma irradiation. 96-well tissue culture plate containing a partially confluent layer (C No. CRL1503) Prepare the following: Add approximately 0.2 mL of primordial germline cell (PGC) suspension to each well. The first subculturing was performed in EG growth medium after 7-10 days, and each well was irradiated with STO mushrooms. Transfer the fibroblasts to one well of a 24-well culture dish prepared in advance. Typically, 7–30 days or 1–4 cycles are performed until a cell morphology consistent with the cell is observed. The culture medium is changed daily and the cells are incubated until the next generation.

[0163] In a particular example, the stem cells are subjected to at least one method described in this specification. Before exposure to one β-cell maturation factor, cells may be undifferentiated (for example, cells assigned to a specific lineage). (Cells that have not been treated). On the other hand, in other examples, at least one β cell as described in this specification. It is desirable to differentiate the stem cells into one or more intermediate cell types before exposure to maturation factors. There is a possibility of this. For example, the stem cells can be distinguished from differentiated cells of embryonic or adult origin. It may exhibit morphological, biological, or physical characteristics of undifferentiated cells, which can be used for this purpose. In some cases, undifferentiated cells are colonies of cells with a high nucleus / cytoplasmic ratio and prominent nuclei. In the case of the aforementioned stem cells, this may be evident in the two-dimensional microscopic field. However, often (for example, where no undifferentiated cells are virtually absent), or differentiated cells It may be used in the presence of. In certain examples, the stem cells are used when the stem cells proliferate and To promote further differentiation, they may be cultured in the presence of appropriate nutrients and, if applicable, other cells. For example, embryonic fibroblasts or fibroblast-like cells may support the proliferation of the stem cells. The fibroblasts may be present in the culture. The fibroblasts are present in one stage of stem cell proliferation. It may be done, but it is not necessary at every stage. For example, the fibroblasts are used in the initial culture. It is added to the stem cell culture at one stage, and in one or more subsequent culture stages, it is added to the stem cells. It's not necessary.

[0164] Stem cells used in all aspects of the present invention may be any type of tissue (e.g., embryonic tissue, e.g.) For example, these could be any cells obtained from fetal or pre-fetal tissue or adult tissue. In this case, stem cells are offspring of various cell types, for example, the three germ layers (endoderm, mesoderm, and ectoderm). Features that become possible under suitable conditions to produce at least one of all derivatives of the leaf These cell types may be provided in the form of established cell lines, or otherwise. These may be obtained directly from primary embryonic tissue and may be used immediately for differentiation. Cells listed in the embryonic stem cell registry, for example, hESBGN-01, hESBGN -02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6(ES C ell International);Miz-hES1(MizMedi Hosp ital-Seoul National University);HSF-1, HS F-6(University of California at San Fran Cisco; and H1, H7, H9, H13, H14 (Wisconsin Alum) ni Research Foundation(WiCell Research I This includes (institute). In some embodiments, it is converted to mature insulin-positive cells. The source of human stem cells or pluripotent stem cells used in scientifically induced differentiation is human embryos. It did not include destruction.

[0165] In another embodiment, the stem cells may be isolated from a tissue, such as a solid tissue. In the embodiment, the tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart or This is cardiac tissue. In other embodiments, the tissue may be, for example, umbilical cord blood, placenta, etc., without limitation. , bone marrow or cartilage.

[0166] The desired stem cells are those described in Thomson et al. (1998) Science. Human embryonic stem (hES) cells described in 282:1145; embryonic stem cells derived from other primates. For example, rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844), marmoset trunk slender cells (Thomson et al. (1996) Biol. Reprod. 55:2 54); and human embryonic germline (hEG) cells (Shambloft et al., P roc. Natl. Acad. Sci. USA 95:13726, 1998 Examples include various types of embryonic cells, as illustrated by ). Lineage-related stem cells, for example, Germ layer stem cells and other early cardiogenic cells are also included (Reyes et al. (2001) )Blood 98:2615-2625;Eisenberg & Bader(19 96) See Circ Res. 78(2):205-16, etc. ). The aforementioned stem cells. This applies to any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, etc. It may be obtained from mice, rats, hamsters, primates, etc. In some embodiments, The embryo is for the source of pluripotent cells used in the methods and compositions disclosed in this specification. It was not destroyed.

[0167] ES cells are undifferentiated when they have not been assigned to a specific differentiation line. It is possible. Such cells distinguish them from differentiated cells of embryonic or adult origin. Morphological characteristics are presented. Undifferentiated ES cells are easily recognizable to those skilled in the art and are typically clear. Two-dimensional microscopic view of cell colonies with a high nucleus / cytoplasmic ratio and prominent nuclei. This is evident in the field. Undifferentiated ES cells are used as markers to detect the presence of undifferentiated cells. It expresses genes that can be used for negative selection. It can be used as a maker. For example, U.S. Patent Application Publication No. 2003 / 0224411 Details;Bhattacharya(2004)Blood 103(8):2956-6 4; and see Thomson (1998) above. Each is described by reference in this application. It will be incorporated into the detailed documentation. Human ES cell lines are undifferentiated non-human primate ES cells and stage-specific cells. Different embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81 and Human EC cells express cell surface markers that characterize them, including alkaline phosphatase. The globo series glycolipid GL7 has the SSEA-4 epitope, and glob It is formed by adding sialic acid to the o-series glycolipid GbS. Leeds glycolipid GbS has the SSEA-3 epitope. Therefore, GL7 is SSE It reacts with antibodies against both A-3 and SSEA-4. The aforementioned undifferentiated human ES cell line is Differentiated cells do not stain with SSEA-1, but stain strongly with SSEA-I. Methods for proliferating S cells in an undifferentiated state are described in WO99 / 20741, WO01 / 51616 and It is listed in WO03 / 020920.

[0168] A mixture of cells from suitable sources of endothelial, muscle, and / or nerve stem cells is used in mammals. It can be recovered from the donor by methods known in the field. A suitable source is hematopoietic microscopy. The environment. For example, preferably, kinetic (i.e., mobilized) circulating peripheral blood It is extracted from the subject. Alternatively, bone marrow is extracted from mammals that have undergone autologous transplantation, for example, hi Stem cells may be obtained from patients. In some embodiments, stem cells are, for example, U.S.7. Disclosed in specifications No. 390,484 and No. 7,429,488, from Cytori The CELUTION® SYSTEM can be used to obtain from the target adipose tissue. The patent is incorporated in its entirety by reference into this specification.

[0169] In some embodiments, human umbilical cord blood cells (HUCBCs) are used in the method disclosed in this specification. It is useful for human UBC cells. Human UBC cells are recognized as a rich source of hematopoietic and mesenchymal progenitor cells. (Broxmeyer et al., 1992 Proc. Natl.) Acad. Sci. USA 89:4109-4113). Previously, umbilical cord blood and fetus Umbilical cord blood was considered waste that is normally discarded at the birth of a child. Umbilical cord blood cells are transplantable. As a source of viable stem cells and progenitor cells, and for malignant diseases (i.e., acute lymphoblastic leukemia) (Hematological diseases, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and neuroblastoma) and Bone marrow-reconstituted cells for the treatment of non-malignant diseases, such as Fanconi anemia and aplastic anemia. Used as a source (Kohli-Kumar et al., 1993 Br J. Haematol. 85:419-422;Wagner et al., 1992 Blood 79;1874-1881;Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78;Lu e et al., 1995 Cell Transplantation 4:493-5 03). Another advantage of HUCBCs is that these cells have immature immune systems that are very similar to fetal cells. It is infectious. The aforementioned immature immunity significantly reduces the risk of rejection by the host. (Taylor & Bryson, 1985J. Immunol. 134:1) 493-1497). Human umbilical cord blood can be proliferated in tissue culture, containing mesoderm and hematopoietic precursors. Includes progenitor cells and endothelial cell precursors (Broxmeyer et al., 1992) Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol 85:419-422;Wagner et al., 1992 Blood 7 9;1874-1881;Lu et al., 1996 Crit. Rev. O ncol. Hematol 22:61-78;Lu et al., 1995 C ell Transplantation 4:493-503;Taylor & B ryson, 1985J. Immunol. 134:1493-1497;Bro xmeyer, 1995 Transfusion 35:694-702;Chen et al., 2001 Stroke 32:2682-2688;Nieda et al., 1997 Br. J. Haematology 98:775-7 77;Erices et al., 2000 Br. J. Haematolog (y 109:235-242). The total amount of hematopoietic primordial cells in umbilical cord blood is the same as, or It surpasses that. Furthermore, highly proliferative hematopoietic cells are found in HUCBC eight times more abundantly than in bone marrow. It expresses blood markers, such as CD14, CD34, and CD45 (Sanchez -Ramos et al., 2001 Exp. Neur. 171:109-1 15;Bicknese et al., 2002 Cell Transplant ation 11:261-264;Lu et al., 1993 J. Exp. Med. 178:2089-2096).

[0170] In another embodiment, pluripotent cells are located in the hematopoietic microenvironment, for example, preferably the peripheral environment of mammals. Cells from the mononuclear fraction of blood, circulating peripheral blood, umbilical cord blood, bone marrow, fetal liver, or yolk sac. The aforementioned stem cells, particularly neural stem cells, can also be obtained from the central nervous system, for example, the meninges. ru.

[0171] In another embodiment, pluripotent cells are located in the embryoid body. The embryoid body is a simple protea ES cells are collected by enzyme digestion, and small clumps of undifferentiated human ESCs proliferate in suspension culture. It is formed by enabling this process. Differentiation is induced by the recovery of the conditioned medium. The embryoid bodies are seeded onto a semi-solid substrate. Differentiated cells are formed in approximately 7 days to 4 weeks. This may be observed later. Live, differentiating cells from in vitro cultures of stem cells. This is selected from similar structures that provide partially separated embryoid bodies or cell aggregates. The aggregate containing the desired cells substantially maintains the cells against cell contact in the aggregate. The phenotypic characteristics are selected using the following method.

[0172] In another embodiment, the stem cells are reprogrammed stem cells, for example, somatic cells or These may be stem cells obtained from differentiated cells. In such embodiments, dedifferentiated stem cells Cells are, for example, not limited to, newly generated cells, tumor cells and cancer cells, or induced reprogrammed cells. Lamb cells, for example, may be induced pluripotent stem cells or iPS cells.

[0173] Cloning and cell culture

[0174] Examples of molecular genetics and genetic engineering that may be used in the technologies described in this specification. One such method is, for example, Molecular Cloning: A Laboratories y Manual,(Sambrook et al., Cold Spring H arbor);Gene Transfer Vectors for Mammali an Cells(Miller & Calos eds.); and Current Protocols in Molecular Biology(FM Aus Found in the latest edition of Ubel et al. eds., Wiley & Sons. To obtain. Cell biology, protein chemistry, and antibody technology, for example, Current Prot ocols in Protein Science(JE Colligan et al. eds., Wiley & Sons);Current Proto cols in Cell Biology(JS Bonifacino et (al., Wiley & Sons) and Current protocols i n Immunology(JE Colligan et al. eds., It can be found in Wiley & Sons.) Examples of reagents for genetic engineering, chloroform The training vectors and kits are, for example, BioRad, Stratagene, Inv Itrogen, ClonTech, and Sigma-Aldrich Co. are commercially available. In some cases, this may be the case.

[0175] Appropriate cell culture methods include, for example, Culture of Animal Cells: A Manual of Basic Technique(RI Freshne y ed., Wiley & Sons);General Techniques of Cell Culture(MA Harrison & IF R ae, Cambridge Univ. Press) and Embryonic St. em Cells:Methods and Protocols(K. Turkse Cell culture as commonly described in the latest edition of (n ed., Humana Press) It can be found in the law. Suitable tissue culture supplies and reagents are, for example, Gibco / BRL. Nalgene-Nunc International, Sigma Chemica It is commercially available from l Co. and ICN Biomedicals.

[0176] Pluripotent stem cells are cultured under conditions that promote proliferation without promoting differentiation, and in our field It can be continuously grown in culture by the user. Typical serum-containing ES medium is 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% specified fetal dorsal Infant serum (FBS, Hyclone) or serum substitute (WO98 / 30679), 1% Non-essential amino acids, 1 mM L-glutamine and 0.1 mM β-mercaptoethanol It is composed of human bFGF added at 4 ng / mL immediately before use (WO99 / 20 741, Geron Corp.). Traditionally, ES cells are feeder cells, typically They are cultured on a layer of fibroblasts obtained from embryonic or fetal tissue.

[0177] Geron scientists found that pluripotent SCs can be maintained in an undifferentiated state even without feeder cells. We found that we could obtain a suitable cell substrate for the environment of feeder-free culture, especially Extracellular matrix, such as Matrigel® or laminin. In terms of type, enzymatic digestion stops before the cells are completely dispersed (for example, collagen (Approximately 5 minutes with -ze IV). Then, the cluster of approximately 10 to 2,000 cells is further dispersed. The seeds are sown directly onto the substrate without any further action.

[0178] Feeder-free culture involves using a nutrient medium containing factors that support cell proliferation without differentiation. It is more supported. Such factors are secreted by cells that secrete such factors, for example, irradiated (Approximately 4,000 rads) Primary mouse embryonic fibroblasts, telomerized mouse fibroblasts By culturing fibroblast-like cells obtained from cells or pPS cells with the aforementioned culture medium, It may be introduced into the culture medium. The medium may be serum-free medium, for example, a 20% serum substitute and Approximately 5-6 × 10 units were added to KO DMEM containing 4 ng / mL bFGF. 4 pieces / cm - 2 The medium can be acclimatized by seeding the feeder at a density of 1-2 days. Furthermore, bFGF is added and used to support pluripotent SC cultures for 1-2 days. The characteristics of the feeder-free culture method are described in International Publication No. 01 / 51616; and X u et al., Nat. Biotechnol. 19:971, 2001. Further consideration is being given to this matter.

[0179] Under microscopic observation, ES cells exhibit a high nucleus-to-cytoplasm ratio, prominent nuclei, and are almost indistinguishable. It appears in small colonies due to intercellular junctions that cannot be formed. Primate ES cells are st Dispecific embryonic antigens (SSEA) 3 and 4, as well as Tra-1-60 and Tra-1-81 The marker is expressed using the specified antibody (Thomson et al.). al., Science 282:1145, 1998). Mouse ES cells are SS As a positive control for EA-1, as well as SSEA-4 and Tra-1 It can be used as a negative control for -60 and Tra-1-81. SEA-4 is consistently present in human embryonic cancer (hEC) cells. In pluripotent SCs In vitro differentiation involves the development of SSEA-4, Tra-1-60, and Tra-1-81. This results in current loss and increased expression of SSEA-1. Increased expression of SSEA-1 is It is also found in undifferentiated hEG cells.

[0180] Stem cell line β cell (SC-β)

[0181] In some embodiments, this disclosure provides stem cell-derived β-cells (SC-β). The SC-β cells shown share many of the discriminative properties of native β cells, but in certain aspects (e.g.) For example, they differ in their gene expression profiles. In some embodiments, the SC-β particles The cells are unnatural. As used in this specification, "unnatural" means that the SC-β cells are unnatural. In certain aspects, naturally occurring β-cells, i.e., cells that are quite different from natural β-cells This means that, however, these significant differences typically indicate specific functional differences. This relates to the structural characteristics that can give rise to SC-β cells, for example, the genes of SC-β cells. The expression pattern should be understood to be different from that of natural β cells. The SC-β cells are They behave in a manner similar to natural beta cells, but with altered functions compared to natural beta cells. In some cases, this may be the case (for example, it may have been improved). For example, as shown in Figure 2E, Compared to the frequency of natural β-cells, SC-β-cells are more frequently responsive to 20 mM glucose. Responding to the data disclosed in this specification, the differences between SC-β cells and native β cells are as follows: Based on this, it should be obvious to those skilled in the art.

[0182] The SC-β cells of this disclosure share many characteristic features of β cells that are important for the function of normal β cells. It has. In some embodiments, the SC-β cells are subjected to glucose in vitro. It exhibits a strong insulin secretion (GSIS) response. In some embodiments, the SC-β cells are It shows the GSIS response in vivo. In some embodiments, the SC-β cells are i The GSIS response is shown in vitro and in vivo. In some embodiments, the above The GSIS response is similar to the endogenous GSIS response of mature pancreatic β-cells. In some embodiments, The SC-β cells respond to at least one glucose challenge with GSIS. The answer is given. In some embodiments, the SC-β cells undergo at least two consecutive gluco- The SC-β cells exhibit a GSIS response to the challenge. In some embodiments, the SC-β cells It exhibits a GSIS response to at least three consecutive glucose challenges. In the embodiment of the part, the GSIS response is intrinsically in response to multiple glucose challenges. It is similar to the GSIS response of human pancreatic islets. In some embodiments, the GSIS response is similar to that of pre- The GSI is observed immediately after transplantation of the cells into humans or animals. In some embodiments, the GSI The S response is observed within approximately 24 hours of transplanting the cells into humans or animals. In the administration method, the GSIS response occurs within approximately one week of transplanting the cells into a human or animal. Observed. In some embodiments, the GSIS response transfers the cells to humans or animals. Observed within approximately two weeks of planting. In some embodiments, compared to low glucose concentrations, Characterized by the percentage of insulin secreted in response to high glucose concentrations. The stimulation index of the aforementioned cells is similar to that of endogenous mature pancreatic β-cells. In the application form, the SC-β cells exhibit a stimulation index greater than 1. In some embodiments, The SC-β cells exhibit a stimulation index of 1 or more. In some embodiments, the SC-β cells This exhibits a stimulation index greater than 1.1. In some embodiments, the SC-β cells are 1. It exhibits a stimulation index of 1 or greater. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. The index is shown. In some embodiments, the SC-β cells exhibit a stimulation index of 2 or more. In this embodiment, the SC-β cells are at least 2.1, 2.2, 2.3, 2.4, 2 0.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3 0.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4 It exhibits a stimulation index of 0.5, 4.6, 4.7, 4.8, 4.9, or 5.0 or higher.

[0183] In some embodiments, the SC-β cells respond to cytokines, It exhibits tokine-induced apoptosis. In some embodiments, the SC-β cells are inter - Leukin-1β (IL-β), interferon-γ (INF-γ), tumor necrosis factor - Response to cytokines selected from the group consisting of α(TNF-α) and combinations thereof The answer demonstrates cytokine-induced apoptosis.

[0184] In some embodiments, insulin secretion from the SC-β cells is controlled by known antidiabetic agents ( For example, antidiabetic agents that act on β-cells ex vivo or in vitro. and / or generally enhanced response to antidiabetic agents in vivo. This disclosure considers any known antidiabetic agent. In some embodiments, the SC-β Insulin secretion from cells is enhanced in response to secretagogues. In terms of form, the secretion-promoting substance is an incretin mimetic, sulfonylurea, meglitinide, and A selection is made from a group consisting of combinations of these.

[0185] In some embodiments, the SC-β cells are monohormone. The SC-β cells exhibit a morphology similar to that of endogenous mature pancreatic β cells. In some embodiments, the SC-β cells contain crystalline insulin granules. The SC-β cells are similar to the insulin granules of endogenous mature pancreatic β cells, as observed under electron microscopes. The image shows encapsulated crystalline insulin granules under microscopic observation. In some embodiments, the SC-β The cells exhibit slow replication. In some embodiments, the SC-β cells exhibit slow replication. In some embodiments, the SC-β cells are subjected to a slow but prolactin treatment. In the response to the above, when measured by staining with C-peptide and Ki67, This demonstrates improved speed replication.

[0186] In some embodiments, the SC-β cells respond to glucose intracellularly Ca 2+ To increase. In some embodiments, the SC-β cells are endogenous mature pancreatic β Glucose-stimulated Ca, similar to cellular GSCF 2+ This shows the flow (GSCF). Partial implementation Morphologically, the SC-β cells are endogenous mature pancreatic cells that have undergone multiple glucose challenges. A method similar to the GSCF of visceral β cells, at least three consecutive glucose challenges This shows the GSCF response to the range.

[0187] In some embodiments, the SC-β cells are subjected to insulin, C-peptide, PDX1, M AFA, NKX6-1, PAX6, NeuroD1, Glucokinase (GCK), SLC2 A1, PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB Selected from the group consisting of 3A, GAD2, and PTPRN, it is immobilized on endogenous mature pancreatic β-cells. It expresses at least one of the markers present.

[0188] In some embodiments, the SC-β cells are a)i) glucagon (GCG) and ii) A hormone selected from the group consisting of somatostatin (SST); b)i) amylase and ii) Glandular cell markers selected from the group consisting of carboxypeptidase A (CPA1) c)i)α-cells selected from the group consisting of GCG, Arx, Irx1 and Irx2 d) i) CFTR and ii) Sox9 are selected tubular cell markers from the group, At least one marker selected from the group consisting of (e.g., endogenous mature pancreatic β cells) It does not express markers that are not expressed by [the relevant factor].

[0189] The present invention is limited to the starting cells from which the SC-β cells are obtained. If not intended, differentiation occurs in vitro from any starting cell. The typical initiating cell is not limited to insulin-positive endocrine cells or any of their precursors. The body, for example, Nkx6-1 positive primordial pancreatic cells, Pdx1 positive primordial pancreatic cells, and pluripotent stem cells. Examples include cells, embryonic stem cells, and induced pluripotent stem cells. In some embodiments, the SC- Beta cells are reprogrammed cells, partially reprogrammed cells (i.e., induced A partially reprogrammed cell exists in an intermediate state between pluripotent cells and the somatic cells from which they are derived. Gram-derived, somatic cells (e.g., fibroblasts), converted cells, in vit Differentiation occurs in ro. In some embodiments, the SC-β cells disclosed in this specification are Differentiated in vitro from insulin-positive endocrine cells or their precursors. Obtain. In some embodiments, the SC-β cells are Nkx6-1 positive pancreatic progenitor cells, Pd From a precursor selected from the group consisting of x1-positive pancreatic progenitor cells and pluripotent stem cells, in v Differentiation occurs in ITR. In some embodiments, the pluripotent stem cells are embryonic stem cells and Selected from the group consisting of SC-β cells and induced pluripotent stem cells. In some embodiments, the SC-β cells Alternatively, the pluripotent stem cells from which the SC-β cells are obtained are human. In some embodiments, The SC-β cells are human.

[0190] In some embodiments, the SC-β cells are not genetically modified. Morphologically, the SC-β cells, in the absence of genetic recombination of cells, are similar to native β cells. They acquire features that are commonly shared with each other. In some embodiments, the SC-β cells are genes It has been rearranged.

[0191] In some embodiments, the insulin produced per SC-β cell is high glucose In a 30-minute incubation at a concentration (e.g., ex vivo), cells 10 There is at least 0.5 μIU per 00 units.

[0192] In some embodiments, the insulin produced per SC-β cell is high glucose During incubation at a concentration of 1,000 cells for 30 minutes, at least 1, At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 μIU. In some embodiments, the SC-β Insulin produced per cell is obtained after 30 minutes of incubation at a high glucose concentration. In this case, the dose is between 0.5 μIU and 10 μIU per 1000 cells. Morphologically, the insulin produced per SC-β cell is 3 at high glucose concentrations. During a 0-minute incubation period, the concentration is approximately 2.5 μIU per 1000 cells.

[0193] In certain aspects, this disclosure provides cell lines including the SC-β cells described in this specification. In some embodiments, the SC-β cells stably express insulin. In some embodiments, the SC-β cells maintain their distinct morphology for at least 30 passages. Without any change, freezing, thawing, and amplification with doubling times of 24 and 44 hours are possible. can.

[0194] Generation of SC-β cells

[0195] Aspects of this disclosure relate to generating SC-β cells (e.g., pancreatic β cells). Specifically, at least one of the SC-β cells or its precursor, for example, as described in this specification. The pancreatic precursor produced based on the indicated method is a mixture or combination of various cells, e.g. For example, Pdx1-positive pancreatic precursors, and pancreatic precursors that co-express Pdx1 and NKX6-1. Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., β-like cells) and A mixture of cells such as ncerin-positive endocrine cells and / or other pluripotent cells or stem cells. It may include.

[0196] The aforementioned at least one SC-β cell or its precursor is a stem cell or a pluripotent cell. It can be produced based on any appropriate culture protocol that differentiates it to the desired stage of differentiation. In some embodiments, the at least one SC-β cell or its precursor is the few Differentiate at least one pluripotent cell into at least one SC-β cell or its precursor. To culture at least one pluripotent cell for a suitable period and under appropriate conditions. It is produced by [the following mechanism].

[0197] In some embodiments, the at least one SC-β cell or its precursor is SC- It is a substantially pure collection of β cells or their precursors. In some embodiments, SC-β cells The cell or its precursors contain a mixture of pluripotent or differentiated cells. In the application morphology, the aggregate of SC-β cells or their precursors is also embryonic stem cells or pluripotent cells. Some do not contain or lack iPS cells.

[0198] In some embodiments, somatic cells, such as fibroblasts, are taken from a subject, for example, a tissue biopsy. For example, isolated from skin biopsy, etc., and reprogrammed to induce pluripotent stem cells for further differentiation. For use in the compositions and methods described in this specification, at least One of the SC-β cells or its precursor may be produced. In some embodiments, somatic cells, For example, fibroblasts are maintained in culture by methods known to those skilled in the art, and in some embodiments They are then proliferated before being converted to SC-β cells by the method disclosed in this specification.

[0199] In some embodiments, the at least one SC-β cell or its precursor is Maintained in culture by known methods, and in some embodiments, as disclosed in this specification. Depending on the method, they proliferate before being converted to SC-β cells.

[0200] Furthermore, at least one SC-β cell or its precursor, for example, a pancreatic precursor, is responsible It may originate from any mammalian species. Non-limiting examples include mice, cattle, monkeys, pigs, and horses. Examples include bovine or human cells. For clarity and simplicity, the present specification is used. The description of the method means at least one mammalian SC-β cell or its precursor, All methods described in this specification involve at least one SC-β cell of another cell type or It can be easily applied to the precursor. In some embodiments, the at least one SC-β Cells or their precursors are obtained from human organisms.

[0201] Induction of differentiation of pluripotent stem cells into adult endoderm cells

[0202] Aspects of this disclosure include adult endoderm cells. Adult endoderm cells useful in this specification Cells can be obtained from any source, or live based on any appropriate protocol. This can be achieved. In some embodiments, pluripotent stem cells, such as iPSCs or hES cells, are used. C differentiates into endodermal cells. In some embodiments, the endodermal cells are gastrulatal cells, Pd x1-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells They are further differentiated into insulin-positive endocrine cells, and subsequently induced into SC-β cells or To ripen.

[0203] In some embodiments, the stem cells may be seeded on a new substrate, or previously The culture medium may be replaced to remove extracellular matrix or soluble factors that inhibit differentiation. This is sometimes called the "direct differentiation method," and is referred to in International Publication No. 01 / 51616. Fret and the specification of U.S. Patent Application Publication No. 2002 / 0019046 use the term as a general term. It is stated therein. The patent is incorporated in its entirety by reference into this specification. To avoid potential complications in the differentiation process caused by feeder cells Therefore, the direct differentiation method, which begins with feeder-free culture of stem cells, is usually preferred. The step involves placing undifferentiated stem cells into a suspension culture. The aforementioned suspension culture is often, These will cause aggregates of differentiated cells and aggregates of undifferentiated cells. For example. The stem cells were collected by simple collagenase digestion, separated into clusters, and then separated into non-adherent cells. The aggregates can be subcultured in a cell culture plate. The aggregates are supplied with nutrients every few days, and then They can be collected after an appropriate period, typically 4-8 days. Depending on the conditions, aggregation is common. This begins by forming a heterogeneous collection of cell types, including endodermal cells at substantial frequency. Next, the aggregates are dispersed and injected with laminin or phytophosphate for the next stage of the differentiation process. The seeds are re-seeded on a substrate such as bronnectin, or, for example, on a non-adhesive plate and suitable It can be subcultured in suspension culture using a suitable culture medium.

[0204] Direct differentiation or differentiation in aggregates is indicated by appropriate markers, e.g., U.S. 7,32 The presence of endodermal cells is monitored using the devices listed in Specification No. 6,572. It is possible. In some preferred embodiments, differentiation is performed using markers such as Sox17. The presence of germ layer cells can be monitored. If a sufficient proportion of endoderm is obtained, the cells will They can be reseeded or manipulated in other ways to initiate another stage of differentiation. In this situation, cells are maintained in micro-clusters (e.g., 50 to 5,000 cells). If present, cell differentiation or maintenance may be improved. Further differentiation as considered in this disclosure The stages are shown in Figure 1.

[0205] In some embodiments, adult endoderm cells are used to produce pluripotent stem cells, as defined in U.S. Law 8,507. Contacting with the compound of formula (I) described in Specification No. 274 ("'274 Patent") For example, it is produced by culturing. The said patent is incorporated herein by reference. The compound of formula (I) described in the aforementioned '274 patent is a cell-permeable small molecule, and Regulate cellular processes by regulating signaling pathways, gene expression, or metabolism. can be effectively used in stem cell differentiation protocols. Small molecules are of high quality and purely synthetic, can be conveniently supplied or removed, and offer great potential for therapeutic applications. High-throughput screening has been carried out to identify novel small molecules that can support the cardiogenic specification and differentiation of specific cell types, particularly neural and muscle cells, of ES cells (Chen et al., 2006; Desbordes et al., 2008), mouse ES cells (Wu et al., 2004) or neural progenitor cells (Diamandis et al., 2007) (reviewed in Ding and Schultz, 2004). The compounds of formula (I) from the '274 patent are expected to be used to differentiate pluripotent stem cells into somatic endoderm cells. t al., 2006;Desbordes et al., 2008)、マウスE S細胞(Wu et al., 2004)または神経始原細胞(Diamandis et al., 2007)の心原性仕様及び特定細胞種、特に、神経及び筋肉の細胞の 誘導(Ding and Schultz, 2004に概説)を支持し得る新規な小分 子を特定するのに行われている。前記‘274特許からの式(I)の化合物は、多能性幹 細胞を成体型内胚葉細胞に分化させるのに使用され得るのが期待される。

[0206] In some embodiments, the compounds of formula (I) from the '274 patent

[0207]

Chemical formula

[0208] include formula (I).

[0209] Wherein,

[0210] R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or cycloalkenyl, each optionally substituted, and / or interrupted in the backbone by one or more O, N, S, S(O) and C(O ), and can be interrupted by one or more O, N, S, S(O) and C(O ), and / or interrupted in the backbone by one or more O, N, S, S(O) and C(O );

[0211] R3 and R 4 These are independently H, halogen, alkyl, alkenyl, alkynyl, and aldehyde. Coxy, aryl, heteroaryl, cyclyl, or cyclyl, respectively, in the case of It can be replaced, or R 3 and R 4 The carbon to which they are attached Together, they form cyclils that are substituted in the case of heterocyclils; and,

[0212] L is C1-C 10 Alkyrenyl, C2- 10 Alkenylenyl or C2- 10 Alki It is nirenyl, and each may be substituted in some cases, and / or bone Within the bracket, there may be interruptions by one or more O, N, S, S(O), and C(O). Cut.

[0213] In some embodiments, the compound of formula (I) from the aforementioned '274 patent is as follows: include.

[0214] [ka]

[0215] In some embodiments, the compound of formula (I) from the aforementioned '274 patent is as follows: include.

[0216] [ka]

[0217] The aforementioned '274 patent next describes a method for confirming the identification of obtained adult-type endoderm cells. Furthermore, methods for isolating, separating, propagating, and further differentiating adult endoderm are described. All of the aforementioned adult endoderms are described in this specification in a manner that will be understood by those skilled in the art. It can be used in the compositions and methods described.

[0218] In some embodiments, at least some of the aggregated pluripotent cells are divided into adult endoderm cells. By transforming it, for example, the aggregate of pluripotent cells is i) TGF-β superfamily ii) Contact with at least one growth factor from and a WNT signaling pathway activator This induces the differentiation of at least some of the aforementioned pluripotent cells into adult endodermal cells. Adult-type endodermal cells can be obtained. In this case, the adult-type endodermal cells are adult It expresses at least one marker specific to the endoderm type.

[0219] This disclosure relates to the TGF- that induces the pluripotent stem cells to differentiate into adult endoderm cells. Any growth factor from the β superfamily (e.g., alone or with WNT signaling) Consider using the TG (in combination with a transmission path activator). In some embodiments, the TG The at least one growth factor from the F-β superfamily comprises activin A. In some embodiments, the at least one of the TGF-β superfamily Growth factors include growth differentiation factor 8 (GDF8).

[0220] This disclosure relates to any WNT that induces the pluripotent stem cells to differentiate into adult endoderm cells. Signaling pathway activators (for example, alone, or the TGF-β superfluid) Consider using it in combination with growth factors from Millie. In some embodiments, the WN The T signaling pathway activator includes CHIR99021. In some embodiments, The WNT signaling pathway activator includes the Wnt3a recombinant protein.

[0221] Those skilled in the art will understand that the concentration of the active ingredient used (e.g., growth factor) may change. It will be understood. In some embodiments, the pluripotent cells are 10 ng / mL-1000 ng At concentrations between g / mL, the at least one of the TGF-β superfamily The cells are brought into contact with growth factors. In some embodiments, the pluripotent cells are brought into contact with a concentration of 100 ng / mL. At a certain degree, the at least one growth factor from the TGF-β superfamily is in contact with the TGF-β superfamily. In some embodiments, the pluripotent cells are administered at concentrations of 20 ng / mL, 30 ng / mL, and 40 ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL or At a concentration of 90 ng / mL, at least one of the TGF-β superfamily It is brought into contact with the growth factor. In some embodiments, 91 ng / mL, 92 ng / mL, 93 ng / mL, 94ng / mL, 95ng / mL, 96ng / mL, 97ng / mL, 98 At concentrations of ng / mL or 99 ng / mL, the preceding TGF-β superfamily The cells are brought into contact with at least one growth factor. In some embodiments, the pluripotent cells are brought into contact with 1 10ng / mL, 120ng / mL, 130ng / mL, 140ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, or 190ng / mL At a concentration of L, the at least one growth factor from the TGF-β superfamily and Bring to contact. In some embodiments, the pluripotent cells are given 10¹ ng / mL, 10² ng / mL, 103ng / mL, 104ng / mL, 105ng / mL, 106ng / mL, 1 At concentrations of 0.7 ng / mL, 10.8 ng / mL, or 10.9 ng / mL, the TGF-β solution is administered. Contact with at least one growth factor from the super family.

[0222] In some embodiments, the pluripotent cells are subjected to a concentration between 1.4 μg / mL and 140 μg / mL. The WNT signaling pathway activator is brought into contact with the WNT signaling pathway activator at a certain concentration. In some embodiments, The pluripotent cells were subjected to the WNT signaling pathway activator at a concentration of 14 μg / mL. To bring into contact with. In some embodiments, the pluripotent cells are given 2 μg / mL, 3 μg / mL, 4μg / mL, 5μg / mL, 6μg / mL, 7μg / mL, 8μg / mL, 9μg / m At concentrations of L, 10 μg / mL, 11 μg / mL, 12 μg / mL, or 13 μg / mL, It is brought into contact with the WNT signaling pathway activator. In some embodiments, the multi-functional Sex cells, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL g / mL, 20μg / mL, 21μg / mL, 22μg / mL, 23μg / mL, 24μ g / mL, 25μg / mL, 26μg / mL, 27μg / mL, 28μg / mL, 29μ At a concentration of g / mL or 30 μg / mL, the WNT signaling pathway activator is contacted. Allow to touch. In some embodiments, the pluripotent cells are given 13.1 μg / mL, 13.2 μg / mL, 13.3μg / mL, 13.4μg / mL, 13.5μg / mL, 13.6μg At concentrations of / mL, 13.7μg / mL, 13.8μg / mL, or 13.9μg / mL, It is brought into contact with the WNT signaling pathway activator. In some embodiments, the multi-functional sex cells, 14.1μg / mL, 14.2μg / mL, 14.3μg / mL, 14.4μ g / mL, 14.5μg / mL, 14.6μg / mL, 14.7μg / mL, 14.8μ The WNT signaling pathway activator at a concentration of g / mL or 14.9 μg / mL. Make contact with it.

[0223] Generally speaking, the pluripotent cells are adult endoderm cells of at least some of the pluripotent cells. Maintained in a suitable culture medium for a sufficient period to induce differentiation (e.g., suspension culture) Typical suitable culture media are shown in Table 1 below.

[0224] [Table 1]

[0225] In some embodiments, a culture medium suitable for differentiating pluripotent cells into adult endoderm cells. This includes S1 medium.

[0226] In some embodiments, contact of the pluripotent cells is achieved in a suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In this state, the period is 3 days. In some embodiments, the TGF-β superfamin The aforementioned at least one growth factor and WNT signaling pathway activator from Lee, It is added to the suspension culture on the first day. The aforementioned at least one growth factor from Lee is supplemented on day 2 during the suspension culture. In one embodiment, the WNT signaling pathway activator is used in the suspension culture for 2 days. It is not replenished in the eyes. In some embodiments, the WNT signaling pathway activator is It is removed from the suspension culture on the second day. In some embodiments, the TGF-β superf At least one growth factor from the family is supplemented on day 2 during the suspension culture. The WNT signaling pathway activator is removed from the suspension culture on day 2. or not supplemented during the suspension culture. In some embodiments, the TGF-β super - At least one growth factor from the family or the WNT signaling pathway None of the cutiverters are replenished on the third day during the suspension culture. In some embodiments, The at least one growth factor from the TGF-β superfamily and the WNT Both signaling pathway activators are removed from the suspension culture on day 3.

[0227] The above method involves the differentiation of at least one pluripotent cell in a cell aggregate into an adult endodermal cell. Inducible. Generally, any pluripotent cells can be used by the method described in this specification. They can then differentiate into adult endoderm cells. In some embodiments, the pluripotent cells are induced Includes pluripotent stem cells. In some embodiments, the pluripotent cells include embryonic stem cells. In this embodiment, the pluripotent cells include human cells.

[0228] In some embodiments, at least some of the aggregated pluripotent cells are differentiated into adult endoderm cells. This involves using i) activin A and ii) CHIR99021 to aggregate pluripotent cells. By bringing them into contact, differentiation of at least some of the pluripotent cells in the aggregate into adult endodermal cells is induced. This is achieved by the following method. In this case, the adult endoderm cells are intrinsically related to the adult endoderm. It expresses at least one marker.

[0229] Other methods for producing adult endoderm cells are known in the art, for example, U.S. Patent Application Publication No. 2006 / 0003446 (G. Keller, et al.) .);No. 2006 / 0003313 (K. D'Amour, et al.,), Same No. 2005 / 0158853 (K. D'Amour, et al.,) and the same Specification No. 2005 / 0260749 (Jon Odorico, et al.) explains The methods revealed are listed below. The relevant parts of the said patent are incorporated into this specification by reference. .

[0230] In some embodiments, adult endoderm cells produced by the method disclosed herein. These are Nodal, Tmprss2, Tmem30b, St14, Spink3, and Sh3g. l2, Ripk4, Rab15, Npnt, Clic6, Cldn8, Cacna1b, A group consisting of Bnipl, Anxa4, Emb, FoxA1, Sox17, and Rbm35a. It expresses at least one marker selected from. In this case, the at least one The expression of the two markers is compared to the pluripotent cells from which it is obtained, and to the adult endodermal cells. Within this context, it is upregulated by a statistically significant amount. In some embodiments, the present specification is Adult endoderm cells produced by the disclosed method are Gata4, SPARC, AFP At least one marker selected from the group consisting of and Dab2 is obtained before it is obtained. It is not expressed in statistically significant amounts in pluripotent cells. In some embodiments, Adult endoderm cells produced by the method disclosed in the patent application are Zicl, Pax6, At least one marker selected from the group consisting of Flk1 and CD31 is obtained In the aforementioned pluripotent cells, it is not expressed in statistically significant amounts.

[0231] In some embodiments, adult endoderm cells produced by the method disclosed herein. This provides a statistically significant amount of higher levels to the pluripotent cells from which it is obtained. It has phosphorylation of Smad2. In some embodiments, by the method disclosed in this specification The adult-type endoderm cells produced have the ability to form the digestive tract in vivo. In some embodiments, adult endoderm produced by the method disclosed herein. Cells can differentiate into cells that have a morphology specific to intestinal cells. In this case, the morphology specific to intestinal cells Cells possessing this express FoxA2 and / or Claudin 6. Some implementations In this state, adult endodermal cells produced by the method disclosed in this specification are of endodermal origin. These cells can further differentiate into other cells.

[0232] In some embodiments, the aggregation of pluripotent cells occurs before any differentiation or during the first stage of differentiation. They are cultured in the presence of at least one β-cell maturation factor. Any pluripotent stem cells For example, human pluripotent stem cells or human iPS cells or pluripotent cells as described in this specification. One of the sex stem cells or other suitable pluripotent stem cells may be used. In some embodiments, The β-cell maturation factors described in this specification are present in the culture medium of the aggregate of pluripotent stem cells. It is possible, or during the proliferation (e.g., replication or growth) of the aggregate of pluripotent stem cells, It may be added as a bolus or periodically. In certain cases, the aggregation of pluripotent stem cells Before any differentiation, they may be exposed to at least one β-cell maturation factor. In other cases, pluripotency Stem cell aggregation may also occur if exposed to at least one β-cell maturation factor during the first stage of differentiation. stomach.

[0233] Induction of differentiation of adult endoderm cells into gastrulatic cells

[0234] Aspects of this disclosure include gastrulatum cells. Useful gastrulatum cells in this specification are any It can be obtained from a source or generated based on any suitable protocol. In some embodiments, adult endoderm cells differentiate into gastrulatal cells. Examples of cells include Pdx1-positive primordial pancreatic cells, NKX6-1-positive primordial pancreatic cells, and Ngn3-positive cells. The progenitor cells of endocrine disorders further differentiate into insulin-positive endocrine cells, and subsequently into SC-β cells. To induce or mature.

[0235] In some embodiments, at least some of the aggregated adult endoderm cells are separated into gastrullous cells. By doing so, for example, adult endoderm cells can be converted into fibroblast growth factor (FGF) cells. Contacting at least one growth factor from Millie with at least a portion of the adult-type endoembryon By inducing the differentiation of lobe cells into gastrullous cells, gastrullous cells can be obtained. In this case, the gastrulatic cells express at least one marker specific to the gastrulatic cells. ru.

[0236] This disclosure relates to the FGF family that induces adult endoderm cells to differentiate into gastrullous cells. - Consider using any growth factor from (for example, alone or in combination with other factors) Consider. In some embodiments, the at least one growth factor from the FGF family The product contains keratinocyte growth factor (KGF). In some embodiments, the FGF formula is used. The at least one growth factor from Milly includes FGF2. In some embodiments, The at least one growth factor from the FGF family includes FGF8B. In this embodiment, the at least one growth factor from the FGF family is FGF Includes 10. In some embodiments, the at least one component from the FGF family The growth factor includes FGF21.

[0237] Those skilled in the art will understand that the concentration of the growth factors used may vary. In one embodiment, the adult endoderm cells are concentrated between 5 ng / mL and 500 ng / mL. At a certain degree, contact with at least one growth factor from the FGF family. In the embodiment, the adult endoderm cells are divided into 10 ng / mL, 15 ng / mL, and 20 ng / In concentrations of mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, or 40 ng / mL , to be brought into contact with at least one growth factor from the FGF family. Partial implementation Morphologically, the adult endoderm cells were divided into 60 ng / mL, 65 ng / mL, and 70 ng / mL. , 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL Alternatively, at a concentration of 100 ng / mL, the at least one component from the FGF family In contact with growth factors. In some embodiments, the adult endoderm cells are treated with 41 ng / mL. 42ng / mL, 43ng / mL, 44ng / mL, 45ng / mL, 46ng / mL, The FGF family at concentrations of 47 ng / mL, 48 ng / mL, or 49 ng / mL. In some embodiments, the adult form is brought into contact with at least one of the growth factors. Germ layer cells, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, or 59 ng / mL At a certain concentration, it is brought into contact with at least one growth factor from the FGF family. In this embodiment, the adult endodermal cells are subjected to the FGF famine at a concentration of 50 ng / mL. The at least one growth factor from Lee is brought into contact with it.

[0238] In some embodiments, the adult endodermal cells are cultured in a suitable culture medium.

[0239] Generally speaking, the adult endoderm cells are gastrulated from at least some of the adult endoderm cells. They are maintained in a suitable culture medium for a period sufficient to induce differentiation into tubular cells (e.g., suspended (Turbid culture). Typical appropriate culture media are shown in Table 2 below.

[0240] [Table 2]

[0241] In some embodiments, a culture medium suitable for differentiating adult endoderm cells into gastrullous cells. This includes S2 medium.

[0242] In some embodiments, contact of the adult endoderm cells is achieved in suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is between 2 and 5 days. In some embodiments, the period is , for 3 days. In some embodiments, the suspension culture is replenished once every 2 days.

[0243] In some embodiments, at least some of the aggregated adult endodermal cells are converted into gastrullary cells. By differentiating them, for example, by bringing the adult endodermal cells into contact with KGF, at least Furthermore, by inducing the differentiation of some of the aforementioned adult endoderm cells into gastrulatal cells, adult endoderm cells Leaf cells can be obtained. In this case, the gastrullary cells are less specific to the adult endoderm. Both express one marker.

[0244] Induction of differentiation of gastrullous cells into Pdx1-positive pancreatic progenitor cells

[0245] Aspects of this disclosure include Pdx1-positive pancreatic progenitor cells. Pdx useful in this specification 1. Positive pancreatic progenitor cells can be obtained from any source or based on any appropriate protocol. They can be generated accordingly. In some embodiments, gastrulatal cells differentiate into Pdx1-positive progenitor pancreatic cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are, for example, NKX6-1-positive. Further differentiation into pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, and insulin-positive endocrine cells. Then, they are induced into or mature into SC-β cells.

[0246] In some embodiments, at least some of the aggregated gastrulatic cells are Pdx1-positive pancreatic progenitor cells. By differentiating them, for example, gastrulatal cells can be transformed into i) at least one bone morphogenetic tan ii) Protein (BMP) signaling pathway inhibitors, at least one from the FGF family iii) at least one growth factor, iv) at least one SHH pathway inhibitor thinoic acid (RA) signaling pathway activator; and v) at least one protein By contacting with the kinase C activator, at least some of the gastrulatinous cells were found to be positive for Pdx1. By inducing differentiation into Pdx1-positive primordial pancreatic cells, it is possible to obtain Pdx1-positive primordial pancreatic cells. Yes, it is possible. In this case, the Pdx1-positive pancreatic progenitor cells express Pdx1.

[0247] This disclosure describes how to induce gastrullous cells and differentiate them into Pdx1-positive progenitor pancreatic cells, any B MP signaling pathway inhibitors (e.g., alone or from the FGF family) Each of the following: one growth factor, at least one SHH pathway inhibitor, and at least one retinoid Acid signaling pathway activator and at least one protein kinase C activator Consider using the BMP signal in any combination with the BMP. In some embodiments, the BMP signal transmission The pathway inhibitors include LDN193189.

[0248] This disclosure relates to the FG method for inducing gastrullous cells to differentiate them into Pdx1-positive pancreatic progenitor cells. Any growth factor from the F family (e.g., alone or with at least one BMP) Signaling pathway inhibitors, at least one SHH pathway inhibitor, at least one retino Acid signaling pathway activator and at least one protein kinase C activator Consider using the FGF fami The aforementioned at least one growth factor from Lee includes keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is It is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21.

[0249] This disclosure describes how to induce gastrullous cells to differentiate into Pdx1-positive progenitor pancreatic cells, any S HH pathway inhibitors (e.g., alone or inhibiting at least one BMP signaling pathway) Harmful agent, at least one growth factor from the FGF family, at least one retinoid Acid signaling pathway activator and at least one protein kinase C activator Consider using (in any combination with ). In some embodiments, the SHH pathway inhibitor This includes Sant1.

[0250] This disclosure describes how to induce gastrullous cells to differentiate into Pdx1-positive progenitor pancreatic cells, any R A signaling pathway activator (e.g., alone or with at least one BMP sequence) Gunal signaling pathway inhibitors, at least one growth factor from the FGF family, at The combination of one SHH pathway inhibitor and at least one protein kinase C activator Consider using (in combination of intents). In some embodiments, the RA signaling pathway Tibeta contains retinoic acid.

[0251] This disclosure describes how to induce gastrullous cells to differentiate into Pdx1-positive progenitor pancreatic cells, using any P KC activator (e.g., alone or in at least one BMP signaling pathway) Inhibitors, at least one growth factor from the FGF family, at least one SHH cell (In any combination of a pathway inhibitor and at least one RA signaling pathway activator) Consider using the following. In some embodiments, the PKC activator includes a PdbU. In some embodiments, the PKC activator includes a TPB.

[0252] Those skilled in the art will understand that the concentration of the active ingredient used (e.g., growth factor) may change. It will be understood. In some embodiments, the gastrulatum cells are between 20 nM and 2000 nM. The BMP signaling pathway inhibitor is brought into contact with the BMP signaling pathway inhibitor at the concentration of the BMP signaling pathway inhibitor. In some embodiments, the Gastrullous cells were subjected to 3040nM, 50nM, 60nM, 70nM, 80nM, 90nM, and 1 00nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM At concentrations of 170 nM, 180 nM, or 190 nM, the BMP signaling pathway is inhibited. The agent is brought into contact with the gastrulatum cells. In some embodiments, the gastrulatum cells are subjected to 191 nM, 192 nM, and 1 93nM, 194nM, 195nM, 196nM, 197nM, 198nM, or 199 The BMP signaling pathway inhibitor is brought into contact with the BMP signaling pathway inhibitor at an nM concentration. In some embodiments, The aforementioned gastrulatic cells were subjected to 300nM, 400nM, 500nM, 600nM, 700nM, and 8 00nM, 900nM, 1000nM, 1100nM, 1200nM, 1300nM, 1 400nM, 1500nM, 1600nM, 1700nM, 1800nM or 1900 The BMP signaling pathway inhibitor is brought into contact with the BMP signaling pathway inhibitor at an nM concentration. In some embodiments, The aforementioned gastrulatic cells were divided into 210nM, 220nM, 230nM, 240nM, 250nM, and 2 At concentrations of 60 nM, 270 nM, 280 nM, or 290 nM, the BMP signaling Contact with a pathway inhibitor. In some embodiments, the gastrulatum cells are treated with a concentration of 200 nM. The BMP signaling pathway inhibitor is then brought into contact with the BMP signaling pathway inhibitor.

[0253] In some embodiments, the gastrulatum cells are concentrated to a concentration between 5 ng / mL and 500 ng / mL. Then, it is brought into contact with at least one growth factor from the FGF family. In the application method, the gastrulatinous cells were administered at 10 ng / mL, 15 ng / mL, 20 ng / mL, and 2 At concentrations of 5 ng / mL, 30 ng / mL, 35 ng / mL, or 40 ng / mL, the above F The GF family is brought into contact with at least one of the growth factors. In some embodiments, the GF family is brought into contact with the growth factor. The gastrulatic cells were given 60 ng / mL, 65 ng / mL, 70 ng / mL, and 75 ng / mL. L, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL At a concentration of g / mL, the at least one growth factor from the FGF family is brought into contact with the In some embodiments, the gastrulatum cells are given 41 ng / mL, 42 ng / mL, and 43 ng / mL, 44ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, 48 At a concentration of ng / mL or 49 ng / mL, at least from the FGF family The cells are brought into contact with one growth factor. In some embodiments, the gastrulatum cells are treated with 51 ng / mL. , 52ng / mL, 53ng / mL, 54ng / mL, 55ng / mL, 56ng / mL The FGF family at concentrations of 57 ng / mL, 58 ng / mL, or 59 ng / mL. - to be brought into contact with at least one of the growth factors from the gastrulatum. In some embodiments, the gastrulatum The cells were subjected to a concentration of 50 ng / mL of the FGF family, which contained at least one of the components from the FGF family. Bring it into contact with the long factor.

[0254] In some embodiments, the gastrulatic cells are subjected to a concentration between 0.1 μM and 0.5 μM. The gastrulation cells are brought into contact with at least one SHH pathway inhibitor. In some embodiments, the gastrulation cells are brought into contact with at least one SHH pathway inhibitor. The cells were divided into concentrations of 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, and 0. 16μM, 0.17μM, 0.18μM, 0.19μM, 0.2μM, 0.21μM, 0 At concentrations of 0.22 μM, 0.23 μM, or 0.24 μM, the at least one SHH pathway The gastrulatum cells are brought into contact with a pathway inhibitor. In some embodiments, the gastrulatum cells are treated with 0.26 μM, 0.2 7μM, 0.28μM, 0.29μM, 0.30μM, 0.31μM, 0.32μM, 0 .33μM, 0.34μM, 0.35μM, 0.36μM, 0.37μM, 0.38μM , 0.39μM, 0.40μM, 0.41μM, 0.42μM, 0.43μM, 0.44 Concentrations of μM, 0.45μM, 0.46μM, 0.47μM, 0.48μM, and 0.49μM Then, it is brought into contact with the at least one SHH pathway inhibitor. In some embodiments, the original Intestinal cells are brought into contact with at least one of the SHH pathway inhibitors at a concentration of 0.25 μM. ru.

[0255] In some embodiments, the gastrulatum cells are subjected to a concentration between 0.01 μM and 1.0 μM. The RA signaling pathway activator is brought into contact with the gastroenteric cell. In some embodiments, the gastroenteric cell The cells were divided into concentrations of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, and 0. At concentrations of 0.7 μM, 0.08 μM, or 0.09 μM, the RA signaling pathway activation Bring to contact with beta. In some embodiments, the gastrulatum cells are mixed with 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0.60 μM, 0.70 μM, 0.80 μM or The RA signaling pathway activator is brought into contact with the 0.90 μM concentration. In this application method, the gastrulatum cells are subjected to the RA signaling pathway activation at a concentration of 0.1 μM. Bring Beta into contact with it.

[0256] In some embodiments, the gastrulatum cells are PK at a concentration between 50 nM and 5000 nM. The cells are brought into contact with a C activator. In some embodiments, the gastrulatic cells are brought into contact with a 100 nM 1 50nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM , at concentrations of 460 nM, 470 nM, 480 nM, or 490 nM, the PKC activator Bring the gastrulatum cells into contact with the gastrulatum. In some embodiments, the gastrulatum cells are given 491nM, 492nM, 493nM, 494nM, 495nM, 496nM, 497nM, 498nM or 49 The PKC activator is brought into contact with the gastrula at a concentration of 9 nM. In some embodiments, the gastrula is brought into contact with the gastrula. Tube cells were subjected to 600nM, 700nM, 800nM, 900nM, 1000nM, and 1100 nM, 1200nM, 1300nM, 1400nM, 1500nM, 1600nM, 17 At concentrations of 00nM, 1800nM, 1900nM, or 2000nM, the PKC acti In some embodiments, the gastrulatum cells are brought into contact with beta. , 503nM, 504nM, 505nM, 506nM, 507nM, 508nM or 5 09nM, 510nM, 520nM, 530nM, 540nM, 550nM, 560nM , at concentrations of 570 nM, 580 nM, or 590 nM, in contact with the PKC activator. In some embodiments, the gastrulatum cells are subjected to the PKC actinate at a concentration of 500 nM. Bring Beta into contact with it.

[0257] Generally speaking, the gastrulatic cells are Pdx1-positive pancreatic cells, at least some of the gastrulatic cells. They are maintained in a suitable culture medium for a sufficient period to induce differentiation into progenitor cells (for example, (Suspension culture). Typical suitable culture media are shown in Table 3 below.

[0258] [Table 3]

[0259] In some embodiments, S3 medium is suitable for differentiating gastrulatal cells into progenitor cells of the pancreas. It can be used as a culture medium.

[0260] In some embodiments, contact with the gastrulatous cells is achieved in a suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In this configuration, the above period is at least 2 days. In some embodiments, the suspension culture is performed every It is replenished daily.

[0261] In some embodiments, at least some of the gastrulatal cells in the aggregate become Pdx1-positive pancreatic cells By differentiating into progenitor cells, for example, the gastrulatum cells can be converted to i) LDN193189, ii) KGF, iii) Sant1; iv) RA and iv) PdbU are brought into contact with a small amount By inducing differentiation of some of the aforementioned gastrulatic cells into Pdx1-positive progenitor pancreatic cells , gastrulation cells can be obtained. In this case, the Pdx1-positive pancreatic progenitor cells are Pdx It expresses 1.

[0262] Induction of differentiation of Pdx1-positive pancreatic primordial cells into NKX6-1+ pancreatic primordial cells

[0263] Aspects of this disclosure include NKX6-1 positive pancreatic progenitor cells. The present specification includes N KX6-1 positive pancreatic progenitor cells can be obtained from any source or any suitable protozoan. It can be generated based on NKX6. In some embodiments, Pdx1-positive pancreatic progenitor cells are NKX6 -1-positive progenitor cells are differentiated. In some embodiments, the NKX6-1-positive progenitor cells are differentiated. The cells are further divided into, for example, Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells. They are transformed and subsequently induced or mature into SC-β cells.

[0264] In some embodiments, the production of NKX6-1 positive pancreatic progenitor cells from Pdx1 positive pancreatic progenitor cells. The method of production is to create Pdx1-positive pancreatic primordial cells (for example, under conditions that promote cell cluster formation). a) A cell aggregate containing cells from at least one of the fibroblast growth factor (FGF) family. b) at least two growth factors, including a sonic hedgehog pathway inhibitor, β-cell maturation Factors, and, if applicable, c) low concentrations of retinoic acid (RA) signaling pathway activation By exposing the pancreatic tissue to contact with the pancreatic tissue for at least 5 days, at least one Pdx1-positive pancreatic tissue in the collection This includes inducing differentiation of visceral primordial cells into NKX6-1 positive pancreatic primordial cells, and the NKX 6-1-positive pancreatic progenitor cells express NKX6-1.

[0265] In some embodiments, Pdx1-positive pancreatic origins are produced under conditions that promote cell cluster formation. The cells i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitors, and, if applicable, iii) low concentrations of RA signaling pathway activators The Pdx1-positive pancreatic progenitor cells were brought into contact with the Pdx1-positive pancreatic progenitor cells for a period of 5 days. By inducing differentiation into dx1-positive, NKX6-1-positive pancreatic progenitor cells, the aforementioned Pd x1-positive, NKX6-1-positive pancreatic progenitor cells can be obtained. In this case, the Pdx Pancreatic progenitor cells that are positive for Pdx1 and NKX6-1 express Pdx1 and NKX6-1. .

[0266] In some embodiments, Pdx1-positive pancreatic progenitor cells are i) obtained from a small amount of the FGF family ii) at least one growth factor, ii) at least one SHH pathway inhibitor, and iii) RA SIGN By bringing the Pdx1-positive pancreatic primordial cells into contact with the Nal transmission pathway activator, at least some of the Pdx1-positive pancreatic primordial cells are brought into contact with the Nal transmission pathway activator. By inducing differentiation of cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, The aforementioned Pdx1-positive, NKX6-1-positive pancreatic progenitor cells can be obtained. In this case, Pancreatic progenitor cells that are positive for Pdx1 and NKX6-1 express Pdx1 and NKX6-1. They are doing it.

[0267] In some embodiments, the Pdx1-positive pancreatic progenitor cells are produced from an aggregate of pluripotent cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are produced from an aggregate of iPS cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are an aggregate of ESC cells. They are produced. In some embodiments, the Pdx1-positive pancreatic progenitor cells are adult endoderm cells. They are produced from a collection of cells. In some embodiments, the Pdx1-positive progenitor pancreatic cells are gastrulat. It is produced from an aggregate of tubular cells.

[0268] This disclosure describes how to induce Pdx1-positive progenitor pancreatic cells and differentiate them into NKX6-1-positive progenitor pancreatic cells. To induce any growth factor from the FGF family (for example, alone or at least) Another SHH pathway inhibitor, or optionally, at least one retinoic acid signaling pathway. Consider using it in any combination with a transmission path activator. In some embodiments, The aforementioned at least one growth factor from the FGF family is a keratinocyte growth factor ( Includes KGF. In some embodiments, at least one component from the FGF family. The growth factor is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. .

[0269] This disclosure describes how to induce Pdx1-positive progenitor pancreatic cells and differentiate them into NKX6-1-positive progenitor pancreatic cells. To induce any SHH pathway inhibitor (e.g., alone or from the FGF family) At least one growth factor or at least one retinoic acid signaling pathway activation Consider using it in any combination with beta. In some embodiments, the SHH pathway is blocked. The harmful agent contains Sant1.

[0270] This disclosure describes how to induce Pdx1-positive progenitor pancreatic cells and differentiate them into NKX6-1-positive progenitor pancreatic cells. To induce any RA signaling pathway activator (e.g., alone or FGF) with at least one growth factor and at least one SHH pathway inhibitor from the family Consider using any combination. In some embodiments, the RA signaling pathway Cutibeta contains retinoic acid.

[0271] In some embodiments, the method involves the cell aggregate (e.g., Pdx1-positive progenitor cells of the pancreas) This includes contacting ) with at least one further β-cell maturation factor. Some embodiments Therefore, the aforementioned at least one further β-cell maturation factor is derived from at least one of the EGF family. It also contains one growth factor. In some embodiments, the method is the Pdx1-positive pancreatic origin. This involves contacting cells with at least one growth factor from the EGF family. The disclosure includes (for example, at least one growth factor from the FGF family, at least one SHH pathway inhibitors, and optionally, at least one RA signaling pathway activator (With any combination of ) Pdx1-positive progenitor cells and NKX6-1-positive progenitor cells Consider using any growth factor from the EGF family that promotes differentiation into cells. In one embodiment, the at least one growth factor from the EGF family is beta Contains cerulin. In some embodiments, at least one of the EGF family The growth factors include EGF.

[0272] Those skilled in the art will understand that the concentration of the active ingredient used (e.g., growth factor) may change. This will be understood. In some embodiments, the Pdx1-positive pancreatic progenitor cells are 1 ng / mL - At a concentration between -100 ng / mL, the at least one component from the FGF family In some embodiments, the Pdx1-positive pancreatic progenitor cells are brought into contact with growth factors. mL, 10ng / mL, 15ng / mL, 20ng / mL, 25ng / mL, 30ng / The FGF family is obtained at concentrations of mL, 35 ng / mL, or 40 ng / mL. Contact with at least one growth factor. In some embodiments, the Pdx1-positive pancreas The progenitor cells were divided into 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, and 80 ng / mL. g / mL, 85ng / mL, 90ng / mL, 95ng / mL, or 100ng / mL At a certain concentration, it is brought into contact with at least one growth factor from the FGF family. In this embodiment, the Pdx1-positive pancreatic progenitor cells are administered at 41 ng / mL, 42 ng / mL, 43ng / mL, 44ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, At a concentration of 48 ng / mL or 49 ng / mL, the least from the FGF family In some embodiments, the Pdx1-positive pancreatic progenitor cells are brought into contact with a single growth factor. , 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL, 55ng / m At concentrations of L, 56 ng / mL, 57 ng / mL, 58 ng / mL, or 59 ng / mL, Contact with at least one growth factor from the FGF family. Partial implementation In this state, the Pdx1-positive pancreatic progenitor cells are subjected to the FGF famine at a concentration of 50 ng / mL. The at least one growth factor from Lee is brought into contact with it.

[0273] In some embodiments, the Pdx1-positive pancreatic progenitor cells are divided into 0.1 μM and 0.5 μM groups. The mixture is brought into contact with at least one SHH pathway inhibitor at an intermediate concentration. In some embodiments, The Pdx1-positive pancreatic progenitor cells were administered at concentrations of 0.11 μM, 0.12 μM, 0.13 μM, and 0. 14μM, 0.15μM, 0.16μM, 0.17μM, 0.18μM, 0.19μM, At concentrations of 0.2 μM, 0.21 μM, 0.22 μM, 0.23 μM, or 0.24 μM, The Pdx is brought into contact with at least one of the SHH pathway inhibitors. In some embodiments, the Pdx 1 Positive pancreatic progenitor cells, 0.26 μM, 0.27 μM, 0.28 μM, 0.29 μM, 0 .30μM, 0.31μM, 0.32μM, 0.33μM, 0.34μM, 0.35μM , 0.36μM, 0.37μM, 0.38μM, 0.39μM, 0.40μM, 0.41 μM, 0.42 μM, 0.43 μM, 0.44 μM, 0.45 μM, 0.46 μM, 0. At concentrations of 47 μM, 0.48 μM, and 0.49 μM, the inhibition of at least one SHH pathway was observed. The agent is brought into contact with the cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are brought into contact with the cells at a concentration of 0.25 μM. The solution is brought into contact with at least one of the SHH pathway inhibitors at the specified concentration.

[0274] In some embodiments, the Pdx1-positive pancreatic progenitor cells are diluted to a concentration of 0.01 μM-1.0 μM. The RA signaling pathway activator is brought into contact with the activator at an intermediate concentration. In some embodiments, The Pdx1-positive pancreatic progenitor cells were administered at concentrations of 0.02 μM, 0.03 μM, 0.04 μM, and 0. At concentrations of 0.5 μM, 0.06 μM, 0.07 μM, 0.08 μM, or 0.09 μM, The RA signaling pathway activator is brought into contact with the Pdx1. Positive pancreatic progenitor cells were collected at concentrations of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, and 0. At concentrations of 60 μM, 0.70 μM, 0.80 μM, or 0.90 μM, the RA signal It is brought into contact with the signaling pathway activator. In some embodiments, the Pdx1-positive pancreatic primordial cells are brought into contact with the signaling pathway activator. The cells are brought into contact with the RA signaling pathway activator at a concentration of 0.1 μM.

[0275] In some embodiments, the Pdx1-positive pancreatic progenitor cells are administered at a dose of 2 ng / mL-200 ng / At concentrations between mL, the at least one growth factor from the EGF family is brought into contact with the In some embodiments, the Pdx1-positive pancreatic progenitor cells are divided into 3 ng / mL, 4 ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10 ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 16 At concentrations of ng / mL, 17ng / mL, 18ng / mL, or 19ng / mL, the EG Contact with at least one growth factor from the F family. In some embodiments, The aforementioned Pdx1-positive pancreatic progenitor cells were subjected to concentrations of 30 ng / mL, 35 ng / mL, and 40 ng / mL. 45ng / mL, 50ng / mL, 55ng / mL, 60ng / mL, 65ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, At a concentration of 95 ng / mL or 100 ng / mL, the small amount from the EGF family In some embodiments, the Pdx1-positive pancreatic primordial cells are brought into contact with at least one growth factor. cells, 21ng / mL, 22ng / mL, 23ng / mL, 24ng / mL, 25ng / At concentrations of mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, or 29 ng / mL , to be brought into contact with at least one growth factor from the EGF family. Partial implementation Morphologically, the Pdx1-positive pancreatic progenitor cells are subjected to the EGF formulation at a concentration of 20 ng / mL. The material is brought into contact with at least one of the growth factors from Millie.

[0276] Generally, the Pdx1-positive pancreatic progenitor cells are at least a portion of the P in the aggregate. The division of dx1-positive pancreatic progenitor cells into the aforementioned Pdx1-positive, NKX6-1-positive pancreatic progenitor cells. It is maintained in a suitable culture medium for a period sufficient to induce transformation. Typical suitable culture medium These are shown in Table 3 above. In some embodiments, the conditions that promote cell cluster formation are This includes suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is at least 5 days. In some embodiments, The suspension culture is replenished once every two days. In some embodiments, the β-cell maturation factor is replenished every two days. It will be refilled once.

[0277] In some embodiments, the protein kinase C activator is used in the suspension culture for 5 days. It is not added during the suspension. In some embodiments, the activator of protein kinase C is the same as the suspension. It is removed 5 days before the start of the culture. In some embodiments, the protein kinase C The cutiverter includes PdbU. In some embodiments, the BMP signaling pathway inhibitor is The suspension culture is not added for 5 days. In some embodiments, the BMP signaling pathway The inhibitor is removed from the suspension culture 5 days prior. In some embodiments, the BMP Gunal signaling pathway inhibitors include LDN193189.

[0278] In some embodiments, at least 10% of the Pdx1-positive pancreatic progenitor cells in the collection This induces differentiation into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells. Morphologically, at least 95% of the aforementioned Pdx1-positive progenitor pancreatic cells are induced to be Pdx1 positive. It differentiates into NKX6-1 positive originating pancreatic cells.

[0279] Generally, any Pdx1-positive pancreatic progenitor cell is Pdx1-positive and NKX6-1-positive. They can differentiate into pancreatic progenitor cells. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are It expresses Pdx1, NKX6-1 and / or FoxA2.

[0280] In some embodiments, Pdx1-positive pancreatic origins are produced under conditions that promote cell cluster formation. The cells i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitors, and, if applicable, iii) low concentrations of RA signaling pathway activators The Pdx1-positive pancreatic progenitor cells were brought into contact with the Pdx1-positive pancreatic progenitor cells for a period of 5 days. By inducing differentiation into dx1-positive, NKX6-1-positive pancreatic progenitor cells, the aforementioned Pd x1-positive, NKX6-1-positive pancreatic progenitor cells can be obtained. In this case, the Pdx Pancreatic progenitor cells that are positive for Pdx1 and NKX6-1 express Pdx1 and NKX6-1. .

[0281] In some embodiments, under conditions that promote cell cluster formation, the Pdx1-positive pancreas The progenitor cells are given i) at least one growth factor from the FGF family, ii) at least One SHH pathway inhibitor, and optionally, iii) RA signaling pathway activator Then, contact was made once every two days for a period of five days, and at least some of the Pdx1 positive samples in the aforementioned collection were detected. By inducing the differentiation of pancreatic primordial cells into NKX6-1 positive pancreatic primordial cells, at least Some of the aforementioned Pdx1-positive origin pancreatic cells are Pdx1-positive, NKX6-1-positive origin pancreatic cells. By differentiating these cells, NKX6-1 positive pancreatic progenitor cells can be obtained. In this case, the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1. .

[0282] In some embodiments, at least some of the Pdx1-positive progenitor cells in the aggregate are P By differentiating them into dx1-positive, NKX6-1-positive pancreatic progenitor cells, for example, the aforementioned P i) At least one growth factor from the FGF family, i i) at least one SHH pathway inhibitor, and optionally iii) RA signaling pathway By bringing the road activator into contact with at least a portion of the Pdx1-positive pancreatic origin in the assembly, By inducing differentiation of cells into NKX6-1-positive pancreatic progenitor cells, Pancreatic primordial cells can be obtained. In this case, the NKX6-1 positive pancreatic primordial cells are P It expresses dx1 and NKX6-1.

[0283] Induction of differentiation of NKX6-1+ pancreatic progenitor cells into insulin+ endocrine cells.

[0284] Aspects of this disclosure include insulin-positive endocrine cells. Useful insulin in this specification Phosphorus-positive endocrine cells can be obtained from any source or based on any appropriate protocol. It can be produced accordingly. In some embodiments, NKX6-1 positive pancreatic progenitor cells are insulin-positive. They differentiate into insulin-positive endocrine cells. In some embodiments, the insulin-positive endocrine cells are, for example, They are further differentiated by induction or maturation into SC-β cells.

[0285] In some embodiments, the production of insulin-positive endocrine cells from NKX6-1 positive pancreatic progenitor cells is carried out. The manufacturing method involves NKX6-1 positive pancreatic cells (for example, under conditions that promote cell cluster formation). Cell aggregates including progenitor cells were subjected to a) TGF-β signaling inhibitors and b) thyroid hormones By contacting at least two β-cell maturation factors, including the Gunal signaling pathway activator, To insulin-positive endocrine cells from at least one NKX6-1-positive pancreatic progenitor cell during aggregation This includes inducing differentiation of insulin-producing pancreatic cells. In this case, the insulin-producing pancreatic cells produce insulin. It is manifesting.

[0286] This disclosure describes how to induce differentiation of NKX6-1-positive pancreatic progenitor cells and insulin-positive endocytes. Any TGF-β signaling pathway inhibitor that causes differentiation into secretory cells (for example, alone or This is in combination with other β-cell maturation factors, such as thyroid hormone signaling pathway activators. Consider using (in combination). In some embodiments, the TGF-β signaling pathway is TG Includes F-β receptor type I kinase signaling. In some embodiments, the TGF-β receptor type I kinase is used. Gunal signaling pathway inhibitors include Alk5 inhibitors II.

[0287] This disclosure describes how to induce differentiation of NKX6-1-positive pancreatic progenitor cells and insulin-positive endocytes. Any thyroid hormone signaling pathway activator that differentiates into secretory cells (e.g., simple In Germany, or in combination with other β-cell maturation factors, e.g., TGF-β signaling pathway inhibitors. Consider using (in combination). In some embodiments, the thyroid hormone signaling pathway The activator contains triiodothyronine (T3).

[0288] In some embodiments, the method involves the cell aggregate (e.g., NKX6-1 positive pancreatic origin). The process involves contacting the cells with at least one further β-cell maturation factor. Morphologically, the method involves the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, i)S ii) HH pathway inhibitors, ii) RA signaling pathway activators, iii) γ-secretase Inhibitors, iv) at least one growth factor from the epidermal growth factor (EGF) family, and And depending on the circumstances, this may include (v) contact with at least one protein kinase inhibitor. nothing.

[0289] In some embodiments, the at least one further β-cell maturation factor is a γ-secretary This disclosure includes an insulin-positive inhibitor. This disclosure relates to the aggregation of NKX6-1 positive pancreatic progenitor cells into insulin-positive cells. Any γ-secretase inhibitor capable of inducing differentiation into secretory cells (e.g., alone or TGF-β signaling pathway inhibitors and / or thyroid hormone signaling pathway inhibitors Consider using it in combination with any of the tibers. In some embodiments, the γ-segment The cretase inhibitor includes XXI. In some embodiments, the γ-secretase inhibitor This includes DAPT.

[0290] In some embodiments, the at least one further β-cell maturation factor is part of the EGF family. —Contains at least one growth factor from. This disclosure relates to NKX6-1 positive pancreatic initial cells during a biomass. Any of the EGF family capable of inducing differentiation of progenitor cells into insulin-positive endocrine cells Growth factors (e.g., alone, or TGF-β signaling pathway inhibitors and / or Consider using it in combination with any of the thyroid hormone signaling pathway activators. In some embodiments, the at least one growth factor from the EGF family is , containing betacellin. In some embodiments, at least one from the EGF family One of the growth factors is EGF.

[0291] In some embodiments, the at least one further β-cell maturation factor is used in low concentrations of retino This disclosure includes an acid (RA) signaling pathway activator. Inducing differentiation of progenitor cells into insulin-positive endocrine cells, any RA signal TGF-β signaling pathway activators (e.g., alone or as TGF-β signaling pathway inhibitors) (and / or in combination with any of the thyroid hormone signaling pathway activators) Consider using it. In some embodiments, the RA signaling pathway activator is RA Includes.

[0292] In some embodiments, the at least one further β-cell maturation factor is sonic hedge This disclosure includes a Hogg (SHH) pathway inhibitor. This disclosure relates to the differentiation of NKX6-1 positive pancreatic progenitor cells. Any SHH pathway inhibitor (e.g.,) induces differentiation into insulin-positive endocrine cells. Used alone, or as a TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway inhibitor. Consider using it in combination with any of the transmission path activators. In some embodiments The SHH pathway inhibitor includes Sant1.

[0293] In some embodiments, the cell aggregate (e.g., NKX6-1 positive progenitor cells) is glucoc To be exposed to the sun.

[0294] In some embodiments, the cell aggregate is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell aggregate is brought into contact with the protein kinase inhibitor. In some embodiments, the cell aggregate is brought into contact with the protein kinase inhibitor. This disclosure describes how NKX6-1 positive pancreatic progenitor cells in aggregation can be converted into insulin-positive endocrine cells. Any protein kinase inhibitor capable of inducing differentiation (e.g., alone or with TG) F-β signaling pathway inhibitors and / or thyroid hormone signaling pathway activators Consider using it in combination with any of the following. In some embodiments, the protein Nase inhibitors include staurosporine.

[0295] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are used in a small number of cases. ii) SHH pathway inhibitors, iii) RA signaling pathway activators, iii) γ- secretase inhibitors, iv) TGF-β signaling pathway inhibitors, v) TH signaling pathway Pathway activators and vi) at least one from the epidermal growth factor (EGF) family When exposed to growth factors, at least some Pdx1-positive, NKX6-1-positive pancreatic primordial cells were detected. It induces differentiation of cells into Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. This allows us to obtain insulin-positive endocrine cells. In this case, the Pdx1 positive cells are obtained. Sex, NKX6-1 positive, insulin positive endocrine cells: Pdx1, NKX6-1, NKX 2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, S It expresses LC2A3 and / or insulin.

[0296] Those skilled in the art will understand that the concentration of the active ingredient used (e.g., growth factor) may change. They will understand.

[0297] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are expressed in a concentration of 100 nM-100 μM. The device is brought into contact with at least one TGF-β signaling pathway inhibitor at a concentration between the specified values. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are used at a concentration of 10 μM. In some embodiments, The aforementioned NKX6-1 positive pancreatic progenitor cells were subjected to 100nM, 200nM, 300nM, and 400nM treatments. At concentrations of M, 500nM, 600nM, 700nM, 800nM, or 900nM, the above Contact with at least one TGF-β signaling pathway inhibitor. In some embodiments, The NKX6-1 positive pancreatic progenitor cells were divided into 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, and 7 μM concentrations. At concentrations of μM, 8 μM, or 9 μM, the at least one TGF-β signaling pathway In some embodiments, the NKX6-1 positive pancreatic progenitor cells are brought into contact with the inhibitor. 1μM, 9.2μM, 9.3μM, 9.4μM, 9.5μM, 9.6μM, 9.7μM, At a concentration of 9.8 μM or 9.9 μM, the at least one TGF-β signaling pathway In some embodiments, the NKX6-1 positive pancreatic progenitor cells are brought into contact with a pathway inhibitor. 1 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, This was in contact with at least one TGF-β signaling pathway inhibitor at a concentration of 19 μM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are mixed at 10.1 μM, 10 .2μM, 10.3μM, 10.4μM, 10.5μM, 10.6μM, 10.7μM, At a concentration of 10.8 μM or 10.9 μM, the at least one TGF-β signaling pathway Bring it into contact with a pathway inhibitor.

[0298] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are divided into 0.1 μM-10 μM groups. At an intermediate concentration, it is brought into contact with the thyroid hormone signaling pathway activator. In the administration method, the NKX6-1 positive pancreatic progenitor cells were administered at a concentration of 1 μM, and the thyroid hormone was used. It is brought into contact with the signaling pathway activator. In some embodiments, the NKX6-1 Positive pancreatic progenitor cells were collected in concentrations of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, and 0.6 μM. At concentrations of 0.7 μM, 0.8 μM, or 0.9 μM, the thyroid hormone signaling pathway In some embodiments, the NKX6-1 positive pancreatic progenitor cells are brought into contact with the pathway activator. These were given in concentrations of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, and 1. At concentrations of 7 μM, 1.8 μM, or 1.9 μM, the γ-thyroid hormone signaling pathway The activator is brought into contact with the NKX6-1 positive pancreatic progenitor cells. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are brought into contact with the activator. At concentrations of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM, The thyroid hormone signaling pathway activator is brought into contact with the thyroid hormone signaling pathway activator.

[0299] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are divided into 0.1 μM-10 μM groups. The γ-secretase inhibitor is brought into contact with the NK at an intermediate concentration. In some embodiments, the NK X6-1-positive pancreatic progenitor cells were contacted with the γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are expressed in a 0.2 μM, 0.3 μM solution. M, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, or 0.9 μM The γ-secretase inhibitor is brought into contact with the NKX6 at a specified concentration. -1 positive pancreatic progenitor cells were measured in concentrations of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, and 1.5 μM. At concentrations of M, 1.6 μM, 1.7 μM, 1.8 μM, or 1.9 μM, the γ-secret The NKX6-1-positive pancreatic progenitor cells are brought into contact with an enzyme inhibitor. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are brought into contact with an enzyme inhibitor. At concentrations of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM, The gamma-secretase inhibitor is brought into contact with the gamma-secretase inhibitor.

[0300] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are administered at a dose of 2 ng / mL-200n In a concentration between g / mL, the at least one growth factor from the EGF family is brought into contact with the EGF family. To be brought into contact. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are given at 3 ng / mL. 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / m L, 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / m At concentrations of L, 16 ng / mL, 17 ng / mL, 18 ng / mL, or 19 ng / mL, Contact with at least one growth factor from the EGF family. Partial implementation In this state, the NKX6-1 positive pancreatic progenitor cells were administered at 30 ng / mL, 35 ng / mL, and 40 ng / mL, 45ng / mL, 50ng / mL, 55ng / mL, 60ng / mL, 65 ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90 At concentrations of ng / mL, 95ng / mL, or 100ng / mL, the EGF family is They are brought into contact with at least one of the aforementioned growth factors. In some embodiments, the NKX6- 1. Positive pancreatic progenitor cells, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25ng / mL, 26ng / mL, 27ng / mL, 28ng / mL or 29n At a concentration of g / mL, the at least one growth factor from the EGF family is brought into contact with the In some embodiments, the NKX6-1 positive pancreatic progenitor cells are concentrated at 20 ng / mL. The at least one growth factor from the EGF family is brought into contact with the at least one growth factor at a certain temperature.

[0301] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are expressed in a range of 0.01 μM to 1.0 μM. The RA signaling pathway activator is brought into contact with the activator at a concentration between M. (Some embodiments) Then, the NKX6-1 positive pancreatic progenitor cells were measured in concentrations of 0.02 μM, 0.03 μM, and 0.04 μM. Concentrations of M, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, or 0.09 μM At a certain degree, the RA signaling pathway activator is brought into contact with the RA signaling pathway activator. In some embodiments, the NKX6-1 positive pancreatic progenitor cells were administered at concentrations of 0.20 μM, 0.30 μM, 0.40 μM, and 0.0 At concentrations of 5 μM, 0.60 μM, 0.70 μM, 0.80 μM, or 0.90 μM, the above It is brought into contact with the RA signaling pathway activator. In some embodiments, the NKX6- 1. Positive pancreatic progenitor cells were subjected to the RA signaling pathway activator at a concentration of 0.1 μM. Make contact.

[0302] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are expressed in a range of 0.01 μM to 1.0 μM. At concentrations between M, it is brought into contact with a low concentration of RA signaling pathway activator. Morphologically, the NKX6-1 positive pancreatic progenitor cells were divided into 0.02 μM, 0.03 μM, and 0.0 4 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, or 0.09 μM At this concentration, it is brought into contact with a low concentration of the RA signaling pathway activator. Some implementations In this state, the NKX6-1 positive pancreatic progenitor cells were subjected to concentrations of 0.20 μM, 0.30 μM, and 0.40 μM. μM, 0.05μM, 0.60μM, 0.70μM, 0.80μM or 0.90μM The low concentration of the RA signaling pathway activator is brought into contact with the activator at a certain concentration. (Some embodiments) Next, the NKX6-1 positive pancreatic progenitor cells were subjected to a low concentration of RA cells at a concentration of 0.1 μM. Establish contact with the Gnar transmission pathway activator.

[0303] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are used in concentrations of 0.1 μM and 0.5 μM. The at least one SHH pathway inhibitor is brought into contact with the at least one SHH pathway inhibitor at a concentration between the specified values. Then, the NKX6-1 positive pancreatic progenitor cells were divided into 0.11 μM, 0.12 μM, and 0.13 μM concentrations. M, 0.14μM, 0.15μM, 0.16μM, 0.17μM, 0.18μM, 0.1 9 μM, 0.2 μM, 0.21 μM, 0.22 μM, 0.23 μM, or 0.24 μM The at least one SHH pathway inhibitor is brought into contact with the concentration. In some embodiments, NKX6-1 positive pancreatic progenitor cells were measured at concentrations of 0.26 μM, 0.27 μM, 0.28 μM, and 0. 29μM, 0.30μM, 0.31μM, 0.32μM, 0.33μM, 0.34μM, 0.35μM, 0.36μM, 0.37μM, 0.38μM, 0.39μM, 0.40μM M, 0.41μM, 0.42μM, 0.43μM, 0.44μM, 0.45μM, 0.4 At concentrations of 6 μM, 0.47 μM, 0.48 μM, and 0.49 μM, at least one of the above S In some embodiments, the NKX6-1 positive pancreatic progenitor cells are brought into contact with the HH pathway inhibitor. This is brought into contact with at least one of the SHH pathway inhibitors at a concentration of 0.25 μM.

[0304] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are divided between 10 nM and 1 μM. The protein kinase inhibitor is brought into contact with the NKX at a specified concentration. In some embodiments, the NKX 6-1 Positive pancreatic progenitor cells were contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are given a concentration of 20 nM, 30 nM. At concentrations of M, 40nM, 50nM, 60nM, 70nM, 80nM, or 90nM, the above The protein kinase inhibitor is brought into contact with the NKX6-1 positive pancreas. In some embodiments, the NKX6-1 positive pancreas is brought into contact with the protein kinase inhibitor. Organ progenitor cells were subjected to 110nM, 120nM, 130nM, 140nM, 150nM, and 160 The protein kinase inhibitory effect was achieved at concentrations of nM, 170nM, 180nM, or 190nM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are brought into contact with the agent. M, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM or The protein kinase inhibitor is brought into contact with the protein kinase inhibitor at a concentration of 900 nM.

[0305] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are divided between 1 mM and 50 mM. In some embodiments, the NKX6-1 positive pancreatic origin is brought into contact with glucose. The cells are brought into contact with glucose at a concentration between 25 mM.

[0306] In some embodiments, under conditions that promote cell cluster formation, the Pdx1 positive Sex, NKX6-1 positive pancreatic progenitor cells, i) TGF-β signaling pathway inhibitors, b) Thyroid hormone signaling pathway activators, and optionally c) at least one ii) SHH pathway inhibitors, ii) RA signaling pathway activators, iii) γ-secretaries X inhibitors and vi) at least one growth factor from the epidermal growth factor (EGF) family Then, by exposing them to contact once every two days, for a period between the 5th and 7th day, at least some of the Pdx1 positive cells were detected. NKX6-1-positive pancreatic progenitor cells, Pdx1-positive, NKX6-1-positive, insulin-positive. By inducing differentiation of endocrine cells, at least some of the Pdx1-positive NK cells are produced. Among the X6-1 positive pancreatic progenitor cells, those that are Pdx1 positive, NKX6-1 positive, and insulin positive By differentiating them into secretory cells, the aforementioned insulin-positive endocrine cells can be obtained. In this case, the endocrine cells that are Pdx1-positive, NKX6-1-positive, and insulin-positive are Pd x1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, It expresses Znt8, SLC2A1, SLC2A3, and / or insulin.

[0307] In some embodiments, for example, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are used. i) at least one SHH pathway inhibitor, ii) RA signaling pathway activator, iii) γ-secretase inhibitors, vi) TGF-β signaling pathway inhibitors, v) TH At least one signaling pathway activator and epidermal growth factor (EGF) family By contacting one growth factor, at least some of the Pdx1-positive, NKX6-1-positive cells Division of pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells By inducing the transformation of at least some of the Pdx1-positive and NKX6-1-positive cells in the aggregate, Pancreatic progenitor cells are differentiated into Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells. By doing so, the insulin-positive endocrine cells can be obtained. In this case, the Endocrine cells that are Pdx1-positive, NKX6-1-positive, and insulin-positive are Pdx1, NKX6-1 , NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2 It expresses A1, SLC2A3, and / or insulin.

[0308] Generally, the cell aggregate is at least one NKX6-1 positive pancreatic cell in the aggregate. Appropriate culture for a sufficient period to induce differentiation of visceral primordial cells into insulin-positive endocrine cells. It is maintained in the culture medium. Typical culture media are shown in Table 4.

[0309] [Table 4]

[0310] In some embodiments, BE5 medium is used to prepare NKX6-1 positive pancreatic progenitor cells for insulin-positive It can be used as a culture medium suitable for differentiating endocrine cells. In some embodiments, Suitable culture media are shown in Table 5.

[0311] In some embodiments, conditions that promote cell cluster formation include suspension culture. In the embodiment, the number of cells co-expressing C-peptide and Nkx6-1 during the period is Includes a period sufficient to maximize. In some embodiments, the period is at least 5 days. In some embodiments, the period is between 5 and 7 days. The aforementioned period is at least 7 days. In some embodiments, the suspension culture is supplemented daily. The cells are filled (e.g., β-cell maturation factors). In some embodiments, between day 5 and day 7 The number of cells co-expressing C-peptide and Nkx6-1 is maximized over a given period.

[0312] In some embodiments, at least 15% of the collection are NKX6-1 positive pancreatic origins. The cells are induced to differentiate into insulin-positive endocrine cells.

[0313] In some embodiments, at least 99% of the NKX6-1 positive pancreatic origin in the collection The cells are induced to differentiate into insulin-positive endocrine cells.

[0314] Insulin + Induction of endocrine cell maturation into SC-β cells

[0315] Aspects of this disclosure include SC-β cells. Useful SC-β cells in this specification are, It can be obtained from a source of choice, or generated based on any appropriate protocol. In this configuration, insulin-positive endocrine cells are induced to mature into SC-β cells.

[0316] In some aspects, the disclosure relates to mature glucose-positive endocrine cells from insulin-positive endocrine cells. The present invention provides a method for generating responsive β-cells. The method includes (for example, promoting cell cluster formation) Under certain conditions, a) a cell aggregate containing insulin-positive endocrine cells, and transforming growth factor β(T GF-β) signaling inhibitors, b) thyroid hormone (TH) signaling pathway activators By contacting at least two β-cell maturation factors including the β-, at least one of the aggregate To induce in vitro maturation of insulin-positive endocrine cells into SC-β cells. include.

[0317] The aspects of this disclosure are similar in shape and function to endogenous mature β-cells, but also Regardless, this includes generating SC-β cells that are distinct from natural β cells. Cells may show a response to at least one glucose challenge. Some implementations In this state, the SC-β cells respond to at least two consecutive glucose challenges The response is observed. In some embodiments, the SC-β cells undergo at least three consecutive glucose This shows the response to the course challenge. In some embodiments, the SC-β cells are multiple Similar to the endogenous response of human pancreatic islets to a glucose challenge, multiple (for example) In some embodiments, the S C-β cells respond to two consecutive glucose challenges with insulin It is capable of releasing or secreting. In some embodiments, the SC-β cells undergo three consecutive cycles In response to a glucose challenge, insulin can be released or secreted. In some embodiments, the SC-β cells are subjected to four consecutive glucose challenges. In the response, insulin can be released or secreted. In some embodiments, SC-β cells respond to five consecutive glucose challenges with insulin They are capable of releasing or secreting. In some embodiments, the SC-β cells are continuously In response to a series of glucose challenges, insulin is released or secreted. In some embodiments, the cells are as described in the examples of this specification. Intracellular Ca 2+ By deciding whether to repeatedly increase them, they become continuous. An assay can be used to determine whether or not it responds to a glucose challenge.

[0318] In some embodiments, the morphology of the SC-β cells is similar to that of endogenous β cells. In some embodiments, the SC-β cells are glucose-stimulated in vitro. It exhibits a phosphorus secretion (GSIS) response. In some embodiments, the SC-β cells are in v The GSIS response in ivo is shown. In some embodiments, the SC-β cells are in vi The GSIS response in vivo and / or in vivo is shown. In some embodiments, the above In vitro and / or in vitro GSIS response is related to endogenous mature β cells. This is similar to the GSIS response of cells. In some embodiments, the SC-β cells are similar to endogenous β cells. It shows an in vitro (GSIS) response similar to the GSIS response in cells. Morphologically, the SC-β cells exhibit an in vivo response similar to the GSIS response of endogenous β cells. The GSIS response is shown at o. The GSIS response is observed immediately after transplantation into a human or animal subject. This may be observed in some embodiments. In some embodiments, the GSIS response is observed in the SC-β cells. Observations are made within two weeks of implantation in a human or animal subject. In some embodiments, the The GSIS response is observed within two weeks of transplanting the SC-β cells into a human or animal subject. In some embodiments, the GSIS response of the SC-β cells is performed on the SC-β cells. Transplanted into human or animal subjects, for 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks. 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 1 By 7 weeks, 18 weeks, 6 months, 7 months, 8 months, 9 months, 10 months, and 11 months, This is observed for more than one year.

[0319] In some embodiments, the SC-β cells are at least mature endogenous pancreatic β cells. One marker is presented. Typical markers are not limited to Pdx1 and HNF. 6, Ptf1a, Sox9, FoxA2, ​​Nkx2.2, Ngn3 and NKX6-1 are In some embodiments, HNF6, Ptf1a, Sox9, FoxA2, ​​Nkx 2.2 The expression of the marker selected from the group consisting of Ngn3 and NKX6-1 is The pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent cells) from which the SC-β cells are obtained. In contrast, the amount is statistically significant in the SC-β cells.

[0320] This disclosure describes how to induce insulin-positive endocrine cells to differentiate and / or develop into SC-β cells. To mature, any TGF-β signaling pathway inhibitor (e.g., alone or less) Both involve one thyroid hormone (TH) signaling pathway activator, or, in some cases, Consider using it in any combination with a protein kinase inhibitor. In some embodiments, The TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor is Alk5 inhibitor II include.

[0321] This disclosure describes how to induce insulin-positive endocrine cells to differentiate and / or develop into SC-β cells. To mature any thyroid hormone signaling pathway activator (e.g., alone or also) This involves at least one TGF-β signaling pathway inhibitor, or optionally, a protein Consider using it in any combination with a kinase inhibitor. In some embodiments, the above The serotonin signaling pathway activator includes T3.

[0322] In some embodiments, the Pdx1-positive, NKX6-1-positive, and insulin-positive cells are used. In some cases, it is brought into contact with a protein kinase inhibitor. In some embodiments, the Pd x1-positive, NKX6-1-positive, insulin-positive endocrine cells, the protein kinase Avoid contact with the inhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive, and Endocrine cells positive for nsrin are brought into contact with the protein kinase inhibitor. This disclosure is, Inducing insulin-positive endocrine cells to differentiate and / or mature into SC-β cells, A protein kinase inhibitor (e.g., alone or with at least one TGF-β) With signaling pathway inhibitors and / or thyroid hormone signaling pathway activators Consider using (any combination). In some embodiments, the protein kinase inhibitor The agent contains staurosporine.

[0323] In some embodiments, the method involves the cell aggregate (e.g., insulin-positive endocrine cells). This includes contacting the product with at least one further β-cell maturation factor.

[0324] In some embodiments, the at least one further β-cell maturation factor is related to the cystic fibrosis membrane. The method includes a conductance regulatory factor (CFTR) inhibitor. In some embodiments, the method is The aforementioned Pdx1-positive, NKX6-1-positive, and insulin-positive endocrine cells were treated with CFTR inhibitors. This includes bringing the cells into contact with the cells. The disclosure describes how to induce insulin-positive endocrine cells to form SC-β cells. Any CFTR inhibitor (e.g., alone or in small amounts) that causes differentiation and / or maturation of cells. At least one TGF-β...

Claims

[Claim 1] The invention described in the specification.

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