Method for promoting differentiation of first cell into second cell, and method for identifying treatment that promotes said differentiation

Abstract

The present disclosure provides a method for identifying a treatment that promotes differentiation of a first cell into a second cell. The present disclosure also provides pancreatic beta cells or cells that are capable of differentiating into pancreatic beta cells. In the present disclosure, the pancreatic beta cells or cells that are capable of differentiating into pancreatic beta cells have a deletion of a specific gene, whereby the same preferably exhibit a high capability of differentiating into pancreatic beta cells, or exhibit a capability of differentiating into pancreatic beta cells exhibiting a glucose-dependent C-peptide secretion ability.

Description

A method for promoting differentiation of a first cell into a second cell, and a method for identifying a treatment that promotes said differentiation

[0001] The present disclosure relates to a method for promoting differentiation of a first cell into a second cell, and a method for identifying a treatment that promotes said differentiation.

[0002] Pancreatic β cells play an important role in secreting insulin and regulating blood glucose levels. Loss of function or destruction of these cells causes serious diseases such as type 1 diabetes and type 2 diabetes. Currently, islet transplantation is performed as an effective therapy for these diabetic patients, but the shortage of available donor islet cells limits its widespread use (Velazco-Cruz et al., 2019). Accordingly, technologies for artificially generating β cells using human pluripotent stem cells (hPSCs) and the like have attracted attention, but existing differentiation induction methods still have issues in the purity and functionality of the generated β cells.

[0003] In recent studies, methods for inducing β cells from hPSCs in vitro have been reported (Veres et al., 2019; Velazco-Cruz et al., 2019). Veres et al. pointed out that the cellular heterogeneity that occurs when inducing differentiation of pancreatic β cells (SC-β cells) from human ES cells or human iPS cells in vitro is a problem, and used single-cell RNA sequencing to evaluate cellular identity in detail. As a result, they discovered that in addition to β cells, α cells, and non-endocrine cells, previously unknown serotonin-producing intestinal chromaffin cell-like (SC-EC) cells are generated. In addition, Veres et al. reported a method of sorting SC-β cells from the differentiation-induced cell population using CD49a (ITGA1) as a surface marker, and purifying SC-β cells with a high purity of 80% (Veres et al., 2019).

[0004] Meanwhile, Velazco-Cruz et al. addressed the problem of functional immaturity of SC-β cells using conventional methods by specifically regulating the inhibition of the TGF-β pathway, controlling the size of cell clusters, and using a serum-free culture medium. This allowed them to create SC-β cells exhibiting dynamic insulin secretion in both the early and second phases, similar to human islet cells, and demonstrating excellent blood glucose-lowering effects in diabetic animal models (Velazco-Cruz et al., 2019).

[0005] On the other hand, the transcription factor ARX is known to play a crucial role in determining the fate of pancreatic endocrine cells, including α cells, in inducing β cells. Gage et al. (PLoS One. 2015 Dec 3;10(12):e0144100) showed that knocking out the ARX gene from human ES cells significantly reduced the generation of insulin-producing and glucagon-producing cells, while significantly increasing the differentiation of somatostatin-producing cells, reporting that ARX is an important factor in determining the fate of pancreatic endocrine cells. Furthermore, Courtney et al., using a mouse model, showed that specifically deleting the second exon of the Arx gene in glucagon-expressing cells in pancreatic α cells resulted in the conversion of α cells into functional β-cell-like cells that produce insulin, even in adulthood, and that new endocrine cells were continuously replenished from ductal epithelial progenitor cells (Courtney et al., 2013). In these studies, gene disruption is achieved by the deletion of all or part of exon 1 or 2, or by a frameshift. Thus, gene disruption is usually achieved by deleting or frameshifting a portion of the coding region, and does not typically involve the deletion of the entire coding region.

[0006] Gage et al. reported that when they knocked out the ARX gene in human ES cells and attempted to differentiate them into pancreatic endocrine cells, not only were glucagon-producing cells reduced by approximately 90%, but insulin-producing cells were also reduced by approximately 65%, with somatostatin-producing cells accounting for approximately 94% of the endocrine cells. This result suggests that disruption of the ARX gene also adversely affects the generation of insulin-producing cells, and it has been thought that efficient induction of pancreatic β-cells by disrupting the ARX gene is difficult.

[0007] Furthermore, Chakravarthy et al. (Cell Metab. 2017; 25(3):622-634) reported that when the Arx gene was conditionally knocked out (single KO) in adult mouse pancreatic α cells, some α cells were converted to insulin-positive cells, but the conversion was incomplete. More than 60% of α cells remained insulin-unexpressing even after 12 weeks, and the converted cells did not express MafA, a β-cell maturation marker. Chakravarthy et al. showed that only by specifically knocking out the Dnmt1 gene in addition to the Arx gene (double KO) in α cells could 50-80% of α cells be efficiently converted to insulin-positive, PDX1-positive, and NKX6.1-positive cells. Brief description of the invention

[0008] This disclosure provides a method for promoting the differentiation of a first cell into a second cell, a method for specifying a treatment to promote such differentiation, for example, pancreatic β-cells or cells capable of differentiating into pancreatic β-cells, a composition containing such cells, and a method for producing the same. This disclosure also clarifies the usefulness of ARX disruption in the differentiation of pancreatic β-cells. More specifically, the inventors have found that under appropriate differentiation conditions, the efficiency of differentiation into pancreatic β-cells can be increased by disrupting only the ARX gene. Furthermore, in recent years, with the explosive spread of CRISPR / Cas genome editing technology, gene disruption libraries using CRISPR / Cas genome editing technology have been created, but these only involve generating indels in a part of the coding region or deleting a part of the coding region, and deletion of the entire coding region has hardly been attempted from the standpoint of editing efficiency. Therefore, this disclosure also provides an invention in which the treatment for promoting differentiation is gene disruption, and the gene disruption particularly includes deletion of the entire coding region.

[0009] (1) A method for culturing cells in vitro, comprising culturing first cells having disruption of the ARX gene or CDX2 gene under conditions for differentiation into pancreatic β cells to obtain a cell population including second cells, wherein the first and second cells are selected from cells selected from the group consisting of pluripotent stem cells, endodermal cells (DE), foregut endoderm cells (PGT), pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β cells, or from pancreatic α cells, and the second cells are in a later differentiation stage than the first cells. (2) The method according to (1) above, wherein at least 10% of the cell population are INS-positive and GCG-negative pancreatic β cells. (3) The method according to (1) or (2) above, wherein the second cells are NKX6.1-positive, INS-positive, GCG-negative, and SST-negative. (4) The method according to any one of (1) to (3) above, wherein the second cells have glucose-responsive C-peptide secretory ability. (5) The method according to any one of (1) to (4) above, wherein the first cell is a pluripotent stem cell, a confirmed endoderm cell, a primitive intestinal / posterior foregut cell, or a pancreatic progenitor cell, and the second cell is a pancreatic endocrine cell or a functional pancreatic β-cell, and the first cell or pancreatic progenitor cell derived from the first cell is cultured in the presence of an ALK5 inhibitor and a γ-secretase inhibitor to differentiate into an endocrine cell. (6) The method according to (5) above, wherein the differentiation conditions further include culturing the endocrine cell in serum-free medium to mature into a functional pancreatic β-cell. (7) The method according to (6) above, wherein the first cell or cell derived from the first cell is cultured as a three-dimensional sphere. (8) The method according to (7) above, further comprising the step of dissociating the cells, removing the undissociated clusters, and then re-aggregating the cells between differentiating into an endocrine cell and maturing the cells. (9) A composition comprising cells having disruption of the ARX gene, selected from cells selected from the group consisting of pluripotent stem cells, confirmed endoderm cells, primitive intestinal / posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β cells. (10) The composition according to (9) above, wherein the cells are selected from the group consisting of pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β cells.(11) The composition according to (9) or (10) above for use in transplanting to a subject. (12) The composition according to (9) or (10) above for use in the method according to any one of (1) to (8) above.

[0010] The present disclosure may provide the following inventions: [1A] A method for identifying a treatment that promotes the differentiation of first cells into second cells, comprising: culturing first cells subjected to treatment under appropriate differentiation conditions or modified conditions thereof for inducing differentiation into second cells; and identifying a treatment that (i) increases the efficiency of differentiation from first cells subjected to the treatment into second cells compared to first cells not subjected to the treatment; (ii) decreases the efficiency of differentiation into cell types other than second cells compared to first cells not subjected to the treatment; or (iii) improves the function of second cells, wherein the first cells are isolated cells subjected to the treatment and have the ability to differentiate into second cells. [2A] The method according to [1A], wherein the treatment is a treatment that deletes a gene coding region, and the first cells have a deletion of a gene coding region. [3A] The method according to [1A] or [2A], wherein the treatment comprises treatment with a compound. [4A] The method according to any one of [1A] to [3A] above, wherein the first cell is a pluripotent cell, a tissue stem cell, or a progenitor cell. [5A] The method according to any one of [1A] to [4A] above, wherein the second cell is an endodermal, mesodermal, or ectoderm cell. [6A] The method according to any one of [1A] to [5A] above, wherein the second cell is a pancreatic β-cell. [7A] The method according to any one of [1A] to [6A] above, wherein the deleted gene encodes a factor that promotes differentiation into cells other than the second cell. [8A] The method according to any one of [1A] to [7A] above, wherein the first cell is a pluripotent stem cell or a mesenchymal stem cell. [9A] The method according to any one of [1A] to [8A] above, wherein the appropriate differentiation conditions are conditions in which a compound that promotes differentiation is used, and the modified conditions are conditions in which the compound is not used. [10A] The method according to any one of [1A] to [9A] above, further comprising: evaluating the stability of the differentiation state of the second cells and / or the differentiation into a third cell and the stability of the differentiation state in the body of a subject administered with a genetically modified second cell; and identifying a treatment to improve the stability of the differentiation state of the second cells and / or the differentiation into a third cell and the stability of the differentiation state.

[0011] The present disclosure may also provide the following inventions: [1B] A composition comprising isolated genetically modified cells, (1) the genetically modified cells having disruption (preferably deletion, more preferably full-length deletion) of one or more genes encoding factors expressed in pancreatic endocrine cells other than pancreatic β-cells, (2) the genetically modified cells comprising one or more cells selected from the group consisting of pluripotent stem cells, definitive endoderm (DE), primitive gut tube, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells, or cells in a differentiation process between two of the said cells, (3) the genetically modified cells having either one or both of the following properties: (i) increased ability to differentiate into pancreatic β-cells, and (ii) increased ability to differentiate into pancreatic β-cells with increased insulin secretion capacity. [2B] The composition according to [1B] above for use in differentiating into pancreatic β cells in vitro. [3B] The composition according to [1B] or [2B] above, wherein the deleted gene comprises one or more genes encoding transcription factors selected from the group consisting of CDX2 and ARX. [4B] The composition according to any one of [1B] to [3B] above, wherein the isolated genetically modified cells are pluripotent stem cells and have a deletion of the gene encoding CDX2. [5B] The composition according to any one of [1B] to [3B] above, wherein the isolated genetically modified cells are pluripotent stem cells and have a deletion of the gene encoding ARX. [6B] The composition according to [1B] above, wherein the isolated genetically modified cells include pancreatic endocrine cells or functional pancreatic β cells and have a deletion of the gene encoding CDX2. [7B] The composition according to [1B] above, wherein the isolated genetically modified cells include pancreatic endocrine cells or functional pancreatic β cells and have a deletion of the gene encoding ARX.

[0012] The present disclosure may also provide the following inventions: [1C] A library of cloned cells, each cell having a genetic modification, each genetic modification of each cell comprising the deletion of an entire coding region of one or more genes. [2C] The library according to [1C], comprising 10 or more types of cloned cells. [3C] The library according to [1C] or [2C], each genetic modification comprising the deletion of an entire coding region of two or more genes. [4C] The library according to any one of [1C] to [3C], wherein the genetic modifications include gene disruption, and 50% or more of the gene disruptions include the deletion of an entire coding region of one or more genes. [5C] The library according to any one of [1C] to [4C], wherein the genetic modifications include gene disruption, and all of the gene disruptions include the deletion of an entire coding region of one or more genes. A library according to any of [1C] to [5C] above, wherein the [6C] cell (first cell) has the ability to differentiate into a specific cell (second cell), and the gene codes for a factor that is not expressed in the first cell, is not expressed in cells other than the second cell induced from the first cell, and is not expressed during the differentiation process from the first cell to the second cell.

[0013] The present disclosure may also provide the following inventions: [1D] A cloned cell having a genetic modification, wherein the genetic modification of each cell includes the deletion of the entire coding region of two or more genes. [2D] The cell according to [1D], wherein the genetic modification includes the deletion of the entire coding region of three or more genes. [3D] The cell according to [1D] or [2D], wherein the genetic modification includes the deletion of the entire coding region of five or more genes. [4D] The cell according to any one of [1D] to [3D], wherein the cell (first cell) has the ability to differentiate into a specific cell (second cell), and the gene codes for a factor that is not expressed in the first cell, is not expressed in cells other than the second cell induced from the first cell, and is not expressed during the differentiation process from the first cell to the second cell.

[0014] This document shows the differentiation potential of genetically modified cells into various pancreatic endocrine cells. It also shows the results of insulin secretion tests on pancreatic β-cells obtained from genetically modified cells. The first half of an example of preparing a multi-gene modified cell library is shown. The second half of an example of preparing a multi-gene modified cell library is shown. An example of the process of introducing modified barcode sequences and common sequences for primer binding is shown. An example of the process of decoding modified barcode sequences for each cell by high-throughput sequencing is shown. The document describes a six-step process for inducing β-cell differentiation from pluripotent stem cells in vitro, the relationship between the differentiation stages of cells during the process and stages 1-6, and the results of estimating the cell abundance ratio in the resulting mature β-cells by single-cell RNA sequencing. The inset shows data for mature β-cells induced from wild-type iPSCs. The scheme of the transplantation experiment into mice is shown. The scheme of the glucose tolerance test in the example is shown. The types and presence or absence of ARX mutations in iPS cells, and the ratio of INS+ / GCG- and the ratio of GCG+ cells at the end of stage 5 (S5 end) and the end of stage 6 (S6 end) are shown. Results for two strains each for the wild type and the ARX mutant are shown. Blood glucose levels during the glucose tolerance test are shown. Changes in blood C-peptide concentration during the glucose tolerance test are shown. Changes in blood glucagon concentration during the glucose tolerance test are shown. Cellular marker expression at the completion of Stage 4 is shown. Cellular marker expression at the completion of Stage 5 is shown. Cellular marker expression at the completion of Stage 6 is shown.

[0015] In this specification, the singular form (a, an, the) includes the plural form unless it is clearly understood from the context that it should be interpreted differently.

[0016] In this specification, the term “comprising” has an open-ended meaning and, unless explicitly specified otherwise, does not exclude the possibility of including other elements or processes in addition to those described. Furthermore, “comprising” may also include the forms “consist essentially of” or “consist of.”

[0017] In this specification, “isolated” means that a substance (e.g., nucleic acid, protein, antibody, cell) has been separated from other components that are naturally bound to or present in its native state. The term “isolated” does not necessarily imply complete purity.

[0018] In this specification, “subjects” refers to mammals, including humans, and is not particularly limited to, but includes, for example, rodents such as mice and rats, domesticated animals such as pigs, goats, llamas, sheep, and cattle, companion animals such as dogs and cats, birds such as chickens, and primates such as monkeys. In this specification, mammals may preferably be humans. In this specification, mammals may also be non-human animals.

[0019] In this specification, “cell” may refer to the cell of interest, preferably a human cell. The cell may be a pluripotent stem cell, an endodermal cell, or a pancreatic β-cell or a cell capable of differentiating into a pancreatic β-cell. More specifically, the cell may be one or more cells selected from the group consisting of pluripotent stem cells (e.g., embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)), endodermal cells (Definitive Endoderm; DE), foregut endoderm cells (Primitive Gut Tube), pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and pancreatic β-cells (preferably functional pancreatic β-cells), or a cell in a differentiation process between two of these cells. Here, “functional” in the context of pancreatic β-cells means that the pancreatic β-cell secretes insulin in a glucose-dependent manner (or exhibits glucose-dependent insulinotropic secretion (GSIS)).

[0020] In this specification, the differentiation pathway from pluripotent stem cells to functional pancreatic β-cells may proceed along the following stages. Cells at each stage may be identified by the expression patterns of the markers described below. However, the marker expression patterns are representative for identifying these cells and are not limited to them. (1) Pluripotent stem cells (before the start of Stage 1): May be OCT4-positive, NANOG-positive, and / or SSEA3-positive. Pluripotent stem cells include embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells). (2) Definitive Endoderm cells (DE) (Stage 1): May be CXCR4-positive, SOX17-positive, and / or FOXA2-positive. Endoderm cells are also called definitive endoderm cells. Stage 1 may be performed by culturing pluripotent stem cells in the presence of activin A and a Wnt pathway agonist (e.g., CHIR99021). (3) Primitive Gut Tube (PGT) cells (Stages 2-3): May be HNF1B positive and / or HNF4A positive. In the posterior foregut stage, they may be PDX1 positive, HNF6 positive, SOX9 positive and NKX6.1 negative. Stage 2 may include culture in the presence of keratinocyte growth factor (KGF), and Stage 3 may include culture in the presence of retinoic acid and SANT1. Primitive gut cells are also called foregut endoderm cells. (4) Pancreatic progenitor cells (Stages 3-4): May be PDX1 positive and NKX6.1 positive. Co-expression of PDX1 and NKX6.1 is characteristic of pancreatic progenitor cells that have the ability to generate functional pancreatic β-cells producing a single hormone. PDX1-positive and NKX6.1-negative cells readily differentiate into polyhormone cells (cells that express multiple hormones simultaneously) and have a low ability to differentiate into functional β-cells. GP2 is known as a surface marker for pancreatic progenitor cells. Stage 4 may include culture in the presence of KGF, SANT1, retinoic acid, and activin A. (5) Endocrine progenitor cells (first half of Stage 5): May be NEUROG3-positive (transient), NKX2.2-positive, NEUROD1-positive, and / or chromogranin A (CHGA)-positive. SOX9 may be decreased. Sustained expression of SOX9 may indicate a failure of commitment to the endocrine line.Stage 5 may include culture in the presence of an ALK5 inhibitor, a γ-secretase inhibitor (e.g., GSXXI or DAPT) and thyroid hormone T3. (6) Pancreatic endocrine cells (later part of Stage 5): May express at least one of INS, GCG, SST and / or CHGA. Pancreatic endocrine cells may include pancreatic β cells (INS positive), pancreatic α cells (GCG positive), pancreatic δ cells (SST positive), and intestinal chromaffin cells (SLC18A1 positive). (7) Functional pancreatic β cells (SC-β cells) (Stage 6): May be NKX6.1 positive and INS positive, and preferably GCG negative and SST negative. Functional pancreatic β cells may have glucose-responsive insulin secretion ability (GSIS) or C-peptide secretion ability. CD49a (ITGA1) is known as a surface marker for functional pancreatic β cells. Stage 6 may include culture in serum-free medium. During the transition from Stage 5 to Stage 6, a reaggregation process may be performed in which cell spheres are dissociated, undissociated clusters are removed, and then reaggregated.

[0021]

[0022] The above stage classifications are illustrative, and the correspondence of cell types included in each stage may vary depending on the type of differentiation protocol. For example, stage 3 cells may correspond to posterior foregut cells or to the early stage of pancreatic progenitor cells, depending on the differentiation protocol. In this specification, the differentiation stages of the first and second cells may be determined not by stage numbers, but by the expression patterns of the markers described above. For example, cells may be identified by the cell names in Table 1, by the stages in Table 1, or by the cell names in Table 1 and markers characteristic of the stage. Those skilled in the art will understand what kind of cells the above names, stages, and markers represent and how they can be obtained.

[0023] In this specification, “therapeutic dose” means an amount sufficient to produce a desired therapeutic effect (e.g., reduction, elimination, or prevention of disease symptoms) in a subject requiring treatment, without causing unacceptable toxicity or side effects.

[0024] In this specification, "sequence identity" refers to the percentage of identical residues when two sequences are optimally aligned. Sequence identity can be calculated using standard alignment algorithms such as BLAST or ClustalW.

[0025] In this specification, “differentiation potential” means the ability of a cell to differentiate into a specific type of cell or tissue under specific stimuli or culture conditions. In this specification, “differentiation potential” refers, for example, to the ability of a cell (e.g., ES cells, iPS cells, mesenchymal stem cells, etc.) to differentiate into at least one type of mature cell, progenitor cell, or tissue.

[0026] In this specification, "insulin" refers to a molecule consisting of two peptide chains, A and B, linked by a disulfide bond, which is synthesized as proinsulin in the β-cells of the pancreas and produced by the enzymatic cleavage of C-peptide. Functionally, it refers to a peptide hormone that is involved in blood glucose regulation and, in the body, acts particularly on the liver, muscles, and adipose tissue to promote glucose uptake from the blood. In the body, insulin is produced when proinsulin is synthesized in the β-cells of the pancreas, and then the C-peptide is cleaved by proteolytic enzymes. The amount of insulin produced is estimated by measuring the amount of insulin or C-peptide.

[0027] In this specification, "pancreatic cells" includes all cells of pancreatic origin unless otherwise specified, and encompasses endocrine cells present in the pancreatic islets (islets of Langerhans), including insulin-secreting β-cells, glucagon-secreting α-cells, somatostatin-secreting δ-cells, PP-cells that secrete pancreatic polypeptides, serotonin-secreting intestinal chromaffin cells (EC cells), and exocrine cells that secrete digestive enzymes (acinate cells), as well as their progenitor cells, stem cells, or cells obtained by induced differentiation.

[0028] In this specification, "pancreatic β-cells" refer to a type of endocrine cell found in the pancreatic islets (islets of Langerhans) that regulates blood glucose levels by synthesizing and secreting proinsulin and producing and secreting mature insulin. Unless otherwise specified, this specification includes not only naturally occurring pancreatic β-cells but also β-cell-like cells derived and differentiated from stem cells and progenitor cells. Pancreatic β-cells can be identified by confirming the presence or absence of expression of pancreatic β-cell-specific markers (e.g., insulin, proinsulin, C-peptide, PDX-1, NKX6.1, MAFA, etc.) by immunohistochemistry, RT-PCR, or flow cytometry, or by glucose stimulation tests to evaluate insulin secretion capacity.

[0029] In this specification, "alpha cells" refer to a type of endocrine cell found in the pancreatic islets (islets of Langerhans) that primarily produces and secretes the peptide hormone glucagon, and is involved in the elevation and regulation of blood glucose levels. Alpha cells can be identified by the presence of markers that are specifically or selectively expressed in alpha cells (for example, glucagon, ARX, IRX2, etc.). Unless otherwise specified, this specification also includes alpha cell-like cells that are induced and differentiated from stem cells or progenitor cells, express these markers, and have the ability to produce glucagon.

[0030] In this specification, "δ cells" refer to a type of endocrine cell present in the pancreatic islets (islets of Langerhans) that primarily produces and secretes the peptide hormone somatostatin, and is involved in the suppression and regulation of hormone secretion within the pancreatic islets and the gastrointestinal tract. δ cells can be identified by the presence of markers specifically or selectively expressed by δ cells (e.g., somatostatin or HHEX). Unless otherwise specified, this specification also includes δ cell-like cells that are induced and differentiated from stem cells or progenitor cells, express these markers, and have the ability to produce somatostatin.

[0031] In this specification, "pancreatic polypeptide cells" or "PP cells" refer to a type of endocrine cell present in the pancreatic islets (islets of Langerhans) that primarily produces and secretes pancreatic polypeptides (PP) and is involved in regulating gastrointestinal motility and secretory function. PP cells can be identified by the presence of markers that are specifically or selectively expressed by PP cells (e.g., pancreatic polypeptides or specific transcription factors (e.g., PAX6)). Unless otherwise specified, this specification also includes PP cell-like cells that are induced and differentiated from stem cells or progenitor cells, express these markers, and have the ability to produce pancreatic polypeptides.

[0032] In this specification, "pancreatic endocrine cells" refers to a group of cells located within the pancreatic islets (islets of Langerhans) that primarily regulate blood glucose levels and gastrointestinal function by producing and secreting hormones. Specifically, this includes β-cells that secrete insulin, α-cells that secrete glucagon, δ-cells that secrete somatostatin, and PP-cells that secrete pancreatic polypeptides.

[0033] In this specification, “transcription factor” refers to a protein that has the ability to promote the transcription of a target gene and is involved in regulating gene expression. Transcription factors typically regulate gene expression by specifically or selectively binding to specific base sequences on DNA (e.g., promoters, enhancers, etc.). In pancreatic cells, transcription factors include, but are not limited to, PDX-1, NKX6.1, NKX2.2, Neurod1, MAFA, MAFB, HHEX, and PAX6 {these genes are preferably not targeted for disruption}. Unless otherwise specified, this specification includes not only these native transcription factors but also their variants that have substantially the same function as these native proteins.

[0034] In this specification, “deletion” refers to the loss or removal of part or all of the gene or genomic sequence in question. More specifically, in nucleic acid sequences such as genomic DNA, cDNA, and RNA, this includes cases where part or all of the base sequence is missing or removed, resulting in either complete loss of function of the gene (e.g., complete deletion) or partial reduction or modification of function (e.g., partial deletion). Furthermore, “deletion” includes both those artificially induced by gene editing technologies (e.g., CRISPR-Cas9, ZFN, TALEN, etc.) and those resulting from natural mutations. The disruption or deletion may preferably be disruption or deletion in all alleles (more preferably a deletion of the entire length). A gene includes a coding region.

[0035] In this specification, "CDX2" refers to a type of homeobox transcription factor, a protein involved in the differentiation of intestinal epithelial cells and the regulation of intestinal-specific gene expression. Materially, it includes proteins that possess the amino acid sequence encoded by the CDX2 gene, and functionally, it includes proteins that bind to specific DNA sequences of target genes and exhibit transcriptional regulatory activity. Human CDX2 may have amino acid sequences registered, for example, NCBI Reference Sequence: NP_001256.4 or NCBI Reference Sequence: NP_001341629.1, or UniProtKB Q99626. CDX2 may include proteins (especially native proteins) that have 90% or more sequence identity with the above amino acid sequences and possess the function of CDX2.

[0036] In this specification, "ARX" refers to a type of homeodomain transcription factor encoded by the Aristaless-related homeobox gene (ARX gene), a protein that plays an important role in the differentiation and development of pancreatic α-cells and the nervous system. Human ARX may have an amino acid sequence registered, for example, in NCBI Reference Sequence: NP_620689.1 or UniProtKB Q96QS3. ARX may include proteins (especially native proteins) that have 90% or more sequence identity with the above amino acid sequence and possess the function of ARX.

[0037] As used herein, "mesenchymal stem cell" (MSC) refers to a stromal progenitor cell having self-renewal ability and pluripotency, and means a cell that has the ability to differentiate into multiple mesodermal lineage cells such as osteoblasts, chondrocytes, and adipocytes under standard culture conditions. MSCs can be isolated from various tissues such as bone marrow, adipose tissue, umbilical cord, placenta, and dental pulp, and generally include a group of cells having phenotypic characteristics of positive expression of CD73, CD90, CD105, and substantially negative or low expression of hematopoietic markers CD34, CD45, CD11b, CD14, CD19, CD79α and HLA-DR. As used herein, "mesenchymal stem cell" is intended to include not only naturally occurring MSCs, but also cells with equivalent properties produced by induced differentiation, reprogramming, genetic manipulation, or the like.

[0038] <Method for identifying a treatment that promotes the transformation of a first cell into a second cell> According to the present disclosure, a method for identifying a treatment that promotes the transformation of a first cell into a second cell is provided. The first cell and the second cell differ in the differentiation stage.

[0039] The method of the present disclosure may be an in vitro method.

[0040] In certain embodiments, the first cell is more undifferentiated than the second cell, and the first cell has the ability to transform into the second cell (e.g., the ability to differentiate). The method of the present disclosure comprises culturing the first cell that has been subjected to the above treatment under appropriate differentiation conditions or modified conditions thereof for inducing differentiation into the second cell (in particular, differentiating into the second cell, or attempting to differentiate into the second cell).

[0041] The aforementioned treatment is, for example, a compound treatment or gene modification, preferably gene disruption, for example, disruption that causes 20% or less, 10% or less, 5% or less, or complete loss of the function of the gene, more preferably deletion of a region including the entire coding region of the gene {the deletion may include the coding region of another gene, but preferably does not include the coding region of another gene}. The compound is not particularly limited, but includes, for example, physiologically active substances such as proteins (e.g., secretory factors, hormones, etc.), nucleic acids, and small molecule compounds (signaling agonists and antagonists, etc.). The gene is not particularly limited, but may be, for example, a gene encoding a transcription factor or a factor of a signaling pathway. In a preferred embodiment, the transcription factor or factor of a signaling pathway may promote differentiation into cells of a lineage other than the second cell. In a preferred embodiment, the transcription factor or factor of a signaling pathway may suppress differentiation into the second cell. The gene is not particularly limited, but may be, for example, a secretory factor (autocrine secretory factor or paracrine secretory factor). Secreted factors can be differentiation-promoting factors, either promoting differentiation into cells other than the second cell, or destabilizing the differentiation state of the second cell.

[0042] Prior art has reported that knocking out the ARX gene in human ES cells significantly reduces the number of insulin-producing cells (Gage et al., 2015). However, the present inventors have found that, under appropriate induction processes (particularly cells selected from the group consisting of pluripotent stem cells, confirmed endoderm cells, primitive intestinal / posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells, as described later), INS-positive and GCG-negative functional pancreatic β-cells can be efficiently obtained from cells with ARX gene disruption without a reduction in INS-positive cells. This finding indicates that the combination of ARX gene disruption and the above-mentioned cells or induction processes is effective in inducing pancreatic β-cells.

[0043] In a preferred embodiment, the aforementioned treatment is disruption of the ARX gene. ARX is a transcription factor that promotes differentiation into pancreatic α cells, and disruption of the ARX gene can suppress differentiation into α cells and increase the efficiency of differentiation into pancreatic β cells. Disruption of the ARX gene may include, but is not limited to, deletion of the entire coding region, nonsense mutations, or frameshift mutations.

[0044] Without being bound by theory, in the differentiation protocols used in the prior art, the resulting cells were immature polyhormonal cells that simultaneously express insulin and glucagon, whereas under the differentiation conditions of the present disclosure, the use of an ALK5 inhibitor at an appropriate timing promotes the specification of endocrine progenitor cells to the β lineage, the γ-secretase inhibitor inhibits Notch signals to promote endocrine differentiation, and the maturation step in a serum-free medium is considered to be particularly advantageous for acquiring glucose-responsive insulin secretion capacity. Under these mature differentiation conditions, disruption of the ARX gene functions as a "bypass block" that selectively blocks differentiation into the α cell lineage, improving the efficiency of differentiation into the β cell lineage.

[0045] The genes to be disrupted can be selected as follows: One or more genes can be selected and disrupted from a group of genes that satisfy the following conditions: they are not expressed in the first cell, they are not expressed during the differentiation process from the first cell to the second cell, they are not expressed in the second cell, and they are expressed in cells that can differentiate from the first cell but are not the second cell. This is because the gene disruption does not affect the acquisition of the second cell, and it may be possible to suppress the differentiation of cells that can differentiate from the first cell but are not the second cell. In one preferred embodiment, the gene to be disrupted may further satisfy the condition that it codes for a transcription factor or a signaling pathway factor. Alternatively, in one preferred embodiment, the gene to be disrupted may further satisfy the condition that it codes for a secretion factor. In another preferred embodiment, the factor to be disrupted may be a factor whose disruption significantly affects the expression levels of multiple genes. For example, the factor to be disrupted may be a factor whose disruption significantly affects the expression levels of two, three, four, five, six, seven, eight, nine, or ten or more other genes. For example, a disrupted factor may be one whose disruption more than doubles or reduces the expression levels of two, three, four, five, six, seven, eight, nine, or ten or more other genes to less than 50%.

[0046] Such genes, in some embodiments, are not, for example, factors for maintaining pluripotency. Examples of factors for maintaining pluripotency include Oct4, Nanog, and Sox2. Also, in some embodiments, such genes are not, for example, factors essential for cell proliferation. Furthermore, such genes may contain one or both of ARX and CDX2, or may not contain one or both of ARX and CDX2. In some preferred embodiments, such genes do not significantly interfere with the normal function of the target cell.

[0047] In one embodiment, a library of cells having one or more gene disruptions that satisfy the above criteria is provided. This library includes, for example, two or more, three or more, four or more, five or more, ten or more, twenty or more, thirty or more, forty or more, or fifty or more types of cells, each cell having a different gene disruption. The disruption may preferably be a deletion of the entire coding region of a gene.

[0048] In one preferred embodiment, each cell in this library has a deletion of the entire coding region of a gene. That is, in one preferred embodiment, all cells in this library have a deletion of the entire coding region of a gene. In one preferred embodiment, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, or all of the gene-disrupting cells in this library are disrupted by a deletion of the coding region. In one embodiment, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, or all of the disrupted genes in the library are disrupted by a deletion of the coding region. Any or all of the cells in this library may have deletions of the entire coding region of two different genes. A library having deletions of the entire coding region of two different genes is not particularly limited, but may be prepared, for example, by a method for preparing a library including multiple gene-modified cells as described later.

[0049] In one preferred embodiment, a cell having multiple gene disruptions, wherein each gene disruption involves the deletion of an entire coding region. The multiple gene disruptions are not particularly limited but may include, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more gene disruptions. The multiple gene disruptions are not particularly limited but may include, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer gene disruptions.

[0050] Appropriate differentiation conditions for inducing differentiation into a second cell include culture conditions suitable for obtaining a second cell from a first cell. These culture conditions may include one-step or multi-step differentiation conditions.

[0051] The modified conditions are conditions obtained by modifying the appropriate differentiation conditions, and may be conditions that promote differentiation induction into a second cell more than the appropriate differentiation conditions, conditions that promote differentiation induction into a second cell equivalent to the appropriate differentiation conditions, conditions that promote differentiation induction into a second cell less efficiently than the appropriate differentiation conditions, or conditions that make differentiation induction into a second cell impossible.

[0052] This disclosure may identify treatments that enable differentiation into a second cell even when the modified conditions include conditions that would otherwise prevent differentiation into a second cell, and may also identify treatments that improve differentiation into a second cell. This determination of differentiation-enabling treatments under modified conditions may allow for the exclusion of compounds previously considered important for differentiation and their replacement with other compound treatments or genetic modifications.

[0053] In some embodiments, the first cell is a pluripotent cell, tissue stem cell, or progenitor cell. In some preferred embodiments, the first cell may be an induced pluripotent stem cell (iPS cell). In some preferred embodiments, the first cell may be a mesenchymal stem cell. The first cell preferably has the ability to differentiate into multiple differentiation lineages, and the treatments in this disclosure may completely or partially restrict this differentiation ability to increase the efficiency of differentiation induction into a second cell or to improve the function of the second cell.

[0054] The second cell is an endodermal, mesodermal, or ectoderm cell. In a preferred embodiment, the second cell may be an endodermal cell (e.g., hepatocytes and pancreatic β-cells). In one embodiment, the second cell is a neuronal cell such as neural stem cells, motor neurons, sensory neurons, dopaminergic neurons, and cholinergic neurons; a glial cell such as astrocytes, oligodendrocytes, and microglia; cardiovascular cells such as cardiac stem cells, cardiac progenitor cells, cardiomyocytes (ventricular myocytes and atrial myocytes), cardiac pacemaker cells, vascular endothelial cells, and vascular smooth muscle cells; endocrine cells such as pancreatic β-cells, pancreatic α-cells, thyroid follicular cells, and pituitary hormone-producing cells; digestive system cells such as hepatocytes, bile duct cells (e.g., bile duct epithelial cells), intestinal epithelial cells, and gastric epithelial cells; respiratory system cells such as alveolar epithelial cells (type I or type II), bronchial epithelial cells (e.g., ciliated cells or goblet cells), and tracheal smooth muscle cells; hematopoietic stem cells, erythrocytes (erythroid progenitor cells) Hematopoietic cells and immune cells such as platelet cells (megakaryocytes), T cells (T lymphocytes), B cells (B lymphocytes), NK cells (natural killer cells), dendritic cells, macrophages, and neutrophils; skeletal muscle cells such as skeletal muscle cells (myotubes), osteoblasts, chondrocytes, and tendon cells (tendonblasts); skin cells such as epidermal keratinocytes, melanocytes, and dermal fibroblasts; sensory cells such as retinal pigment epithelial cells (RPE cells), photoreceptor cells (cone cells, rod cells), photoreceptor progenitor cells, and hair cells (auditory cells of the inner ear); urogenital cells such as renal tubular epithelial cells, glomerular podocytes, and germ cells (male or female primordial germ cells); corneal cells such as corneal epithelial cells and corneal stem cells; and cells similar to these cells, as well as cells having the same function.

[0055] In one embodiment, the first cell may be a pluripotent stem cell (e.g., an iPS cell), and the second cell may be a pancreatic β-cell.

[0056] The coding regions of genes on the genome are typically a few kilobytes to several hundred kilobytes in size. For very large genes, the coding region can reach up to 1 megabyte. Deletions of such large regions can be created, for example, by the method described in US2024 / 0052337A, which is incorporated herein by reference. Knockdown is difficult to achieve complete loss of expression levels, and partial deletion and indel-based knockout may yield different results than the deletion of the entire coding region. In contrast, while the technique of deleting the entire coding region exists, it is generally not used for knockout due to its complexity and inefficiency; instead, partial deletion and indel-based knockout are used exclusively. Partial deletion and indel-based knockout are considered sufficient for effective knockout. Therefore, the deletion of the entire coding region is not typically attempted during knockout.

[0057] The method disclosed herein may be used to obtain first and second cells having multiple genetic modifications. The method disclosed may be used to introduce multiple genetic modifications into the first cell with the aim of improving the induction efficiency of the second cell from the first cell. The method disclosed may also be used to identify a second genetic modification that promotes the differentiation induction of the second cell from the first cell having the first genetic modification.

[0058] The method of this disclosure can be used, for example, to increase the number of second cells obtained when the quality of the second cells is improved and the number of second cells obtained decreases. The method of this disclosure can also be used, for example, to improve the quality of second cells obtained when the number of second cells obtained is improved and the quality of the second cells obtained decreases. Some of these cells may be cells whose differentiation into other cells is suppressed when administered into the body. The method of this disclosure can also be used to identify such cells. The method of identifying the differentiation-stabilizing process of this disclosure is described in detail below.

[0059] <Method for identifying a process to stabilize the differentiation state of the cells in this disclosure> Second cells with gene deletions, or third cells obtained by further differentiating the second cells, may be more likely to maintain the characteristics of the second cells and the third cells after being administered into the body of a subject. Therefore, the above method of this disclosure can be used to construct a technique for stabilizing the characteristics of cells after administration. For example, when pancreatic β cells are administered in vivo, they may change into intestinal cells. For example, by suppressing the differentiation of cells into intestinal cells, it is possible to suppress the change of administered cells into unintended cells in vivo. Therefore, in this disclosure, it may be beneficial to disrupt factors that promote the change into unintended cells.

[0060] In the method disclosed herein, the second cell does not need to be differentiated from the first cell, but may be a cell obtained by differentiating from the first cell.

[0061] In some respects, the method of the present disclosure may be a method for identifying a process that stabilizes the differentiation state of a second cell. The method of the present disclosure may be an in vivo method.

[0062] The method of this disclosure may include administering a second cell that has been subjected to a treatment to a target, and identifying a treatment that suppresses the unintended differentiation of the second cell into an unintended cell type after transplantation, compared to a second cell that has not been subjected to the treatment. Unintended differentiation into an unintended cell type may include, in the case of expecting the maintenance of the second cell after administration, a change from the second cell to a cell other than the second cell, or in the case of expecting differentiation from the second cell to a third cell, a change to a cell other than the third cell. For example, the second cell may be a progenitor cell of the third cell, and it may be expected that the administered second cell will differentiate into the third cell and stabilize as the third cell. An example of the second cell is a progenitor cell of pancreatic β-cells, and an example of the third cell is a pancreatic β-cell.

[0063] The subjects may be humans or non-human animals, such as non-human mammals, such as non-human primates, rodents, rabbits, guinea pigs, hamsters, dogs, cats, pigs, goats, sheep, and cows (e.g., laboratory animals).

[0064] Pancreatic β-cells having the genetic modifications described herein can be used for transplantation into a subject. In some embodiments, the pancreatic β-cells may be transplanted subcapsularly, subcutaneously, or into the hepatic portal vein of the subject. The pancreatic β-cells can maintain glucose-responsive C-peptide secretion for at least 100 days, preferably at least 130 days, after transplantation.

[0065] In one embodiment, pancreatic β-cells having the genetic modification of the present disclosure secrete C-peptide in response to glucose loading after transplantation into a subject, while glucagon secretion is substantially undetectable. This characteristic may indicate that disruption of the ARX gene reduces the contamination of the transplanted cell population with α-cells, and / or suppresses the conversion of pancreatic β-cells to α-cells after transplantation. Therefore, the pancreatic β-cells of the present disclosure can contribute to improving the quality of cell therapy, not only in terms of the stability of insulin secretion after transplantation, but also in terms of suppressing unintended glucagon secretion.

[0066] A composition comprising pancreatic β-cells having disruption of the ARX gene as disclosed herein may be provided as a cell composition for use in the treatment of diabetes (including type 1 diabetes and type 2 diabetes).

[0067] The aforementioned treatment is, for example, a compound treatment or gene modification, preferably gene disruption, and more preferably deletion of a region including the entire coding region of a gene. The compound is not particularly limited, but includes, for example, physiologically active substances such as proteins (e.g., secretory factors, hormones, etc.), nucleic acids, and small molecule compounds (e.g., signaling agonists and antagonists). The gene is not particularly limited, but may be, for example, a gene encoding a transcription factor or a signaling pathway factor. In a preferred embodiment, the transcription factor or signaling pathway factor may promote differentiation into a cell lineage other than the second cell. In a preferred embodiment, the transcription factor or signaling pathway factor may suppress differentiation into the second cell. The gene may encode a factor that promotes differentiation of the second cell into a cell other than the third cell. Alternatively, the gene may encode a factor that suppresses differentiation of the second cell into the third cell. Alternatively, the gene may encode a factor that promotes dedifferentiation of the third cell. Alternatively, the gene may encode a factor that inhibits the stabilization of the third cell. In a preferred embodiment, the factor may be a transcription factor, a signaling pathway factor, or a secretory factor.

[0068] The second cell is an endodermal, mesodermal, or ectoderm cell. In a preferred embodiment, the second cell may be an endodermal cell (e.g., hepatocytes and pancreatic β-cells). In a particular embodiment, the second cell may be a neuronal cell such as a motor neuron, sensory neuron, dopaminergic neuron, and cholinergic neuron; a glial cell such as astrocytes, oligodendrocytes, and microglia; cardiovascular cells such as cardiomyocytes (ventricular myocytes and atrial myocytes), cardiac pacemaker cells, vascular endothelial cells, and vascular smooth muscle cells; endocrine cells such as pancreatic β-cells, pancreatic α-cells, thyroid follicular cells, and pituitary hormone-producing cells; digestive system cells such as hepatocytes, bile duct cells (e.g., bile duct epithelial cells), intestinal epithelial cells, and gastric epithelial cells; respiratory system cells such as alveolar epithelial cells (type I or type II), bronchial epithelial cells (e.g., ciliated cells or goblet cells), and tracheal smooth muscle cells; hematopoietic stem cells, red blood cells Examples include hematopoietic cells and immune cells such as blood cells (erythroid progenitor cells), platelet cells (megakaryocytes), T cells (T lymphocytes), B cells (B lymphocytes), NK cells (natural killer cells), dendritic cells, macrophages, and neutrophils; skeletal muscle cells such as skeletal muscle cells (myotube cells), osteoblasts, chondrocytes, and tendon cells (tendonblasts); skin cells such as epidermal keratinocytes, melanocytes, and dermal fibroblasts; sensory cells such as retinal pigment epithelial cells (RPE cells), photoreceptor cells (cone cells, rod cells), and hair cells (auditory cells of the inner ear); and urogenital cells such as renal tubular epithelial cells, glomerular podocytes, and germ cells (male or female primordial germ cells); or their progenitor cells.

[0069] In some embodiments, the methods of the present disclosure may include evaluating the stability of the differentiation state of a second cell having a genetic modification (particularly deletion of an entire coding region of a gene) in the body of a subject (human or non-human subject) administered with the second cell, and / or differentiation into a third cell and the stability of that differentiation state.

[0070] The assessment of differentiation stability may include measuring not only the target secretory factor (e.g., insulin or C-peptide) but also untargeted secretory factors (e.g., glucagon). For example, when administered to pancreatic β-cells, if the glucagon concentration in the body fluid is substantially undetectable or decreased compared to a control (e.g., a subject administered cells without gene disruption), it indicates that the transplanted pancreatic β-cells have not differentiated into α-cells, and thus demonstrates high differentiation stability of the pancreatic β-cells. In one embodiment, the differentiation stability of pancreatic β-cells can be assessed by observing an increase in glucose-responsive C-peptide secretion and the absence of a substantial increase in glucagon secretion in a glucose tolerance test.

[0071] The stability of the differentiation state of the second cell, and / or the differentiation into the third cell and the stability of that differentiation state, can be evaluated by invasive or non-invasive methods. If the second or third cell is a secretory cell, the stability of the differentiation state of the second cell, and / or the differentiation into the third cell and the stability of that differentiation state, can be evaluated by measuring the factors secreted by the cell. For example, if the second or third cell is a pancreatic β-cell, measuring the insulin concentration in body fluid (especially in the blood) indicates that stability of the differentiation state of the second cell, and / or the differentiation into the third cell and the stability of that differentiation state, indicates high stability of the differentiation state of the second cell, and / or the differentiation into the third cell and the stability of that differentiation state, while a decrease in concentration indicates low stability of the differentiation state of the second cell, and / or the differentiation into the third cell and the stability of that differentiation state. If the decrease in the concentration of secretory factors is suppressed compared to the control, it indicates that the stability of the differentiation state of the second cell, and / or the differentiation into the third cell and the stability of that differentiation state, is higher than in the control (e.g., cells without the gene disruption).

[0072] Secretory cells include pancreatic β-cells that secrete insulin, pancreatic α-cells that secrete glucagon, thyroid follicular cells that secrete thyroid hormones, pituitary hormone-producing cells that secrete pituitary hormones (e.g., ACTH, TSH, growth hormone, prolactin, etc.), hepatocytes that secrete blood proteins, albumin, and coagulation factors, alveolar type II epithelial cells that secrete pulmonary surfactant, goblet cells that secrete airway mucus, dendritic cells and macrophages that secrete cytokines and hormones, plasma cells that secrete antibodies, osteoblasts that secrete bone matrix proteins, chondrocytes that secrete cartilage matrix, retinal pigment epithelial cells that secrete trophic factors and cytokines, and renal tubular epithelial cells that secrete hormones such as erythropoietin.

[0073] If the secretory cells are pancreatic β-cells that secrete insulin, pancreatic α-cells that secrete glucagon, thyroid follicular cells that secrete thyroid hormones, pituitary hormone-producing cells that secrete pituitary hormones, plasma cells that produce blood proteins, albumin, and antibodies, as well as hepatocytes that secrete coagulation factors, and renal tubular epithelial cells that secrete hormones such as erythropoietin, then the secretory factors can be measured in a blood sample.

[0074] Accordingly, in some embodiments, the method of the present disclosure may include measuring secretion factor levels in the body fluids (preferably blood samples, e.g., serum, plasma, whole blood, etc.) of a subject (human or non-human subject) to which a second cell having a genetic modification (particularly deletion of an entire gene coding region) has been administered, wherein maintenance or decrease in secretion factor levels indicates maintenance or decrease in the stability of the second cell or a third cell differentiated from the second cell, respectively. In some embodiments, the method of the present disclosure may include administering a second cell having a genetic modification (particularly deletion of an entire gene coding region) to a subject (human or non-human subject), and then measuring secretion factor levels in the subject's body fluids (preferably blood samples, e.g., serum, plasma, whole blood, etc.), wherein maintenance or decrease in secretion factor levels indicates maintenance or decrease in the stability of the second cell or a third cell differentiated from the second cell, respectively. Since embryonic surfactant is secreted into the alveolar space or airways, the body fluids of that portion may be the target of measurement. Goblet cells secrete mucus onto the surface of the bronchial or intestinal lumen, so the mucus on these surfaces can be used for measurement.

[0075] The selection of genes to be disrupted can be carried out according to the guidelines described above.

[0076] <Cells of the Disclosure> The Disclosure provides a first cell and a second cell having the aforementioned genetic modification. The first cell can be used to induce differentiation into the second cell. Accordingly, the Disclosure provides a composition comprising the first cell for use in inducing differentiation into the second cell. The Disclosure also provides a composition comprising the second cell for use in administration to a subject. In some embodiments, the first cell may be a pluripotent stem cell, preferably an iPS cell. In some embodiments, the second cell may be a pancreatic β-cell or its progenitor cell.

[0077] <Compositions of the Disclosure> The Disclosure provides compositions comprising isolated genetically modified cells. The compositions of the Disclosure can be used, for example, to obtain pancreatic β-cells by differentiating them in vitro.

[0078] The isolated genetically modified cells may be (α) pancreatic β-cells or (β) cells capable of differentiating into pancreatic β-cells. Cells capable of differentiating into pancreatic β-cells are not particularly limited, but may include, for example, one or more cells selected from the group consisting of pluripotent stem cells, definitive endoderm (DE), primitive gut tubes, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells, or cells in the differentiation process between two of the said cells.

[0079] In a preferred embodiment, isolated genetically modified cells may have one or both of the following characteristics: (i) increased ability to differentiate into pancreatic β-cells, and (ii) increased ability to differentiate into pancreatic β-cells with increased insulin secretion. In a preferred embodiment, isolated genetically modified cells have genetic modifications to have one or both of the above characteristics (i) and (ii).

[0080] Genetic modification may involve disruption (e.g., deletion, frameshift mutation, missense mutation, or nonsense mutation) of factors expressed in pancreatic cells other than pancreatic β-cells (e.g., pancreatic endocrine cells other than pancreatic β-cells). In a preferred embodiment, the disruption may be a deletion, more preferably a deletion of the entire gene.

[0081] In one preferred embodiment, the gene comprises a gene encoding one or more transcription factors expressed in pancreatic endocrine cells. Examples of transcription factors expressed in pancreatic endocrine cells (excluding pancreatic β-cells) include ARX, CDX2, MafB, Brn4 (POU3F4), Hhex, Pax4, Nkx2.2, Neurogenein3, NeuroD1, Isl1, Rfx6, and Insm1. The expression of these genes is necessary for differentiation into pancreatic endocrine cells (excluding pancreatic β-cells) or their normal function, and therefore, disruption is expected to reduce the influence of contamination by pancreatic endocrine cells (excluding pancreatic β-cells) in the resulting composition.

[0082] In some aspects, genes may be essential factors for differentiation into pancreatic endocrine cells (excluding pancreatic β-cells).

[0083] In one preferred embodiment, the gene comprises one or more genes selected from the group consisting of CDX2 and ARX.

[0084] In one preferred embodiment, the cells are cells having the ability to differentiate into pancreatic β-cells, the gene containing CDX2, and (ii) having the ability to differentiate into pancreatic β-cells with increased insulin secretion capacity. The increased insulin secretion capacity may be an increase in insulin secretion capacity compared to pancreatic β-cells without disruption of the CDX2 gene, differentiated in vitro under the same conditions.

[0085] In one preferred embodiment, the cells are cells capable of differentiating into pancreatic β-cells, and the gene contains ARX, which (i) increases the ability to differentiate into pancreatic β-cells. The increased differentiation ability may be compared to pancreatic β-cells without disruption of the ARX gene, which are differentiated in vitro under the same conditions.

[0086] In one preferred embodiment, the cells include pancreatic β-cells with increased insulin secretion capacity. In another embodiment, the cells include pancreatic β-cells with increased insulin secretion capacity, and the gene includes CDX2. The gene may further include genes other than CDX2 (e.g., HNF1α, HNF4α, PBX1, etc.), provided that the additional genes do not significantly inhibit insulin secretion due to CDX2 deletion.

[0087] In one preferred embodiment, the cells include pancreatic β-cells. In another embodiment, the cells include pancreatic β-cells and the gene includes ARX. In this embodiment, preferably, the composition contains a higher proportion of pancreatic β-cells compared to cells without ARX deletion. The gene may further include genes other than ARX (e.g., TLE1, TLE4, PAX6, DLX1, DLX2, etc.), provided that the additional genes do not significantly inhibit the improvement in the proportion of pancreatic β-cells due to ARX deletion.

[0088] The disclosure may also provide isolated pancreatic β-cells or a cell population containing said pancreatic β-cells having disruption of the ARX gene and being NKX6.1 positive, INS positive, GCG negative, and SST negative. The said pancreatic β-cells preferably have glucose-responsive C-peptide secretion ability. The said pancreatic β-cells may have a proportion of INS-positive cells equivalent to or greater than that of pancreatic β-cells obtained under the same differentiation conditions from cells without disruption of the ARX gene.

[0089] In one embodiment, in insulin-positive, PDX1-positive, and NKX6.1-positive cells, the ARX gene is disrupted, but preferably the Dnmt1 gene is not disrupted or is functional. In one embodiment, the ARX gene is disrupted, but the Dnmt1 gene is not disrupted or is functional. In one embodiment, the cells are selected from a group consisting of pluripotent stem cells, endodermal cells, foregut endoderm, pancreatic progenitor cells, and endocrine progenitor cells (particularly the first half of S5), and the ARX gene may or may not be disrupted, and the Dnmt1 gene may or may not be disrupted. In one embodiment, the cells are selected from a group consisting of endocrine progenitor cells (particularly the first half of S5), pancreatic endocrine cells (particularly the second half of S5), and functional pancreatic β-cells, and the ARX gene may be disrupted, and the Dnmt1 gene may not be disrupted or may be functional. In one embodiment, the cells have disrupted Dnmt1 and ARX genes.

[0090] <Method for producing the compositions of the present disclosure> The method for producing the compositions of the present disclosure may be used to produce the compositions of the present disclosure. The method for producing the compositions of the present disclosure may include differentiating starting cells under appropriate differentiation conditions to obtain pancreatic β-cells.

[0091] The following describes an example of a method for obtaining pancreatic β-cells using pluripotent stem cells as starting cells, but the manufacturing method disclosed herein is not limited to the following.

[0092] The manufacturing method disclosed herein may include one or more of six differentiation steps, from Stage 1 to Stage 6. Stage 1 may include a differentiation step from pluripotent stem cells to endodermal cells. Stage 2 may include a differentiation step from endodermal cells to foregut endoderm cells. Stage 3 may include a differentiation step from foregut endoderm cells to PDX1-positive pancreatic progenitor cells. Stage 4 may include a differentiation step from PDX1-positive pancreatic progenitor cells to NKX6.1-positive pancreatic progenitor cells. Stage 5 may include a differentiation step from NKX6.1-positive pancreatic progenitor cells to endocrine cells. Stage 6 may include a differentiation step from endocrine cells to β cells (preferably functional β cells).

[0093] In one preferred embodiment, the differentiation is carried out in three-dimensional sphere culture. Pluripotent stem cells are seeded on a suspension culture plate, and spheres (cell spheres) are formed by agitation culture (e.g., a rotating orbital shaker), after which differentiation is induced in the sphere state. Compared to two-dimensional sheet culture, three-dimensional sphere culture maintains intercellular paracrine and juxtaclin signaling and reproduces a pancreatic islet-like three-dimensional microenvironment. This culture form can contribute to the differentiation and functional maturation of endocrine cells.

[0094] The manufacturing method of the present disclosure may preferably include a stage 6 differentiation step. The stage 6 differentiation step may include culturing pancreatic endocrine cells in the presence of thyroid hormone T3 and a γ-secretase inhibitor (DAPT). This step is preferably carried out in the absence of a TGF-β inhibitor. The pancreatic endocrine cells include pancreatic β cells.

[0095] The manufacturing method of the present disclosure may preferably include a stage 5 differentiation step. The stage 5 differentiation step may include culturing NKX6.1-positive pancreatic progenitor cells in the presence of an ALK5 inhibitor, thyroid hormone T3, and a γ-secretase inhibitor (DAPT).

[0096] The manufacturing method of the present disclosure may preferably include a Stage 4 differentiation step. The Stage 4 differentiation step may include culturing PDX1-positive pancreatic progenitor cells in the presence of noggin, epidermal growth factor (EGF), and a TGF-β pathway inhibitor (e.g., an ALK5 inhibitor).

[0097] The manufacturing method of the present disclosure may preferably include a Stage 3 differentiation step. The Stage 3 differentiation step may include culturing foregut endoderm cells in the presence of fibroblast growth factor (FGF) 7, retinoic acid, SANT1, noggin, and EGF.

[0098] The manufacturing method of the present disclosure may preferably include a Stage 2 differentiation step. The Stage 2 differentiation step may include culturing endodermal cells in the presence of keratinocyte growth factor (KGF) and a Wnt inhibitor (e.g., IWR-1).

[0099] The manufacturing method of the present disclosure may preferably include a Stage 1 differentiation step. The Stage 1 differentiation step may include culturing pluripotent stem cells in the presence of activin A and a Wnt pathway agonist (e.g., CHIR99021).

[0100] In one preferred embodiment, a re-aggregation step is performed between differentiation into endocrine cells and maturation into functional pancreatic β-cells. This step involves dissociating the cell spheres into single cells, removing undissociated clusters, and then reseeding to form new spheres. This re-aggregation step is performed, for example, by dissociating the cells using enzymatic treatment (e.g., TrypLE), removing undissociated clusters through a cell strainer (e.g., 100 μm), and then reseeding at a predetermined cell density. The re-aggregation step can enrich the endocrine system cells and reform spheres of uniform size, thereby promoting the functional maturation of pancreatic β-cells in the subsequent maturation step.

[0101] As described above, pancreatic β-cells can be produced from one or more cells selected from the group consisting of pluripotent stem cells, definitive endoderm (DE), primitive gut tube, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells, or from cells in the differentiation process between two of the aforementioned cells. Furthermore, those skilled in the art can obtain pancreatic β-cells by techniques well known in this art. For example, see Veres A. et al., Nature, 569, pages 368-373 (2019) and Velazco-Cruz L. et al., Stem Cell Report, 12(2):1-15 (2019).

[0102] <Characteristics of cells with ARX gene disruption at each differentiation stage> The following describes the characteristics of cells with ARX gene disruption at each differentiation stage.

[0103] (A) Pancreatic progenitor cell stage (stage 4) Cells with disruption of the ARX gene can differentiate into pancreatic progenitor cells. Pancreatic progenitor cells with disruption of the ARX gene may have the same level of PDX1 and NKX6.1 expression as pancreatic progenitor cells without disruption of the ARX gene.

[0104] In one embodiment, the first or second cell is a pancreatic progenitor cell having a disruption of the ARX gene. In another embodiment, the first or second cell is a PDX1-positive pancreatic progenitor cell having a disruption of the ARX gene. In yet another embodiment, the first or second cell is an NKX6.1-positive pancreatic progenitor cell having a disruption of the ARX gene.

[0105] In one embodiment, the first or second cell is a pancreatic progenitor cell having a disruption of the ARX gene, and being PDX1-positive and NKX6.1-positive.

[0106] In one embodiment, the first or second cell is a pancreatic progenitor cell having a disruption of the ARX gene, and being PDX1-positive, NKX6.1-positive, and NEUROG3-negative.

[0107] In one embodiment, the first or second cell is a pancreatic progenitor cell having a disruption of the ARX gene, and being PDX1-positive, NKX6.1-positive, CHGA-negative, and INS-negative.

[0108] In one embodiment, the first or second cell is a pancreatic progenitor cell having a disruption of the ARX gene, and being PDX1-positive, NKX6.1-positive, and GP2-positive.

[0109] In one embodiment, the first or second cell is a stage 4 cell having a disruption of the ARX gene. In another embodiment, the first or second cell is a stage 3 or stage 4 cell having a disruption of the ARX gene.

[0110] In one embodiment, the first or second cell is a stage 4 cell that has a disruption of the ARX gene, is PDX1-positive and NKX6.1-positive.

[0111] In one embodiment, the first or second cell is a stage 4 cell having a disruption of the ARX gene, and is PDX1-positive, NKX6.1-positive, CHGA-negative, and INS-negative.

[0112] (B) Endocrine progenitor cell / pancreatic endocrine cell stage (stage 5) Pancreatic progenitor cells with disruption of the ARX gene can differentiate into pancreatic endocrine cells via the endocrine progenitor cell stage. A cell population containing pancreatic endocrine cells with disruption of the ARX gene may have a lower proportion of GCG-positive cells (α cells) and a maintained or increased proportion of INS-positive cells (β cells) compared to a cell population obtained under the same differentiation conditions from cells without disruption of the ARX gene.

[0113] In one embodiment, the first or second cell is an endocrine progenitor cell having a disruption of the ARX gene. In another embodiment, the first or second cell is a pancreatic endocrine cell having a disruption of the ARX gene.

[0114] In one embodiment, the second cell is an INS-positive pancreatic endocrine cell with a disruption of the ARX gene.

[0115] In one embodiment, the second cell is a pancreatic endocrine cell that has a disruption of the ARX gene, is INS-positive, and GCG-negative.

[0116] In one embodiment, the second cell is a pancreatic endocrine cell that has a disruption of the ARX gene, is INS-positive, GCG-negative, and SST-negative.

[0117] In one embodiment, the second cell is a pancreatic endocrine cell that has a disruption of the ARX gene, is NKX6.1 positive, and is INS positive.

[0118] In one embodiment, the second cell is a pancreatic endocrine cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, and GCG negative.

[0119] In one embodiment, the second cell is a pancreatic endocrine cell that has a disruption of the ARX gene and is NKX6.1 positive, INS positive, GCG negative, and SST negative.

[0120] In one embodiment, the second cell is a pancreatic endocrine cell having a disruption of the ARX gene, and is NKX6.1 positive, INS positive, GCG negative, SST negative, and SLC18A1 negative.

[0121] In one embodiment, the second cell is an endocrine progenitor cell that has a disruption of the ARX gene, is CHGA-positive, and is NKX6.1-positive.

[0122] In one embodiment, the second cell is an endocrine progenitor cell having a disruption of the ARX gene, being CHGA-positive, NKX6.1-positive, and SOX9-negative or low-expressing.

[0123] In one embodiment, the second cell is a stage 5 cell with disruption of the ARX gene. In another embodiment, the second cell is an early stage 5 cell (corresponding to an endocrine progenitor cell) with disruption of the ARX gene. In yet another embodiment, the second cell is an late stage 5 cell (corresponding to a pancreatic endocrine cell) with disruption of the ARX gene.

[0124] In one embodiment, the second cell is a stage 5 cell that has a disruption of the ARX gene, is INS-positive, and GCG-negative.

[0125] In one embodiment, the second cell is a stage 5 cell with a disruption of the ARX gene, and is NKX6.1 positive, INS positive, GCG negative, and SST negative.

[0126] In one embodiment, the second cell is a stage 5 cell that has a disruption of the ARX gene, is CHGA-positive and NKX6.1-positive.

[0127] In one embodiment, the proportion of GCG-positive cells in a cell population containing stage 5 cells with ARX gene disruption is 70% or less, 50% or less, 30% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or below the detection limit, compared to the proportion of GCG-positive cells in a stage 5 cell population obtained from cells without ARX gene disruption under the same differentiation conditions.

[0128] In one embodiment, the proportion of somatostatin-positive (SST+) cells in a cell population containing stage 5 cells with ARX gene disruption is 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, or 6% or less in a stage 5 cell population obtained from cells without ARX gene disruption under the same differentiation conditions, for example, 3% to 7%.

[0129] In one embodiment, the proportion of SLC18A1-positive (SLC18A1+) cells in a cell population containing stage 5 cells with ARX gene disruption is 20% or less, 15% or less, or 10% or less in a stage 5 cell population obtained from cells without ARX gene disruption under the same differentiation conditions, for example, 5% to 11%.

[0130] In one embodiment, the proportion of INS-positive cells in a cell population containing stage 5 cells with ARX gene disruption is at least 80%, at least 90%, at least 95%, at least 100%, or at least 110% of the proportion of INS-positive cells in a stage 5 cell population obtained from cells without ARX gene disruption under the same differentiation conditions.

[0131] In one embodiment, the proportion of INS-positive cells in a cell population containing stage 5 cells with ARX gene disruption may be at least 10%, 15%, 20%, or 25% (e.g., 20% to 30%) of a stage 5 cell population obtained from cells without ARX gene disruption under the same differentiation conditions. In another embodiment, the proportion of INS-positive, GCG-negative, and SST-negative cells in a cell population containing stage 5 cells with ARX gene disruption may be at least 10%, 15%, or 20% (e.g., 20% to 25%) of a stage 5 cell population obtained from cells without ARX gene disruption under the same differentiation conditions.

[0132] (C) Functional pancreatic β-cell stage (stage 6) Pancreatic endocrine cells with disruption of the ARX gene can mature into functional pancreatic β-cells. Functional pancreatic β-cells with disruption of the ARX gene may have glucose-responsive insulin secretion ability or C-peptide secretion ability, and may maintain such secretion ability for a long period after transplantation to the target.

[0133] In one embodiment, the second cell is a functional pancreatic β-cell with disruption of the ARX gene. In another embodiment, the second cell is an SC-β-cell with disruption of the ARX gene.

[0134] (ii) In a defined form by cell name × marker, the second cell is a functional pancreatic β-cell with disruption of the ARX gene and positive for INS.

[0135] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is INS-positive, and GCG-negative.

[0136] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is INS-positive, GCG-negative, and SST-negative.

[0137] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is NKX6.1 positive, and is INS positive.

[0138] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, and GCG negative.

[0139] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, GCG negative, and SST negative.

[0140] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, GCG negative, SST negative, and SLC18A1 negative.

[0141] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene and is NKX6.1 positive, INS positive, PDX1 positive, GCG negative, and SST negative.

[0142] In one embodiment, the second cell is a stage 6 cell having a disruption of the ARX gene.

[0143] In one embodiment, the second cell is a stage 6 cell that has a disruption of the ARX gene, is INS-positive, and GCG-negative.

[0144] In one embodiment, the second cell is a stage 6 cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, and GCG negative.

[0145] In one embodiment, the second cell is a stage 6 cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, GCG negative, and SST negative.

[0146] In one embodiment, the second cell is a stage 6 cell that has a disruption of the ARX gene and is NKX6.1 positive, INS positive, PDX1 positive, GCG negative, SST negative, and SLC18A1 negative.

[0147] In one embodiment, the second cell is a functional pancreatic β-cell having disruption of the ARX gene and glucose-responsive C-peptide secretion ability.

[0148] In one embodiment, the second cell is a functional pancreatic β-cell having a disruption of the ARX gene, being NKX6.1 positive, INS positive, GCG negative, and SST negative, and possessing glucose-responsive C-peptide secretion ability.

[0149] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is INS-positive and GCG-negative, and exhibits glucose-responsive C-peptide secretion ability after transplantation into the subject.

[0150] In one embodiment, the second cell is a functional pancreatic β-cell that has a disruption of the ARX gene, is NKX6.1 positive, INS positive, GCG negative, and SST negative, and maintains glucose-responsive C-peptide secretion for at least 100 days after transplantation into the subject.

[0151] In one embodiment, the proportion of GCG-positive cells in a cell population containing stage 6 cells with ARX gene disruption is 70% or less, 50% or less, 30% or less, or 10% or less of the proportion of GCG-positive cells in a stage 6 cell population obtained from cells without ARX gene disruption under the same differentiation conditions.

[0152] In one embodiment, the proportion of INS-positive and GCG-negative cells in a cell population containing stage 6 cells with ARX gene disruption is at least 80%, at least 90%, at least 95%, at least 100%, or at least 110% of the proportion in a stage 6 cell population obtained from cells without ARX gene disruption under the same differentiation conditions.

[0153] In one embodiment, the second cell is a functional pancreatic β-cell having disruption of the ARX gene, which secretes C-peptide in response to glucose loading after transplantation into a subject and substantially does not secrete glucagon. In another embodiment, the cell population containing the functional pancreatic β-cells shows increased glucose-responsive C-peptide secretion and no substantial increase in plasma glucagon concentration when a glucose loading test (e.g., intraperitoneal glucose tolerance test) is performed after transplantation into a subject. This characteristic indicates that the transplanted cells stably maintain their identity as β-cells, and that differentiation or dedifferentiation to α-cells is suppressed. In another embodiment, this characteristic is maintained for at least 100 days, preferably at least 130 days, after transplantation.

[0154] The cell population obtained by the method disclosed herein may include cells characterized by the expression patterns of the following markers.

[0155] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are INS-positive cells.

[0156] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are INS-positive and GCG-negative cells.

[0157] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are INS-positive, GCG-negative, and SST-negative cells.

[0158] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are INS-positive and NKX6.1-positive cells.

[0159] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are NKX6.1 positive, INS positive, and GCG negative.

[0160] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are NKX6.1 positive, INS positive, GCG negative, and SST negative.

[0161] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are NKX6.1 positive, INS positive, GCG negative, SST negative, and SLC18A1 negative cells. In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cell population are NKX6.1 positive, INS positive, GCG negative, and with respect to SST, the proportion of SST-positive cells in the cell population is 30%, 25%, or 20% or less (e.g., 10-20%), and with respect to SLC18A1, the proportion of SLC18A1-positive cells in the cell population is 40%, 35%, or 30% or less (e.g., 20-30%).

[0162] In some embodiments, the proportion of INS-positive cells in the cell population is not lower than the proportion of INS-positive cells in a cell population obtained under the same differentiation conditions from cells without ARX gene disruption. In some embodiments, the proportion is at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the proportion obtained from cells without ARX gene disruption. In some embodiments, the proportion exceeds the proportion obtained from cells without ARX gene disruption. These embodiments can also be applied to INS-positive and GCG-negative cells, INS-positive and NKX6.1-positive cells, or NKX6.1-positive, INS-positive, GCG-negative and SST-negative cells instead of INS-positive cells.

[0163] In one embodiment, the proportion of GCG-positive cells (α cells) in the cell population is lower than the proportion of GCG-positive cells in a cell population obtained under the same differentiation conditions from cells that do not have the ARX gene disruption. In one embodiment, the proportion of GCG-positive cells is 70% or less, 50% or less, 30% or less, 20% or less, or 10% or less of the proportion of GCG-positive cells obtained from cells that do not have the ARX gene disruption.

[0164] In one embodiment, the proportion of GCG-positive cells in the cell population is 10% or less, 5% or less, 3% or less, or 1% or less.

[0165] In one embodiment, the proportion of SLC18A1-positive cells (intestinal chromaffin cells) in the cell population is lower than the proportion of SLC18A1-positive cells in a cell population obtained under the same differentiation conditions from cells without the disruption of the gene. This embodiment preferably relates to cells having the disruption of the CDX2 gene.

[0166] In one embodiment, the pancreatic β-cells in the cell population have glucose-responsive insulin secretion (GSIS) or C-peptide secretion. Glucose responsiveness may mean that the amount of insulin or C-peptide secreted under high glucose stimulation (e.g., about 20 mM) is at least 1.5 times, at least 2.0 times, at least 2.5 times, or at least 3.0 times the amount secreted under low glucose stimulation (e.g., about 2 mM). Glucose responsiveness can be evaluated by in vitro static GSIS, in vitro dynamic GSIS (perifusion), or in vivo glucose tolerance testing (e.g., intraperitoneal glucose tolerance test (ipGTT)).

[0167] In one embodiment, the pancreatic β-cells in the cell population maintain glucose-responsive C-peptide secretion ability after transplantation to the subject. In another embodiment, the C-peptide secretion ability is maintained for at least 30 days, at least 60 days, at least 90 days, at least 100 days, or at least 130 days after transplantation.

[0168] The obtained pancreatic β-cells can be tested for glucose-dependent insulin secretion (GSIS) capacity. Testing for GSIS capacity can be carried out, for example, as follows: Pancreatic β-cells are incubated in Krebs buffer containing a low concentration of glucose (e.g., approximately 2.8 mM to approximately 3 mM), and then incubated in Krebs buffer containing a high concentration of glucose (e.g., approximately 20 mM) for, for example, 1 hour to introduce glucose stimulation to the cells. Subsequently, the pancreatic β-cells are incubated in Krebs buffer containing a high concentration of glucose (e.g., approximately 20 mM) for, for example, 30 minutes, and the C-peptide level in the supernatant is measured. These tests can be performed under temperature conditions of 37°C. The C-peptide level can be determined by measurement methods well known to those skilled in the art, such as ELISA or alpha-LISA.

[0169] In one embodiment, genetically modified cells can be used as the starting cells in the above. Therefore, a composition comprising starting cells is provided for use in obtaining pancreatic β-cells by the method of the present disclosure.

[0170] The starting cells may be one or more cells selected from the group consisting of pluripotent stem cells, definitive endoderm (DE), primitive gut tube, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells, or cells in the differentiation process between two of the aforementioned cells, which can then differentiate into pancreatic β-cells through the necessary stages 1 to 6.

[0171] The starting cells may also be cells capable of differentiating into any of the cells in the differentiation process to pancreatic β-cells described above, such as mesenchymal stem cells, which can then be differentiated into any of the cells in the differentiation process to pancreatic β-cells and then differentiated into pancreatic β-cells through the necessary stages, for example, stages 1 to 6.

[0172] The starting cells may be genetically modified pluripotent stem cells. The starting cells may also be, for example, genetically modified mesenchymal stem cells. Details of the genetic modifications are as described above.

[0173] <Method for Preparing a Multiple Gene Disruption Library> The present disclosure further provides a method for preparing a multiple gene disruption library (see, for example, Figures 3A-3D). The method of the present disclosure includes introducing one different gene modification (first gene modification) into each cell (see, for example, Figure 3A). The gene modification may preferably be a gene deletion (in particular, a deletion of an entire protein-coding region). The gene modification may involve introducing a modified barcode sequence into the modification site (see, for example, Figure 3C). The modified barcode sequence is a sequence specific to the allele and the modification, and the modified barcode sequence is linked to the content of the modification, allowing the modified barcode sequence to be identified from the content of the modification, and allowing the content of the modification to be identified from the modified barcode sequence.

[0174] In one preferred embodiment, each modified barcode sequence may have a common primer-binding sequence on both sides of its adjacent regions that can be hybridized with a universal primer (see, for example, Figure 3D). This allows all modified barcode sequences to be amplified using a single set of universal primers. The amplified product can then be used for sequencing.

[0175] For example, to induce a gene deletion in a cell, genome editing technology can be used in the presence of donor DNA to cause a cut in the cell genome so that the gene is deleted, and the gene can be replaced with a sequence from the donor DNA. The donor DNA has an upstream homology arm (HA) that can induce homologous recombination with the gene and homologous recombination with the upstream region of the gene, and a downstream HA that can homologous recombination with the downstream region of the gene, and between the upstream HA and the downstream HA, for example, a modified barcode sequence. This makes it possible to cause the gene deletion and insert the modified barcode sequence into the deletion site. The region encoding a desired protein or the like may be included between the upstream HA and the downstream HA of the donor DNA, and this region may contain mutations that change the amino acid sequence, and by using such donor DNA, the encoding region can be inserted into the genome.

[0176] The method of the present disclosure includes cloning cells into which each cell has been introduced with a single gene modification. The method of the present disclosure includes mixing the cloned and sufficiently grown cells with cells into which other gene modifications have been introduced to form a cell pool containing cells into which each cell has been introduced with a different single gene modification. The method of the present disclosure may include introducing a further different gene modification (a second gene modification) into each cell in the resulting cell pool (see, for example, Figure 3A). This gene modification can be introduced in the same way as introducing the first gene modification as described above. This can result in a single cell pool containing cells, each having a different first gene modification and a different second gene modification. The method of the present disclosure may further include cloning cells from the pool containing cells, each having a different first gene modification and a different second gene modification (see, for example, Figure 3A).

[0177] The method of this disclosure may further include subjecting the modified barcode sequence to sequencing. By sequencing, the modified barcode sequence can be deciphered to determine what genetic modifications have been made to the cells. This allows for the formation of a library of cells, each having multiple different genetic modifications.

[0178] In one embodiment, the method of the present disclosure may include a cell-specific cell barcode in the primers used to amplify the modified barcode sequence (see, for example, Figures 3A and 3D). The cell barcode may be ligated to the 5' region of the primer. By amplifying the modified barcode portion with a primer having a cell-specific cell barcode, the amplified product will have a different cell barcode and modified barcode sequence for each cell. The resulting amplified product can be mixed and subjected to sequencing, and the modified barcode sequence can be understood from the decoded sequence, and the modified barcode sequence will be accompanied by the sequence of the cell barcode to which it was ligated (see, for example, Figure 3D). Thus, sequencing can reveal what modified barcode sequence a particular cell had. Sequence decoding can be performed, for example, by a next-generation sequencer in, for example, one or two runs (units of decoding process). A next-generation sequencer can decode multiple nucleic acid fragments simultaneously. Next-generation sequencers are not particularly limited, but examples include sequencers using the Illumina method (MiSeq, NextSeq, NovaSeq, etc.), which enable high-precision and high-throughput sequence decoding using bridge PCR and reversible terminator methods. Those skilled in the art can appropriately select a sequencing method and achieve sequence decoding according to conventional methods.

[0179] In this way, a library of multiple gene-modified cell lines can be obtained, which includes cells in which the modifications specific to each cell type are clearly defined.

[0180] In the method for identifying a treatment that promotes the transformation or differentiation of a first cell into a second cell as described above, the first cell may be a multiple genetically modified cell, and the multiple genetically modified cell line library obtained herein can be used as a library of cells having one or more disruptions in genes that satisfy the above criteria. The multiple genetically modified cell line library contains a set of cells with a vast number of combinations of genetic modifications, each of which may exhibit different differentiation characteristics. Users of the library can select cells with desirable characteristics from among them and use them in the desired experiment.

[0181] Example 1: Destruction of ARX 1. Materials and Methods (1) Undifferentiated hiPSCs cultured on a two-dimensional plate pre-coated with sphere-forming vitronectin (Thermofisher) were dissociated, and 5 × 10⁻⁶ 6 Cells were re-seed into 6-well suspension culture plates at a density of cells / well (Greiner Bio One #657185), cultured at 37°C for 24 hours, and sphere formation was achieved using a 95 rpm rotating orbital shaker.

[0182] (2) Pancreatic differentiation process Undifferentiated RPChiPS771-3G cells were cultured and differentiated according to a published protocol (Velazco-Cruz et al., Stem Cell Rep., Vol.12: 351-365, 2019) with some modifications (see, for example, Figure 4A). Briefly, undifferentiated hiPSCs cultured on a two-dimensional plate pre-coated with vitronectin were dissociated and 5 × 10⁻⁶ cells were cultured. 6 Cells were replicated in a 6-well suspension culture plate at a cell / well density (Greiner Bio One #657185) and cultured at 37°C for 24 hours, followed by sphere formation using a 95 rpm rotating orbital shaker. Differentiation was initiated by replacing the medium with Stage 1 (DE) medium. On day 3, the medium was changed to Stage 2 (PGT) medium. On day 6, the medium was changed to Stage 3 (PP1) medium and cultured for 1 day. On day 7, the medium was switched to Stage 4 (PP2) medium and cultured for 5 days. On day 12, the medium was switched to Stage 5 (EN) medium. On day 20, cell sphere reaggregation was performed, and the medium was switched to Stage 6 (SC-β) medium. Cell spheres were washed with PBS and incubated in TrypLE Select Enzyme (Thermofisher) at 37°C for 20 minutes. Spheres were mechanically dissociated using a P1000 pipette. Subsequently, the cells were quenched in Stage 6 medium, spun down, washed with Stage 6 medium + Y27632 (10 μM, Fujifilm Wako), and then immersed in Stage 6 medium + Y27632 for approximately 1 × 10⁶ cells. 6 The cells were resuspended and passed through a 100 μm CellStrainer (pluriS elect) to remove any remaining undissociated clusters.

[0183] Count the dissociated single cells and measure 3–5 × 10⁶ cells per well in Stage 6 medium + Y27632. 6 Cells were seeded at the specified cell density into 6-well suspension culture plates and cultured in an incubator at 37°C with agitation at 95 rpm. In Stage 6 medium, half of the medium was changed every 48 hours, and incubation continued until day 30.

[0184] Stage 1 (DE): Differentiation was initiated by changing the medium with MCDB 131 (Gibco) supplemented with 0.44 g / L glucose (Otsuka Pharmaceutical Co., Ltd.), 0.5% FAF-BSA (Proliant Biotics), 0.002% ITS-X (Gibco), 3 μM CHIR99021 (Fujifilm Wako), 100 ng / mL recombinant human Activin-A (Api), and 10 μM Y27632. On days 1 and 2, the medium was changed to Stage 1 (DE) medium without CHIR99021 and Y27632.

[0185] Stage 2 (PGT): On days 3 and 4, the culture medium was replaced with MCDB 131 supplemented with 0.44 g / L glucose, 0.5% FAF-BSA, 0.002% ITS-X, 50 ng / mL KGF (Fujifilm Wako), and 0.25 mM L-ascorbic acid 2-phosphate (A2P, Sigma).

[0186] Stage 3 (PP1): On day 6, the medium was replaced with MCDB 131 supplemented with 0.44 g / L glucose, 0.5% FAF-BSA, 0.5% ITS-X, 50 ng / mL KGF, 0.25 μM SANT-1 (Sigma), 500 nM PdBU (Nacalai tesque), 0.2 μM LDN193189 (Stemgent), 2 μM RA (Fujifilm-Wako), 0.25 mM A2P, and 10 μM Y27632.

[0187] Stage 4 (PP2): From day 7 to day 12, the culture medium was replaced with MCDB 131 supplemented with 0.44 g / L glucose, 0.5% FAF-BSA, 0.5% ITS-X, 50 ng / mL KGF, 0.25 μM SANT-1, 100 nM RA, 0.25 mM A2P, 5 ng / mL activin A, and 10 μM Y27632.

[0188] Stage 5 (EN): From day 12 to day 16, cells were cultured in MCDB 131 supplemented with 3.6 g / L glucose, 0.5% FAF-BSA, 0.5% ITS-X, 10 μM Alk5i (Tocris), 1 μM γ-secretase inhibitor XXI (GSXXI) (Sigma), 20 ng / ml betacellin (Pepro Tech, Inc.), 100 nM RA, 0.25 mM A2P, 1 μM L-3,3',5-Triiodothyron, 0.25 μM SANT-1, and 10 μg / ml heparin (Sigma). On days 16-20, cells were cultured in the same medium without SANT-1, and the RA concentration was changed from 100 nM to 25 nM.

[0189] Stage 6 (SC-β): From day 20, cells were cultured in MCDB 131 supplemented with 0.44 g / L glucose, 2% FAF-BSA, 10 μg / mL heparin, 1 μM ZnSO4, 0.1% trace element A (Corning), and 0.1% trace element B (Corning). Cells were replenished with half fresh medium every other day. For single-cell RNA-seq analysis, vitamin C (Sigma-A) at 44 μg / mL and T3 at 1 μM were used instead of A2P.

[0190] (3) For analysis of immunocytochemically differentiated hiPSC spheres, cells were dissociated, re-seeded in 96-well plates, cultured overnight at 37°C, and then subjected to immunostaining. Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 1% Triton X-100 (Nacalai Tesque), and then blocked with 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted with 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamino-2-phenylindole (DAPI) (Roche Diagnostics). Guinea pig anti-INSULIN (Agilent Dako, IR002), mouse anti-GLUCAGON (monoclonal, clone K79bB10; Sigma-Aldrich), rabbit anti-SLC18A1 (ATLAS ANTIBODIES, HPA063797), and rabbit anti-SOMATOSTATIN (Sigma-Aldrich, SAB4502861) were used as primary antibodies. Secondary antibodies conjugated with Alexa Fluor488, 647, or CF568 were used to visualize the primary antibodies. Positive cells versus all cells (DAPI-positive cells) were quantified using the ImageXpress Micro cellular imaging system (Molecular Devices).

[0191] (4) C-peptide release assay: The culture medium was changed on day 20 of differentiation induction, and the culture supernatant was collected immediately afterward and 1 hour after the change. The concentration of C-peptide in the collected culture supernatant was measured using HISCL-800 (SYSMEX). The obtained measurements were normalized as the secretion amount per cell.

[0192] (5) Quantitative and statistical analysis Individual data are expressed or represented as mean ± standard deviation (SD). Each experiment was performed using biological replication (≧3). Group differences were analyzed by one-way ANOVA and Dunnett's multiple comparisons tests. Statistical significance is indicated as follows: *: p < 0.05, **: p < 0.01, ***: P < 0.001, and ****: P < 0.0001.

[0193] 2. Results: Spheres were collected at the end of Stage 5, and the cells were dissociated to measure the proportions of β cells (INS+ / GCG- / SST-), EC cells (SLC18A1+), and α cells (GCG+). As shown in Figure 1, spheres obtained by differentiation from CDX2 KO iPS cells (clones #1-3-6 and #1-3-7) and ARX KO iPS cells (clones #2-2-1 and #2-2-2) showed a decrease in EC cells and α cells, respectively. On the other hand, β cells increased in spheres obtained by differentiation from ARX KO iPS cells. This result is in contrast to Gage et al. (2015), which found that partial deletion of ARX significantly reduced the generation of insulin-producing cells. The cell marker expression at the completion of Stage 4, Stage 5, and Stage 6 is shown in Figures 6A, 6B, and 6C, respectively.

[0194] Next, spheres were collected at the end of Stage 5, and a C-peptide release assay was performed. The amount of C-peptide released from spheres differentiated from CDX2 KO iPS cells was significantly increased compared to the unmodified group.

[0195] Example 2: KO format of ARX (6) Comparison of different knockout methods for the ARX gene Knockout of the ARX gene was produced using the following two methods: (a) Full deletion: A mutant in which the entire coding region of the ARX gene was deleted. (b) Frameshift mutation (23bp FS): A mutant in which a frameshift was introduced by deleting 23 base pairs in exon 1 of the ARX gene. This mutant is the ARX mutant produced and validated by Gage et al. (2015). For each mutant, cells were harvested at the end of stage 5 (endocrine cells) (day 20) and the end of stage 6 (functional β cells) (day 30) according to the 6-step differentiation protocol described in (2) above, and the proportion of β cells (INS+ / GCG- / SST-) and α cells (GCG+) was measured by immunocytochemistry as described in (3) above. Unmodified cells (WT) were used as a control.

[0196] (7) Subcapsular transplantation of stem cell-derived pancreatic endocrine spheroids Stem cell-derived pancreatic endocrine spheroids differentiated to stage 6 (day 30) according to the 6-stage differentiation protocol described in (2) above (2.5 × 10 6 A quantity equivalent to cells was collected from the culture vessel (bioreactor) according to the scheme shown in Figure 4B and washed twice with RPMI1640 medium (Fujifilm Wako). The spheroids were suspended in a small amount of the same medium for the third time and allowed to settle naturally. The spheroids were then concentrated by centrifugation for a short time using a small microcentrifuge, being careful not to crush them. The concentrated spheroids were injected subcapsulate into the kidney capsule of 8-week-old male NOG mice (NOD / Shi-scid, IL-2Rγ^null) using a polyethylene tube. The tube insertion site in the kidney capsule was sealed with a surgical adhesive derived from coagulation factors, the kidney was returned to the body, and the myotomy site was sutured. Subsequently, suture clips were inserted into the skin incision site to close it, and after confirming skin adhesion one week later, the suture clips were removed. The transplantation groups consisted of spheroids derived from unmodified iPSCs (WT group) and spheroids derived from ARX-KO iPSCs (ARX-KO group), with a sham surgery group serving as the control.

[0197] (8) Intraperitoneal glucose tolerance test (ipGTT) To evaluate glucose metabolism capacity after transplantation, an ipGTT was performed on day 131 post-transplant. As shown in Figure 4C, mice were fasted overnight (approximately 14 hours), and then 10% glucose injection solution (Otsuka Pharmaceutical) was administered intraperitoneally at a dose of 2 g per kg of body weight. Blood was collected from the tail vein immediately before administration (0 minutes, Fasting) and 2 hours after administration (2h post-challenge), and blood glucose levels were measured using a simple blood glucose meter (Glutest Mint II, Sanwa Chemical Research Institute).

[0198] (9) Simultaneously with the measurement of plasma hormone concentrations, blood samples for hormone analysis were collected from the tail vein. The collected blood was immediately centrifuged at 4°C (1,500 × g, 15 minutes), and the resulting plasma was collected in low-protein-adsorption tubes (PROTEOSAVE, Sumitomo Bakelite). The samples were frozen and stored at -30°C until measurement. Plasma C-peptide and glucagon concentrations were measured using a commercially available ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer's protocol. A microplate reader (Varioskan, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure absorbance, and the concentration of each sample was calculated based on a calibration curve. For steady-state blood hormone concentration measurements, blood collection was performed 2 hours after the start of light (between 9:00 AM and 11:00 AM) to account for diurnal variation.

[0199] 3. Comparison of ARX Knockout Methods (Full-Length Deletion vs. Frameshift Mutation) At the end of Stage 5 (Day 20) and the end of Stage 6 (Day 30), the proportion of INS-positive and GCG-positive cells in spheres differentiated from unmodified cells (WT), ARX full-length deletion mutants (ARX KO Full del), and ARX frameshift mutants (ARX KO 23bp FS) was measured. As shown in Figure 5A, in both ARX KO Full del and ARX KO 23bp FS, GCG-positive cells (α cells) decreased compared to WT, but no significant decrease in INS-positive cells (β cells) was observed. This result was in contrast to the report by Gage et al. (2015) of a significant decrease in INS-producing and GCG-producing cells when ARX was knocked out by exon 1 frameshift (23bp and 41bp deletion). Furthermore, no clear difference was observed between Full del and FS.

[0200] In the protocol by Gage et al. (2015), differentiation induction was completed in 26 days, and the resulting cells were mainly immature polyhormone cells expressing both insulin and glucagon. In contrast, in this example, differentiation into functional pancreatic β-cells exhibiting glucose-responsive insulin secretion (GSIS) was achieved by following the 6-stage differentiation protocol (totaling 30 days) described in (2) above, particularly by the addition of an ALK5 inhibitor, T3, γ-secretase inhibitor (GSXXI), and betacellin in stage 5, and by the addition of high-concentration FAF-BSA (2%), heparin, ZnSO4, and trace elements in stage 6. Furthermore, the cell reaggregation process on day 20 (TrypLE dissociation, removal of undissociated clusters by Cell Strainer, and re-seeding) contributed to the enrichment and maturation of endocrine cells. It is thought that these differences in the differentiation protocol led to the maintenance of β-cell differentiation even under ARX knockout. This embodiment demonstrates that, under appropriate differentiation conditions (i.e., the six-step differentiation protocol described above or an equivalent differentiation protocol capable of achieving maturation into functional β-cells), disruption of the ARX gene (including deletion of the entire coding region and frameshift) is an effective treatment that enhances the efficiency of differentiation into β-cells and suppresses differentiation into α-cells.

[0201] 4. In vivo functional evaluation by transplantation of ARX-KO iPSC-derived SC-β cells. Unmodified iPSC-derived spheroids (WT group) and ARX-KO iPSC-derived spheroids (ARX-KO group) differentiated to stage 6 according to the method described in (7) above were transplanted subcapsulate of normal immunodeficient NOG mice (2.5 × 10⁻¹⁴). 6 (cells / mouse). On day 131 post-transplant, ipGTT was performed according to the methods described in (8) and (9) above.

[0202] The blood glucose measurement results are shown in Figure 5B. In the Sham group, an increase in blood glucose levels was observed at 2 hours post-challenge compared to the fasting stage. In the ARX-KO group, a blood glucose response to glucose loading was observed.

[0203] The results of plasma C-peptide concentration measurements are shown in Figure 5C. The C-peptide value at the fasting time was used as the baseline, and the difference (ΔC-peptide) from the C-peptide value at each time point was calculated. An increase in C-peptide secretion in response to glucose loading (2h post-challenge) was confirmed in the ARX-KO group. This result indicates that stage 6 cells (SC-β cells) derived from ARX-KO iPSCs engrafted in vivo for a long period of 131 days after transplantation and maintained glucose-responsive insulin (C-peptide) secretion ability. In contrast, as shown in Figure 5D, almost no glucagon secretion was detected in WT and ARX-KO iPSC-derived stage 6 cells (SC-β cells).

[0204] This example demonstrates that functional pancreatic β-cells induced from pluripotent stem cells with disrupted ARX genes using the six-step differentiation protocol described above maintain a stable differentiation state that sustains glucose-responsive C-peptide secretion over the long term in vivo. This result provides a concrete example for evaluating the stability of the differentiation state of the second cells (pancreatic β-cells) in the body of subjects who have been administered these genetically modified cells.

Claims

1. A method for culturing cells in vitro, comprising culturing a first cell having disruption of the ARX gene or CDX2 gene under conditions for differentiation into pancreatic β-cells to obtain a cell population including a second cell, wherein the first and second cells are selected from cells selected from the group consisting of pluripotent stem cells, endodermal cells (DE), foregut endoderm cells (PGT), pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells, and the second cell has a later differentiation stage than the first cell.

2. The method according to claim 1, wherein at least 10% of the cell population are INS-positive and GCG-negative pancreatic β-cells.

3. The method according to claim 1 or 2, wherein the second cell is NKX6.1 positive, INS positive, GCG negative, and SST negative.

4. The method according to any one of claims 1 to 3, wherein the second cell has glucose-responsive C-peptide secretion ability.

5. The method according to any one of claims 1 to 4, wherein the first cell is a pluripotent stem cell, a confirmed endoderm cell, a primitive intestinal / posterior foregut cell, or a pancreatic progenitor cell, the second cell is a pancreatic endocrine cell or a functional pancreatic β-cell, and the method comprises differentiating the first cell or a pancreatic progenitor cell derived from the first cell into an endocrine cell by culturing it in the presence of an ALK5 inhibitor and a γ-secretase inhibitor.

6. The method according to claim 5, wherein the differentiation conditions further include culturing the endocrine cells in serum-free medium to mature them into functional pancreatic β-cells.

7. The method according to claim 6, wherein the first cells or cells derived from the first cells are cultured as three-dimensional spheres.

8. The method according to claim 7, further comprising the step of dissociating cells, removing undissociated clusters, and then re-aggregating the cells between differentiating them into endocrine cells and maturing them.

9. A composition comprising cells having disruption of the ARX gene, selected from cells selected from the group consisting of pluripotent stem cells, confirmed endoderm cells, primitive intestinal / posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells.

10. The composition according to claim 9, wherein the cells are selected from the group consisting of pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, and functional pancreatic β-cells.

11. The composition according to claim 9 or 10 for use in transplantation to a target.

12. The composition according to claim 9 or 10 for use in the method according to any one of claims 1 to 8.

13. A library of cloned cells, each cell having a genetic modification, the genetic modification of each cell comprising the deletion of an entire coding region of one or more genes.

14. The library according to claim 13, comprising 10 or more types of cloned cells.

15. The library according to claim 13, wherein each gene modification includes the deletion of the entire coding region of two or more genes.

16. The library according to claim 13, wherein the gene modification includes gene disruption, and 50% or more of the gene disruption includes the deletion of the entire coding region of one or more genes.

17. The library according to claim 13, wherein the gene modification includes gene disruption, and all gene disruptions include deletion of the entire coding region of one or more genes.

18. The library according to claim 13, wherein a cell (first cell) has the ability to differentiate into a specific cell (second cell), and the gene codes for a factor that is not expressed in the first cell, is not expressed in cells other than the second cell induced from the first cell, and is not expressed during the differentiation process from the first cell to the second cell.

19. Cloned cells having genetic modifications, wherein the genetic modifications in each cell involve the deletion of the entire coding region of two or more genes.

20. The cell according to claim 19, wherein the gene modification includes the deletion of the entire coding region of each of three or more genes.

21. The cell according to claim 19, wherein the gene modification includes the deletion of the entire coding region of each of five or more genes.

22. The cell according to claim 19, wherein the cell (first cell) has the ability to differentiate into a specific cell (second cell), and the gene encodes a factor that is not expressed in the first cell, is not expressed in cells other than the second cell induced from the first cell, and is not expressed during the differentiation process from the first cell to the second cell.

23. A method for identifying a treatment that promotes the differentiation of first cells into second cells, comprising: culturing first cells subjected to the treatment under appropriate differentiation conditions or modified conditions thereof for inducing differentiation into second cells; and identifying a treatment that (i) increases the efficiency of differentiation from first cells subjected to the treatment into second cells, (ii) decreases the efficiency of differentiation into cell types other than second cells, or (iii) improves the function of second cells, wherein the first cells are isolated cells subjected to the treatment and have the ability to differentiate into second cells.

24. The method according to claim 23, wherein the process is a process that deletes the coding region of a gene, and the first cell has a deletion of the coding region of the gene.

25. The method according to claim 23, wherein the treatment includes treatment with a compound.

26. The method according to claim 23, wherein the first cell is a pluripotent cell, a tissue stem cell, or a progenitor cell.

27. The method according to claim 23, wherein the second cell is a cell of the endodermal lineage, mesodermal lineage, or ectoderm lineage.

28. The method according to claim 23, wherein the second cell is a pancreatic β-cell.

29. The method according to claim 23, wherein the deleted gene encodes a factor that promotes differentiation into cells other than the second cell.

30. The method according to claim 23, wherein the first cell is a pluripotent stem cell or a mesenchymal stem cell.

31. The method according to claim 23, wherein the appropriate differentiation conditions are conditions for using a compound that promotes differentiation, and the modified conditions are conditions for not using the compound.

32. The method according to claim 23, further comprising: evaluating the stability of the differentiation state of the second cells and / or the differentiation into a third cell and the stability of the differentiation state in the body of a subject administered with a second genetically modified cell; and identifying a treatment to improve the stability of the differentiation state of the second cells and / or the differentiation into a third cell and the stability of the differentiation state.

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