A method for screening in vitro populations of stem cell-derived beta-like cells and their novel markers.

Abstract

The present invention relates to a method for screening beta-like cells in an in vitro cell population of pluripotent stem cell-derived cells, comprising identifying beta-like cells that express specific markers or combinations thereof to predict the in vivo functionality of the cells prior to transplantation. The present invention also relates to an in vitro population of pluripotent stem cell-derived beta-like cells, wherein the beta-like cells comprise one or more markers that are not present in naturally occurring human beta cells or the expression levels of the markers differ from those of naturally occurring human beta cells.

Description

Technical Field

[0001] The present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells by identifying beta-like cells that express one or more specific markers. The present invention also relates to an in vitro population of pluripotent stem cell-derived cells comprising beta-like cells that express one or more markers that are not present in natural human beta cells or have an expression level different from that of natural human beta cells.

Background Art

[0002] Patients with type 1 diabetes can be treated using transplantation of islets from human donors, and some patients achieve insulin independence. However, donor islets are scarce and have variable quality, and thus pluripotent stem cell-derived beta-like cells offer an attractive alternative to islets.

[0003] During in vitro differentiation, human pluripotent stem cells are differentiated using various chemical and biological factors, resulting in heterogeneous islet-like cell clusters containing different endocrine cell types.

[0004] Classical beta-cell markers include insulin (INS), as well as standard transcription factors such as PDX1 and NKX6.1. Traditionally, these markers have been used to identify stem cell-derived beta-like cells. While all stem cell-derived beta-like cells may appear phenotypically similar to native human beta cells based on the expression of classical beta-cell markers in vitro, they differ because not all stem cell-derived beta-like cells express and secrete insulin after transplantation. Classical beta-cell markers are insufficient to identify stem cell-derived beta-like cells in vitro that maintain insulin expression and secretion after transplantation. Some classical beta-cell markers, such as PDX1, may phenotype similar to native human beta cells before transplantation, but may alter their phenotype after transplantation, leading to undesirable cell populations or even negative correlations with in vivo function, resulting in the selection of in vitro differentiation protocols from stem cells to beta-like cells.

[0005] Identifying in vitro populations of stem cell-derived beta cells that maintain insulin expression and secretion after transplantation is essential for the product performance, evaluation, and production of differentiated beta-like cells. Therefore, there is a need to identify novel markers that define in vitro populations of stem cell-derived beta-like cells that maintain insulin expression and secretion after transplantation. [Overview of the Initiative]

[0006] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0007] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, the markers being present in native human beta cells.

[0008] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, wherein at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and the expression level of the markers is, on average, at least about 1 log-fold change compared to the expression of the markers in native human beta cells.

[0009] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, including beta-like cells, for use in the treatment of type 1 diabetes.

[0010] In one embodiment, the present invention relates to a cell composition comprising an in vitro population of pluripotent stem cell-derived cells, including beta-like cells obtained by the present invention, and a cell culture medium.

[0011] In one embodiment, the present invention relates to an implantable device comprising an in vitro population of pluripotent stem cell-derived cells, including beta-like cells obtained by the present invention.

[0012] In one embodiment, the present invention relates to a method for treating type 1 diabetes, comprising administering an in vitro population of pluripotent stem cell-derived cells, including beta-like cells obtained by the present invention, to a person in need of such treatment.

[0013] In one embodiment, the present invention relates to a cryopreserved in vitro population of pluripotent stem cell-derived cells, including beta-like cells obtained by the present invention.

[0014] In one embodiment, the present invention provides an in vitro population of pluripotent stem cells, including beta-like cells or insulin-producing cells, prior to transplantation.

[0015] The present invention can also solve further problems that may become apparent from the disclosure of exemplary embodiments. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1A is a graph showing the T-distributed stochastic neighbor embedding (tSNE) projections of all cells sampled from eight different (modified) protocols for obtaining stem cell-derived cells and from cadaveric human pancreatic islet cells. Figure 1B is a graph showing the tSNE projections of 26 cell clusters, each cluster consisting of cells with similar gene expression profiles. [Figure 2] Figure 2 is a graph showing stem cell-derived cluster numbers on the X-axis and beta cell scores on the Y-axis. This shows that stem cell-derived beta-like cell cluster numbers 14, 15, and 26 are nearly identical to native human beta cell cluster number 16, based on human beta cell gene module scoring for all cells. [Figure 3] Figure 3 is a graph showing the native pancreatic islet subtypes on the X-axis and the beta cell score on the Y-axis. All cells with high scores in the beta cell gene set were classified as native human beta cells. [Figure 4]Figure 4 is a graph showing gene expression profiles specific to an in vitro population of stem cell-derived beta-like cells. Native human beta cells (cluster 16) were compared to stem cell-derived beta-like cells (clusters 14, 15, and 26). The median UPT (a measure of RNA quality) of native human beta cells was equal to 0. The median UPT of stem cell-derived beta-like cells was greater than that of native human beta cells. The plots show the mean expression difference between primary or native human beta cells and stem cell-derived beta-like cells using box plots, and expression in individual cells using single dots. Below each column, the percentage of cells showing no expression is shown. [Figure 5] Figure 5 shows stem cell-derived cells before and after transplantation in vivo. This indicates that some stem cell-derived cells do not remain insulin-positive after in vivo exposure. NKX6.1, insulin, and glucagon staining of stem cell-derived cells before and after transplantation shows that while the cells appear similar to native human beta cells before transplantation, insulin expression and secretion are significantly different after transplantation. [Figure 6] Figure 6 is a graph showing the percentage of cells in clusters 14, 15, and 26 on the X-axis, and the mean human C-peptide 8 weeks after transplantation on the Y-axis. This shows a positive correlation between stem cell-derived beta-like cells and human C-peptide secreted 8 weeks after in vivo exposure. C-peptide levels measured in transplanted SCID-beige animals with cells generated using different (modified) differentiation protocols show a positive correlation with the accumulation size of clusters 14, 15, and 26. [Figure 7] Figure 7 is a graph showing that the classical beta cell markers NKX6.1, SYT13, NKX2.2, PDX1, PDX1+ / NKX6.1+, and PAX4 have a negative correlation with mean human C-peptide 8 weeks after transplantation in SCID mice. [Figure 8]Figure 8a) The upper panel shows a graph indicating that SLC30A8 is rich in stem cell-derived beta cell populations. Figure 8a) The lower panel shows a graph indicating widespread expression of PDX1. Figure 8b) The upper panel shows a graph indicating that SLC30A8 is positively correlated with mean human c-peptide 8 weeks after transplantation in SCID mice. Figure 8b) The lower panel shows a graph indicating that PDX1 is negatively correlated with mean human c-peptide 8 weeks after transplantation in SCID mice. [Figure 9] Figure 9a) shows that ACVR1C is not expressed in native human beta cells. Figure 9b) shows that ACVR1C marks a subset of cells in stem cell-derived cell cultures. Figure 9c) is a graph showing that ACVR1C expression is positively correlated with in vivo function. [Figure 10] Figure 10a) The upper panel is a tSNE plot showing high expression of PCDH7 in clusters 14, 15, and 26. Figure 10a) The lower panel is a tSNE plot showing high expression of PCP4 in clusters 14, 15, and 26. Figure 10b) The upper panel is a graph showing the positive correlation between PCDH7 and mean human C-peptide after 8 weeks of in vivo exposure in SCID-beige mice. Figure 10b) The lower panel is a graph showing the positive correlation between PCP4 and mean human C-peptide after 8 weeks of in vivo exposure in SCID-beige mice. [Figure 11] Figure 11a) is a tSNE plot showing G6PC2 expression. Figure 11b) is a graph showing that high expression of G6PC2 mRNA, as measured by nanostrings, correlates with in vivo insulin secretion in the form of circulating mean human C-peptide 8 weeks after transplantation in SCID mice. Figure 11c) is a graph showing that high expression of G6PC2 mRNA, as measured by nanostrings, correlates with in vitro insulin secretion in the form of human C-peptide secreted during glucose exendin 4 and IBMX / forskolin attack inoculation. [Modes for carrying out the invention]

[0017] The present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, which includes identifying beta-like cells that express one or more specific markers. The markers and methods of the present invention enable the pre-transplant identification of beta-like cells that maintain insulin expression and secretion after transplantation. This is possible because stem cell-derived beta-like cells identified according to the present invention do not change their phenotype after transplantation, unlike stem cell-derived beta-like cells identified based on the expression of classical beta cell markers.

[0018] Furthermore, the present invention provides the application of specific novel markers or classical beta cell markers in the identification of stem cell-derived beta-like cells that maintain insulin expression and secretion after transplantation.

[0019] Furthermore, the present invention enables the identification of beta-like cells that maintain insulin expression and secretion after transplantation by detecting markers that are not present in natural human beta cells or have a higher expression level in an in vitro population of stem cell-derived beta cells than in natural human beta cells.

[0020] In one aspect according to the present invention, marker genes and their combinations are used for protein-based assays such as proteomics, flow cytometry, immunohistochemistry, or RNA-based assays such as RNA sequencing, qPCR probe-based detection, etc., but not limited to these, for identifying or purifying stem cell-derived beta cells that maintain insulin expression and secretion after transplantation, and for other analytical methods or screening methods to evaluate differentiation efficiency, quality, and performance.

[0021] In one aspect, the present invention relates to a method for screening for beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising identifying beta-like cells that express NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0022] In one aspect, the present invention relates to a method for screening for beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising identifying beta-like cells that express NKX6.1 in combination with one or more markers selected from FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, DLK1, and MAFB.

[0023] In one aspect, the present invention relates to a method for screening for beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising identifying beta-like cells that express NKX6.1 in combination with one or more markers selected from CHRNA3, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, DLK1, and MAFB.

[0024] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with one or more markers selected from CHRNA3, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PLAGL1, EGFL7, RAD21, RTN1, LBH, and DLK1.

[0025] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with ACVR1C.

[0026] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with FREM2.

[0027] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with CHRNA3.

[0028] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with DCC.

[0029] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with SOX11.

[0030] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with MARCKSL1.

[0031] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with BASP1.

[0032] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with STARD10.

[0033] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with AMBP.

[0034] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with ST6GALNAC5.

[0035] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with HMGCS1.

[0036] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with ELAVL2.

[0037] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with PCP4.

[0038] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with PCDH7.

[0039] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with NEFL.

[0040] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with PLAGL1.

[0041] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with EGFL7.

[0042] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with RAD21.

[0043] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with RTN1.

[0044] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with PLXNA2.

[0045] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with LBH.

[0046] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with NEFM.

[0047] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with SLC30A8.

[0048] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with DLK1.

[0049] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with MAFB.

[0050] In one embodiment, the present invention relates to a method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, the method comprising the step of identifying beta-like cells expressing NKX6.1 in combination with ISL1.

[0051] Methods for evaluating the expression of protein and nucleic acid markers in in vitro cell populations include qualitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blotting, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Asubel et al., eds. 2001 supplement)), immunoassays such as immunohistochemistry of section materials, Western blotting, and, for markers accessible in intact cells, flow cytometry (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).

[0052] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0053] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0054] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1, wherein the beta-like cells maintain insulin expression and secretion after transplantation.

[0055] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1, wherein the beta-like cells maintain insulin expression and secretion after transplantation.

[0056] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, DLK1, and MAFB.

[0057] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, DLK1, and MAFB, wherein the beta-like cells maintain insulin expression and secretion after transplantation.

[0058] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from CHRNA3, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, DLK1, and MAFB.

[0059] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from CHRNA3, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, DLK1, and MAFB, wherein the beta-like cells maintain insulin expression and secretion after transplantation.

[0060] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from CHRNA3, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PLAGL1, EGFL7, RAD21, RTN1, LBH, and DLK1.

[0061] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from CHRNA3, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PLAGL1, EGFL7, RAD21, RTN1, LBH, and DLK1, wherein the beta-like cells maintain insulin expression and secretion after transplantation.

[0062] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with ACVR1C.

[0063] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with FREM2.

[0064] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with CHRNA3.

[0065] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with DCCs.

[0066] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with SOX11.

[0067] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with MARCKSL1.

[0068] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with BASP1.

[0069] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with STARD10.

[0070] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with AMBP.

[0071] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with ST6GALNAC5.

[0072] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with HMGCS1.

[0073] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with ELAVL2.

[0074] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with PCP4.

[0075] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with PCDH7.

[0076] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with NEFL.

[0077] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with PLAGL1.

[0078] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with EGFL7.

[0079] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with RAD21.

[0080] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with RTN1.

[0081] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with PLXNA2.

[0082] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with LBH.

[0083] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with NEFM.

[0084] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with SLC30A8.

[0085] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with DLK1.

[0086] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with MAFB.

[0087] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, the population comprising at least 15% beta-like cells expressing NKX6.1 in combination with ISL1.

[0088] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, the markers being present in native human beta cells.

[0089] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 85% of the beta-like cells contain SOX11, a marker that is not present in native human beta cells.

[0090] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 75% of the beta-like cells contain FREM2, a marker that is not present in native human beta cells.

[0091] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, wherein at least 85% of the beta-like cells contain DCCs, and the marker is not present in native human beta cells.

[0092] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 75% of the beta-like cells contain BASP1, the marker which is not present in native human beta cells.

[0093] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 80% of the beta-like cells contain CHRNA3, and this marker is not present in native human beta cells.

[0094] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 55% of the beta-like cells contain ELALV2, the marker which is not present in native human beta cells.

[0095] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, wherein at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and the expression level of the markers is, on average, at least about 1 log-fold different from the expression level of the markers in native human beta cells.

[0096] In one embodiment, the average log-fold change is equal to the average log-fold change between the target cell cluster and all other cell clusters.

[0097] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 95% of the beta-like cells contain ACVR1C, and the expression level of the marker is, on average, at least 2 log-fold higher than the expression level of the marker in native human beta cells.

[0098] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 95% of the beta-like cells contain MARCKSL1, and the expression level of the marker is, on average, at least about 2 log-fold higher than the expression level of the marker in native human beta cells.

[0099] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 95% of the beta-like cells contain STARD10, and the expression level of the marker is, on average, at least 1 log-fold different from the expression level of the marker in native human beta cells.

[0100] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 90% of the beta-like cells contain AMBP, and the expression level of the marker is, on average, at least 2 log-fold higher than the expression level of the marker in native human beta cells.

[0101] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 85% of the beta-like cells contain ST6GALNAC5, and the expression level of the marker is, on average, at least about 1 log-fold higher than the expression level of the marker in native human beta cells.

[0102] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 80% of the beta-like cells contain HMGCS1, and the expression level of the marker is, on average, at least about 1 log-fold higher than the expression level of the marker in native human beta cells.

[0103] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells in which at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, wherein the expression level of the markers is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than the expression level of the markers in native human beta cells.

[0104] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, including beta-like cells that maintain insulin expression and secretion after transplantation.

[0105] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, including beta-like cells, wherein the cell population exhibits a glucose-stimulated insulin secretion (GSIS) response after transplantation.

[0106] In one embodiment, the response is an in vitro GSIS response. In another embodiment, the response is an in vivo GSIS response. In another embodiment, the GSIS response is similar to that of native human beta cells.

[0107] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, including beta-like cells, wherein one or more markers show a positive correlation with mean plasma c-peptide levels after transplantation.

[0108] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells including beta-like cells, wherein the beta-like cells exhibit a decrease in blood glucose after transplantation.

[0109] In one embodiment, the present invention relates to an in vitro population of pluripotent stem cell-derived cells, including beta-like cells, wherein the beta-like cells are glucose-responsive.

[0110] In one embodiment, the present invention relates to an in vitro population of stem cell-derived cells including beta-like cells, wherein the beta-like cells are glucose-responsive after transplantation.

[0111] In one embodiment, the present invention relates to an in vitro population of stem cell-derived cells including beta-like cells, wherein the beta-like cells exhibit enhanced C-peptide levels after transplantation.

[0112] In one embodiment, the present invention relates to an in vitro population of stem cell-derived cells including beta-like cells, wherein the beta-like cells exhibit improved insulin levels after transplantation.

[0113] In one embodiment according to the present invention, an in vitro population of stem cell-derived cells, including beta-like cells, shows a decrease in blood glucose after transplantation.

[0114] In one embodiment, the present invention relates to an in vitro cell culture or composition comprising a cell population of pluripotent stem cell-derived cells expressing NKX6.1 in combination with at least 15% beta-like cells, in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1, and a cell culture medium.

[0115] In one embodiment, the present invention relates to an in vitro cell culture or composition comprising a cell population of pluripotent stem cell-derived cells in which at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, and a cell culture medium, wherein the markers are not present in native human beta cells.

[0116] In one embodiment, the present invention relates to an in vitro cell culture or composition comprising a cell population of pluripotent stem cell-derived cells in which at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and a cell culture medium, wherein the expression level of the markers is, on average, at least 1 log-fold higher than the expression level of the markers in native human beta cells.

[0117] In one embodiment, the cells, stem cells, stem cell-derived cells, and / or stem cell beta-like cells of the present invention are human cells.

[0118] In one embodiment, the stem cells are embryonic stem cells or induced pluripotent stem cells.

[0119] In one embodiment, the present invention relates to an implantable device comprising an in vitro population of stem cell-derived cells, wherein at least 15% of the beta-like cells express NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0120] In one embodiment, the present invention relates to an implantable device comprising an in vitro population of stem cell-derived cells, wherein at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, the markers being absent in native human beta cells.

[0121] In one embodiment, the present invention relates to an implantable device comprising an in vitro population of stem cell-derived cells, wherein at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and the expression level of the markers is, on average, at least 1 log-fold different from the expression level of the markers in native human beta cells.

[0122] In one embodiment, the present invention relates to a method for treating type 1 diabetes, the method comprising administering an in vitro population of stem cell-derived cells to a person in need of such treatment, wherein at least 15% of the beta-like cells express NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0123] In one embodiment, the present invention relates to a method for treating type 1 diabetes, the method comprising administering an in vitro population of stem cell-derived cells to a person in need of treatment, wherein at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, the markers not present in native human beta cells.

[0124] In one embodiment, the present invention relates to a method for treating type 1 diabetes, the method comprising administering an in vitro population of stem cell-derived cells to a person in need of such treatment, wherein at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and the expression level of the markers is, on average, at least 1 log-fold change compared to the expression of the markers in native human beta cells.

[0125] In one embodiment, the present invention relates to a cryopreserved population of stem cell-derived cells, wherein at least 15% of beta-like cells express NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1.

[0126] In one embodiment, the present invention relates to a cryopreserved population of stem cell-derived cells, wherein at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, the markers being absent in native human beta cells.

[0127] In one embodiment, the present invention relates to a cryopreserved population of stem cell-derived cells, wherein at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and the expression level of the markers is, on average, at least 1 log-fold different from the expression level of the markers in native human beta cells.

[0128] Definition: Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. The practice of the present invention will, unless otherwise indicated, utilize conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art.

[0129] It should be noted that all headings and subheadings used herein are for convenience only and should not be construed as limiting the invention.

[0130] The use of any and all examples or illustrative phrases presented herein (e.g., "such as") is solely intended to clarify the invention and, unless otherwise requested, does not limit the scope of the invention. Nothing in this specification should be construed as indicating that any unclaimed element is essential for the practice of the invention.

[0131] Throughout this application, the terms “method” and “protocol” can be used interchangeably to refer to the process of differentiating cells.

[0132] Furthermore, as used herein, “and / or” means and encompasses any and all possible combinations of one or more of the items described herein, as well as the absence of any combination when interpreted as an alternative ("or"). Moreover, the present invention also intends that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0133] As used herein, "a" means "one or more".

[0134] As used herein, the term “about” means plus or minus 10%, for example, plus or minus 5%, unless otherwise indicated.

[0135] As used herein, the term “cell population” refers to a defined group of cells, which may be in vitro or in vivo. In preferred embodiments, the cell population according to the present invention is an in vitro cell population.

[0136] stem cells Stem cells are undifferentiated cells defined by their ability at the single-cell level to both self-replicate and differentiate to produce progeny cells, including self-replicating progenitor cells, non-replicating progenitor cells, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm), and to generate tissues from multiple germ layers after transplantation, contributing to substantially most, if not all, of the tissue after injection into a blastocyst.

[0137] Stem cells are classified according to their developmental potential as follows: (1) totipotent (meaning they can give rise to all embryonic and extraembryonic cell types), (2) pluripotent (meaning they can give rise to all embryonic cell types), (3) multipotent (meaning they can give rise to subsets of cell lineages, all within a specific tissue, organ, or physiological system (e.g., hematopoietic stem cells (HSCs) can produce offspring including HSCs (self-replicating), blood cell-restricted pluripotent progenitor cells, and all cell types and elements that are normal components of blood (e.g., platelets))), (4) minimally pluripotent (meaning they can give rise to a more restricted subset of cell lineages than multipotent stem cells), and (5) unipotent (meaning they can give rise to a single cell lineage (e.g., spermatogonial stem cells)).

[0138] Human pluripotent stem cells As used herein, “human pluripotent stem cells” (hPSCs) refer to cells that may originate from any human source and, under appropriate conditions, are capable of producing offspring of different cell types that are derivatives of all three germ layers (endoderm, mesoderm, and ectoderm). hPSCs may have the ability to form teratomas in 8-12 week old SCID mice and / or to form identifiable cells of all three germ layers in tissue culture. The definition of human pluripotent stem cells includes human embryonic stem cells (hESCs) (see, e.g., Thomson et al. (1998), Heins et al. (2004)) and induced pluripotent stem cells (iPSCs) (see, e.g., Yu et al. (2007), Takahashi et al. (2007)). Various methods and other embodiments described herein may require or utilize hPSCs from various sources. For example, suitable hPSCs for use may be obtained from developing embryos. In addition, suitable hPSCs may be obtained from established cell lines and / or human induced pluripotent stem (hiPS) cells.

[0139] ES cell lines can also be derived from single blastomeres without destroying the extrauterine embryo and without affecting clinical outcomes (Chung et al. (2006) and Klimanskaya et al. (2006)).

[0140] Blastocyst-derived stem cells As used herein, the term “blastocyst-derived stem cells” is expressed as BS cells, and the human form is called “hBS cells.” In the literature, the cells are often referred to as embryonic stem cells, more specifically, human embryonic stem cells (hESCs). Therefore, the pluripotent stem cells used in the present invention may be embryonic stem cells prepared from blastocysts, as described in WO03 / 055992 and WO2007 / 042225, or commercially available hBS cells or cell lines. However, it is further conceivable that any human pluripotent stem cells may be used in the present invention, including differentiated adult cells that are reprogrammed into pluripotent cells by treating adult cells with certain transcription factors such as OCT4, SOX2, NANOG, and LIN28, as disclosed in Yu, et al. (2007), Takahashi et al. (2007), and Yu et al. (2009).

[0141] Human embryonic stem cells As used herein, the terms “human embryonic stem cell” or “hESC” refer to stem cells derived from the inner cell mass of a human embryo. Human embryonic stem cells can self-regenerate indefinitely in an undifferentiated state. Furthermore, human embryonic stem cells are pluripotent, meaning they can differentiate into derivatives of all three major germ layers: the ectoderm, endoderm, and mesoderm.

[0142] Human induced pluripotent stem cells As used herein, the terms “human induced pluripotent stem cells” or “hIPSCs” refer to stem cells produced by reprogramming somatic cells to return them to an embryoid pluripotent state. Induced pluripotent stem cells can self-regenerate indefinitely in an undifferentiated state. Induced pluripotent stem cells can also differentiate into all derivatives of the three major germ layers: ectoderm, endoderm, and mesoderm.

[0143] differentiation As used herein, “differentiation” refers to the process by which a cell progresses from an undifferentiated state to a differentiated state, or from a differentiated state to a more differentiated state. A cell may be in a differentiated state, but is not fully differentiated into a particular cell type.

[0144] The term "differentiation factor" refers to compounds added to pancreatic cells that enhance their differentiation into endocrine cells, which also include insulin-producing beta cells.

[0145] In one embodiment, cell differentiation includes culturing cells in a culture medium containing one or more differentiation factors.

[0146] Definitive endoderm cells (DE cells) Embryonic endoderm cells are characterized by the expression of the marker SOX17. Further markers for DE are FOXA2 and CXCR4.

[0147] "SOX17" (SRY-box 17), as used herein, is a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the control of embryonic development and the determination of cell fate.

[0148] "FOXA2" (Forkhead Box A2), as used herein, is a member of the Forkhead class of DNA-binding proteins.

[0149] "CXCR4" (CXC-motif chemokine receptor 4), as used herein, is a CXC chemokine receptor specific to stromal cell-derived factor 1.

[0150] Non-limiting examples of DE induction protocols include the conventional D'Amour protocol (Novocell, Nature Biotec 2006, 2008) and the protocol described in WO2012 / 175633 (which is incorporated herein in its entirety by reference).

[0151] Pancreatic endoderm cells (PE cells) Pancreatic endoderm cells are characterized by the expression of at least 5% NKX6.1+ / PDX1+ double-positive markers. Further markers for PE are PTF1A and CPA1.

[0152] As used herein, "PDX1" refers to a homeodomain transcription factor involved in pancreatic development.

[0153] "NKX6.1" is a member of the NKX transcription factor family, as used herein.

[0154] When used herein, "PTF1A" is a component of the pancreatic transcription factor 1 complex (PTF1), a protein known to play a role in mammalian pancreatic development.

[0155] "CPA1," as used herein, is a member of the carboxypeptidase A family of zinc metalloproteinases. This enzyme is produced in the pancreas.

[0156] The protocol described in WO2014 / 033322 is incorporated herein by reference in its entirety.

[0157] Endocrine progenitor cells (EP cells) Endocrine progenitor cells are characterized by the expression of markers NGN3, NeuroD, and NKX2.2, which are characteristic of EP cells whose endocrine cell fate is determined by the endocrine cell fate.

[0158] When used herein, "NGN3" is a member of the neurogenin family of basic loop-helix-loop transcription factors.

[0159] "NKX2.2" and "NKX6.1" are members of the NKX transcription factor family, as used herein.

[0160] As used herein, "NeuroD" refers to a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors.

[0161] The protocol described in WO2015 / 028614 is incorporated herein by reference in its entirety.

[0162] Natural human beta cells As used herein, the terms “native human beta cells” or “primary beta cells” refer to cells in the human body that produce insulin in response to glucose or secretagogues.

[0163] stem cell derived cells As used herein, the terms “stem cell-derived cells” or “pluripotent stem cell-derived cells” refer to cells obtained by the differentiation of pluripotent stem cells that have the potential for further differentiation or are terminally differentiated cells, but do not necessarily exhibit insulin secretion after transplantation.

[0164] Stem cell-derived beta cells As used herein, “beta-like cells,” “stem cell-derived beta cells,” “SC-β cells,” “stem cell-derived beta-like cells,” or “stem cell-derived beta cells in vitro” refers to cells derived from stem cells that express at least one marker of native human beta cells, such as PDX1, NKX6.1, or INS, and exhibit glucose-responsive in vitro and / or in vivo insulin secretion similar to native insulin secretion. The protocol described in WO2017 / 144695 is incorporated herein by reference in its entirety.

[0165] screening As used herein, the term “screening” means detecting, identifying, purifying, selecting, or characterizing cells or cell populations based on the presence of one or more cells having a particular phenotype of a genotype. Phenotypes and genotypes may be established based on the expression of markers. In one embodiment, identifying means classifying cells according to the expression levels of markers that characterize the cells. In preferred embodiments, screening according to the present invention is performed in vitro.

[0166] Expression level As used herein, the term “expression level” refers to the degree of gene expression and / or gene product activity in a cell. Expression levels may be determined in any absolute or normalized units (relative to a known expression level of a control reference).

[0167] marker As used herein, the term “marker” refers to a spontaneously occurring, identifiable expression produced by a cell that may correlate with certain characteristics of the cell and serve to identify, predict, or characterize a cell or cell population. Markers may sometimes be called genes. Markers may also be in the form of mRNA or proteins, for example, proteins on the cell surface.

[0168] As used herein, the term “expression” in reference to a marker refers to the absence or presence of a detectable molecule in a cell. In some embodiments, the molecule being expressed is mRNA or a protein. The expression of a marker can be detected at any preferred level, such as mRNA or protein levels. Those skilled in the art will readily understand that cells can be defined by the positive or negative expression of a marker, that is, the characteristics and state of cells may be equally correlated based on the expression and absence of a particular marker. When referring to a specific marker, the presence or absence of expression may be indicated by a + (plus) or a - (minus), respectively.

[0169] As used herein, "ACVR1C" refers to activin A receptor type 1C.

[0170] As used herein, "AMBP" is an alpha-1-microglobulin / bikunin precursor.

[0171] As used herein, "BASP1" refers to brain-abundant membrane-bound signaling protein 1.

[0172] As used herein, "CHRNA3" refers to the nicotinic alpha-3 subunit of a cholinergic receptor.

[0173] As used herein, "DCC" refers to the gene encoding the Netrin 1 receptor.

[0174] As used herein, "DLK1" is a delta-like non-standard notch ligand.

[0175] As used herein, "EGFL7" refers to EGF-like domain multiple 7.

[0176] As used herein, "ELAVL2" refers to embryonic lethal, abnormally visual, fruit fly-like 2.

[0177] As used herein, "FREM2" refers to FRAS1-associated extracellular matrix 2.

[0178] As used herein, "G6PC2" refers to glucose-6-phosphatase catalytic subunit 2.

[0179] As used herein, "HMGCS1" refers to 3-hydroxy-3-methylglutaryl-CoA synthase 1.

[0180] As used herein, "ISL1" refers to ISL LIM Homeobox 1.

[0181] As used herein, "LBH" refers to LBH regulators of the WNT signaling pathway.

[0182] As used herein, "MAFB" refers to MAF bZIP transcription factor B.

[0183] As used herein, "MARCKSL1" means MARCKS-like 1.

[0184] As used herein, "NEFL" refers to neurofilament light.

[0185] As used herein, "NEFM" refers to neurofilament medium.

[0186] As used herein, "PCDH7" refers to protocadherin 7.

[0187] As used herein, "PCP4" refers to Purkinje cell protein 4.

[0188] As used herein, "PLAGL1" refers to PLAG1-like zinc finger 1.

[0189] As used herein, "PLXNA2" refers to Plexin A2.

[0190] As used herein, "RAD21" refers to the RAD21 cohesin complex component.

[0191] As used herein, "RTN1" refers to Reticulone 1.

[0192] As used herein, "SLC30A8" is a member 8 of the solute carrier family 30.

[0193] As used herein, "SOX11" refers to SRY-box transcription factor 11.

[0194] As used herein, "STARD10" is a StAR-related lipid transport domain-containing 10.

[0195] As used herein, "ST6GALNAC5" refers to ST6 N-acetylgalactosaminid alpha-2,6-sialyltransferase 5.

[0196] Insulin expression or production Insulin expression or production is the ability of a cell to synthesize insulin protein and / or insulin RNA. In preferred embodiments, the beta-like cells of the present invention maintain insulin expression after transplantation.

[0197] Insulin secretion Insulin secretion is the ability of cells to release insulin. In preferred embodiments, the beta-like cells of the present invention maintain insulin secretion after transplantation.

[0198] Culture medium / composition A solid, liquid, or semi-solid medium designed to support cell proliferation. Various types of commercial culture media are used to grow various types of cells. In a preferred embodiment, the cell culture or composition according to the present invention is an in vitro cell culture or composition.

[0199] Unless otherwise indicated herein, terms presented in the singular form also include plural situations.

[0200] The present invention can be further described by the following non-limiting embodiments. 1. A method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, comprising the step of identifying beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1. 2. The method according to Embodiment 1, wherein beta-like cells are screened by RNA-seq. 3. The method according to Embodiment 1 or 2, wherein beta-like cells are screened using qPCR, nested PCR, ddPCR, or a combination thereof. 4. An in vitro population of pluripotent stem cell-derived cells, wherein the population includes at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1. 5. An in vitro population of pluripotent stem cell-derived cells according to Embodiment 4, wherein the population comprises at least 15% beta-like cells expressing NKX6.1 in combination with one or more markers selected from ACVR1C, FREM2, CHRNA3, DCC, SOX11, MARCKSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, MAFB, and ISL1, and the beta-like cells maintain insulin expression and secretion after transplantation. 6. An in vitro population of pluripotent stem cell-derived cells according to Embodiment 4 or 5, wherein at least 55% of the beta-like cells contain one or more markers selected from SOX11, FREM2, DCC, BASP1, CHRNA3, and ELALV2, and the markers are not present in native human beta cells. 7. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 4 to 6, wherein at least 85% of the beta-like cells contain SOX11, and the marker is not present in native human beta cells. 8. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 4 to 6, wherein at least 75% of the beta-like cells contain FREM2, and the marker is not present in native human beta cells. 9. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 4 to 6, wherein at least 85% of the beta-like cells contain DCCs, and the marker is not present in native human beta cells. 10. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 4 to 6, wherein at least 75% of the beta-like cells contain BASP1, and the marker is not present in native human beta cells. 11. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 4 to 6, wherein at least 80% of the beta-like cells contain CHRNA3, and the marker is not present in native human beta cells. 12. An in vitro population of pluripotent stem cell-derived cells according to Embodiments 4-6, wherein at least 55% of the beta-like cells contain ELALV2, and the marker is not present in native human beta cells. 13. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 4 to 9, wherein at least 75% of the beta-like cells contain SOX11, FREM2, and DCC, and the markers are not present in native human beta cells. 14. An in vitro population of pluripotent stem cell-derived cells according to any one of Embodiments 10 to 12, wherein at least 55% of the beta-like cells contain BASP1, CHRNA3, and ELAVL2. 15. In vitro population pluripotent stem cell-derived cells according to Embodiment 4 or 5, wherein at least 80% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, AMBP, ST6GALNAC5, and HMGCS1, and the expression level of said markers is, on average, at least about 1 log-fold different from the expression level of said markers in native human beta cells. 16. In vitro population pluripotent stem cell-derived cells according to Embodiment 15, wherein at least 90% of the beta-like cells contain one or more markers selected from ACVR1C, MARCKSL1, STARD10, and AMBP, and the expression level of the markers is, on average, at least about 1 log-fold different from the expression level of the markers in native human beta cells. 17. In vitro population pluripotent stem cell-derived cells according to Embodiment 15 or 16, wherein at least 95% of the beta-like cells contain ACVR1C, and the expression level of the marker is, on average, at least 2 log-fold higher than the expression level of the marker in native human beta cells. 18. In vitro population pluripotent stem cell-derived cells according to Embodiment 15 or 16, wherein at least 95% of the beta-like cells contain MARCKSL1, and the expression level of the marker is, on average, at least about 2 log-fold higher than the expression level of the marker in native human beta cells. 19. In vitro population pluripotent stem cell-derived cells according to Embodiment 15 or 16, wherein at least 95% of the beta-like cells contain STARD10, and the expression level of the marker is, on average, at least 1 log-fold higher than the expression level of the marker in native human beta. 20. In vitro population pluripotent stem cell-derived cells according to Embodiment 15 or 16, wherein at least 90% of the beta-like cells contain AMBP, and the expression level of the marker is, on average, at least 2 log-fold higher than the expression of the marker in native human beta. 21. In vitro population pluripotent stem cell-derived cells according to Embodiment 15, wherein at least 85% of the beta-like cells contain ST6GALNAC5, and the expression level of the marker is, on average, at least about 1 log-fold higher than the expression level of the marker in native human beta. 22. In vitro population pluripotent stem cell-derived cells according to any one of Embodiment 15, wherein at least 80% of the beta-like cells contain HMGCS1, and the expression level of the marker is, on average, at least about 1 log-fold higher than the expression level of the marker in native human beta. 23. An in vitro population of pluripotent stem cell-derived cells according to any one of prior embodiments 4 to 22, wherein the cells further express G6PC2. 24. An in vitro population according to any one of Embodiments 1 to 23, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. 25. An in vitro population of pluripotent stem cell-derived cells according to any one of the prior embodiments 4 to 24, wherein the population, including beta-like cells, maintains insulin expression and secretion after transplantation. 26. An in vitro population of pluripotent stem cell-derived cells according to any one of prior embodiments 4 to 25, wherein the population containing beta-like cells exhibits a glucose-stimulated insulin secretion (GSIS) response after transplantation. 27. An in vitro population of pluripotent stem cell-derived cells according to any one of the prior embodiments 4 to 26, wherein one or more markers show a positive correlation with mean plasma c-peptide levels after transplantation. 28. An in vitro population of pluripotent stem cell-derived cells according to any one of the prior embodiments 4 to 27, wherein the population containing beta-like cells exhibits a decrease in blood glucose after transplantation. 29. An in vitro population of pluripotent stem cell-derived cells as described in any one of the prior embodiments 4 to 28, for use as a pharmaceutical. 30. An in vitro population of pluripotent stem cell-derived cells according to any one of the prior embodiments 4 to 28, for use in the treatment of diabetes. 31. An in vitro population of pluripotent stem cell-derived cells as described in any one of the prior embodiments 4 to 28, for use in the treatment of type 1 diabetes. 32. A cell culture or composition comprising pluripotent stem cell-derived cells as described in any one of the prior embodiments 4 to 28, and a cell culture medium. 33. An implantable device comprising an in vitro population of pluripotent stem cell-derived cells as described in any one of the prior embodiments 4 to 28. 34. A method for treating type 1 diabetes, comprising administering an in vitro population of pluripotent stem cell-derived cells described in any one of the prior embodiments 4 to 28 to a person in need of treatment therefor. 35. A cryopreserved cell culture comprising an in vitro population of pluripotent stem cell-derived cells as described in any one of the prior embodiments 4 to 28. 36. A cryopreserved population of pluripotent stem cell-derived cells according to Embodiment 35, wherein at least 50%, 60%, 70%, 80%, or 90% of the cell population are viable.

[0201] List of Abbreviations +ve:positive AGN: AGN 193109 ALK5: Activin receptor-like kinase, BC: Beta cell DE: Embryonic endoderm EP: Endocrine progenitor cell GCG: Glucagon hBS: Human blastocyst-derived stem hBSC: Human blastocyst-derived stem cells hES: Human embryonic stem hESC: Human Embryonic Stem Cells hiPSC: Human induced pluripotent stem cells hPSC: Human pluripotent stem cells LDN:LDN193189 PAX4: Paired Box 4 PE: Pancreatic endoderm RT: room temperature SCID: Severe combined immunodeficiency SYT13: Synaptotagmin 13 T3: Triiodothyronine

[0202] Example 1 A subpopulation of stem cell-derived beta-like cells contributes to insulin secretion after transplantation. Stem cell-derived cells are heterogeneous based on gene expression, molecular properties, glucose sensing, and insulin secretion. The objective of this experiment was to identify subpopulations of stem cell-derived or beta-like cells that contribute to insulin production after transplantation. To enhance protocol-related cellular heterogeneity, human embryonic stem cells (hESCs) were differentiated into endocrine cell clusters using eight different modifications of the stepwise differentiation protocol provided in Table 1 below. The hESCs were directed towards the embryonic endoderm (DE), then the pancreatic endoderm (PE), and finally endocrine progenitor cells (EP) to generate insulin-producing beta-like cells as described in Japanese Patent Publications WO / 2012 / 175633, WO2014 / 033322, WO2015 / 028614, and WO2017 / 144695 (all incorporated herein by reference). [Table 1] To identify subpopulations of stem cell-derived or beta-like cells that contribute to insulin expression and secretion after transplantation, cell clusters obtained from each of eight (modified) differentiation protocols were analyzed using pre-transplant single-cell RNA sequencing. Cells were dissociated into single cells using actase (Stem Cell Technologies), and gene expression profiles for single cells were obtained for 2000–3000 cells per protocol using RNA sequencing. Human pancreatic islets were also included as a reference to native human beta cells. Gene expression data for all cells were integrated across all samples to make the data comparable between conditions, and dimensionality reduction and clustering were applied so that cells with similar gene expression profiles were grouped independently of the protocol used to generate the cells. A total of 26 distinct cell populations were identified (Figure 1b), revealing that all protocols generated similar cell populations at very different rates (Figure 1a). To identify populations that closely resembled native human beta cells (cluster 16), each of the 26 identified clusters was compared to the native beta cell gene set. Clusters 14, 15, and 26 received scores similar to the human beta cell cluster (cluster 16) and were therefore identified as the beta-like cell populations that most closely resembled native human beta cells (Figure 2).

[0203] To determine post-transplant insulin expression in animals, numerous differentiated cell clusters (corresponding to 3000 pIEQ) obtained using each of eight (modified) differentiation protocols were transplanted subcapsulate into the kidneys of immunodeficient SCID-beige mice in groups of 10 animals per protocol. Human C-peptide levels were measured in serum samples 8 weeks after transplantation using human C-peptide ELISA (enzyme-linked immunosorbent assay) to assess insulin secretion from engrafted human cells. By plotting the mean circulating human C-peptide levels in transplanted animals against the percentage of cells (out of total cell number) in clusters 14, 15, and 26, a positive correlation was observed between the percentage of cells in these clusters and in vivo insulin secretion (Figure 6).

[0204] In conclusion, it was found that only a subpopulation of stem cell-derived cells, i.e., beta-like cells, resembled native human beta cells and contributed to insulin secretion after transplantation. A list of genes describing and identifying this subpopulation or beta-like cell (clusters 14, 15, and 26) is provided in Tables 1, 2, and 3; please refer to Examples 2–4 provided below. Identifying and characterizing this population is essential for optimizing the differentiation protocol from stem cells to beta cells and ultimately obtaining highly effective beta cell therapy, i.e., the ability of cells to normalize blood glucose.

[0205] Example 2: Conventional beta cell markers do not predict insulin secretion after transplantation. To demonstrate that conventional beta-cell markers do not predict in vivo efficacy, we measured the expression of well-known beta-cell markers such as NKX6.1, PDX1, PAX4, NKX2.2, and SYT13, as well as the co-expression of PDX1+ / NKX6.1+, in cell populations obtained from eight modified protocols, using either nanostrings for mRNA detection or flow cytometry for protein detection. None of these markers were found to have a positive correlation with mean c-peptide levels in animals 8 weeks after transplantation (Figures 7 and 8b, bottom panel).

[0206] Single-cell sequence analysis identified several markers previously associated with beta cells, including SLC30A8. These markers are listed in Table 2 below. [Table 2]

[0207] To demonstrate how SLC30A8 is distinguished from another well-known beta-cell marker, PDX1, in stem cell-derived cells, their expression was shown across all identified clusters (Figure 8a). High expression of SLC30A8 was found in the most beta-like cells (clusters 14, 15, and 26), as well as in native beta cells, and some expression was also found in clusters 24 and 25, which also express GCG. In contrast, PDX1 is expressed in beta cells and the most beta-like cells (clusters 14, 15, and 26), but also in almost all other clusters. To show how SLC30A8 and PDX1 correlate with post-transplant insulin secretion, levels of SLC30A8 and PDX1 mRNA transcripts were measured on nanostrings in eight modified protocols and correlated to mean in vivo C-peptide levels 8 weeks after transplantation. SLC30A8 was positively correlated with circulating human C-peptide levels after transplantation (Figure 8b, upper panel), while PDX1 was negatively correlated (Figure 8b, lower panel). The expression of PDX1 in almost all cell clusters explains why this marker is negatively correlated with in vivo insulin secretion. PDX1 is expressed in native beta cells and the most beta-like cells from stem cell cultures (cells in clusters 14, 15, and 26), but its expression in other clusters indicates that this marker cannot identify cells that mature into fully functional beta cells after transplantation. Since certain classical beta cell markers have been shown not to predict insulin secretion after transplantation, we consider the application of the markers shown in Table 2 above to be novel.

[0208] To further evaluate the cell composition before and after transplantation, immunohistochemistry (IHC) was performed on kidney sections containing pre-transplant clusters and post-transplant engrafted human cells. Conventional markers were used to identify alpha and beta cells, and staining was performed for C-peptide, NKX6.1, and glucagon. From all eight protocols, a high percentage of cells co-expressed C-pep / NKX6.1 before transplantation (Figure 5, left panel), but only a subset of cells maintained this co-expression of C-pep / NKX6.1 after transplantation (Figure 5, right panel).

[0209] In conclusion, most conventional beta cell markers were shown not to predict post-transplant insulin secretion from in vitro stem cell-derived endocrine cell cultures. The use of these conventional markers for cell screening or cell selection may lead to optimization toward undesirable cell subpopulations that may resemble pre-transplant beta cells but whose phenotypes change after exposure to the in vivo environment.

[0210] Example 3 Stem cell-derived beta-like cells are similar to, but not identical to, natural beta cells. To identify genes expressed in stem cell-derived beta-like cells rather than in native human beta cells, gene expression in these stem cell-derived beta-like cells was measured by single-cell RNA sequencing and compared to gene expression in native human beta cells.

[0211] To obtain gene expression from native human beta cells, single-cell gene expression data were pooled from two human islet samples derived from two different human donors. To identify these cells within the islets as native human beta cells, each cell from these samples was scored against the gene sets of native islet subtypes: alpha, beta, delta, epsilon, gamma, duct, acinus, endothelium, stellate, ductal, macrophage, mast, and Schwann cells (Figure 3). Cells with the highest scores for beta cells were clustered together for comparison with clusters 14, 15, and 26. Comparing the two groups led to the identification of gene sets specific to stem cell-derived beta-like cells (Table 3). [Table 3]

[0212] In conclusion, we identified genes that distinguish stem cell-derived beta-like cells from human native beta cells and demonstrated that these cells are not identical to their native counterparts.

[0213] Example 4 ACVR1C is a novel pancreatic endocrine cell marker in stem cell-derived beta cells, and its expression correlates with insulin secretion after maturation in vivo. ACVR1C is on the list of novel genes expressed in clusters 14, 15, and 26 (Table 4), and these clusters were identified as the most similar to native human beta cells among all the clusters. [Table 4]

[0214] By using the method described in Example 3, ACVR1C was identified as specific to stem cell-derived beta-like cells because it was expressed in over 99% of cells in clusters 14, 15, and 26, and not in 80% of native human beta cells. The remaining 20% ​​of cells showed low ACVR1C expression (Figure 9a). ACVR1C was highly expressed in clusters 14, 15, and 16, but also showed some expression in other clusters located in other pancreatic endocrine subtypes, such as pancreatic alpha cells and pancreatic somatostatin cells (Figure 9b). To demonstrate that ACVR1C expression was positively correlated with in vivo insulin secretion, levels of ACVR1C mRNA transcript were measured on nanostrings and correlated to the mean in vivo c-peptide levels 8 weeks after transplantation (Figure 9c).

[0215] In conclusion, we identified ACVR1C as a novel and specific marker for in vivo C-peptide levels in stem cell-derived beta-like cells 8 weeks after transplantation. C-peptide is equivalent to insulin. Human C-peptide is measured because it may originate solely from transplanted cells and not from potential externally administered treatments using insulin. Therefore, ACVR1C can be used to qualify and optimize protocols for stem cell-derived beta-like cells.

[0216] Example 5 PCDH7 is an extracellular marker that is most specific to the beta-like cell population, while PCP4 is an intracellular marker. Both PCDH7 and PCP4 were expressed in cell clusters 14, 15, and 26 (Table 4), and these clusters in our single-cell RNA sequencing dataset were identified as the most similar to native human beta cells among all clusters. Compared to ACVR1C, PCDH7 and PCP4 showed expression specific to clusters 14, 15, and 26. In any of the other identified clusters, PCDH7 and PCP4 expression was absent or minimal (Figure 10a). The specificity of these markers makes them suitable for predicting insulin secretion after transplantation and elutes them to their ability to be used to identify and quantify stem cell-derived beta-like cells for use as quality markers. With respect to ACVR1C, levels of PCDH7 and PCP4 mRNA transcripts were measured on nanostrings and correlated this to mean circulating human c-peptide levels in vivo in mice 8 weeks after transplantation (Figure 10b). Both markers were found to positively correlate with insulin secretion in vivo at 8 weeks.

[0217] Since PCDH7 is identified as an extracellular marker expressed almost exclusively in stem cell-derived beta-like cells, it can be further used to purify stem cell-derived beta cells from heterogeneous cell populations. This can then be used for further characterization or to improve the efficacy and safety of the final cell therapy product. In conclusion, PCDH7 and PCP4 were identified as specific to stem cell-derived beta cells.

[0218] Example 6 G6PC2 is a marker correlated with in vivo insulin production and in vitro insulin release. To identify markers that could be used for further efficacy assessment of stem cell-derived beta-like cells, the ability of cells to secrete insulin in vitro was measured for all eight protocol modifications. Insulin secretion was measured using a perfusion system, with cells initially inoculated with 10 mM glucose, followed by a second inoculation with 10 mM glucose + 1000 nM exendin 4, and then a third inoculation with IBMX and forskolin. The total amount of insulin secreted during the inoculation was defined as the area under the perfusion curve and plotted against markers positively correlated with in vivo insulin production. G6PC2 was found to be expressed in native human beta and alpha cells (clusters 16 and 22, respectively, in single-cell RNA sequencing datasets), as well as in subsets of cells within the most beta-like cell clusters 14, 15, and 26 (Figure 11a). To demonstrate that G6PC2 expression positively correlated with in vivo insulin production, levels of G6PC2 mRNA transcript were measured on nanostrings and correlated to the mean in vivo c-peptide levels 8 weeks after transplantation (Figure 11b). Finally, to validate its use as a potential potency marker of insulin secretion, G6PC2 expression was compared to the area under the curve from in vitro insulin secretion studies (Figure 11c).

[0219] In conclusion, GCPC2 was identified as a marker expressed in stem cell-derived beta-like cells that correlates with in vitro insulin secretion, and is therefore useful for measuring in vitro efficacy or in vitro differentiation of stem cells into beta cells.

[0220] While certain features of the present invention are illustrated and described herein, many modifications, substitutions, alterations, and equivalents will be conceivable to those skilled in the art. It should therefore be understood that the appended claims are intended to encompass all modifications and alterations that fall within the true spirit of the present invention.

Claims

1. A method for screening beta-like cells in an in vitro population of pluripotent stem cell-derived cells, comprising the step of identifying beta-like cells expressing NKX6.1 and ACVR1C.

2. The method according to claim 1, wherein the beta-like cells are screened by RNAseq.

3. The method according to claim 1 or 2, further comprising the step of identifying beta-like cells that express G6PC2.

4. The method according to any one of claims 1 to 3, further comprising the step of identifying one or more markers selected from FREM2, CHRNA3, DCC, SOX11, MARCSL1, BASP1, STARD10, AMBP, ST6GALNAC5, HMGCS1, ELAVL2, PCP4, PCDH7, NEFL, PLAGL1, EGFL7, RAD21, RTN1, PLXNA2, LBH, NEFM, SLC30A8, DLK1, and MAFB.

5. The method according to any one of claims 1 to 4, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

6. The method according to any one of claims 1 to 5, wherein the beta-like cells maintain insulin expression and secretion after transplantation.

7. The method according to any one of claims 1 to 6, wherein the beta-like cells exhibit a glucose-stimulated insulin secretion (GSIS) response after transplantation.

8. The method according to any one of claims 1 to 7, wherein the beta-like cells exhibit a decrease in blood glucose after transplantation.

9. The method according to any one of claims 1 to 8, wherein ACVR1C shows a positive correlation with the mean plasma c-peptide level after transplantation.

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