Method for culturing human pancreatic islet cells

Abstract

The present invention provides a method for culturing human pancreatic islet cells, the method comprising culturing human pluripotent stem cell-derived pancreatic islet cells in a medium containing B27 or ITS-X.

Description

Method for culturing human pancreatic islet cells

[0001] The present invention relates to a method for efficiently expanding or producing human pluripotent stem cell-derived pancreatic islet cells, preferably human pancreatic islet progenitor-like cells.

[0002] Diabetes mellitus develops due to an absolute or relative deficiency of insulin secreted by pancreatic islet β cells in the pancreas. Adult pancreatic islet cells have almost no self-renewal capacity, and therefore their function cannot be regenerated. Pancreatic islet transplantation is a medical treatment that enables diabetic patients to maintain stable blood glucose levels by transplanting pancreatic islet tissue. However, the shortage of donor is a serious problem, particularly in Japan, and the development of methods for regenerating pancreatic islet function using various approaches is urgently needed.

[0003] Embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts and induced pluripotent stem cells (iPSCs), generated by introducing reprogramming factors from somatic cells, can differentiate into all three germ cell layers. Taking advantage of this property, human ESCs and iPSCs (hESCs / iPSCs) can be differentiated into the desired functional cells and used in regenerative medicine and cell therapy. However, studying human embryonic development is extremely difficult due to significant ethical issues and limited sample numbers. In such cases, human iPSCs offer an extremely useful tool.

[0004] In recent years, the establishment of islet-like insulin-producing cells by inducing differentiation of hESCs / iPSCs has been reported, but the technology for generating large quantities of functional insulin-positive islet cells has not yet been established, and there are concerns about rejection and autoimmune reactions. The inventors have focused on the self-renewal capacity of pancreatic islet cells before and after birth and have reported a self-expansion technique for pancreatic islet cells (Patent Document 1, Non-Patent Document 1). However, further improvements in the expansion efficiency of human iPSC-derived islet cells are desired for application in cell transplantation therapy for diabetes patients.

[0005] International Publication No. 2021 / 117840

[0006] Nature Metabolism, Vol.4, 254-268 (2022)

[0007] Therefore, an object of the present invention is to provide a method for efficiently expanding or producing human pluripotent stem cell-derived pancreatic islet cells, preferably human pancreatic islet progenitor cell-like cells.

[0008] The present inventors have discovered that the self-proliferation ability of human islet cells is lower than that of mouse islet cells, and have investigated an efficient method for culturing human iPSC-derived islet cells expressing the MycL gene, i.e., human islet progenitor cell-like cells. In the process, they have found that culturing human islet progenitor cell-like cells in media with or without specific supplementary factors significantly improves their self-proliferation ability. Based on these findings, the present inventors have conducted further research, resulting in the completion of the present invention.

[0009] That is, the present invention provides the following: [1] A method for culturing human pancreatic islet cells, comprising culturing human pluripotent stem cell-derived islet cells in a medium containing B27 or ITS-X. [2] The culture method according to [1], wherein the pancreatic islet cells are islet progenitor-like cells expressing MycL. [3] The culture method according to [1] or [2], wherein the pluripotent stem cells are induced pluripotent stem cells. [4] The culture method according to any one of [1] to [3], wherein the medium further contains an FGF signaling pathway activator. [5] The culture method according to [4], wherein the FGF signaling pathway activator is one or more substances selected from the group consisting of FGF2, FGF7, and FGF10. [6] The culture method according to any one of [1] to [5], wherein the medium does not contain EGF and / or contains an EGF signaling pathway inhibitor. [7] The culture method according to [6], wherein the EGF signaling pathway inhibitor is afatinib and / or sepatinib. [8] The culture method according to any one of [1] to [7], wherein the culture medium further contains one or more agents selected from the group consisting of a GLP1 signaling pathway activator, a FOXO1 inhibitor, and an adenylate cyclase activator. [9] The culture method according to [8], wherein the GLP1 signaling pathway activator is Exendin 4.

[10] The culture method according to [8], wherein the FOXO1 inhibitor is AS-1842856.

[11] The culture method according to [8], wherein the adenylate cyclase activator is Forskolin.

[12] The culture method according to any one of [1] to

[11] , wherein the culture medium does not contain A83-01 and / or Noggin.

[13] The culture method according to any one of [1] to

[12] , wherein the culture medium does not contain serum and / or T3.

[14] The culture method according to any one of [1] to

[13] , wherein the culture medium is suspension culture.

[15] The culture method according to

[14] , wherein the cells are cultured in suspension in a culture plate that has been subjected to low-adhesion treatment.

[16] The culture method according to

[15] , wherein the culture plate has a V-bottom.

[17] The culture method according to any one of [1] to

[16] , wherein the cells are cultured in the absence of Matrigel.

[18] A method for producing human islet precursor-like cells, comprising the following steps (1) to (3): (1) a first step of differentiating human pluripotent stem cells into pancreatic islet cells, (2) a second step of increasing MycL gene expression in the islet cells to produce islet precursor-like cells, and (3) a third step of culturing by the culture method described in any one of [1] to

[17] .

[19] Human islet precursor-like cells produced by the production method described in

[18] .

[20] The culture method described in any one of [1] to

[17] , wherein the medium does not contain EGF.

[21] A method for producing human islet precursor-like cells, comprising the following steps (1) to (3): (1) a first step of differentiating human pluripotent stem cells into pancreatic islet cells, (2) a second step of increasing MycL gene expression in the islet cells to produce islet precursor-like cells, and (3) a third step of culturing by the culture method described in

[20] .

[22] Human islet progenitor-like cells produced by the production method according to

[21] .

[23] The culture method according to

[20] , wherein the culture method is a method for selectively culturing, purifying, enriching, or concentrating human pancreatic islet cells.

[0010] According to the present invention, it is possible to more efficiently expand and culture human pluripotent stem cell-derived pancreatic islet cells, preferably human pancreatic islet progenitor cell-like cells.

[0011] Figure 1 shows the culture scheme for inducing islet progenitor-like cells from iPS cell-derived β cells by lentiviral MycL gene transfer, and the cell counts after induction. Figure 2 shows the culture scheme for inducing islet progenitor-like cells from iPS cell-derived β cells by lentiviral MycL gene transfer, and the culture scheme for inducing islet progenitor cells from iPS cell-derived β cells. Figure 3 shows the results of single-cell RNA sequencing analysis of amplified uninfected β cells, islet progenitor-like cells, and islet progenitor cells, followed by clustering using UMAP. Figure 4 shows representative markers for islet progenitor-like cells, islet progenitor cells, and non-target cells. Figure 5 shows that CHGA and MycL-mCherry are highly expressed in islet progenitor-like cell clusters and low in non-target cell clusters, and that MECOM is highly expressed in non-target cell clusters and low in islet progenitor-like cell clusters. Figure 6 shows a culture scheme for exploring conditions that increase viable cell counts and islet progenitor-like cell markers and reduce the expression of non-target cell markers in medium compositions supplemented with various factors. For the evaluation, pre-expanded islet progenitor-like cells were seeded onto PrimeSurface Plate 96V (Sumitomo Bakelite; product number MS-9096V) and cultured in suspension. The added factors were evaluated. Figure 7 shows the results of an investigation into the combination of basal media (DMEM / F12, RPMI1640) and additives (B27, ITS-X, FBS, T3) to increase viable cell counts and islet progenitor-like cell markers and reduce non-target cell markers and differentiation markers. Figure 8 shows the results of an investigation into the effects of additive factors (A83-01, EGF, FGF10, Noggin, B27) on increasing viable cell counts and islet progenitor-like cell markers and reducing non-target cell markers. Figure 9 shows the results of an investigation into the use of an additive factor (Extendin 4) to increase viable cell counts and islet progenitor-like cell markers and reduce non-target cell markers. Figure 10 shows the results of an investigation into the use of additive factors (FGF7, FGF2) to increase viable cell counts and islet progenitor-like cell markers and reduce non-target cell markers.Figure 11 shows the results of an investigation into additive factors (forskolin, AS-1842856) for increasing viable cell counts and islet progenitor-like cell markers and decreasing non-target cell markers. Figure 12 shows the results of an investigation into additive factors (afatinib, sepatinib) for increasing viable cell counts and islet progenitor-like cell markers and decreasing non-target cell markers. Figure 13 shows a culture scheme for inducing islet progenitor-like cells from iPS cell-derived β cells by MycL gene transfer using Sendai virus. Figure 14 shows cell counts after inducing islet progenitor-like cells from iPS cell-derived β cells by MycL gene transfer using Sendai virus. Figure 15 shows the results confirming that expanded islet progenitor cells have the potential to re-maturate into insulin-expressing cells (positive cells).

[0012] 1. Method for Culturing Human Pancreatic Islet Cells The present invention provides a method for culturing human pancreatic islet cells (hereinafter referred to as the "culturing method of the present invention"), which comprises culturing human pluripotent stem cell-derived islet cells in a medium containing B27 or ITS-X. In one aspect of the present invention, the pancreatic islet cells are islet progenitor-like cells that express MycL.

[0013] (1) Human Pluripotent Stem Cell-Derived Pancreatic Islet Cells. As used herein, "islet-like cells" refer to pancreatic islet cells into which the MycL gene or its gene product has been introduced. Furthermore, as used herein, cells expressing the MycL gene obtained during the differentiation of stem cells into pancreatic islet cells and / or islet-like cells transitioned to a proliferation phase due to forced expression of the MycL gene are referred to as "islet progenitor-like cells." Furthermore, by halting the expression of the MycL gene and / or by reducing the expression of the MycL gene, these cells can be differentiated into cells capable of producing insulin (hereinafter also referred to as "insulin-producing cells"). Typically, transient expression of the MycL gene increases the number of islet-like cells and allows them to differentiate into cells capable of producing insulin. Regarding the markers expressed in each cell, "islet progenitor-like cells" are positive for at least one (two, three, four, five, or six) of the genes selected from, for example, CHGA, Fev, Pax4, Cck, Cdk4, NGN3, and Mki67. In addition, when focusing on the proteins expressed, they are also characterized by a decrease in insulin and glucagon production, or an increase in somatostatin production, compared to that observed in normal pancreatic islets.

[0014] Pancreatic islet cells, commonly referred to as islets of Langerhans, are cell clusters called pancreatic islets that control the endocrine function of the pancreas. They account for approximately 1-2% of the total cells in the pancreas. Pancreatic islet cells are primarily composed of five types of cells: α cells, β cells, δ cells, ε cells, and PP cells. The primary islet cell type is β cells, which occupy the center of the pancreas, particularly in mice. β cells account for approximately 60-80% of pancreatic islets and secrete insulin, which enables glucose transport to most cells in the body. On the other hand, α cells account for approximately 10-30% of pancreatic islets and secrete glucagon, which enables glucose release from the liver to maintain normal blood glucose levels. δ cells account for approximately 5-10% of pancreatic islet cells and secrete somatostatin, which further regulates glucose levels. ε cells and PP cells (approximately 5-10% of pancreatic islet cells) secrete ghrelin and pancreatic polypeptide, respectively. Pancreatic polypeptide-producing cells (approximately 5-10% of the islet cells) release hormones that modify exocrine and gastrointestinal function. Other islet cell types also exist, including endothelial cells, neural cells, and progenitor cells.

[0015] As used herein, "pancreatic islet cells" include the aforementioned α cells, β cells, δ cells, ε cells, and PP cells, as well as their precursor islet progenitor cells, intermediate cells generated during islet cell development and during the differentiation of pluripotent stem cells into islet cells or islet progenitor cells, and cells into which the MycL gene has been introduced (islet-like cells, MycL-expressing islet progenitor-like cells). Here, "intermediate cells" refer to cells whose fate is committed to differentiating into islet cells. Examples of cell markers for islet progenitor cells include one or more (two, three, four, five, or six) selected from the group consisting of PDX1, PTF1a, NKX6.1, Fev, Pax4, and CcK.

[0016] As used herein, "pluripotent stem cells" refer to cells that have both the ability to differentiate into all cells that make up a living organism (pluripotency) and the ability to produce daughter cells with the same differentiation potential through cell division (self-replication ability).

[0017] Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing for the formation of teratomas containing cells of each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0018] Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and embryonic tumor cells (EC cells), but are not limited thereto as long as they have both pluripotency and the ability to self-replicate. In the culture method of the present invention, iPS cells (more preferably human iPS cells) are preferably used.

[0019] iPS cells are cells that have artificially acquired pluripotency and self-renewal capabilities by contacting somatic cells (e.g., fibroblasts, skin cells, lymphocytes, etc.) with nuclear reprogramming factors. iPS cells were first discovered by introducing nuclear reprogramming factors consisting of Oct3 / 4, Sox2, Klf4, and c-Myc into somatic cells (e.g., fibroblasts, skin cells, etc.) (Cell, 126: pp. 663-676, 2006). Since then, many researchers have made various improvements to the combinations of reprogramming factors and the methods for introducing these factors, and a variety of methods for producing iPS cells have been reported.

[0020] The nuclear reprogramming factor may be composed of any substance, such as a proteinaceous factor or a nucleic acid encoding the same (including in a form incorporated into a vector), or a low molecular weight compound, as long as it is a substance (or substances) that can induce cells with pluripotency and self-renewal ability from somatic cells such as fibroblasts. When the nuclear reprogramming factor is a proteinaceous factor or a nucleic acid encoding the same, preferred examples include the following combinations (hereinafter, only the names of the proteinaceous factors are given): (1) Oct3 / 4, Klf4, Sox2, c-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18. Klf4 can be replaced with Klf1, Klf2, or Klf5. c-Myc can be replaced with T58A (active mutant), N-Myc, or L-Myc.) (2) Oct3 / 4, Klf4, Sox2 (3) Oct3 / 4, Klf4, c-Myc (4) Oct3 / 4, Sox2, Nanog, Lin28 (5) Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Lin28 (6) Oct3 / 4, Klf4, Sox2, bFGF (7) Oct3 / 4, Klf4, Sox2, SCF (8) Oct3 / 4, Klf4, c-Myc, Sox2, bFGF (9) Oct3 / 4, Klf4, c-Myc, Sox2, SCF

[0021] Among these combinations, when the therapeutic use of the resulting iPS cells is considered, typically, the combination of four factors (1), the combination of three factors (2), and the combination of six factors (5) are preferred. Of these, the combination of four factors (1) and the combination of three factors (2) are typically more preferred. On the other hand, when the therapeutic use of iPS cells is not considered (for example, when they are used as a research tool for drug discovery screening, etc.), typically, the four factors Oct3 / 4, Klf4, Sox2, and c-Myc, or the five factors obtained by adding Lin28 or Nanog to these four factors, are preferred.

[0022] Pluripotent stem cells in which genes on chromosomes have been modified using known genetic engineering techniques can also be used in the culture method of the present invention. The pluripotent stem cells may be cells into which a marker gene (e.g., a fluorescent protein gene such as the mCherry gene) has been introduced together with a target gene (e.g., the MycL gene) using a known method, making it possible to identify that the target gene has been introduced using expression of the marker gene as an indicator.

[0023] The pancreatic islet cells cultured by the culture method of the present invention are preferably pancreatic islet cells obtained by inducing differentiation from the above-mentioned pluripotent stem cells, i.e., pluripotent stem cell-derived pancreatic islet cells (in one embodiment, pluripotent stem cell-derived β cells).

[0024] When using pluripotent stem cell-derived islet cells in the culture method of the present invention, those skilled in the art can produce pluripotent stem cell-derived islet cells according to known information (e.g., Diabetes 2020; 69: 634-646, Nature. 2019 May; 569(7756): 368-373, WO2019099725A, WO2019169351A, etc.). In detail, they can be produced according to the method described in the first step of the production method of the present invention described below.

[0025] Pluripotent stem cell-derived pancreatic islet cells are also available commercially, such as the Cellartis hiPSC Beta Cells (from ChiPSC12) Kit (Takara; product code Y10100) and the Cellartis hiPSC Beta Cells (from ChiPSC22) Kit (Takara; product code Y10106).

[0026] Furthermore, the pancreatic islet cells cultured by the culture method of the present invention are preferably pancreatic islet precursor cell-like cells that express MycL.

[0027] As used herein, the MycL (also referred to as "L-Myc") gene is a member of the Myc gene family, which includes the c-Myc gene and MycN (the "N-Myc" gene). Human cDNA sequence information for the MycL gene can be obtained by referring to the NCBI accession number (e.g., NM 001033081), and those skilled in the art can easily isolate the cDNA.

[0028] Pancreatic islet progenitor cell-like cells can be obtained by introducing an isolated MycL gene into pancreatic islet cells and expressing the MycL gene, or by introducing an isolated MycL gene into pluripotent stem cells and inducing the differentiation of the pluripotent stem cells into pancreatic islet cells and expressing the MycL gene, according to known procedures. Specifically, they can be produced according to the method described in the second step of the production method of the present invention, which will be described later.

[0029] Pancreatic islet cells isolated from donors or pluripotent stem cell-derived islet cells may contain cells (also referred to herein as "non-target cells") that are close to the lineage of pancreatic islet cells, such as enterochromaffin cells, pancreatic duct cells, or pancreatic exocrine cells, during the isolation process or the process of inducing differentiation of pluripotent stem cell-derived islet cells. When the MycL gene is introduced into islet cells contaminated with non-target cells, MycL-expressing islet cells (MycL-expressing islet progenitor-like cells), non-MycL-expressing islet cells, and cells differentiated from islet progenitor-like cells (pancreatic islet progenitor cells), as well as MycL-expressing non-target cells and non-MycL-expressing non-target cells, may appear depending on the gene introduction efficiency or subsequent culture. Examples of markers for islet progenitor-like cells include CHGA; non-target cell markers include MECOM, PRSS1, SPINK1, 4A4, and SOX9; and islet progenitor cell markers include NKX6.1. These markers can be used to validate media that promote the growth of islet progenitor-like cells and suppress the growth of non-target cells and islet progenitor cells.

[0030] (2) Culture Medium Containing B27 or ITS-X As described in the Examples below, we investigated basal media and additives (supplements) that can increase viable cell numbers and islet progenitor-like cell markers while decreasing non-target cell markers and differentiation markers. We found that culturing islet progenitor-like cells in media containing the medium supplement B27 enhanced cell proliferation compared to culture in media without B27. In particular, cell proliferation was maximized by combining the basal medium DMEM / F12 with the additive B27. Furthermore, gene expression analysis demonstrated gene expression of the islet progenitor-like cell marker CHGA, while the non-target cell marker MECOM was below the detection limit, suggesting that a B27-containing medium is a basal condition that maximizes the proliferation of islet progenitor-like cells. Therefore, in one aspect, the medium used in the culture method of the present invention contains B27.

[0031] The concentration of B27 in the medium used in the culture method of the present invention is not particularly limited, but is typically 0.1 to 10%, preferably 0.5 to 5% (2% in one embodiment).

[0032] B27 can be purchased from a culture medium manufacturer (e.g., Thermo Fisher Scientific, product number 12587010), and its composition is shown in Table 1.

[0033]

[0034] Furthermore, similar to B27, when islet progenitor-like cells were cultured in a medium containing the medium supplement ITS-X as an additive, cell proliferation was promoted compared to when the cells were cultured in a medium without ITS-X, although the effect was more limited than with B27. Thus, in one embodiment, the medium used in the culture method of the present invention contains ITS-X.

[0035] The concentration of ITS-X in the medium used in the culture method of the present invention is not particularly limited, but is typically 10 to 0.1%, preferably 5 to 1%, and even more preferably 2%.

[0036] ITS-X can be purchased from a culture medium manufacturer (e.g., Gibco, product number 51500-056), and its composition is shown in Table 2.

[0037]

[0038] As described in the Examples below, further investigations were conducted to determine whether additives could increase viable cell counts and islet progenitor-like cell markers while decreasing non-target cell markers. The results confirmed that (i) the medium contained an FGF (fibroblast growth factor) signaling pathway activator, (ii) the medium contained an EGF signaling pathway inhibitor, (iii) the medium contained a GLP1 signaling pathway activator, (iv) the medium contained a FOXO1 inhibitor, (v) the medium contained an adenylate cyclase activator, (vi) the medium did not contain EGF (epidermal growth factor), (vii) the medium did not contain A83-01 and / or Noggin, (viii) the medium did not contain serum and / or T3, and combinations of (i) to (viii) had the above-mentioned effects.

[0039] Therefore, the culture medium used in the culture method of the present invention may further contain one or more agents selected from the group consisting of an FGF signaling pathway activator, an EGF signaling pathway inhibitor, a GLP1 signaling pathway activator, a FOXO1 inhibitor, and an adenylate cyclase activator. The culture medium used in the culture method of the present invention may also be an EGF-free medium, for example, a medium that does not contain EGF but contains an EGF signaling pathway inhibitor. The culture medium used in the culture method of the present invention may also be a medium that does not contain an EGF signaling pathway activator. The culture medium used in the culture method of the present invention may also be a medium that does not contain A83-01 and / or Noggin, or a medium that does not contain a TGFβ family signaling pathway inhibitor. The culture medium used in the culture method of the present invention may also be a medium that does not contain serum and / or T3.

[0040] (3) Culture Medium Containing an FGF Signaling Pathway Activator As described in the Examples below, when islet progenitor-like cells were cultured in a medium containing no FGF10 as an additive, the number of viable cells and the expression of the non-target cell markers MECOM and PRSS1 were unchanged compared to when the cells were cultured in a medium containing FGF10, but the expression of the islet progenitor-like cell marker CHGA was reduced. Furthermore, when islet progenitor-like cells were cultured in a medium containing FGF2 or FGF7 as an additive, the number of viable cells increased and the expression of the islet progenitor-like cell marker CHGA increased compared to when the cells were cultured in a medium containing no FGF10. Therefore, the culture medium used in the culture method of the present invention may further contain an FGF signaling pathway activator.

[0041] As used herein, the term "FGF signaling pathway activator" is not particularly limited as long as it is a substance that can enhance the signaling pathway mediated by FGF. Examples of FGF signaling pathway activators include proteins belonging to the FGF family, FGF receptor agonists, anti-FGF receptor agonist antibodies, and FGF partial peptides, and preferably proteins belonging to the FGF family.

[0042] The FGF signaling pathway activator may be a protein known to those skilled in the art, specifically, one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF7 (also known as keratinocyte growth factor (KGF)), FGF8, FGF10, etc., preferably one or more selected from the group consisting of FGF2, FGF7, and FGF10.

[0043] The concentration of the FGF signaling pathway activator in the medium can be appropriately set depending on the substance used. For example, when FGF2 is used as the FGF signaling pathway activator, the concentration is typically 10 ng / ml to 500 ng / ml (in one embodiment, 50 ng / ml). When FGF7 or FGF10 is used, the concentration is typically 10 ng / ml to 500 ng / ml (in one embodiment, 100 ng / ml). When using an FGF signaling pathway activator other than these, the concentration of the FGF signaling pathway activator in the medium can be appropriately selected. In one embodiment, the concentration may be such that the FGF signaling pathway activation ability is equivalent to that exhibited by FGF2, FGF7, or FGF10 at a concentration of 10 ng / ml to 500 ng / ml. Those skilled in the art can measure the FGF signaling pathway activation ability using known methods.

[0044] (4) Culture Medium Containing an EGF Signaling Pathway Inhibitor As described in the Examples below, when islet progenitor-like cells are cultured in a medium containing afatinib or sepatinib as an added factor, the expression of the islet progenitor-like cell marker CHGA is increased and the expression of the non-target cell markers MECOM and PRSS1 is decreased compared to when the cells are cultured in a medium not containing afatinib or sepatinib. Therefore, the culture medium used in the culture method of the present invention may further contain an EGF signaling pathway inhibitor.

[0045] As used herein, the term "EGF signaling pathway inhibitor" is not particularly limited as long as it is a substance that inhibits the signaling pathway mediated by EGF. Examples of EGF signaling pathway inhibitors include substances that act directly on EGF (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding EGF (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of EGF receptors to EGF, and substances that inhibit physiological activities resulting from signal transduction by EGF receptors (e.g., EGF receptor inhibitors, EGF receptor tyrosine kinase inhibitors), with EGF receptor inhibitors being preferred. EGF receptors form dimers of ErbB1, 2, 3, or 4, and multiple combinations exist. EGF receptor inhibitors are preferably inhibitors that inhibit multiple ErbB combinations (pan-inhibitors).

[0046] Proteins known to those skilled in the art can be used as EGF signaling pathway inhibitors, specifically, for example, one or more selected from the group consisting of afatinib, sepatinib, gefitinib, erlotinib, dacomitinib, osimertinib, etc., preferably afatinib and / or sepatinib.

[0047] The concentration of the EGF signaling pathway inhibitor in the medium can be set appropriately depending on the substance used. For example, when afatinib or sepatinib is used as the EGF signaling pathway inhibitor, the concentration is usually 0.1 nM to 1000 nM, 1 nM to 1000 nM, or greater than 10 nM to 1000 nM (in one embodiment, 100 nM). When an EGF signaling pathway inhibitor other than these is used, the concentration of the EGF signaling pathway inhibitor in the medium is selected appropriately. In one embodiment, the concentration may be such that the inhibitor exhibits an EGF signaling pathway inhibitory ability equivalent to that exhibited by afatinib or sepatinib at the above concentrations. The EGF signaling pathway inhibitory ability can be measured by those skilled in the art using known methods.

[0048] (5) Medium Containing a GLP1 Signaling Pathway Activator As described in the Examples below, when islet progenitor-like cells are cultured in a medium containing Exendin 4 as an additive, the number of viable cells and the expression of the islet progenitor-like cell marker CHGA are increased compared to when the cells are cultured in a medium not containing Exendin 4. Therefore, the medium used in the culture method of the present invention may further contain a GLP1 signaling pathway activator.

[0049] As used herein, the term "GLP1 signaling pathway activator" is not particularly limited as long as it is a substance that can enhance the signaling pathway mediated by GLP1. Examples of the GLP1 signaling pathway activator include proteins belonging to the GLP1 family, GLP1 receptor agonists, anti-GLP1 receptor agonist antibodies, and GLP1 partial peptides, with GLP1 receptor agonists being preferred.

[0050] The GLP1 signaling pathway activator may be a protein or compound known to those skilled in the art, specifically, one or more selected from the group consisting of Exendin-4, GLP1, Lixisenatide, Liraglutide, Semaglutide, etc., preferably Exendin-4.

[0051] The concentration of the GLP1 signaling pathway activator in the medium can be appropriately set depending on the substance used. For example, when Exendin-4 is used as the GLP1 signaling pathway activator, the concentration is typically 100 ng / ml to 500 ng / ml, or 400 ng / ml to 500 ng / ml (in one embodiment, 418.5 ng / ml). When a GLP1 signaling pathway activator other than these is used, the concentration of the GLP1 signaling pathway activator in the medium is appropriately selected. In one embodiment, the concentration may be such that the GLP1 signaling pathway activation ability is equivalent to the GLP1 signaling pathway activation ability exhibited by Exendin-4 at the above concentrations. The GLP1 signaling pathway activation ability can be measured by those skilled in the art using known methods.

[0052] (6) Culture Medium Containing a FOXO1 Inhibitor As described in the Examples below, when islet progenitor-like cells were cultured in a medium containing AS-1842856 as an additive, the expression of non-target cell markers MECOM and PRSS1 was reduced compared to when the cells were cultured in a medium without AS-1842856. Therefore, the culture medium used in the culture method of the present invention may further contain a FOXO1 inhibitor.

[0053] As used herein, the term "FOXO1 inhibitor" is not particularly limited as long as it is a substance that can suppress the transcriptional activity of the transcription factor FOXO1. Examples of FOXO1 inhibitors include substances that act directly on FOXO1 (e.g., proteins, antibodies, aptamers, etc.) and substances that suppress the expression of the gene encoding FOXO1 (e.g., antisense oligonucleotides, siRNA, etc.), and preferably substances that act directly on FOXO1.

[0054] The FOXO1 inhibitor may be any compound known to those skilled in the art, such as one or more selected from the group consisting of AS-1842856, FOXO1-IN-3, FOXO1-IN-8, and FOXO1-IN-10, preferably AS-1842856.

[0055] The concentration of the FOXO1 inhibitor in the medium can be set appropriately depending on the substance used. For example, when AS-1842856 is used as the FOXO1 inhibitor, the concentration is typically 0.01 μM to 1 μM (in one embodiment, 0.1 μM). When a FOXO1 inhibitor other than those is used, the concentration of the FOXO1 inhibitor in the medium can be selected appropriately. In one embodiment, the concentration may be such that the FOXO1 inhibitor exhibits FOXO1 inhibitory activity equivalent to that exhibited by AS-1842856 at the above-mentioned concentration. FOXO1 inhibitory activity can be measured by those skilled in the art using known methods.

[0056] (7) Medium Containing Adenylate Cyclase Activator As described in the Examples below, when islet progenitor-like cells are cultured in a medium containing forskolin as an added factor, the expression of the non-target cell marker MECOM is reduced compared to when the cells are cultured in a medium without forskolin. Therefore, the medium used in the culture method of the present invention may further contain an adenylate cyclase activator.

[0057] As used herein, the term "adenylate cyclase activator" is not particularly limited as long as it is a substance that can enhance the activity of an adenylate cyclase activator. Examples of adenylate cyclase activators include substances that act directly on adenylate cyclase (e.g., proteins, antibodies, aptamers, etc.).

[0058] The adenylate cyclase activator may be any compound known to those skilled in the art, specifically, one or more selected from the group consisting of forskolin, colforsin daropate, etc., preferably forskolin.

[0059] The concentration of the adenylate cyclase activator in the medium can be set appropriately depending on the substance used. For example, when forskolin is used as the adenylate cyclase activator, the concentration is typically 0.01 μM to 1 μM (0.1 μM in one embodiment). When using other adenylate cyclase activators, the concentration of the adenylate cyclase activator in the medium can be selected appropriately. In one embodiment, the concentration may be such that the adenylate cyclase activating activity is equivalent to that exhibited by forskolin at the above-mentioned concentrations. Adenylate cyclase activating activity can be measured by those skilled in the art using known methods.

[0060] In addition to the above, the present inventors further investigated additive factors that can increase the number of viable cells and islet progenitor-like cell markers while decreasing non-target cell markers, and as a result, confirmed that EGF, A83-01, and Noggin have the opposite effects. Therefore, in one embodiment, the medium used in the culture method of the present invention does not contain at least one (two or three) selected from the group consisting of EGF, A83-01, and Noggin. In a further embodiment, the medium does not contain an EGF signaling pathway activator and / or a TGFβ family signaling pathway inhibitor.

[0061] (8) EGF Signaling Pathway Activator (EGF)-Free Medium As described in the Examples below, culturing islet progenitor-like cells in a medium that does not contain EGF as an added factor resulted in a decreased number of viable cells compared to culture in a medium that contained EGF, but it reduced the expression of non-target cell markers MECOM and PRSS1 and did not alter the expression of the islet progenitor-like cell marker CHGA. In the Examples below, based on the results of the Examples, culturing islet progenitor-like cells in a medium that does not contain EGF as an added factor increased the proportion (yield) of CHGA-positive cells (target cells) compared to culture in a medium that contained EGF. Therefore, when culturing human islet cells while suppressing the proliferation of non-target cells, it is preferable that the medium does not contain EGF. In one aspect, the culture method of the present invention is a method for selectively culturing human islet cells, a method for purifying human islet cells, a method for enriching human islet cells, and a method for concentrating human islet cells. In one embodiment, the percentage of human pancreatic islet cells (target cells) in total cells after performing a culture method of the present invention (e.g., a method for selectively culturing, purifying, enriching, or concentrating human pancreatic islet cells) can be, for example, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more (e.g., 81%, 81.7%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), or 100%. Therefore, in one embodiment, the medium used in the culture method of the present invention does not contain EGF. In a further embodiment, the medium does not contain an EGF signaling pathway activator.

[0062] As used herein, the term "EGF signaling pathway activator" is not particularly limited as long as it is a substance that can enhance the signaling pathway mediated by EGF. Examples of EGF signaling pathway activators include proteins belonging to the EGF family, EGF receptor agonists, anti-EGF receptor agonist antibodies, and EGF partial peptides, and preferably proteins belonging to the EGF family.

[0063] Examples of the EGF signaling pathway activator include proteins well known to those skilled in the art, specifically, one or more selected from the group consisting of EGF, TGFα, amphiregulin, HB-EGF, betacellulin, etc. In one embodiment of the culture method of the present invention, the EGF signaling pathway activator is EGF.

[0064] As used herein, "free" typically means that the concentration is not detected by a detection method commonly used in measuring EGF signaling pathway activators (e.g., EGF), i.e., is below the threshold value. Furthermore, in one embodiment, when a commercially available medium as described below is used in the culture method of the present invention, a medium known to be free of EGF signaling pathway activators (e.g., EGF) is used, and no EGF signaling pathway activators (e.g., EGF) are added from the outside in any of the culture steps. The same applies to "(9) TGFβ family signaling pathway inhibitors" and "(10) serum- and / or T3-free medium" described below.

[0065] (9) Culture Medium Free of TGFβ Family Signaling Pathway Inhibitors As described in the Examples below, when islet progenitor-like cells were cultured in a medium free of A83-01 as an added factor, the number of viable cells and the expression of the islet progenitor-like cell marker CHGA increased, and the expression of the non-target cell markers MECOM and PRSS1 decreased, compared to when cultured in a medium containing A83-01. Furthermore, when islet progenitor-like cells were cultured in a medium free of Noggin as an added factor, the expression of the non-target cell marker PRSS1 decreased, compared to when cultured in a medium containing Noggin. Therefore, in one embodiment, the culture medium used in the culture method of the present invention does not contain A83-01 and / or Noggin. In a further embodiment, the medium does not contain a TGFβ family signaling pathway inhibitor.

[0066] As used herein, the term "TGFβ family signaling pathway inhibitor" refers to a substance capable of inhibiting signal transduction mediated by the TGFβ family. Examples of the TGFβ family include TGFβ, activin, and bone morphogenetic protein (BMP). The TGFβ family signaling pathway inhibitor is not particularly limited as long as it inhibits the signal transduction pathway caused by the TGFβ family. Examples include substances that act directly on TGFβ (or activin, BMP) (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGFβ (or activin, BMP) (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ receptors (or activin receptors, BMP receptors) to TGFβ (or activin, BMP), and substances that inhibit physiological activity resulting from signal transduction by TGFβ receptors (or activin receptors, BMP receptors) (e.g., inhibitors of TGFβ receptors (or activin receptors, BMP receptors), Smad inhibitors, etc.). In the culture method of the present invention, examples of the TGFβ family signaling pathway inhibitor include TGFβ signaling pathway inhibitors and BMP signaling pathway inhibitors.

[0067] TGFβ signaling pathway inhibitors include compounds well known to those skilled in the art, specifically, A83-01, SB431542, SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208, EW-7197, RepSox, SM16, R268712, IN1130, Galunisertib, AZ12799734, A77-01, KRCA 0008, GSK 1838705, Crizotinib, Ceritinib, ASP 3026, TAE684, AZD3463, TP0427736, TGFBR1-IN-1, TEW-7197, LY3200882, BIBF-0775, and the like. In one embodiment, the medium used in the culture method of the present invention does not contain A83-01.

[0068] Examples of BMP signaling pathway inhibitors include proteins well known to those skilled in the art, specifically, Noggin, Chordin, Follistatin, Gremlin, Inhibin, Twisted Gastrulation, Coco, DAN, etc. In one embodiment, the medium used in the culture method of the present invention does not contain Noggin. In a further embodiment, the medium used in the culture method of the present invention does not contain A83-01 or Noggin.

[0069] (10) Serum- and / or T3-Free Medium As described in the Examples below, when islet progenitor-like cells are cultured in a medium containing 10% FBS and B27 as supplementary factors, the number of viable cells is reduced and the number of differentiated islet progenitor cell markers is increased compared to when cultured in a medium containing only B27. Furthermore, triiodo-L-thyronine (T3) reduced the number of viable cells. Therefore, in one embodiment, the medium used in the culture method of the present invention is serum- and / or T3-free.

[0070] The medium used in the culture method of the present invention can be prepared using a basal medium used for culturing mammalian cells. Examples of the basal medium include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, Ham's F-12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal medium, and mixtures thereof (e.g., DMEM / F-12 medium (a 1:1 mixture of DMEM medium and Ham's F-12 medium)). DMEM / F-12 medium is preferred.

[0071] The serum-free medium used in the culture method of the present invention may also contain a serum substitute. To more easily carry out the culture method of the present invention, commercially available serum substitutes can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (Invitrogen), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0072] The medium used in the culture method of the present invention may contain, as long as the above-mentioned effects are observed, any of a variety of additives, including but not limited to buffers (e.g., HEPES), salts (e.g., inorganic salts such as sodium chloride and sodium bicarbonate) or antioxidants (e.g., 2-mercaptoethanol; in one embodiment, no antioxidants are contained), nutrients such as amino acids (e.g., non-essential amino acids; in one embodiment, no acidic amino acids are contained), fatty acids, sugars, vitamins, lipids, or pyruvic acid, antibiotics (e.g., penicillin, streptomycin), dyes (e.g., phenol red), cytoprotectants (e.g., Poloxamer 188 (0.01-1%, e.g., 0.1%)), substrates for nucleotide synthesis (e.g., thymidine, hypoxanthine), CCK2 receptor signaling pathway activators (e.g., Gastrin 1 (1-10 nM, e.g., 10 nM)), N-acetylcysteine ​​(0.1 mM-10 mM, e.g., 1.25 mM), nicotinamide (1-100 mM, e.g., 10 The basal medium may contain one or more additives appropriately selected from the group consisting of 0.1 mM of basal medium, 0.1 mM of basal medium, 0.1 mM of basal medium, 0.1 mM of basal medium, and 0.5 ...

[0073] (11) Suspension Culture As described in the Examples below, when investigating additive factors that can promote the proliferation of islet progenitor-like cells and suppress the proliferation of non-target cells, islet progenitor-like cells were cultured in suspension on cultureware treated with low adhesion. Suspension culture allows the formation of aggregates of islet progenitor-like cells in each well, mimicking the in vivo developmental pattern of pancreatic islets compared to adherent culture, thereby enabling proliferation while maintaining the characteristics of islet cells. Furthermore, suspension culture allows the size of aggregates to be controlled by controlling the number of viable cells constituting the aggregates, facilitating measurement of aggregate size and cell count, thereby reducing the effort required for quality control. Therefore, in the culture method of the present invention, cells may be cultured under suspension culture conditions.

[0074] As used herein, "suspension culture" refers to culture carried out under conditions in which cells or cell clumps are maintained suspended in a culture medium, i.e., culture under conditions in which strong cell-substratum junctions are not formed between the cells or cell clumps and the culture vessel.

[0075] The culture period for suspension culture is not particularly limited as long as it is a period during which pancreatic islet cells can proliferate. A person skilled in the art can appropriately determine the period during which the required cells can be obtained, but in one embodiment, the culture period is 1 to 180 days, preferably 1 to 120 days, and more preferably 3 to 30 days.

[0076] The cultureware used for suspension culture is not particularly limited as long as it is capable of supporting suspension culture, and those skilled in the art can appropriately determine the appropriate cultureware. Examples of such cultureware include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, spinner flasks, and roller bottles. These cultureware are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive cultureware include those whose surfaces are not artificially treated to improve cell adhesion. Examples of non-cell-adhesive cultureware include those whose surfaces have been artificially treated (low-adsorption treatment) to reduce cell adhesion. As used herein, "low-adsorption treatment" refers to a surface treatment using a superhydrophilic polymer, as described below, that reduces protein adsorption and prevents cell adhesion. Specifically, low-adsorption treatments that reduce cell adhesion include, for example, superhydrophilic treatments using coatings such as 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, Poly(2-hydroxyethyl methacrylate) (Poly-HEMA), and polyethylene glycol (PEG), as well as low-protein adsorption treatments. The culture surface of the cultureware may be flat, U-, or V-shaped, or may have an uneven surface. Preferably, the culture surface of the cultureware is V-shaped. Commercially available cultureware that has undergone low-adsorption treatment may be used, such as PrimeSurface Plate 96V (Sumitomo Bakelite; product number MS-9096V).

[0077] Furthermore, suspension culture may be performed in the absence of an extracellular matrix. Examples of extracellular matrices include Matrigel and laminin. In one embodiment, the culture method of the present invention is performed in the absence of Matrigel. In a further embodiment, the culture method of the present invention is performed in the absence of an extracellular matrix.

[0078] By culturing human pancreatic islet cells using the above-mentioned medium and culture method, a cell expansion effect of approximately 1.5 to approximately 20 times (e.g., approximately 10 times) is observed within one passage (e.g., 7 to 14 days) compared to a control group that does not have MycL introduced.

[0079] 2. Method for Producing Human Pancreatic Islet Cells The present invention also provides a method for producing human islet progenitor-like cells (hereinafter referred to as the production method of the present invention), comprising the following steps (1) to (3): (1) a first step of differentiating human pluripotent stem cells into pancreatic islet cells; (2) a second step of increasing MycL gene expression in the pancreatic islet cells to produce islet progenitor-like cells; and (3) a third step of culturing using the culture method of the present invention.

[0080] The production method of the present invention comprises (1) a first step of differentiating human pluripotent stem cells into pancreatic islet cells. The pluripotent stem cells and pancreatic islet cells may be the cells described in the culture method of the present invention.

[0081] The first step can be carried out by those skilled in the art according to known means (Diabetes 2020;69:634-646, Nature. 2019 May;569(7756):368-373, WO2019099725A, WO2019169351A, etc.), as described in the culture method of the present invention. More specifically, in one embodiment, the first step comprises stage 1 for producing endoderm, stage 2 for producing primitive gut cells, stage 3 for producing Pdx1-positive pancreatic progenitor cells, stage 4 for producing NKX6.1-positive pancreatic progenitor cells, stage 5 for producing Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells, and stage 6 for producing β cells. Below, one embodiment of the differentiation induction method for each stage is shown.

[0082] Stage 1: Endodermal cells can be obtained by culturing pluripotent stem cells in a medium containing a growth factor belonging to the TGF-β superfamily and a WNT signaling pathway activator. In one embodiment, the growth factor belonging to the TGF-β superfamily may be activin A or growth differentiation factor 8 (GDF8). The WNT signaling pathway activator may be CHIR99021 (a GSK3β inhibitor) or Wnt3a protein. The number of days of culture may be any number of days until endoderm cells are produced, but is usually around 3 days (about 2 days, about 3 days, about 4 days, or about 5 days). Those skilled in the art can confirm the induction of endoderm cells using markers expressed in endoderm cells (e.g., Nodal, FoxA1, Sox17, etc.).

[0083] The pluripotent stem cells used in Stage 1 are prepared by recovering previously passaged pluripotent stem cells and dissociating them to single cells or a state close to single cells. To suppress cell death of pluripotent stem cells (particularly human pluripotent stem cells) induced by dissociation, a ROCK inhibitor is preferably added to the medium during dissociation. The concentration of the ROCK inhibitor is sufficient to suppress cell death of pluripotent stem cells induced by dissociation. For example, for Y-27632, such a concentration is typically 0.1 to 200 μM, preferably 2 to 50 μM (10 μM in one embodiment). Treatment with the ROCK inhibitor is typically carried out for one day, and during Stage 1 differentiation induction, the culture may be continued in a medium without a ROCK inhibitor.

[0084] Stage 2: Endodermal cells induced in Stage 1 can be cultured in a medium containing an activator of the FGF signaling pathway to obtain primitive gut cells. In one embodiment, the FGF signaling pathway activator may be keratinocyte growth factor (KGF), FGF2, FGF8, or FGF10. The number of days of culture may be any number of days until primitive gut cells are produced, but is typically around 2 to 3 days (about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days). Those skilled in the art can confirm that primitive gut cells have been induced using markers expressed in primitive gut cells (e.g., HNF1B, HNF4A, FoxA2, ​​etc.) as indicators.

[0085] Stage 3: Pdx1-positive pancreatic progenitor cells can be obtained by culturing the primitive gut cells induced in Stage 2 in a medium containing one or more selected from the group consisting of a BMP signaling pathway inhibitor, a growth factor belonging to the TGF-β superfamily, an FGF signaling pathway activator, an SHH signaling pathway inhibitor, a retinoic acid (RA) signaling pathway activator, and a protein kinase C (PKC) activator. The medium may further contain a ROCK inhibitor. In one embodiment, the BMP signaling pathway inhibitor may be LDN193189 or DMH-1, the growth factor belonging to the TGF-β superfamily may be activin A or GDF8, the FGF signaling pathway activator may be an FGF protein such as keratinocyte growth factor (KGF), FGF2, FGF8, or FGF10, the SHH signaling pathway inhibitor may be Sant1, the retinoic acid (RA) signaling pathway activator may be retinoic acid, the protein kinase C (PKC) activator may be PdBU or TPB, and the ROCK inhibitor may be thiazovivin, Y-27632, or Fasudil / HA1077. The number of days of culture may be any number of days until Pdx1-positive pancreatic progenitor cells are produced, but is usually around 2 to 3 days (about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days). Those skilled in the art can confirm that Pdx1-positive pancreatic progenitor cells have been induced using a marker (Pdx1) expressed in Pdx1-positive pancreatic progenitor cells as an indicator.

[0086] Stage 4: NKX6.1-positive pancreatic progenitor cells can be obtained by culturing the Pdx1-positive pancreatic progenitor cells induced in Stage 3 in a medium containing one or more selected from the group consisting of an FGF signaling pathway activator, an SHH signaling pathway inhibitor, a retinoic acid (RA) signaling pathway activator, a BMP signaling pathway inhibitor, a protein kinase C (PKC) activator, an EGF signaling activator, and nicotinamide. The medium may further contain a growth factor belonging to the TGF-β superfamily or a ROCK inhibitor. Specific examples of these substances are the same as those described in Stage 3. The culture period may be any number of days required to produce NKX6.1-positive pancreatic progenitor cells, but is typically around 5 days (approximately 4, 5, or 6 days). Those skilled in the art can confirm the induction of NKX6.1-positive pancreatic progenitor cells using the marker (NKX6.1) expressed on NKX6.1-positive pancreatic progenitor cells as an indicator.

[0087] Stage 5: Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells can be obtained by culturing the NKX6.1-positive pancreatic precursor cells induced in Stage 4 in a medium containing one or more selected from the group consisting of a TGF-β signaling pathway inhibitor and a thyroid hormone signaling pathway activator. In one embodiment, the TGF-β signaling pathway inhibitor may be Alk5 inhibitor II, and the thyroid hormone signaling pathway activator may be triiodothyronine (T3) or GC-1. The number of days of culture may be any number of days until Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells are produced, but is usually around 7 days (about 5, 6, 7, 8, or 9 days). Those skilled in the art can confirm that Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells have been induced using markers expressed in Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells (Ngn3, Pdx1, NKX6.1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, insulin, etc.).

[0088] The medium may further contain an SHH signaling pathway inhibitor, a RA signaling pathway activator, a γ-secretase inhibitor, a growth factor belonging to the epidermal growth factor (EGF) family, a protein kinase inhibitor, a BMP signaling pathway inhibitor, or a ROCK inhibitor. In one embodiment, the SHH signaling pathway inhibitor may be Sant1, the retinoic acid (RA) signaling pathway activator may be retinoic acid, the γ-secretase inhibitor may be XXI (also known as compound E) or DAPT, the growth factor belonging to the EGF family may be betacellulin or EGF, the protein kinase inhibitor may be staurosporine, the BMP signaling pathway inhibitor may be LDN193189 or DMH-1, and the ROCK inhibitor may be thiazovivin, Y-27632, or Fasudil / HA1077. These substances do not need to be present in the medium throughout the entire culture period of Stage 5. For example, they may be present in the medium only for the first 1, 2, 3, 4, or 5 days. In one embodiment, some of the substances, such as SHH signaling pathway inhibitors, RA signaling pathway activators, and growth factors belonging to the EGF family, may be removed from the medium after the first three days of culture.

[0089] Stage 6: β cells can be obtained by culturing Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells induced in Stage 5 in a maturation medium. Examples of maturation media include NS-GFs medium, MCDB131 medium, DMEM medium, and CMRL medium. In one embodiment, FBS (e.g., about 10%), HSA (e.g., about 1%), BSA (e.g., about 2%), etc. may be added to the medium. These media do not need to contain the above-mentioned differentiation factors. They may also contain the above-mentioned growth factors belonging to the TGF-β superfamily or thyroid hormone signaling pathway activators.

[0090] The production method of the present invention includes a second step (2) of increasing the expression of MycL gene in the pancreatic islet cells to produce islet progenitor cell-like cells. The MycL gene may be any of the genes described in the culture method of the present invention.

[0091] Methods for increasing the expression of the MycL gene in pancreatic islet cells include introducing the MycL gene into pancreatic islet cells.

[0092] The MycL gene to be introduced can be identified by its cDNA nucleotide sequence using its NCBI accession number (e.g., NM 001033081). However, usable MycL genes also include single-stranded or double-stranded DNA and its RNA complement (including RNA replicons). DNA includes, for example, naturally occurring DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. DNA is preferred as the nucleic acid to be introduced.

[0093] The MycL gene preferably has a sequence (polyadenylation (poly A) signal, also called a terminator) downstream of the MycL gene that terminates transcription of mRNA from the gene. For example, a terminator sequence derived from a viral gene or from various mammalian or avian genes can be used. Preferably, a terminator derived from SV40 is used.

[0094] The MycL gene may further contain a drug resistance gene or a marker gene, and pancreatic islet cells into which the nucleic acid has been introduced can be selected using drug resistance, fluorescent activity, or the like as an indicator. Examples of drug resistance genes include the neomycin phosphotransferase II (nptII) gene, the hygromycin phosphotransferase (hpt) gene, and the puromycin-N-acetyltransferase (Pac) gene. Examples of marker genes include, but are not limited to, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. Examples of luciferases encoded by luciferase genes include firefly luciferase, bacterial luciferase, synthetic Renilla luciferase, Oplophorus gracilirostris luciferase (e.g., NanoLuc®), and secreted luciferases. Examples of fluorescent proteins encoded by fluorescent protein genes include green fluorescent proteins such as GFP and EGFP, blue fluorescent proteins such as BFP and TagBFP, and red fluorescent proteins such as RFP, DsRed, and mCherry. Examples of chromogenic enzymes encoded by chromogenic enzyme genes include β-galactosidase, β-glucuronidase, and alkaline phosphatase. These marker genes can be used alone or in combination of two or more.

[0095] The MycL gene, drug resistance gene, and marker gene are preferably under the control of any promoter that can function in pancreatic islet cells. Examples include viral promoters such as the SV40-derived early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, murine leukemia virus (MoMuLV) LTR, and adenovirus (AdV)-derived early promoter, as well as the β-actin gene promoter, PGK gene promoter, and transferrin gene promoter. Furthermore, the drug resistance gene or marker gene preferably also has a poly(A) signal.

[0096] The method for introducing the MycL gene into pancreatic islet cells is not particularly limited, but is typically carried out using a gene transfer vector containing the MycL gene. The MycL gene may be integrated into the genome of the pancreatic islet cells or may exist independently of the genome. Gene transfer vectors that can be used in the second step include viral vectors and plasmid vectors. Viral vectors include retroviral vectors (including lentiviral vectors and pseudotyped vectors), adenoviral vectors, adeno-associated viral vectors, herpesvirus vectors, Sendai viral vectors, and episomal vectors. Transposon expression systems (e.g., PiggyBac system) may also be used. Plasmid vectors include animal cell expression plasmids (e.g., pa1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo).

[0097] In one embodiment, the MycL gene is introduced into pancreatic islet cells using homologous recombination with a viral vector (lentiviral vector or Sendai virus vector). The MycL gene may be knocked into a specific locus on one or both chromosomes by homologous recombination using a viral vector. Examples of specific loci include those with an open chromatin structure, such as the human PPP1R2C locus, which are less susceptible to gene expression suppression after insertion. When using homologous recombination, donor DNA is typically used, and this donor DNA contains at least the MycL gene in the region between the 5' and 3' homology arms (hereinafter also referred to as the "intended insertion region"). Furthermore, simultaneous expression of the MycL gene and a marker gene can be achieved by including an IRES (internal ribosome entry site) or a 2A (e.g., T2A, P2A, E2A, F2A) coding sequence in the intended insertion region.

[0098] Examples of genome editing methods for homologous recombination include a method using zinc finger nucleases (ZFNs), which combine a zinc finger DNA-binding domain with a nonspecific DNA cleavage domain (Japanese Patent No. 4968498), a method using TALENs (TAL effector nucleases), which combine a transcription activator-like (TAL) effector, a DNA-binding module, with a DNA endonuclease (WO2011 / 072246), or a method using the CRISPR-Cas9 system, which combines the DNA sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) with the nuclease Cas protein family, which plays an important role in conjunction with CRISPR (WO2013 / 176772).

[0099] The MycL gene can be introduced into pancreatic islet cells using any of the following methods: calcium phosphate co-precipitation, electroporation, lipofection, retroviral infection, aggregation, microinjection, gene gun (particle gun), and DEAE-dextran.

[0100] The production method of the present invention includes (3) a third step of culturing by the culture method of the present invention. The third step can be carried out according to the culture method of the present invention.

[0101] The production method of the present invention may include a step of removing the introduced MycL gene. "Removing the MycL gene" encompasses not only removing the MycL gene integrated into the genome or present in the cell (e.g., in the form of a viral vector), but also reducing the expression level of the gene. Methods for removing the MycL gene are not limited, and include, for example, episomal vectors (Okita K., et al., Nat Methods 2011 May 8 (5):409-412), temperature-sensitive negative-strand RNA viral vectors (e.g., Sendai virus, Warren L., et al., Cell Stem Cell 2010 Nov 7 (5):618-630), photo-regulated viral vectors (Tahara M., et al., PNAS, vol. 116, pp. 11587-11589, 2019), and PROTAC protein degradation systems. Alternatively, the MycL gene integrated into the genome can be removed using the PiggyBac system, Cre / loxP system, or the above-mentioned genome editing techniques. Temperature-sensitive Sendai virus vectors are prepared by substituting a gene encoding a temperature-sensitive P protein with an amino acid mutation in the viral P protein. In this case, raising the culture temperature (to 37°C) inactivates the temperature-sensitive P protein, halting viral transcription and replication and eliminating the viral vector. PROTAC protein degradation systems are prepared by adding a gene encoding a degron to the viral P protein (or a temperature-sensitive P protein). Adding a bromotag to the culture medium promotes proteasomal degradation of the degron-attached P protein, halting viral transcription and replication and eliminating the viral vector. See, for example, a system in which at least one viral protein encoded in the genome (preferably the P protein or the temperature-sensitive P protein) is degraded and removed by a PROTAC system ( WO2024195811A1 ).A photoregulated viral vector is a viral vector in which a gene encoding a photoswitch protein called Magnet has been introduced (WO2019013258A1), and the expression of a target gene incorporated into the vector can be controlled using blue light (WO2021117840A1).

[0102] Regarding the production method of the present invention, other necessary matters than those mentioned above are as follows: "1. The contents of the method for culturing human pancreatic islet cells are all incorporated by reference."

[0103] 3. Uses of the Human Islet Cells of the Present Invention Because the human islet cells of the present invention can be suitably used in regenerative medicine, in another aspect, a transplantation therapy agent (hereinafter, sometimes referred to as the "transplantation therapy agent of the present invention") containing the human islet cells of the present invention is provided. The present invention also encompasses a method for treating or preventing a disease in which insulin function is insufficient in the body (typically, insulin resistance or decreased insulin secretion), in which an effective amount of the human islet cells of the present invention is administered or transplanted into a subject (human). The disease is typically diabetes, but more specifically, diseases, disorders, or symptoms associated with severe hypoglycemia, type I diabetes (including slowly progressive type 1 diabetes or type 1.5 diabetes), type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, or metabolic syndrome, as well as diseases caused by other specific mechanisms or diseases, such as genetic abnormalities involved in pancreatic beta cell function, genetic abnormalities involved in the insulin action transduction mechanism, and diseases or conditions associated with other diseases or conditions, such as exocrine pancreatic diseases, endocrine diseases, liver diseases, drug- or chemical-induced diseases, infectious diseases, rare pathologies caused by immune mechanisms, and gestational diabetes. Furthermore, diabetic complications caused by diabetes (e.g., diabetic retinopathy, diabetic neuropathy, etc.) can also be included in the indications, as can insulin secretion deficiency resulting from total or partial pancreatectomy associated with pancreatitis or pancreatic cancer.

[0104] The transplantation therapy agent of the present invention can be administered or transplanted into the body of a subject (human) in need thereof. The method of administration or transplantation is not particularly limited as long as the desired therapeutic or prophylactic effect is achieved. It may be parenteral administration, for example, administration by injection or infusion to any location in the body that can respond to glucose, or direct transplantation into a region in the body where human pancreatic islet cells can be fixed at a fixed position. Specifically, it may be administered, for example, intrapancreatically, under the kidney capsule, preferably subcutaneously or intraperitoneally, more preferably intravascularly or intravenously, and even more preferably into the portal vein. In one aspect, the transplantation therapy agent of the present invention is administered into the hepatic portal vein of the subject.

[0105] The human islet cells to be transplanted may be administered in a therapeutically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the transplant recipient. The number of human islet cells to be transplanted is not particularly limited. For example, the number of human islet cells to be transplanted may be 10 × 10 3 Cell ~10×10 12 In one embodiment, the human islet cells of the present invention are transplanted into a subject so that the number of islets is at least 5,000 islet equivalent units (EIN) / kg body weight. Here, 1 EIN is generally equivalent to 1,000 cells. In another embodiment, for a 50 kg subject, 2.0 x 10 7 ~2.0×10 10 Approximately 2.5 x 10 cells, preferably 2.5 x 10 7 ~2.0×10 9 cells, preferably 2.0 x 10 8 ~2.0×10 9 The human pancreatic islet cells of the present invention are administered so that the number of cells reaches approximately 1000. Transplantation and administration may be performed once or multiple times (e.g., 2, 3, 4, 5, etc.) during the course of treatment, etc. When performed multiple times, the number of human pancreatic islet cells to be transplanted may be appropriately changed, and may be set to, for example, 99%, 95%, 90%, etc., of the number of cells transplanted in the first administration.

[0106] When using the human pancreatic islet cells of the present invention in cell transplantation therapy, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" refers to HLA genotypes that are identical to those of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, such as somatic cells with HLA types that match the three loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C, or six loci including HLA-DP and HLA-DQ. If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0107] The transplantation therapy agent of the present invention can be prepared by, for example, mixing with a pharmaceutically acceptable aqueous liquid. Therefore, in one embodiment, there is also provided a method for producing a transplantation therapy agent containing human pancreatic islet cells, etc., which includes a step of formulating the human pancreatic islet cells, etc. of the present invention. Such a production method may also include a step of preparing the human pancreatic islet cells, etc. of the present invention. Furthermore, it may also include a step of preserving the human pancreatic islet cells, etc. of the present invention.

[0108] The pharmaceutically acceptable aqueous liquid that can be contained in the transplantation therapy agent of the present invention can contain, for example, an appropriate selected buffer, isotonicity agent, pH adjuster, antioxidant, chelating agent, etc., within a range that does not affect the viability and physiological activity of the transplanted human pancreatic islet cells.

[0109] Examples of buffers include phosphate buffers, borate buffers, citrate buffers, tartrate buffers, acetate buffers, amino acids, and epsilon-aminocaproic acid. Examples of isotonicity agents include sugars such as D-sorbitol, D-glucose, and D-mannitol, polyhydric alcohols such as glycerin and propylene glycol, salts such as sodium chloride, and boric acid. Examples of chelating agents include sodium edetate and citric acid. Examples of pH adjusters include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or a salt thereof (borax), hydrochloric acid, citric acid or a salt thereof (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or a salt thereof (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or a salt thereof (sodium acetate, ammonium acetate, etc.), and tartaric acid or a salt thereof (sodium tartrate, etc.). Examples of antioxidants include ascorbic acid, glutathione, sodium bisulfite, dried sodium sulfite, sodium pyrosulfite, and tocopherol. Specific examples of "pharmaceutically acceptable aqueous liquids" include aqueous liquids such as physiological saline and isotonic solutions containing glucose and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The transplantation therapy agent of the present invention may be formulated with, for example, a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc.

[0110] The transplantation therapy agent of the present invention may be provided in a cryopreserved state under conditions typically used for cryopreserving cells, and may be thawed immediately before use. In such cases, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO, etc.), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0111] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0112] We explored an efficient method for expanding islet progenitor-like cells, which were generated by expressing MycL in islet cells induced to differentiate from iPS cells. We also explored the optimal additives for the expansion medium of islet progenitor-like cells. The effects of additives were evaluated using indicators of cell expansion effect, gene expression levels in target cells, and gene expression levels in non-target cells, and we investigated the relationships between them.

[0113] Example 1: Induction of Islet Progenitor-Like Cells Using Lentivirus Prior to the expansion and culture of islet progenitor-like cells described below, pancreatic islet cells induced from iPS cells were differentiated and transduced with the MycL gene using lentivirus to induce islet progenitor-like cells. Cellartis hiPSC Beta Cells (from ChiPSC12) Kit (Takara) was used as iPS cell-derived islet cells. After waking the cells as described by the manufacturer and culturing them in the culture medium provided with the kit for 24 hours, they were dispersed with trypsin-EDTA and infected with lentivirus expressing MycL and the fluorescent protein mCherry at an MOI of 30 or 2700. A non-infected control was used. After seeding the cells into an AggreWell 400 24-well plate (StemCell Technologies), they were cultured at 37°C and 5% CO2 for 48 hours to form cell clusters. The medium used was RPMI1640 supplemented with 10% FBS, penicillin, streptomycin, 1 mM N-acetylcysteine, 10 mM nicotinamide, and 4.5 g / L glucose. The resulting cell clusters were then cultured for 14 days to induce islet precursor cells. The cells were cultured in DMEM / F12 medium supplemented with Glutamax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF-10, 10 nM Gastrin1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-spondin1, 2% B27, and 0.1% Poloxamer 188. The glucose concentrations were 2.0 g / L and 4.5 g / L, and the medium was changed every 24 hours. After induction, cell numbers were measured using a Countess (ThermoFisher).

[0114] Example 2: Induction of islet progenitor-like cells using Sendai virus Prior to the expansion and culture of islet progenitor-like cells described below, the MycL gene was introduced using Sendai virus into pancreatic islet cells induced to differentiate from iPS cells. The temperature-sensitive Sendai virus mutant Sev(NHL)L-MYC / TS15ΔF was used as the virus for MycL expression induction at MOIs of 3, 10, 50, 100, and 200. Sev TS15ΔF was also used as a control at MOIs of 3, 10, 50, and 100. A mock condition was used as a non-infected control. For the induction of islet progenitor-like cells, the Cellartis hiPSC Beta Cells (from ChiPSC12) Kit (Takara) was used as the source of iPS cell-derived islet cells. Cells were induced to sleep as described by the manufacturer, then cultured for 24 hours in the culture medium provided with the kit. They were then dispersed with trypsin / EDTA and infected with Sendai virus expressing MycL. They were seeded into AggreWell 400 24-well plates (StemCell Technologies) and cultured for 48 hours at 32°C and 5% CO2 to form cell clusters. The medium used was RPMI 1640 containing 10% FBS, penicillin, streptomycin, 1 mM N-aceyttrium-3-phosphate dehydrogenase (NADPH), 10 mM nicotinamide, and 4.5 g / L glucose. The formed cell clusters were then cultured for 14 days to induce islet progenitor cell development. The cells were cultured in DMEM / F12 medium supplemented with Glutamax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF-10, 10 nM Gastrin1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-spondin1, 2% B27, and 0.1% Poloxamer 188. The glucose concentrations were 2.0 g / L and 4.5 g / L, and the medium was changed every 24 hours under 5% CO2 at 35°C or 37°C. After induction, cell numbers were measured using a Countess (ThermoFisher).

[0115] Example 3: Characterization of Islet Progenitor-Like Cells. Uninfected cells and cells transfected with the MycL gene at an MOI of 30 or 2700 using lentivirus were cultured in Matrigel for 17 days, recovered from the Matrigel, and dispersed into single cells using Tyrpsin to prepare a single-cell gene expression library. The Chromium Next GEM Single Cell 3' Reaget Kits v3.1 (10xGenomics) were used for the preparation of the single-cell gene expression library. A Novaseq6000 was used for sequencing analysis. As a differentiated cell control, cells were awake using the Cellartis hiPSC Beta Cells (from ChiPSC12) Kit (Takara) and cultured for 21 days in the culture medium provided with the kit. The cells were then dispersed into single cells using Tyrpsin and used.

[0116] Example 4: Method for evaluating the expansion of islet progenitor-like cells. Islet progenitor-like cells were dispersed into single cells using trypsin and seeded onto PrimeSurface 96V plates (Sumitomo Bakelite; product number MS-9096V) at 1500 cells / well. Cell clusters were formed by culturing for 48 hours at 37°C and 5% CO2. The medium used was RPMI 1640 containing 10% FBS, penicillin, streptomycin, 1 mM N-aceytlisteine, 10 mM nicotinamide, and a glucose concentration of 4.5 g / L. The glucose concentrations of the culture medium used for the investigation were set to 2.6 g / L and 4.3 g / L, and the medium was changed every 24 hours. After 10 to 14 days of expansion, the number of viable cells was measured using CellTiterGlo (Promega) as an index of ATP concentration. The luminescence value was measured using ARVO (Tecan).

[0117] Example 5: Gene Expression Analysis of Islet Progenitor-Like Cells. After 10 to 14 days of expansion culture, cDNA was prepared using the Cells To CT Kit (TheroFisher). RT-qPCR was performed using Taqman Probes, and the expression of CHGA (target cells), MECOM and PRSS1 (non-target cells), and NKX6.1 (islet progenitor cells) was measured using QuantStudio (ThermoFisher). GAPDH was used as an internal standard.

[0118] Example 6: Factor Search We searched for factors optimal for the expansion and culture of islet progenitor-like cells. Culture experiments were performed under the following conditions with n = 3 to 5. (Culture Experiment 1) The expansion effect of islet progenitor-like cell induction and gene expression analysis were performed using a medium with the composition shown below. Details of the cell expansion cell-like medium are shown in Table 3. The glucose concentration was changed alternately between 4.3 g / L and 2.6 g / L every 24 hours.

[0119]

[0120] (Culture Experiment 2) The expansion of islet progenitor-like cells and gene expression analysis were performed using a medium with the following composition. Details of the cell-expansion cell-like medium are shown in Table 4. The glucose concentration was 4.3 g / L and 2.6 g / L, and the medium was changed alternately every 24 hours.

[0121]

[0122] (Culture Experiment 3) Expansion of islet progenitor-like cells and gene expression analysis were performed using a medium with the following composition. The cell expansion medium consisted of DMEM / F12 supplemented with GlutaMax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF10, 10 nM Gastrin 1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-Spondin 1, 2% B27, and 0.1% Poloxamer 188. Additional factors are listed in Table 5. The glucose concentration was 4.3 g / L and 2.6 g / L, with the medium being alternately changed every 24 hours.

[0123]

[0124] (Culture Experiment 4) Expansion and gene expression analysis of islet progenitor-like cells were performed using the medium composition shown below. The cell expansion medium consisted of DMEM-F12 supplemented with GlutaMax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF10, 10 nM Gastrin 1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-Spondin 1, 2% B27, and 0.1% Poloxamer 188. Additional factors are listed in Table 6. The glucose concentration was 4.3 g / L and 2.6 g / L, with the medium being alternately changed every 24 hours.

[0125]

[0126] (Culture Experiment 5) Expansion of islet progenitor-like cells and gene expression analysis were performed using a medium with the following composition. The cell expansion medium consisted of DMEM-F12 supplemented with GlutaMax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF10, 10 nM Gastrin 1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-Spondin 1, 2% B27, and 0.1% Poloxamer 188. Additional factors were listed in Table 7. The glucose concentration was 4.3 g / L and 2.6 g / L, with the medium being alternately changed every 24 hours.

[0127]

[0128] (Culture Experiment 6) Expansion and gene expression analysis of islet progenitor-like cells were performed using the medium composition shown below. The cell expansion medium consisted of DMEM-F12 supplemented with GlutaMax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF10, 10 nM Gastrin 1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-Spondin 1, 2% B27, and 0.1% Poloxamer 188. The glucose concentrations were 4.3 g / L and 2.6 g / L, with the medium being exchanged alternately every 24 hours.

[0129]

[0130] Example 7: Evaluation of amplification efficiency In each of the two culture experiments described above, the cell masses on any of the culture days from 10 to 17 after the start of the amplification culture were counted for viable cell numbers using CellTiterGlo (Promega) as an index of ATP concentration. Measurements were performed by measuring luminescence values ​​using ARVO (Tecan).

[0131] Example 8: Evaluation of expression of target cell genes and non-target cell genes In each of the two culture experiments described above, cDNA was prepared from cell clumps on any of the culture days between 10 and 17 after the start of amplification culture using the Cells To CT Kit (TheroFisher), and the expression of CHGA, MECOM, PRSS1, and NKX6.1 was measured using Taqman Probe on QuantStudio (Applied Biosystems).

[0132] Example 9: Evaluation of the differentiation potential of expanded islet progenitor cells into insulin-positive cells (Culture Experiment 7) Culture Experiment 7 was performed to confirm the potential of expanded islet progenitor cells to re-maturate into insulin-expressing cells (positive cells). After transfection with the MYLC gene using a temperature-sensitive Sendai virus, the cells were cultured for expansion at 35°C for 16 days in a medium containing DMEM / F12 supplemented with GlutaMax, HEPES, penicillin, streptomycin, 100 ng / mL FGF10, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, and 2% B27 to obtain amplified cell samples. Some of the expanded cell samples were then cultured for 14 days at 37°C in the Cellartis Beta Cell Maintenance Medium included in the Cellartis hiPSC Beta Cells (from ChiPSC12) Kit (Takara) for 2 weeks to reduce MycL gene expression. This cultured sample was used as a post-expansion differentiation culture sample. RNA was extracted from the amplified cell samples and the post-amplified differentiated culture samples, and the expression levels of MycL and insulin (INS) were quantified and evaluated using RT-qPCR with GAPDH as an internal standard.

[0133] Example 10: Establishment of a method for culturing highly pure islet progenitor-like cells (culture experiment 8) In order to identify a medium composition that allows the proliferation of human islet cells (target cells) and does not favor the proliferation of non-target cells, islet progenitor-like cells obtained by the same method as in Example 1 above were cultured in a medium containing EGF and a medium not containing EGF by the same culture method as in Example 6. The proportion (yield) of cells expressing CHGA (target cells) was evaluated for the cells cultured in each medium.

[0134] (Experimental Results) Figure 1 shows the results of an investigation into the induction of islet progenitor-like cells from iPS cell-derived β cells by MycL gene transfection. The culture medium used for induction was DMEM / F12 supplemented with GlutaMax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF10, 10 nM Gastrin1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM Nicotinamide, 1 μg / mL R-Spondin1, 2% B27, and 0.1% Poloxamer 188. The cell numbers after induction are shown in Figure 1. Compared to the uninfected control, MycL gene transfection clearly increased the cell numbers. Previous findings indicated that when inducing expansion in mouse cells, a medium containing RPMI1640 supplemented with 10% FBS, penicillin, streptomycin, 1 mM N-acetylcysteine, and 10 mM nicotinamide was used. However, it was revealed that amplified cells can be induced in human cells under medium conditions different from those used in mouse cells.

[0135] Next, to characterize the amplified cells, we performed gene expression analysis using single-cell RNA-seq (Figure 2). Four conditions were used: uninfected cells (MOCK), cells infected with MycL gene-expressing lentivirus at MOI 30 and MOI 2700, and cells cultured for 21 days in the medium provided with the kit as a differentiated control. RNA-seq data were analyzed and clustered using UMAP, revealing three main populations (Figure 3). The first was a population of islet progenitor-like cells transfected with the MycL gene, expressing markers such as FEV1, which are islet progenitor-like cells. The second was a population of non-target cells with low MycL expression and expressing the pancreatic ductal cell marker Sox9 and the pancreatic exocrine cell marker PRSS1. The third was a differentiated cell population, which was the major cell population under the differentiated control conditions and expressed the islet progenitor cell marker NKX6.1 (Figure 4). These results confirmed that the cell population transduced with the MycL gene by lentivirus was induced into islet progenitor-like cells. On the other hand, in experiments using the Cellartis hiPSC Beta Cells (from ChiPSC12) Kit, a population of non-target cells was also observed.

[0136] A culture method that efficiently expands islet progenitor-like cells without expanding non-target cells is an important technology for ultimately preparing insulin-producing cells. However, no methods for efficiently expanding human islet progenitor-like cells have been reported to date. Therefore, we first utilized single-cell analysis data to maximize cell expansion efficiency and identify gene markers that could distinguish islet progenitor-like cells from potentially contaminating non-target cells. Genes with significant differential expression between islet progenitor-like cells and non-target cell clusters were displayed and analyzed using a violin plot. As a result, we selected CHGA as a marker gene highly expressed in the islet progenitor-like cell cluster and MECOM as a marker gene highly expressed in the non-target cell cluster (Figure 5).

[0137] To efficiently expand islet progenitor-like cells, we used viable cell count and the gene expression levels of the islet progenitor-like cell marker CHGA and the non-target cell marker MECOM as indicators of cell characteristics. We then explored conditions for increasing viable cell count and islet progenitor-like cell markers and decreasing the expression of non-target cell markers in medium compositions supplemented with various factors. For reference, we also obtained gene expression data for the islet progenitor cell marker NKX6.1 in some experimental runs. For factor screening, we used cells subcultured after lentiviral transfection with the MycL gene. For screening, we used a PrimeSurface 96V (Sumitomo Bakelite; product number MS-9096V) (3D culture plate) to seed and culture cells at 1500 cells / well in one clump / well, improving the throughput of factor screening (Figure 6).

[0138] First, in culture experiment 1, we investigated the basal medium and additives. We tested DMEM / F12 + HEPES, which can induce human islet progenitor-like cells, and RPMI1640 + 1 mM Nicotinamide + 1 mM N-Acetylcysteine, which is used to induce mouse islet progenitor-like cells. We also tested 10% FBS, 2% ITS-X, 2% B27, and 1 μM T3 as additives (Figure 7). Cell proliferation was maximized in DMEM / F12 supplemented with B27. Furthermore, gene expression analysis revealed CHGA gene expression, while the non-target cell marker MECOM was below the detection limit, suggesting that this basal medium condition maximizes target cell proliferation. Furthermore, the addition of 10% FBS suppressed cell proliferation, suggesting that it was not optimal, but increased expression of the differentiation marker NKX6.1, suggesting that it may promote differentiation. Although cell proliferation was confirmed even with the addition of ITS-X, the effect was limited compared to B27. It was also revealed that RPMI1640, a basal medium used for mouse cells, has lower proliferation efficiency compared to DMEM / F12. Furthermore, adding T3 alone inhibited cell proliferation. Based on these findings, the medium composition of DMEM / F-12 + HEPES + B27 was identified as a combination of basal proliferation medium and factors more suitable for human cells.

[0139] Next, to examine the effects of supplemental factors, we investigated the effects of A83-01, EGF, FGF10, Noggin, and B27 in culture experiment 2 (Figure 8). Condition 3, in which EGF was not added, showed a decrease in viable cell count, but also a decrease in the expression of the non-target cell markers MECOM and PRSS1. On the other hand, the expression of the islet progenitor-like cell marker CHGA was not decreased, suggesting that EGF may be involved in the expansion of non-target cells. Furthermore, condition 2, in which A83-01 was not added, showed an increase in viable cell count and the expression of the target cell marker CHGA, but a decrease in the expression of the non-target cell marker MECOM and PRSS1. Condition 5, in which Noggin was not added, showed no effect on viable cell count or the expression of the target cell marker CHGA, but a decrease in the expression of the non-target cell marker PRSS1. Condition 4, in which FGF10 was not added, showed no effect on viable cell count or the expression of the non-target cell markers MECOM and PRSS1, but a decrease in the expression of the target cell marker. Based on these results, we identified EGF, A83-01, and Noggin as factors that promote the expansion of non-target cells, and FGF10 as a factor that promotes the expansion of target cells.

[0140] To further optimize the expansion medium, we conducted Culture Experiment 3 (Figure 9), Culture Experiment 4 (Figure 10), Culture Experiment 5 (Figure 11), and Culture Experiment 6 (Figure 12) to identify factors that promote the expansion of target cells and factors that suppress the expansion of non-target cells. As a result, we identified Exendin 4 (condition 2) in Culture Experiment 3 and FGF7 and FGF2 (condition 4) as factors that promote the expansion of target cells, i.e., factors that act on cell expansion and increase the expression of the target cell gene marker CHGA. Furthermore, we identified Forskolin (condition 2) in Culture Experiment 5, AS-1842856 (condition 3), and Afatinib and Sepatinib (conditions 2 and 3) in Culture Experiment 6 as factors that suppress the expansion of non-target cells, i.e., factors that decrease the expression of the non-target cell markers MECOM and PRSS1.

[0141] An outline of an experiment investigating the induction of islet progenitor-like cells from iPS cell-derived β cells by MycL gene transfer using Sendai virus is shown in Figure 13, and the induction results are shown in Figure 14. Cells were infected with Sendai virus expressing MycL at an MOI of 3, 10, 50, 100, or 200, and as controls, uninfected cells and cells infected with the control vector SevTS15ΔF were used at an MOI of 3, 10, 50, or 100. To form aggregates, the cells were cultured at 32°C for 48 hours in RPMI1640 containing 10% FBS, penicillin, streptomycin, 1 mM N-acetylcysteine, 10 mM nicotinamide, and 4.5 g / L glucose. Subsequently, the cells were cultured at 35°C or 37°C for 16 days in DMEM / F12 containing GlutaMax, HEPES, penicillin, streptomycin, 0.5 μM A83-01, 50 ng / mL EGF, 100 ng / mL FGF10, 10 nM Gastrin1, 100 ng / mL Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1 μg / mL R-Spondin1, 2% B27, and 0.1% Poloxamer 188. The cells were counted and the results are shown in Figure 14. Compared to the control non-infection condition, introduction of the MycL gene resulted in Sev MycL MOI-dependent cell proliferation at both 35°C and 37°C culture temperatures, demonstrating that Sendai virus can be used to induce proliferative cells, just like lentivirus.

[0142] To confirm that the expanded islet progenitor cells have the potential to re-maturate into insulin-expressing cells (positive cells), we performed culture experiment 7. The results showed that the expression of MycL decreased and the expression of the maturation marker insulin (INS) increased after expansion and differentiation culture of islet cells (Figure 15). This indicates that islet progenitor-like cells expanded by MycL transfection can re-differentiate and mature by appropriate treatment, such as blocking MycL expression.

[0143] To identify a medium composition that would promote the proliferation of human pancreatic islet cells (target cells) and limit the proliferation of non-target cells, and to establish a method for culturing these cells with high purity, we conducted a culture experiment. After inducing islet progenitor-like cells by lentiviral MycL gene transfer, culturing in EGF-free medium suppressed the proliferation of non-target cells and increased the percentage (yield) of CHGA-expressing cells (target cells) to 81.7%. In contrast, culturing in EGF-containing medium reduced the percentage (yield) of CHGA-expressing cells to only 24.2%. Therefore, culturing in EGF-free medium was found to be effective for achieving high purity and high yield of human pancreatic islet cells (target cells).

[0144] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0145] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include any modifications within the scope of the claims and meanings equivalent to the claims.

[0146] According to the present invention, it is possible to efficiently expand and culture human iPSC-derived pancreatic islet cells that express the MycL gene, opening the door to cell transplantation therapy for diabetes patients.

[0147] This application is based on patent application No. 2024-002180 filed in Japan (filing date: January 10, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for culturing human pancreatic islet cells, comprising culturing human pluripotent stem cell-derived pancreatic islet cells in a medium containing B27 or ITS-X.

2. The culturing method according to claim 1, wherein the pancreatic islet cells are pancreatic progenitor cell-like cells expressing MycL.

3. The culturing method according to claim 1 or 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

4. The culturing method according to any one of claims 1 to 3, wherein the medium further contains an FGF signaling pathway activator.

5. The culturing method according to claim 4, wherein the FGF signaling pathway activator is one or more substances selected from the group consisting of FGF2, FGF7, and FGF10.

6. The culturing method according to any one of claims 1 to 5, wherein the medium does not contain EGF and / or contains an EGF signaling pathway inhibitor.

7. The culturing method according to claim 6, wherein the EGF signaling pathway inhibitor is Afatinib and / or Sepatinib.

8. The culturing method according to any one of claims 1 to 7, wherein the medium further contains one or more agents selected from the group consisting of a GLP1 signaling pathway activator, a FOXO1 inhibitor, and an adenylate cyclase activator.

9. The culturing method according to claim 8, wherein the GLP1 signaling pathway activator is Exendin 4.

10. The culturing method according to claim 8, wherein the FOXO1 inhibitor is AS-1842856.

11. The culturing method according to claim 8, wherein the adenylate cyclase activator is Forskolin.

12. The culturing method according to any one of claims 1 to 11, wherein the medium does not contain A83-01 and / or Noggin.

13. The culturing method according to any one of claims 1 to 12, wherein the medium does not contain serum and / or T3.

14. The culturing method according to any one of claims 1 to 13, characterized by suspension culture.

15. The culturing method according to claim 14, characterized by suspension culture in a culture plate subjected to low-attachment treatment.

16. The culturing method according to claim 15, wherein the culture plate is a V-bottom culture plate.

17. The culturing method according to any one of claims 1 to 16, characterized by culturing in the absence of Matrigel.

18. A method for producing human pancreatic progenitor cell-like cells, comprising the following steps (1) to (3); (1) a first step of differentiating human pluripotent stem cells into pancreatic islet cells, (2) a second step of increasing the expression of the MycL gene in the pancreatic islet cells to produce pancreatic progenitor cell-like cells, and (3) a third step of culturing using the culturing method according to any one of claims 1 to 17.

19. Human pancreatic progenitor cell-like cells produced by the production method according to claim 18.

Images