US 201403297.04A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0329704 A1 Melton et al. (43) Pub. Date: Nov. 6, 2014

(54) MARKERS FORMATURE BETA-CIELLS AND Publication Classification METHODS OF USING THE SAME (51) Int. C. (71) Applicant: President and Fellows of Harvard College, Cambridge, MA (US) CI2O I/68 (2006.01) (52) U.S. C. (72) Inventors: Douglas A. Melton, Lexington, MA CPC ...... CI2O I/6881 (2013.01); C12O 2600/158 (US); Sinisa Hrvatin, Cambridge, MA (2013.01); C12O 2600/136 (2013.01) (US) USPC ...... 506/9: 435/375; 435/6. 12 (21) Appl. No.: 14/228,959 (22) Filed: Mar 28, 2014 (57) ABSTRACT Related U.S. Application Data (60) Provisional application No. 61/806,371, filed on Mar. Markers for mature B-cells and methods of using these mark 28, 2013. ers are disclosed. Patent Application Publication Nov. 6, 2014 Sheet 1 of 43 US 2014/0329704 A1

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MARKERS FORMATURE BETA-CIELLS AND transcriptome of pancreatic cells exhibits a pattern of expres METHODS OF USING THE SAME sion similar to the reference non-f-cell transcriptome. 0007. In some aspects, the present invention provides a RELATED APPLICATIONS method of identifying the functional maturity of B-cells, the 0001. This application claims the benefit of U.S. Provi method comprising: (a) obtaining a putative B-cellor a popu sional Application No. 61/806,371, filed Mar. 28, 2013. The lation of putative B-cells; and (b) detecting an expression entire teachings of the above application are incorporated level in the putative f3-cellor the population of putative B-cells herein by reference. of one or more genes listed in Table 2, Table 3 or Table 4, wherein: (i) an elevated level of expression of one or more GOVERNMENT SUPPORT genes listed in Table 1 or Table 2 in the putative B-cell or the population of putative B-cells indicates that the putative B-cell 0002 This invention was made with government support or the population of putative B-cells are functionally mature under 2 U01 DK072473-07 awarded by the National Insti B-cells; and (ii) an elevated level of expression of one or more tutes of Health. The government has certain rights in the genes listed in Table 3 or Table 4 in the putative B-cell or the invention. population of putative B-cells indicates that the putative B-cell or the population of putative B-cells are functionally imma BACKGROUND OF THE INVENTION ture f3-cells. 0003. The directed differentiation of stem cells has the 0008. In some aspects, the present invention provides a potential to produce B-cells for administration to individuals method of identifying mature B-cells, the method compris Suffering from diseases associated with n-cell abnormality ing: (a) obtaining a putative B-cell or a population of putative (e.g., diabetes). However, existing in vitro differentiation pro B-cells; and (b) detecting an expression level in the putative tocols often produce “B-like cells, which do not have the B-cell or the population of putative B-cells of one or more same functional properties as mature f3-cells. In addition, the genes listed in Table 1 or Table 3, wherein: (i) an elevated complete set of signals and mechanisms governing B-cell level of expression of one or more genes listed in Table 1 or maturation remains unknown. Table 2 in the putative B-cell or the population of putative B-cells indicates that the putative f3-cell or the population of SUMMARY OF THE INVENTION putative B-cells are mature B-cells. 0004. The present invention provides solutions to one or 0009. In some aspects, the present invention provides a more of the problems outlined above. In particular, the method of identifying fetal B-cells, comprising: (a) obtaining present invention provides markers for identifying mature a putative B-cell or a population of putative B-cells; and B-cells and methods of using the markers for identifying (b) detecting an expression level in the putative B-cell or the mature B-cells, methods of identifying agents that modulate population of putative B cells of one or more genes listed in maturity of B-cells (e.g., agents that induce functional B-cell Table 3, wherein: (i) an elevated level of expression of one or maturation in vitro, or agents that induce B-cell maturation in more genes listed in Table 3 in the putative f3-cell or the vivo), methods of modulating disorders associated with population of putative B-cells indicates that the putative B-cell B-cell deficiency, and related compositions and methods. or the population of putative B-cells are fetal B-cells. 0005. In some aspects, the invention provides a method of 0010. In some aspects, the present invention provides a detecting B-cells, the method comprising: (a) obtaining a method of identifying in vitro-differentiated insulin-positive putative B-cell or a population of putative B-cells to be B-like cells, comprising: (a) obtaining a putative B-cell or a assessed; (b) measuring expression of a plurality of genes in population of putative B-cells; and (b) detecting an expression the putative B-cell or the population of putative f3-cells to level in the putative f3-cellor the population of putative B-cells produce a transcriptome of the putative f3-cell or the popula of one or more genes listed in Table 4, wherein: (i) an elevated tion of putative B-cells; (c) comparing the transcriptome of level of expression of one or more genes listed in Table 4 in the putative B-cell or the population of putative f3-cells to a the putative B-cell or the population of putative B-cells indi reference mature B-cell transcriptome exhibiting a pattern of cates that the putative f3-cell or the population of putative expression depicted in FIG. 12; and (d) detecting fB-cells, B-cells are in vitro-differentiated insulin-positive B-like cells. wherein the putative B-cell or a population of putative B-cells 0011. In some aspects, the present invention provides a are mature B-cells if the transcriptome of the putative B-cellor method of distinguishing mature and immature B-cells, com the population of putative B-cells exhibits a pattern of expres prising: (a) obtaining a putative B-cell or a population of sion similar to the reference mature B-cell transcriptome. putative B-cells; (b) measuring expression of a group of genes 0006. In some aspects, the invention provides a method of in the putative B-cell or the population of putative B-cells to distinguishing B-cells and non-f-cells in a sample comprising produce an expression profile of the B-cell or the population pancreatic cells, the method comprising: (a) obtaining a off-cells; (c) comparing the expression profile of the putative sample comprising pancreatic cells; (b) measuring expres B-cell or the population of putative B-cells to any or all of: (i) sion of a plurality of genes in the sample to produce a tran a reference mature f3-cell expression profile selected from the Scriptome of the pancrentic cells; (c) comparing the transcrip group consisting of a first group of genes having higher tome of the pancreatic cells to a reference B-cell expression levels in mature B-cells compared to fetal B-cells, transcriptome exhibiting a pattern of expression depicted in wherein the first group of genes is selected from the group FIG. 12 and/or a reference non-f-cell transcriptome exhibit consisting of STAT4, NPAS2, STAT3, NPAS2, STAT3, ing a pattern of expression depicted in FIG. 13; and (d) PBX3, NR3C2, DDIT3, SIX4, ETV5, SIX2, TP53, BCL6, distinguishing B-cells and non B-cells, wherein (i) the pan MESP1, HOPX, BHLHB3, EPAS1, KLF9, KCNK3, GPI, creatic cells are B-cells if the transcriptome of pancreatic cells CHUB, ALDOA, MAFA, SYT7, IAPP, WNT4, PDK3, exhibits a pattern of expression similar to the reference f-cell KCNK1, SLC2A2, ESR1, G6PC2, and a second group of transcriptome, or (ii) the pancreatic cells are non-B-cells if the genes having higher expression levels in mature B-cells com US 2014/03297.04 A1 Nov. 6, 2014 pared to insulin-positive B-like cells, wherein the second putative B-cells; (b) assessing enrichment of a biological pro group of genes is selected from the group consisting of XBP1, cess to produce a biological process enrichment list of the NFIA, PURA, PDX1, NR3C2, MNX1, GLIS3, EPAS1, putative B-cell or the population of putative B-cells, wherein HSF4, TSHZ3, MAFA, NKX6-1, HOPX, RORC, NFIX, the biological process is selected from the group consisting PEG3, CEBPD, KLF9; STX1A, KCNMA1, PDX1, CHGB, of: (i) generation of precursor metabolites and energy, (ii) MNX1, PCSK2, NKX6.1, GLIS3, KCNK12, KCNK3, oxidation reduction, (iii) Vesicle-mediated transport, (iv) GCGR, KCNK1, SLC30A8, PCSK1, MAFA, ESR1, electron transport chain, (v) monosaccharide metabolic pro SLC2A2, IAPP, G6PC2, STXBP1, KCNH2, KCNMB2, cess, (vi) cell morphogenesis, (vii) cellular component mor UCN3, WNT4; (ii) a reference immature fetal B-cell expres phogenesis, (viii) cell projection organization, (ix) Wnt sion profile selected from the group consisting of a third receptor signaling pathway, (X) cell projection morphogen group of genes having higher expression levels in fetal B-cells esis, (xi) cytoskeleton organization, (xii) Sterol biosynthetic compared to mature B-cells, wherein said third, group of process, (xiii) cholesterol biosynthetic process, (xiv) actin genes is selected from the group consisting of LZTS1, EVI1, filament-based process, (XV) actin cytoskeleton organization, MYCN, FOS, EGR1, RCOR2, TCF3, ASCL2, NOTCH1, (Xvi) Sterol metabolic process, and (Xvii) neuron projection LMO4, PAX4, NFIB, ISX, SOX11, LHX4, ZNF423, SOX8, development; (c) distinguishing mature and immature RFX1, PROX1, HHEX, CSRNP3, LZTR1, SOX4, NKX6.2, B-cells, wherein the putative B-cell or the population of puta COL1A1, PAX4, KCNH6, RIMS3, PROX1, SOX4, ACSS1, tive f3-cells are: (i) mature B-cells if the biological process GHRL, NOTCH1, KCNN3, GCK, PYY, HCN3, KCNJ4, and enrichment list of the putative f3-cell or the population of a fourth group of genes having higher expression levels in putative B-cells indicates that at least one of the biological insulin-positive B-like cells compared to mature B-cells, processes selected from the group consisting of (i), (ii), (iii), wherein said fourth group of genes is selected from the group (iv), and (v) is enriched in the f-cell or the population of consisting of FOXA1, HHEX, NR2F1, FEV, IRX2, SOX11, B-cells; or (ii) immature B-cells if the biological process PAX4, ONECUT2, LMO4, AEBP1, HES6, TGIF2, LZTS1, enrichment list of the putative f3-cell or the population of TCF3, GATA4, ARX, EGR1, RCOR2, CEBPA, ELF4, putative B-cells indicates that at least one of the biological HNF4G, PBX2, ISX, ZNF217, NTS, GAST, RIMS3, processes selected from the group consisting of (vi), (vii), CACNA1E, PYY. SCT, FOXA1, GATA4, KCNH6, ARX, (viii), (ix), (X), (xi), (xii), (xiii), (xiv), (XV), (Xvi), and (Xvii) is DLL3, NOTCH1, IRX2, DPP4, PAX4, ACOX2, KCNB1, enriched in the putative B-cell or the population of putative PROX1, GHRL, SLC2A1, ONECUT2, and SLC2A3; and (d) B-cells. distinguishing mature and immature B-cells, wherein the putative f3-cell or the population of putative f3-cells are: (i) 0014. In some aspects, the present invention provides a mature B-cells if the expression profile of the putative B-cell method of identifying a candidate agent that modulates the or the population of putative B-cells exhibit a pattern of functional maturity of B-cells, comprising: (a) contacting a expression similar to either reference mature B-cell expres B-cell or a population of B-cells with a test agent; (b) moni sion profile; or (ii) immature f3-cells if the expression profile toring expression of a group of genes in the B-cell or the of the putative B-cell or the population of putative B-cells population of B-cells, in the presence of the test agent, to exhibit a pattern of expression similar to either reference produce an expression profile of the B-cell or the population immature B-cell expression profile expression profile. of B-cells; (c) comparing the expression profile of the B-cell or the population of B-cells to: (i) a reference mature f3-cell 0012. In some aspects, the present invention provides a expression profile selected from the group consisting of a first method of distinguishing mature and immature B-cells, com group of genes having higher expression levels in mature prising: (a) obtaining a putative B-cell or a population of B-cells compared to fetal B-cells, wherein the first group of putative B-cells; (b) assessing enrichment of a signaling path genes is selected from the group consisting of KCNK3, GPI, way to produce a signaling pathway enrichment plot of the CHGB, ALDOA, MAFA, SYT7, IAPP, WNT4, PDK3, putative B-cell or the population of putative B-cells, wherein KCNK1, SLC2A2, ESR1, G6PC2, and a second group of the signaling pathway is selected from the group consisting of genes having higher expression levels in mature B-cells com an unfolded protein response signaling pathway, an insulin pared to insulin-positive B-like cells, wherein the second synthesis and secretion signaling pathway, and a metal ion group of genes is selected from the group consisting of SLC transporters signaling pathway; and (c) distinguishing STX1A, KCNMA1, PDX1, CHGB, MNX1, PCSK2, NKX6. mature and immature B-cells, wherein the putative 3-cell or 1, GLIS3, KCNK12, KCNK3, GCGR, KCNK1, SLC30A8, the population of putative B-cells are: (i) mature f3-cells if the PCSK1, MAFA, ESR1, SLC2A2, IAPP, G6PC2, STXBP1, signaling pathway enrichment plot of the B-cell or the popu KCNH2, KCNMB2, UCN3, and WNT4; (ii) a reference lation of B-cells indicates that at least one of the unfolded immature B-cell expression profile selected from the group protein response signaling pathway, the insulin synthesis and consisting of a third group of genes having higher expression secretion signaling pathway, and the metalion SLC transport levels in fetal B-cells compared to mature f3-cells, wherein ers signaling pathway are enriched in the putative B-cell or the said third group of genes is selected from the group consisting population of putative B-cells; or (ii) immature f3-cells if the of NKX6.2, COL1A1, PAX4, KCNH6, RIMS3, PROX1, signaling pathway enrichment plot of the B-cell or the popu SOX4, ACSS1, GHRL, NOTCH1, KCNN3, GCK, PYY, lation of B-cells indicates that none of the unfolded protein HCN3, and KCNJ4, and a fourth group of genes having response signaling pathway, the insulin synthesis and secre higher expression levels in insulin-positive B-like cells com tion signaling pathway, and the metal ion SLC transporters pared to mature B-cells, wherein said fourth group of genes is signaling pathway are enriched in the putative B-cell or the selected from the group consisting of NTS, GAST, RIMS3, population of putative B-cells. CACNA1E, PYY. SCT, FOXA1, GATA4, KCNH6, ARX, 0013. In some aspects, the present invention provides a DLL3, NOTCH1, IRX2, DPP4, PAX4, ACOX2, KCNB1, method of distinguishing mature and immature B-cells, com PROX1, GHRL, SLC2A1, ONECUT2, and SLC2A3; and (d) prising: (a) obtaining a putative B-cell or a population of identifying the test agent as a candidate agent that modulates US 2014/03297.04 A1 Nov. 6, 2014

the functional maturity of B-cells, wherein: (i) the testagent is from the group consisting of (vi), (vii), (viii), (ix), (X), (xi), a candidate agent that induces B-cells to become functionally (xii), (xiii), (xiv), (XV), (Xvi), and (Xvii) is enriched in the mature if the B-cell or the population of B-cells exhibit a B-cell or the population of B-cells. pattern of expression similar to the either reference mature 0017. In some embodiments, the B-cell or the population B-cell expression profile in the presence of the test agent; (ii) of B-cells are obtained from an in vitro source. In some the test agent is a candidate agent that induces B-cells to embodiments, the putative f3-cell or the population of putative become functionally immature if the f-cell or the population B-cells are obtained from an in vitro source. of B-cells exhibit a pattern of expression similar to either 0018. In some embodiments, the in vitro source is a culture reference immature B-cell expression profile. of differentiating stem cells. 0015. In some aspects, the present invention provides a 0019. In some embodiments, the stem cells are selected method of identifying a candidate agent that modulates the from the group consisting of human embryonic stem cells functional maturity of B-cells, comprising: (a) contacting a (hESCs), induced pluripotent stem cells (iPSCs), and combi B-cell or a population off-cells with a test agent; (b) assess nations thereof. In some embodiments, the in vitro source is ing enrichment of a signaling pathway in the presence of the selected from the group consisting of a cell bank, cell line, cell test agent to produce a signaling pathway enrichment plot of culture, cell population, and combinations thereof. In some the B-cell or the population off-cells, wherein the signaling embodiments, the in vitro Source is an artificial tissue or pathway is selected from the group consisting of an unfolded Organ. protein response signaling pathway, an insulin synthesis and 0020. In some embodiments, the cell or population of cells secretion signaling pathway, and a metalion SLC transporters are obtained from an in vivo Source. In some embodiments, signaling pathway; (c) identifying the test agent as a candi B-cellor the population of B-cells are obtained from an in vivo date agent that modulates the functional maturity of B-cells, Source. In some embodiments, the putative B-cellor the popu wherein: (i) the test agent is a candidate agent that induces lation of putative B-cells are obtained from an in vivo source. B-cells to become functionally mature if the signaling path 0021. In some embodiments, the in vivo source is an indi way enrichment plot of the B-cell or the population of B-cells vidual that has received an administration off-cells. In some indicates that at least one of the unfolded protein response embodiments, the in vivo Source is an individual Suffering signaling pathway, the insulin synthesis and secretion signal from a B-cell disorder selected from the group consisting of a ing pathway, and the metal ion SLC transporters signaling disorder associated with immature B-cells, a disorder associ pathway are enriched in the B-cell or the population of ated with destruction of B-cells, a disorder associated with B-cells; or (ii) the test agent is a candidate agent that induces dysfunctional B-cells, and a disorder associated with an insuf B-cells to become functionally immature if the signaling ficient number of B-cells. In some embodiments, the in vivo pathway enrichment plot of the f-cell or the population of Source is an individual Suspected of being in need of func B-cells indicates that none of the unfolded protein response tionally mature B-cells. In some embodiments, the in vivo signaling pathway, the insulin synthesis and secretion signal Source is a tissue or organ obtained from a donor individual. ing pathway, and the metal ion SLC transporters signaling 0022. In some embodiments, the individual is a human or pathway are enriched in the B-cellor the population of B-cells. animal individual. 0016. In some aspects, the present invention provides a 0023. In some embodiments, measuring expression com method of identifying a candidate agent that modulates the prises utilizing a technique selected from the group consisting functional maturity of B-cells, comprising: (a) contacting a of a microarray analysis, RNA-seq RT-PCR, and q-RT-PCR. B-cell or a population off-cells with a test agent; (b) assess 0024. In some embodiments, detecting the expression ing enrichment of a biological process in the presence of the level comprises conducting at least one binding assay to test agent to produce a biological process enrichment list of determine the expression level of the one or more genes. the B-cell or the population of B-cells, wherein the biological 0025. In some embodiments, the methods further com process is selected from the group consisting of (i) generation prise sorting immature and mature B-cells. In some embodi of precursor metabolites and energy, (ii) oxidation reduction, ments, sorting the immature and mature B-cells fluorescence (iii) Vesicle-mediated transport, (iv) electron transport chain, activated cell sorting (FACS). In some embodiments, the (v) monosaccharide metabolic process, (vi) cell morphogen FACS comprises staining at least one antibody specific for a esis, (vii) cellular component morphogenesis, (viii) cell pro putative B-cell Surface marker selected from the group con jection organization, (ix) Wnt receptor signaling pathway, (X) sisting of ABCA3, CD79B, FXYD2, KCNB2, NEGN1, cell projection morphogenesis, (xi) cytoskeleton organiza PTPRU, SLC6A9, ABCC8, CD8A, GCGR, KCNF1, NPR2, tion, (xii) sterol biosynthetic process, (xiii) cholesterol bio ROBO1, SORL1, ABCG1, CDH2, GPR120, KCNG3, synthetic process, (xiv) actin filament-based process, (XV) NRCAM, RTN4, SVOP, ACSL1, CDH22, GPR19, KCNH2, actin cytoskeleton organization, (Xvi) Sterol metabolic pro PCDHA1, SEMA5A, TGFBR3, ATPIB2, CHRNA5, GRIA2, cess, and (Xvii) neuron projection development; and (c) iden KCNMA1, PCDHA3, SERP2, TRPM2, CACNA1H, tifying the test agent as a candidate agent that modulates the CYB561, KCNH1, KCNQ2, PIGU, SLC17A6, TRPM5, functional maturity off-cells, wherein: (i) the test agent is a CADM1, EFNB3, IGSF11, MADD, PLXNA2, SLC43A2, candidate agent that induces B-cells to become functionally TSPAN13, CASR, FFAR1, IL17RB, NEO1, PRRG2, mature if the biological process enrichment list of the B-cell SLC6A6, and UNC5A. or the population off-cells indicates that at least one biologi 0026. In some embodiments, the methods further com cal process selected from the group consisting of (i), (ii), (iii). prise quantifying the sorted B-cells. (iv), and (v) is enriched in the f-cell or the population of 0027. In some embodiments, the methods further com B-cells; or (ii) the test agent is a candidate agent that induces prise preserving the sorted B-cells. B-cells to become functionally immature if the biological 0028. In some embodiments, the methods further com process enrichment list of the B-cell or the population of prise conducting a GSIS assay on the B-cell or population of B-cells indicates that at least one biological process selected B-cells. US 2014/03297.04 A1 Nov. 6, 2014

0029. In some embodiments, the mature B-cell is a human ing of the genes listed in Table 1 (Table 1A, Table 1B, or both B-cell. Tables 1A and 1B) or Table 2 (Table 2A, Table 2B, or both 0030. In some embodiments, the fetal B-cell is a human Tables 2A and 2B). B-cell. 0038. In some embodiments, the at least one marker of 0031. In some embodiments, the insulin-positive B-like B-cell functional maturity comprises a protein marker of cell is derived from human cells selected from the group B-cell functional maturity. consisting of human embryonic stem cells, reprogrammed 0039. In some embodiments, the at least one marker of human Somatic cells, and induced human pluripotent stem B-cell functional maturity comprises an mRNA marker of cells. B-cell functional maturity. 0032. In some aspects, the invention provides a method of 0040. In some aspects, the present invention provides a delivering a molecule of interest to a B-cell or a population of method of detecting immature B-cells comprising conducting B-cells, comprising: contacting the B-cell or the population of at least one binding assay for at least one marker of B-cell B-cells with a composition comprising the molecule of inter functional immaturity in a cell or population of cells, wherein est conjugated to an antibody that binds to a putative B-cell the presence of the at least one marker of B-cell functional Surface marker selected from the group consisting of ABCA3, immaturity in the cell or a population of cells indicates that CD79B, FXYD2, KCNB2, NLGN1, PTPRU, SLC6A9, the cell or population of cells are immature f3-cells. ABCD8, CD8A, KCNF1, NPR2, ROBO1, SORL1, ABCG1, 0041. In some embodiments, the at least one marker of CDH2, GPR120, KCNG3, NRCAM, RTN4, SVOP, ACSL1, B-cell functional immaturity is selected from the group con CDH22, GPR19, KCNH2, PCDHA1, SEMA5A, TGFBR3, sisting of the genes listed in Table 3 (Table 3A, Table 3B, or ATPIB2, CHRNA5, GRIA2, KCNMA1, PCDHA3, SERP2, both Tables 3A and 3B) or Table 4 ((Table 4A, Table 4B, or TRPM2, CACNA1H, CYB561, KCNH1, KCNQ2, PIGU, both Tables 4A and 4B). SLC17A6, TRPM5, CADM1, EFNB3, IGSF11, MADD, 0042. In some embodiments, the at least one marker of PLXNA2, SLC43A2, TSPAN13, CASR, FFAR1, IL17RB, B-cell functional immaturity comprises a protein marker of NE01, PRRG2, SLC6A6, and UNC5A. B-cell functional immaturity. 0033. In some aspects, the present invention provides a 0043. In some embodiments, the at least one marker of method of identifying a candidate agent that modulates dif B-cell functional immaturity comprises an mRNA marker of ferentiation of B-cells, comprising: contacting a cell, popula B-cell functional immaturity. tion of cells, cell line or cell culture with a test agent; and monitoring the cell, population of cells, cell line, or cell BRIEF DESCRIPTION OF THE DRAWINGS culture for expression of one or more B-cell specific transcrip 0044) The patent or application file contains at least one tion factors in the presence of the test agent, wherein the drawing executed in color. Copies of this patent or patent B-cell specific transcription factors are selected from the application publication with color drawings will be provided group consisting of ASCL2, NROB1, SIX4, CHD7, TOX, by the Office upon request and payment of the necessary fee. OLIG1, TSHZ3, DACH1, TSNAX, DACH2, MYT1L, 0045 FIGS. 1A and 1B depict an exemplary six stage PEG3, ZNF10, NDN, ZNF395, ETV5, ZNF540, HOPX, directed differentiation protocol. Cell types shown in FIG. 1A RXRG and ZNF672; and identifying the test agent as a can include: ES-embryonic stem cell; DE-definitive endoderm; didate agent that modulates differentiation of B-cells if the PP pancreatic progenitors; EP endocrine progenitors: cell, population of cells, cell line, or cell culture expresses one EN=endocrine; Media components: AlkSi=aTGFB signaling or more of the B-cell specific transcription factors in the inhibitor that acts via inhibition of activin receptor-like kinase presence of the test agent. 5; FAF-RSA=fatty acid-free bovine serum albumin; 0034. In some aspects, the present invention provides a KGF=keratinocyte growth factor, also known as fibroblast composition for differentiating a precursor cell to a pancreatic growth factor 7; ITS-X-insulin, transferrin, selenium; B-cell, comprising one or more B-cell specific transcription noggin a BMP signaling antagonist that acts via binding of factors selected from the group consisting of ASCL2. BMP-7: PDBuphorbol 12,13-dibutyrate, a protein kinase C NROB1, SIX4, CHD7, TOX, OLIG1, TSHZ3, DACH1, agonist; RA retinoic acid; SANT-1=a sonic hedgehog path TSNAX, DACH2, MYT1L, PEG3, ZNE10, NDN, ZNF395, way antagonist that acts via inhibition of the receptor Smooth ETV5, ZNF540, HOPX, RXRG and ZNF672. ened. FIG. 1B depicts the stepwise differentiation from 0035. In some aspects, the present invention provides a hESCs to pancreatic endocrine cells. The Table shown in FIG. method for differentiating a precursor cell to a pancreatic 1B contains reagents used during each stage of directed dif B-cell, comprising causing the precursor cell to upregulate ferentiation. expression of one or more B-cell specific transcription factors 0046 FIGS. 2A, 2B and 2C demonstrate that human selected from the group consisting of ASCL2, NROB1, SIX4, B-cells display a molecular signature distinct from other islet CHD7, TOX, OLIG1, TSHZ3, DACH1, TSNAX, DACH2, cells. FIG. 2A is a Table showing the results of a global MYT1L, PEG3, ZNE10, NDN, ZNF395, ETV5, ZNF540, analysis for probe sets differentially expressed (514 up, 889 HOPX, RXRG and ZNF672. down) in human islet populations enriched for B-cells com 0036. In some aspects, the present invention provides a pared to those depleted of B-cells. Human islet isolations method of detecting mature B-cells comprising conducting at likely contain contaminating non-islet tissues (e.g. exocrine least one binding assay for at least one marker of B-cell cells, mesenchyme), contributing to the heterogeneity of the functional maturity in a cell or a population of cells, wherein pool. Expression in unsorted islets is shown for comparison. the presence of the at least one marker of B-cell functional Left, listed are the top ten gene ontology terms for non-?3 islet maturity in the cell or a population of cells indicates that the cell-specific genes and all overrepresented terms for B-cell cell or population of cells are mature B-cells. specific genes (qs0.05). Right, row-normalized heatmap of 0037. In some embodiments, the at least one marker of probes shown with samples (columns) in the order of human B-cell functional maturity is selected from the group consist B-cells, other human islet cells, hESC-derived stage 6 cells, US 2014/03297.04 A1 Nov. 6, 2014

and insulin-- stage 6 cells. From this list of probes, array expressed between fetal and adult B cells. FIG. 7B shows signals are displayed for select established markers of non-(3 immunofluorescence staining of hPSC-INS' cells for PDX1, pancreatic cell types (FIG. 2B) and select known transcrip NKX6-1 and MAFA. Scale 100 um. FIG. 7C is alist of 42 tion factors preferentially expressed in B-cells (FIG. 2C). transcription factors that were differentially expressed based 0047 FIGS. 3A, 3B, 3C and 3D demonstrate the results of on microarray data between human adult B cells and hPSC protein analysis of sorted islet populations. FIG. 3A is a bar derived INS cells (fold change >3, p<0.05) and confirmed by graph showing results obtained using the Qproteome FFPE RNAseq between FIUES8-INS" cells and adult B cells (fold tissue kit; the inventors extracted a mean 104 pg protein per change >3). FIG.7D is a Table listing the top 10 most signifi cell, as determined by the micro BCA assay, from fixed cant (Benjamini q value) gene ontology biological processes human islets and hESC-derived pancreatic cultures. Error enriched hPSC-INS" cells over adult B cells. bars are SEM. FIG. 3B is a schematic showing that Pdx1 0052 FIGS. 8A, 8B and 8C demonstrate a dysregulated protein of expected molecular weight was resolved by SDS ER stress response in hESC-derived insulin+ cells. FIG. 8A PAGE and Western blot using lysate from hESC-derived shows results of gene set enrichment analysis using curated stage 6 cells that were paraformaldehyde-fixed and saponin Reactome gene sets identified three canonical pathways sig permeabilized and from cells that were fresh/unfixed. FIG. nificantly underexpressed in hESC-derived stage 6 (S6) insu 3C is a FACS plot depicting humanislets dispersed, fixed, and lin+ cells compared to adult f-cells (FDR q-values 0.05), stained for intracytoplasmic insulin and glucagon, resolving Enrichment plots are ordered from left to right by increasing by FACS clear populations of B- and C-cells. FIG. 3D are q-value. FIG. 8B shows an Ingenuity Pathway Analysis (IPA) schematics depicting Western blots of proteins isolated from map of the endoplasmic reticulum stress pathway. All factors sorted ?- and C-cells. Pdx1 was detected in B-cells but not that overlapped with the Reactome unfolded protein response C-cells, while glucokinase and the housekeeping protein Ran pathway are shown. Members are colored such that green were equally found in B- and C-cells. indicates underexpression in S6 insulin-- cells compared to 0048 FIGS. 4A, 4B, 4C and 4D demonstrate global B-cells, and red indicates overexpression. Disease/function expression (GO) analysis of hESC-derived cells and B-cells. annotations of pathway members are noted in ovals when FIGS. 4A, 4B, 4C and 4D show gene ontology biological relevant to B-cell biology. FIG. 8C is a schematic depicting process annotation clustering for genes up-regulated in S6 genes downstream of XBP1 comprised the most significantly insulin+ cells compared to all unsorted S6 cells (2.6% of inhibited transcription factororiginated gene set in S6 insu probes) (FIG. 4A), unsorted S6 cells compared to the S6 lin+ cells relative to B-cells. 28 of 34 differentially expressed insulin+population (2.3%) (FIG. 4B), B-cells compared to S6 genes had expression direction consistent with XBP1 inhibi insulin+ cells (4.0%) (FIG. 4C), and S6 insulin+ cells com tion. The p-value corresponds to the overlap between all pared to B-cells (4.9%) (FIG. 4D). Shown are the broadest underexpressed genes and the genes regulated by XBP1, as enriched terms that are each representative of a cluster. Num determined in IPA. Asterisks indicate that a gene is repre bers indicate genes in each group with a given annotation. sented in the microarray by multiple probes. 0049 FIGS.5A, 5B and 5C illustrate the relative expres 0053 FIGS.9A,9B and 9C depict the expression of B-cell sion of genes associated with endocrine pancreas develop signature genes in hESC-derived cells. The signature gene ment or function in B-cells and hESC-derived cells. FIG. 5A sets of adult B-cells (458 genes) and non-fi pancreatic cells is a heatmap showing normalized expression values of 71 (775 genes) identified in this study were used to assess hESC established endocrine lineage genes in human B-cells, hESC derived stage 6 (S6) cells. FIG.9A shows that gene set enrich derived stage 6 (S6) cells fresh and unsorted, or S6 insulin-- ment analysis indicated significant enrichment of B-cell cells. Of these genes, 40 were significantly differentially genes in S6 insulin--cells compared to unsorted S6 cells (top expressed between the S6 insulin-- cells and the unsorted panel), and of non-?3 pancreatic genes in unsorted S6 cells population (FIG. 5B), and 35 were differentially expressed over the insulin-H fraction (middle panel). All genes were between S6 insulin-- cells and human fB-cells (FIG. 5C). ranked by signal-to-noise ratio. p<0.001. FIG. 9B depicts Genes are listed by descending bio-weight. hierarchical clustering based on Pearson correlation between 0050 FIGS. 6A, 6B, 6C and 6D depict human B cell centroids demonstrated that S6 insulin-- cells cluster more maturation. FIG. 6A shows FACS plots of human adult islets closely with B-cells than do unsorted S6 cells, but many and human fetal pancreata sorted for INS cells (APC) FIG. differences between S6 insulin-- cells and B-cells remain. A 6B shows differentially expressed transcription factors heatmap is shown with row-normalized expression of all between adult and fetal? cells. FIG. 6C is a bar graph show 1403 probes that comprise the signature sets. FIG. 9C is a ing the relative expression of UCN3 in mouse and human fetal graph showing results of statistical weighted Voting using the and adult? cells. Expression normalized to fetal levels in each 1403 probe signature classified unsorted S6 cells as non-f species. FIG. 6D is a Table listing the top 5 most significant pancreatic cells with modest confidence, but S6 insulin--cells (Benjaminiq value) Gene ontology biological processes rela could not be confidently classified as B-cells. A positive con tively enriched in either adult or fetal B cells. fidence index (maximum 1.0) corresponds to expression 0051 FIGS. 7A, 7B, 7C and 7D show the results of dif similarity with the B-cell signature compared to the non-f- ferential expression between human fB cells and hPSC-de cell signature. Bars are mean-SEM. rived insulin expressing cells. FIG. 7A is a Table listing 152 0054 FIGS. 10A, 10B, 10C and 10D depict B-cell signa pancreatic lineage genes colored for genes that are differen ture transcription factor expression in hESC-derived insulin-- tially over-expressed in adult B cells (red) or hPSC-INS cells cells. FIG. 10A is a list of 49 transcription factors within the (blue). Differential gene expression was calculated based on signature set which were differentially expressed in S6 insu microarray data between human adult B cells and hPSC-INS" lin+ cells compared to B-cells. Of the 20 factors having higher cells (fold change >3, p<0.05) and confirmed by RNAseq expression levels in B-cells than S6 insulin+ cells, 18 are between HUES8-INS" cells and adult B cells (fold change normally preferentially expressed in B-cells. Genes are >3). Asterisk indicates genes that are also differentially ordered by bioweight and expression is displayed as fold US 2014/03297.04 A1 Nov. 6, 2014

change:SEM. FIGS. 10B, 10C and 10D show the results of population of B-cells to a reference mature B-cell transcrip immunohistochemistry, which revealed no co-expression of tome, wherein the B-cell or the population of B-cells are NRX6.1 in c-peptide-- (insulin-producing) cells (FIG. 10B), mature B-cells if the transcriptome of the B-cell or the popu little co-expression of MafA (FIG. 10C), and full co-expres lation of B-cells exhibit a pattern of expression similar to the sion of Pdx1 (FIG. 10D). reference mature B-cell transcriptome. 0055 FIGS. 11A, 11B and 11C demonstrate that hPSC 0061. In some embodiments, a method of detecting derived insulin expressing cells resemble human fetal B cells. mature B-cells comprises (a) obtaining a putative B-cell or a FIG. 11A is a bar graph showing glucose stimulated insulin population of putative f3-cells; (b) measuring expression of a secretion of dispersed cells. In contrast to adult B cells, fetal B plurality of genes in the B-cell or a population of B-cells to cells and hPSC-INS cells both appear functionally immature produce a transcriptome of the B-cell or the population of as indicated by increased basal glucose secretion and lack of B-cells; (c) comparing the transcriptome of the B-cell or the glucose stimulation. FIG. 11B shows hierarchical clustering population of B-cells to a reference mature B-cell transcrip based on microarray global gene expression across all genes tome exhibiting a pattern of expression depicted in FIG. 12; indicated that hPSC-INS' cells cluster closely with human and (d) detecting mature B-cells, wherein the B-cell or the fetal and not adult f cells. Numbers in parentheses indicate population of B-cells are mature B-cells if the transcriptome biological replicates. Lengths in the dendrogram represent of the B-cell or the population of B-cells exhibit a pattern of correlation distances. FIG. 11C shows R values based on expression similar to the reference mature B-cell transcrip microarray data across all genes are shown. Each row and tOne. column represents one sample. R values between biological 0062. In some aspects, the invention provides a method of replicates of adult B cell samples (Adult ins) are on average distinguishing B-cells and non-f-cells in a sample comprising 0.89+0.04. Rf values between sorted hPSC-derived insulin' pancreatic cells, the method comprising: (a) obtaining a stage 6 cells and sorted fetal B cells are 0.88+0.02. The bio sample comprising pancreatic cells; (b) measuring expres logical variation between adult f cells is not statistically sion of a plurality of genes in the sample to produce a tran smaller then the variation between fetal B cells and hPSC Scriptome of the pancreatic cells; (c) comparing the transcrip INS" stage 6 cells (p=0.49). This indicates that a high degree tome of the pancreatic cells to a reference B-cell of similarity between hPSC-INS' cells and human fetal B transcriptome exhibiting a pattern of expression depicted in cells. FIG. 12 and/or a reference non-f-cell transcriptome exhibit 0056 FIG. 12 shows a Table of genes with higher expres ing a pattern of expression depicted in FIG. 13; and (d) sion levels in sorted f cells relative to non B islet cells. distinguishing B-cells and non B-cells, wherein (i) the pan 0057 FIG. 13 shows a Table of genes with higher expres creatic cells are B-cells if the transcriptome of pancreatic cells sion levels in non 13 islet cells relative to sorted B cells. exhibits a pattern of expression similar to the reference f-cell transcriptome, or (ii) the pancreatic cells are non-B-cells if the DETAILED DESCRIPTION OF THE INVENTION transcriptome of pancreatic cells exhibits a pattern of expres 0058. The present invention relates to novel markers for sion similar to the reference non-f-cell transcriptome. The detecting B-cells (e.g., functionally mature B-cells), and present invention contemplates the use of any Suitable methods of using those markers for identifying functionally method to measure the expression of the genes in a putative mature B-cells and distinguishing between immature and B-cell or a population of putative B-cells to produce a tran mature B-cells (e.g., determining whether an in vitro-differ scriptome. Other suitable methods will be apparent to those entiated f-cell has matured). In particular, the work described skilled in the art. In some embodiments of this and other herein provides for the first time the mature B-cell transcrip aspects of the invention, measuring expression comprises tome. In addition, the work described herein provides genes utilizing a technique selected from the group consisting of a which are differentially expressed between mature and imma microarray analysis, RNA-seq RT-PCR, and q-RT-PCR. ture B-cells, signaling pathways which are enriched in mature 0063 As used here, “transcriptome' refers to the collec and immature B-cells, biological processes which are tion of all gene transcripts in a and their appropriate relative enriched in mature and immature B-cells, and putative B-cell abundances given cell (e.g., a B-cell), including both coding cell-surface markers. RNA (mRNAs) and non-coding RNAs (e.g., miRNA, tRNA, 0059. Accordingly, the present invention provides mark linckNAs etc.). ers and methods for identifying the functional maturity of 0064. In some embodiments the disclosure provides a B-cells (e.g., distinguishing between immature and mature mature B-cell transcriptome that may be used for a variety of B-cells in a population), identifying agents that modulate the purposes. In some embodiments the mature B-cell transcrip functional maturity of a B-cell, identifying agents that modu tome comprises expression levels of the genes listed in FIG. late disorders associated with immature B-cells, identifying 12 or counterparts thereof (e.g., orthologs in other organ individuals in need of functionally mature B-cells, selecting isms). In some embodiments measurement of expression lev functionally mature in vitro-differentiated B-cells or (3-like els of the genes or a subset thereof may be used to identify cells for administration to an individual (e.g., transplantation mature B-cells (e.g., as compared with immature B-cells, such of mature B-cells into the individual, e.g., a humanor animal), as fetal B-cells or in vitro-differentiated insulin-positive and identifying whether B-cells that have been administered B-like cells). In some embodiments a Subset comprises at to an individual are mature B-cells in vivo. least 5, 10, 20, 50, 100, 200,300, 400, or more genes listed in 0060. In one aspect, the present invention provides a FIG. 12. Gene expression levels may be measured by mea method of detecting mature B-cells. An exemplary method of Suring mRNA, protein or other gene product. Any Suitable detecting mature B-cells comprises measuring expression of method may be used. In some embodiments gene expression genes in a putative B-cell or a population of putative B-cells to may be measured using RNA-Seq microarray analysis, or produce a transcriptome of the B-cell or a population of quantitative PCR. In some embodiments B-cells or popula B-cells, and comparing the transcriptome of the B-cell or the tions of B-cells are classified based on the mature B-cell US 2014/03297.04 A1 Nov. 6, 2014

transcriptome. For example, whether the B-cells or B-cell “mRNA marker of B-cell functional maturity are used inter population’s transcriptome more closely resembles a mature changeably to refer to mRNA encoded by a gene which is a B-cell transcriptome or not may be determined. Heirarchical marker of B-cell maturity. As used herein, “mRNA marker of clustering or PCA analysis may be used, for example, to B-cell immaturity” and “mRNA marker of B-cell functional determine whether a particular B-cell population (e.g., immaturity are used interchangeably to refer to mRNA colony, culture, cell line, etc.) clusters with mature B-cells as encoded by a gene which is a marker of B-cell immaturity. described herein or clusters with immature B-cells as 0069. As used herein, a “functionally mature f3-cell' and described herein. Mature B-cells which substantially match “mature f3-cell are used interchangeably to refer to a cell that the transcriptome of the mature B-cell transcriptome (e.g., displays one or more markers of B-cell functional maturity that cluster with mature B-cells as described herein), may, for and exhibits an appropriate GSIS response. example, be suitable for administration in Viva. In some 0070 The markers and methods of the present invention embodiments a Mature B-cell transcriptome may be used in are capable of identifying the functional maturity of any identifying compounds or conditions that promote formation B-cell, (3-like cell, or putative B-cell. As used herein “B-like of mature B-cells. For example, compounds or conditions cell refers to a cell that displays at least two markers indica may be used in a differentiation protocol and their effect on tive of a pancreatic B-cell. Such as, pancreas duodenum the transcriptome of pluripotent cells subjected to the differ homeobox-1 (PDX-1), insulin, Somatostatin, glucose trans entiation protocol may be assessed. Compounds that promote porter-2 (GLUT-2), glycogen, amylase, and neurogenin-3 a B-cell transcriptome resembling that of a mature B-cell (NGN-3), or genes having higher expression levels in mature transcriptome may be identified. Such compounds may be B-cells. Markers indicative of pancreatic B-cells also include used in a differentiation protocol to generate B-cells (e.g., morphological characteristics (e.g., spherical shape). functionally mature B-cells). In some embodiments, an R2 0071. As used herein “insulin-positive B-like cell refers correlation can be used to determine whether a cell is a B-cell. to a cell that displays the at least two markers indicative of a In Such embodiments, a cell is a B-cell when compared to the pancreatic B-cell and also express insulin but lack an appro B-cell transcriptome gene expression data the cells have a priate GSIS response. As used herein “in vitro-differentiated correlation coefficient (r) across all genes assessed re-0.9 or insulin-positive B-like cell. “S6 insulin+ cells.” “hPSC-ins+ re-O.95 or re-O.98 or re-O.99. cells.” HUF28-ins+ cells,” and “hESC derived stage 6 (sG) 0065. As used herein, a “functionally immature f3-cell insulin+ cells' are used interchangeably to refer to B-like cells and “immature f3-cell are used interchangeably to refer to a that are positive for insulin expression, and are derived from cell that displays one or more markers of B-cell functional differentiation of stem cells. immaturity or does not display one or more markers off-cell 0072 The markers and methods of the present invention functional maturity, and lacks an appropriate GSIS response. are capable of distinguishing mature and immature B-cells. 0066. As used herein, “marker of B-cell maturity” and Generally, distinguishing between mature and immature “marker of functional B-cell maturity’ are used interchange B-cells can be accomplished by analyzing putative B-cell for ably and refer to a mature B-cell transcriptome, one or more one or more markers of B-cell functional maturity or one or genes having higher expression levels in mature B-cells com more markers of B-cell functional immaturity. On the one pared to fetal B-cells or in-vitro-differentiated insulin-posi hand, if a putative B-cell or a population of putative B-cells tive f3-like cells, one or more signaling pathways which are displays one or more markers of B-cell functional maturity, enriched in mature f3-cells compared to fetal B-cells or in then the B-cell or population of B-cells are likely mature vitro-differentiated insulin-positive B-like cells, or one or B-cells. On the other hand, if a putative B-cell or a population more biological processes which are enriched in mature of putative B-cells displays one or more markers of B-cell B-cells compared to fetal B-cells or in-vitro-differentiated functional maturity, then the B-cell or population of B-cells insulin-positive f3-like cells. are likely immature B-cells. Those skilled in the art will 0067. As used herein, a “marker of B-cell immaturity” and appreciate that confirmation of B-cell functional maturity or “marker of functional (3-cell immaturity’ are used inter functional immaturity can be done by performing a GSIS changeably to refer to one or more genes having higher assay to determine if the putative B-cell or the population of expression levels in fetal B-cells or in vitro-differentiated putative f3-cells exhibits or lacks an appropriate GSIS insulin-positive B-like cells compared to mature B-cells, one response, respectively. or more signaling pathways which are enriched in fetal Genes Differentially Expressed between Mature and Imma B-cells or in vitro-differentiated insulin-positive f3-like cells ture f3-cells compared to mature B-cells, or one or more biological pro 0073. The present invention contemplates distinguishing cesses which are enriched in fetal B-cells or in vitro-differen mature and immature B-cells by detecting the presence or tiated insulin-positive f3-like cells compared to mature absence of expression of one or more genes which are differ B-cells. entially expressed between mature and immature B-cells in a 0068. It is to be understood that the markers of B-cell B-cell, population off-cells, a putative B-cell, or a population maturity and markers off-cell immaturity may be detectably of putative B-cells. expressed in the form of protein and or mRNA within a cell. 0074. In some aspects, the present invention provides a Accordingly, as used herein, protein marker of B-cell matu method of detecting mature B-cells, the method comprising rity” and “protein marker of B-cell functional maturity are conducting at least one binding assay for at least one marker used interchangeably to refer to a protein encoded by a gene off-cell functional maturity in a cell or a population of cells, which is a marker of B-cell maturity. As used herein, “protein wherein the presence of the at least one marker of B-cell marker of B-cell immaturity” and “protein marker of B-cell functional maturity in the cell or a population of cells indi functional immaturity are used interchangeably to refer to a cates that the cell or population of cells are mature B-cells. protein encoded by a gene which is a marker off-cell imma 0075. The present invention contemplates detecting turity. As used herein, “mRNA marker of B-cell maturity” and mature B-cells by detecting the presence of any marker of US 2014/03297.04 A1 Nov. 6, 2014

B-cell functional maturity in a cell or a population of cells. protein antibodies can be generated according to routine pro The cell or population of cells can be a cell or population of tocols, or can be readily obtained from a variety of commer cells Suspected of being B-cells (e.g., a culture differentiating cial sources (e.g., Sigma-Aldrich). Other Suitable techniques stem cells). In some embodiments, the at least one marker of for detecting the presence of proteins in B-cells are apparent B-cell functional maturity is selected from the group consist to those skilled in the art. ing of the genes listed in Table 1 or Table 3. In some embodi I0082 Generally, the presence of elevated levels of mRNA ments, the at least one marker of B-cell functional maturity markers of B-cell functional maturity in a B-cell or population comprises a protein marker of B-cell functional maturity, as of B-cells is indicative that the B-cell or population of B-cells is functionally mature, whereas the absence of the same described herein. In some embodiments, the at least one mRNA markers (or relative lower levels of the same mRNA marker of B-cell functional maturity comprises an mRNA markers) in the B-cell or population off-cells may be indica marker of B-cell functional-maturity, as described herein. tive that the B-cell or population of B-cells is functionally 0076. In some aspects, the present invention provides a immature. Conversely, the presence of elevated levels of method of detecting immature B-cells, the method compris mRNA markers of B-cell functional immaturity in a B-cellor ing conducting at least one binding assay for at least one population of B-cells is indicative that the B-cell or population marker off-cell functional immaturity in a cell or population of B-cells is functionally immature, whereas the absence of of cells, wherein the presence of the at least one marker of the same mRNA markers (or relative lower levels of the same B-cell functional immaturity in the cellor a population of cells mRNA markers) in the B-cell or population of B-cells may be indicates that the cell or population of cells are immature indicative that the f-cell or population of B-cells is function B-cells. ally mature. 0077. The present invention contemplates detecting I0083. It is to be understood that the phrase “elevated levels immature B-cells by detecting the presence of any marker of of mRNA refers to levels of mRNA in a mature B-cell rela B-cell functional immaturity in a cell or a population of cells. tive to an immature f3-cell or levels of mRNA in an immature The cell or population of cells can be a cell or population of B-cell relative to a mature f3-cell. Elevated levels of mRNA cells Suspected of being B-cells (e.g., a culture differentiating may be represented as a fold-change in expression of the mRNA in the mature f3-cell relative to the immature f3-cell, stem cells). and vice versa. 0078. In some embodiments, the at least one marker of I0084. The present invention contemplates detecting the B-cell functional immaturity is selected from the group con presence or absence of elevated levels of mRNA according to sisting of the genes listed in Table 3 or Table 4. In some any technique available to the skilled artisan. In some embodiments, the at least one marker of B-cell functional embodiments of this and other aspects of the invention, immaturity comprises a protein marker of B-cell functional detecting the presence or absence of elevated levels of mRNA immaturity. In some embodiments, the at least one marker of markers off-cell functional maturity orimmaturity in a B-cell B-cell functional immaturity comprises an mRNA marker of or population of B-cells comprises conducting one or more B-cell functional immaturity. hybridization assays. In some embodiments of this and other 0079 Generally, the presence of a protein marker of B-cell aspects of the invention, the one or more hybridization assays functional maturity in a B-cell or population of B-cells is comprise a microarray. In some embodiments of this and indicative that the f-cell or population of B-cells is function other aspects of the invention, the one or more hybridization assay comprises RNA-seq. In some embodiments of this and ally mature, whereas the absence of the same protein marker other aspects of the invention, the one or more hybridization in the f-cell or population of B-cells may be indicative that the assays comprises q-RT-PCR. For the purposes of the inven B-cell or population off-cells is functionally immature. Con tion the hybridization assays described or mentioned herein versely, the presence of a protein marker of B-cell functional are considered binding assays. immaturity in a B-cell or population of B-cells is indicative I0085. In some embodiments of this and other aspects of that the B-cell or population of B-cells is functionally imma the invention, the presence of elevated levels of mRNA mark ture, whereas the absence of the same protein marker in the ers of B-cell functional maturity comprises at least a 2 fold B-cell or population of B-cells may be indicative that the increase, a 3 fold increase, a 4 fold increase, a 5 fold increase, B-cell or population of B-cells is functionally mature. or N-fold increase (where N is a positive integer) in the levels 0080. The present invention contemplates detecting the of the mRNA marker in a mature B-cell relative to the levels presence or absence of protein markers of B-cell functional of the mRNA maker in immature B-cells. maturity or immaturity according to any technique available I0086. In some embodiments of this and other aspects of to the skilled artisan. In some embodiments of this and other the invention, the presence of elevated levels of mRNA mark aspects of the invention, detecting the presence or absence of ers off-cell functional immaturity comprises at least a 2 fold protein markers of B-cell functional maturity or B-cell func increase, a 3 fold increase, a 4 fold increase, a 5 fold increase, tional immaturity comprises immunostaining (e.g., Western or N-fold increase (where N is a positive integer) in the levels blotting, immunohistochemistry, ELISA, etc). In Such of the mRNA marker in an immature B-cell relative to the embodiments, anti-protein marker antibodies targeted to a levels of the mRNA marker in mature f3-cells. particular protein marker of B-cell functional maturity or I0087. In some embodiments of this and other aspects of B-cell functional immaturity can be used to detect the pres the invention, the marker of B-cell functional immaturity does ence or absence of the particular protein marker. For the not include PDX1. In some embodiments of this and other purposes of the invention the immunostaining techniques aspects of the invention, the marker of B-cell functional described or mentioned herein are considered binding assays. immaturity does not include MAFB. In some embodiments of 0081. Such antibodies can include polyclonal antibodies, this and other aspects of the invention, the marker of B-cell monoclonal antibodies, chimeric antibodies, single-chain functional immaturity does not include NKX6.1. antibodies, antibody fragments, humanized antibodies, I0088. In some embodiments of this and other aspects of multi-specific antibodies, and modified antibodies (e.g., the invention, the marker of B-cell functional maturity does fused to a protein to facilitate detection.) Suitable anti-marker not include MAFA. In some embodiments of this and other

US 2014/03297.04 A1 Nov. 6, 2014 11

TABLE 1 B-continued Genes with higher expression levels in mature f-cells compared to fetal B-cells

0091. It is to be understood that the levels of expression of levels of expression of the combination of genes in the cell, one or more of the genes listed in Table 1 are depicted in FIG. culture, cell line, tissue, or population of cells is elevated (for 6C as a fold change in expression of mature B-cells compared example, as depicted in FIG. 6C), the cell, culture, cell line, to fetal B-cells. tissue, or population of cells comprises mature B-cells. 0092. It should also be appreciated that any gene listed in 0094 Exemplary genes which have higher expression lev Table 1 can be used as a marker for detecting mature B-cells els in mature B-cells compared to in vitro-differentiated insu by measuring the level of expression of the gene in a cell, lin-positive B-like cells are listed in Table 2 (Table 2 includes culture, cell line, tissue, or population of cells (e.g., Suspected both Tables 2A and 2B). of being B-cells), wherein if the level of expression of the gene in the cell, culture, cell line, tissue, or population of cells is TABLE 2A elevated (for example, as depicted in FIG. 6C), the cell, cul ture, cell line, tissue, or population of cells comprises mature Genes with higher expression levels in mature B-cells B-cells. compared to in vitro-differentiated insulin-positive B-like cells 0093. Those skilled in the art will also appreciate that any KLF9, CEBPD, PEG3, NFIX, RORC, HOPX, TSHZ3, HSF4, two or more of the genes listed in Table 1 can be used in EPAS1, GLIS3, NR3C2, SIX4, PURA, NFIA, XBP1, CHGB, combinations of up to N genes (where N is a positive integer IAPP, ESR1, GLIS3, MAFA, MNX1, NKX6-1, PDX1, GPI, PCSK1, PCSK2, SLC30A8, STX1A, STXBP1, SYT4, G6PC2, SLC2A2, greater than or equal to 2) as markers for detecting mature KCNA5, KCNH2, KCNK1, KCNK12, KCNK3, KCNMA1, GCGR, B-cells by measuring the levels of expression of the combi UCN3, WNT4 nation of genes in a cell, culture, cell line, tissue, or popula tion of cells (e.g., Suspected of being B-cells), wherein if the TABLE 2B Genes with higher expression levels in mature B-cells compared to in vitro-differentiated insulin-positive B-like cells A2LD1, A2M, A4GNT, AADAC, AADACL1, AAED1, AANAT, AARS, AARSD1, AASDH, ABO19439.68, ABO19441.29, ABCA10, ABCA13, ABCA17P, ABCA5, ABCA6, ABCA8, ABCA9, ABCA9-AS1, ABCB1, ABCB1OP1, ABCB1OP4, ABCB11, ABCB7, ABCC12, ABCC2, ABCC3, ABCC9, ABCD2, ABCE1, ABCG1, ABCG8, ABHD1, ABHD10, ABHD11-AS1, ABHD2, ABHD3, ABHD5, ABHD6, ABI3, ABP1, ABRACL, AC000036.4, AC000110.1, ACOOO124.1, AC002056.3, AC002056.5, AC002064.5, AC002128.4, ACOO2306.1, AC002310.10, AC002314.2, ACOO2398.12, ACOO2398.9, AC002400.1, ACOO2429.5, ACOO2456.2, AC002467.5, ACOO2486.2, ACOO2511.2, ACOO251.1.4, ACOO2519.5, ACOO2519.6, ACOO2553.1, ACOO3O874, AC003092.2, ACOO3101.1, AC003104.1, AC003989.3, AC003991.3, ACOO4014.4, AC004019.10, ACOO4041.2, AC004O69.2, AC004074.3, AC004156.3, ACOO4159.1, AC004240.2, AC004381.7, AC004383.3, ACOO44614, AC004471.9, ACOO4485.3, AC004528.4, AC004562.1, ACOO4696.2, AC004744.3, AC00477.1.1, ACOO4797.1, AC004832.1, ACOO4837.5, ACOO4840.8, AC004851.1, ACOO4854.4, ACOO4854.5, AC004870.5, AC004876.1, AC004878.7, AC004899.3, AC004941.3, ACOO4945.1, AC004988.1, ACOO5027.3, AC005037.1, AC005062.2, AC005076.5, AC005077.8, AC005082.12, AC005102.1, ACOO5229.1, AC005.255.1, ACOO5262.4, ACOO5264.2, ACOO5280.1, ACOO5281.2, ACOO5307.1, ACOO5307.3, AC005336.2, ACOO5336.5, ACOO5351.1, ACOO5355.1, ACOO5356.1, AC005391.2, AC005481.5, ACOO5498.3, ACOO5517.3, AC005532.5, AC005534.9, AC005546.2, ACOO5616.2, ACOO5682.7, ACOO5740.3, ACOO5779.2, ACOO5783.1, ACOO5884.1, ACOO5895.3, ACOO60.01.1, ACOO6019.3, ACOO6019.4, AC006026.10, ACOO6026.13, ACOO6026.9, ACOO6033.22, ACOO6038.4, AC006042.8, ACOO6070.11, AC006116.12, AC006116.13, AC006116.15, AC006116.17, AC006116.21, AC006116.24, AC006116.26, AC006157.2, ACOO6227.1, ACOO6296.3, ACOO6355.3, ACOO6369.2, ACOO6378.2, ACOO6378.3, ACOO6460.2, ACOO6465.3, ACOO6483.5, ACOO6486.1, ACOO6509.7, AC006539.1, ACOO6946. 12, AC007000.11, AC007009.1, AC007009.2, ACOO7014.1, AC007041.2, AC007098.1, AC007099.2, AC007126.1, ACOO7204.2, ACOO7229.3, ACOO7246.3, ACOO7249.3, AC007253.1, AC007255.7, AC007255.8, ACOO7271.3, ACOO7272.3, ACOO7277.3, ACOO7279.2, ACOO7282.6, ACOO7283.4, ACOO7283.5, ACOO7318.5, ACOO7319.1, ACOO7349.5, ACOO7362.1, ACOO7362.3, ACOO7365.3, ACOO7386.3, ACOO7386.4, ACO07389.4, ACO07390.5, ACO07390.6, ACO07392.3, ACO07392.4, ACOO7405.2, ACOO7567.1, AC007618.3, AC007620.3, ACOO7682.1, ACOO7731.1, ACOO7750.5, ACOO7787.2, AC007796.1, AC007842.2, ACOO7879.2, ACOO7879.4, AC007879.5, ACOO7881.1, ACOO7919.18, ACOO7952.1, ACOO7966.1, AC007970.1, ACO08069.2, AC008132.12, AC008147.2, AC008155.1, ACOO8269.2, AC008279.1, ACOO828.0.3, ACOO8427.2, AC008537.2, AC008555.2, US 2014/03297.04 A1 Nov. 6, 2014 12

TABLE 2B-continued Genes with higher expression levels in mature B-cells compared to in vitro-differentiated insulin-positive B-like cells ACOO858.1.1, AC008592.3, ACOO8592.4, ACOO8592.5, ACOO8703.1, ACOO8746.5, ACOO8937.2, AC008937.3, ACOO8984.7, AC008992.2, AC009093.1, ACOO9095.4, ACO09120.10, AC009120.4, ACOO9166.5, ACOO9223.1, ACOO9232.2, AC009236.1, ACOO9236.2, AC009237.1, ACO09237.11, AC009237.8, AC009274.6, AC009299.2, ACOO9299.3, AC0093.02.2, ACOO9303.1, ACOO9475.2, ACOO9487.6, ACOO9495.3, ACOO9495.4, ACOO9499.1, ACOO95O1.4, ACOO9502.1, AC0095.06.1, AC009948.5, AC009948.7, AC00995.0.2, AC009961.5, AC009963.6, AC0099942, AC010.095.5, ACO10127.4, ACO10136.2, ACO10149.4, ACO10226.4, AC010240.2, ACO10240.3, ACO 10336.2, ACO10468.1, ACO10487.1, ACO 10525.4, ACO10525.5, ACO 10525.7, ACO 10547.9, ACO10641.1, ACO10733.4, ACO10878.3, ACO10890.1, ACO10894.5, ACO10969.1, ACO10982.1, ACO11239.1, ACO11242.6, ACO11290.5, ACO11298.2, ACO11322.1, ACO11330.12, ACO11330.5, ACO11330.6, ACO 11385.1, ACO11406.2, ACO11450.2, ACO11477.1, ACO11498.1, ACO11499.1, ACO11526.1, ACO11537.1, ACO 11754.1, ACO11816.3, ACO11891.5, ACO11933.2, ACO12065.5, ACO12066.1, ACO12074.2, ACO12087.2, ACO12146.7, ACO12309.5, ACO12314.6, ACO12317.1, ACO12354.6, ACO12354.8, ACO1236.0.4, ACO12363.4, ACO12442.5, ACO12512.1, ACO 13268.5, ACO13269.3, ACO 13275.2, ACO13468.1, ACO13470.6, ACO 13717.3, ACO 13733.3, ACO15726.1, ACO15849.14, ACO15922.6, ACO15922.7, ACO15923.1, ACO15971.2, ACO15987.1, ACO15987.2, ACO16292.3, ACO16582.2, ACO16586.1, ACO16644.1, ACO16689.1, ACO16700.3, ACO16700.4, ACO16700.6, ACO16716.1, ACO16732.2, ACO16745.1, ACO16831.7, ACO16909.1, ACO16910.1, ACO16912.3, ACO16995.3, ACO17048.3, ACO 17048.4, ACO 17071.1, ACO17074.1, ACO 17079.3, ACO 17079.4, ACO17083.2, ACO 17083.3, ACO17101.10, ACO17104.2, ACO17104.4, ACO18462.2, AC018642.1, ACO18696.4, ACO18696.7, ACO18720.10, AC018735.1, ACO18737.1, AC018737.4, ACO18867.2, ACO18890.6, ACO1905.0.1, ACO19097.7, ACO19117.2, ACO19181.3, ACO1918.6.1, ACO19294.1, AC020571.3, AC020594.5, ACO20900.2, ACO20907.1, ACO22153.1, ACO22182.3, ACO22431.1, ACO22819.3, ACO23085.1, ACO23797.1, ACO24082.3, ACO24560.2, ACO24592.9, ACO24704.2, ACO25287.1, ACO25335.1, ACO25442.3, ACO25750.6, ACO25918.2, ACO26150.6, ACO26150.9, AC026166.1, ACO26202.3, ACO26471.6, ACO34110.1, ACO34154.1, ACO34187.2, ACO34228.4, ACO34229.1, ACO37459.4, AC044860.1, AC046130.1, AC053503.4, ACO55811.1, ACO58791.2, ACO60226.1, AC062028.1, AC062029.1, ACO63976.3, ACO63976.6, ACO64843.1, ACO64843.2, ACO64850.4, ACO64871.3, ACO64872.1, ACO66692.3, ACO67945.4, ACO67950.1, ACO67961.1, ACO68042.1, ACO68134.6, ACO68134.8, ACO68137.2, ACO68279.3, ACO68491.1, ACO68491.3, ACO68492.1, ACO6.8538.2, ACO68754.1, ACO69154.2, ACO69155.1, ACO6920.0.1, ACO69282.6, AC069292.6, ACO69363.1, ACO72031.1, AC073043.2, AC073072.5, AC073109.2, AC073115.7, AC073128.10, AC073135.2, AC073254.1, AC0733 16.1, AC073343.1, AC073343.11, AC073343.2, AC073410.1, AC073479.1, AC073626.2, AC073635.5, AC073834.3, AC073842.18, AC073842.19, AC073934.6, ACO74O11.2, ACO74091.1, ACO74091.13, ACO74182.1, ACO74183.4, ACO74212.3, AC074391.1, ACO78819.1, ACO78852.1, ACO78852.2, ACO788834, ACO78899.1, ACO7914.0.1, ACO79145.4, ACO79250. , ACO79354.6, ACO794.66. , ACO79630.4, ACO79741.2, AC079922.3, ACO83799. , ACO83843.3, ACO83862. , ACO83863.7, ACO83873.4, ACO83899.3, ACO83900. , ACO84125.2, ACO84149. , ACO84809.3, ACO84859. 1, ACO87163.2, ACO87294.2, ACO875.01.1, AC087793. , ACO9004.4.1, ACO90044.2, ACO90286.2, ACO90286.4, AC090420.1, AC090519.7, ACO905874, AC090602.1, ACO90617.1, ACO90952.4, ACO91167.3, ACO91492.2, ACO91633.2, ACO91729.7, ACO91729.8, AC091969. , ACO92106.2, ACO92117. , ACO92155.1, ACO92165.4, ACO92170.1, ACO922.95.4, ACO92574.1, ACO92574.2, ACO92594.1, ACO92597.3, ACO926.10.12, ACO92646 .2, ACO92661.2, ACO92664.1, ACO92667.2, ACO92687.5, ACO92755.4, ACO92811. , ACO92835.2, AC093106.7, AC093142.2, ACO93162.5, ACO93171.1, ACO93390. , ACO93391.2, ACO93510. , ACO93620.5, ACO93627.12, ACO93662.6, ACO93702. , ACO93899.3, ACO94019.4, AC096579.1, ACO96591.1, ACO96649.1, ACO96649.2, ACO96669.1, ACO96669.3, ACO96670.3, ACO96753.1, ACO96753.2, ACO97359. , ACO97381.1, ACO97382. , ACO97467.2, ACO97500. 1, ACO97500.2, ACO97523. ACO97523.2, ACO97635.5, ACO97662.2, ACO97711.1, ACO97721.2, ACO97724.3, ACO98592.6, ACO98614.2, ACO98820.3, ACO98823.3, ACO98828.3, ACO99048. , ACO99344.3, AC099535.4, ACO99544.2, ACO99552.4, ACO.9985.0.1, AC100803. , AC103681.1, AC103740. , AC1038.01.2, AC104024.1, AC104076.3, AC104134.2, AC104135.2, AC104297. , AC104306.1, AC104306.2, AC104306.4, AC104600. , AC10465.0.2, AC104651.2, AC104695.3, AC104841.1, AC104984.4, AC104986. , AC105020.1, AC105052.2, AC105053.4, AC105247.1, AC105396.3, AC105399.2, AC1054.02.1, AC105402.4, AC105461.1, AC106053.1, AC106722.1, AC106827.2, AC107021.1, AC107072.2, AC107081.5, AC107982.4, AC108039.1, AC108066. , AC108448.2, AC108463.1, AC108463.2, AC108463.3, AC108488.3, AC108676. , AC108868.3, AC108868.4, AC108938.2, AC1093.09.4, AC109309.5, AC109826. , AC109826.2, AC110491.1, AC111200.2, AC112229.7, AC113554.1, AC113607. , AC113607.2, AC113607.3, AC114546.1, AC114730.2, AC114730.3, AC114730.5, AC114752.2, AC114755.3, AC114772.1, AC114776.1, AC114947.1, AC116050. , AC116366.5, AC117490.2, AC117834.1, AC11834.4.1, AC12O194.1, AC121336. , AC121336.2, AC123768.3, AC123886.2, AC124890.1, AC126118.1, AC128709. , AC1294.92.6, AC131011.1, AC131097.4, AC133485.1, AC133644.3, AC135776. , AC136289.1, AC137590.1, AC137932.6, AC13843.0.2,

US 2014/03297.04 A1 Nov. 6, 2014 32

TABLE 2B-continued Genes with higher expression levels in mature B-cells compared to in vitro-differentiated insulin-positive B-like cells

US 2014/03297.04 A1 Nov. 6, 2014 34

TABLE 2C-continued Genes differentially expressed between mature B-cells compared to in vitro-differentiated insulin-positive B-like cells M6PRBP1, MAFA, MAMDC2, MAMLD1, MAN1C1, MAP3K6, MAPK12 MAPK15, 3-Mar, MARCKSL1, MATN2, ME3, MEG3, MEP1A, MET, METTL7A, MFAP4, MFGE8, MFNG, MGAT3, MGAT4A, MGAT4C, MGC16291, MGC16384, MGC18216, MGC39900, MIAT, MMP7, MNX1, MR1, MRAP2, MSL3L1, MT1X, MTE, MUC13, MVP, MX1, MYL5, MYLIP, MYO10, MYO16, MYO1D, MYOM1,

OLFM1, ONECUT2, OSBP2, OVGP1, OXCT2, OXGR1, PABPC1L, PAFAH1B3, PALM2, PARM1, PAX4, PAX9, PBX2, PCDHB2, PCSK1, PCSK2, PDE8B, PDK4, PGM5, PHGDH, PHLDA3, PHYHIPL, PIR, PLA2G1B, PLA2G4C, PLCH2, PLCL2,

PPP2R3B, PRAGMIN, PRAMEF19, PRICKLE1, PRKCH, PRKD1, PRNP, PROC, PRODH2, PROM1, PROX1, PRPH, PRR15L, PRSS1, PRSS23, PRSS8, PRUNE2, PSMB8, PTGR1, PTGS2, PTPN3, PTPRD, PTPRT, PURA, PXDN, QPRT, RAB11 FIP5, RAB31, RAB34, RAPH1, RASD1, RASGRP1, RBBP8, RBP4, RBPMS2, RCOR2, RDH12, RENBP, RGL1, RGMA, RGS9, RHBDF1, RHOC, RHOU, RICH2, RIN2, RNASEL, RND3, RNF144, RNF144A, RNF165, ROR2, RORC, RPRM, RPS19BP1, RYR1, S100A10, S100A16, S100A4, SCARA3, SCD5, SCGB2A1, SCIN, SDF2L1, SELENBP1, SELM, SEMA3A, SEMA6C, 9-Sep, SERP1, SERPINA1, SERPINA10, SERPINF1, SERPINF2, SERTAD4, SFRP1, SGMS2, SH3GL2, SHC3, SHISA2, SIRPA, SIX4, SLC12A5, SLC16A9, SLC17 A6,

SNORA12, SNORD114-3, SNX26, SOCS2, SORBS1, SORL1, SOX11, SPATA18, SPATS2L, SPIRE1, SPON2, SRRM4. SRXN1, SSPO, ST6GAL1, ST6GALNAC5, STC2, STEAP2, STMN1, STMN4, STOM, STRA6, SURF4, SVIL, SYK, SYNGR4, SYT13, TAP1, TCF3, TGEBR2, TGEBR3, TGIF2, TGOLN2, TH, THBS3, TICAM2, TIMP2, TLE6, TM7SF2, TMEM109, TMEM163, TMEM190, TMEM27, TMEM56, TMEM61, TMEM86B, TMEM98, TMSB15A, TNFRSF21, TNFRSF25, TOX3, TP53I3, TP53INP1, TPST2, TRIM24, TRIM4, TRIM46, TRIOBP, TRIP6, TRO, TSHZ3, TSPAN14, TSPAN6, TSPYL5, TST, TTLL10, UCP2, UNCSA, UNC5CL,

0100. It should be appreciated that any gene listed in Table lation of cells are similar to the levels of expression of the 2C can be used as a marker for distinguishing between mature same combination of genes in in vitro-differentiated insulin B-cells and immature B-cells by measuring the level of positive f3-like cells then the cell, culture, cell line, tissue, or expression of the gene in a cell, culture, cell line, tissue, or population of cells comprises immature B-cells population of cells (e.g., Suspected of being B-cells), wherein if the level of expression of the gene in the cell, culture, cell 0102. In some aspects, a marker of B-cell functional line, tissue, or population of cells is similar to the level of immaturity includes one, any combination or Sub-combina expression in mature B-cells then the cell, culture, cell line, tion, or all genes which are have a higher expression level in tissue, or population of cells comprises mature B-cells, and fetal B-cells or in vitro-differentiated insulin-positive B-like wherein if the level of expression of the gene in the cell, cells compared to mature B-cells. culture, cell line, tissue, or population of cells is similar to the 0103) In some embodiments of this and other aspects of level of expression in in vitro-differentiated B-cells, then the the invention, genes which have a higher expression level in cell, culture, cell line, tissue, or population of cells comprises fetal B-cells compared to mature B-cells are listed in Table 3 immature B-cells. (Table 3 includes both Tables 3A and 3B) 0101 Those skilled in the art will also appreciate that any two or more of the genes listed in Table 2C can be used in TABLE 3A combinations of up to N genes (where N is a positive integer greater than or equal to 2) as markers for distinguishing mature B-cells from immature B-cells by measuring the levels Genes having higher expression levels in fetal-cells of expression of the combination of genes in a cell, culture, compared to mature f-cells cell line, tissue, or population of cells (e.g., Suspected of being B-cells), wherein if the levels of expression of the combina SOX4, LZTR1, CSRNP3, HHEX, PROX1, RFX1, SOX8, ZNF423, tion of genes in the cell, culture, cell line, tissue, or population LHX4, SOX11, ISX, NFIB, PAX4, LMO4, NOTCH1, ASCL2, TCF3, of cells are similar to the levels of expression of the same RCOR2, EGR1, FOS, MYCNEVI1, LZTS1, GHRL, PYY, HNF1A, combination of genes in mature B-cells then the cell, culture, INSM1, IRX2, ISL1, MYT1, NEUROG3, ACSS1, GCK, HCN3, cell line, tissue, or population of cells comprises mature KCNJ4, COL1A1, FFAR1 B-cells, and wherein if the levels of expression of the combi nation of genes in the cell, culture, cell line, tissue, or popu

US 2014/03297.04 A1 Nov. 6, 2014 37

TABLE 3B-continued Genes having higher expression levels in fetal B-cells compared to mature f-cells

0104. It is to be understood that the levels of expression of genes in the cell, culture, cell line, tissue, or population of one or more of the genes listed in Table 3 are depicted in FIG. cells is elevated (for example, as depicted in FIG. 6C), the 6C as a fold change in expression of mature B-cells compared cell, culture, cell line, tissue, or population of cells comprises to fetal B-cells. mature B-cells. 0105. It should also be appreciated that any gene listed in 0107. In some embodiments of this and other aspects of Table 3 can be used as a marker for detecting immature B-cells the invention, genes having higher expression levels in in or fetal B-cells by measuring the level of expression of the vitro-differentiated insulin-positive B-like cells compared to gene in a cell, culture, cell line, tissue, or population of cells mature f3-cells are listed in Table 4 (Table 4 includes both (e.g., suspected of being B-cells), wherein if the level of Table 4A and Table 4B). expression of the gene in the cell, culture, cell line, tissue, or population of cells is elevated (for example, as depicted in TABLE 4A FIG. 6C), the cell, culture, cell line, tissue, or population of cells comprises immature B-cells or fetal B-cells. Genes having higher expression levels in vitro-differentiated 0106 Those skilled in the art will also appreciate that any insulin-positive f3-like cells compared to mature f-cells two or more of the genes listed in Table 3 can be used in ZNF217, ISX, PBX2, HNF4G, ELF4, CEBPA, RCOR2, MSL3L1, combinations of up to N genes (where N is a positive integer EGR1, TCF3, LZTS1, TGIF2, HES6, AEBP1, LMO4, SOX11, greater than or equal to 2) as markers for detecting immature IRX2, FEV, NR2F1, CHGA, GAST, GCG, GHRL, NTS, PYY, ARX, FOXA1, FOXA3, GATA4, IRX2, ISL1, NOTCH1, ONECUT2, B-cells or fetal f-cells by measuring the levels of expression PAX4, PROX1, SOX4, ACOX2, RIMS3, SLC2A1, SLC2A3, of the combination of genes in a cell, culture, cell line, tissue, CACNA1E, HCN3, KCNB1, KCNJ2, DLL3, DPP4, HHEX, FOXA1 or population of cells (e.g., Suspected of being fB-cells), wherein if the levels of expression of the combination of TABLE 4B Genes having higher expression levels in vitro-differentiated insulin-positive-like cells compared to mature f-cells 7SK, A1CF, A2M-AS1, A2ML1-AS2, A2MP1, A3GALT2P, A4GALT, AAA1, AACS, AACSP1, AAMP, AASS, ABO15752.4, ABC7-423898OON19.1, ABCA11 P. ABCA12, ABCA4, ABCA7, ABCB10, ABCC1, ABCC10, ABCC4, ABCC6, ABCC6P1, ABCC6P2, ABCD1, ABCD4, ABCG4, ABCG5, ABHD11, ABHD11 AS2, ABHD12B, ABHD14A, ABHD14A-ACY1, ABHD15, ABHD9, ABL1, ABLIM2, ABLIM3, ABO, ABT1P1, ABTB1, ABTB2, AC000032.2, AC000078.5, ACOOO120.7, AC002066.1, AC002116.7, AC002310.17, AC002310.7, ACOO2365.1, ACOO2398.5, ACOO2472.11, ACOO2472.13, ACOO2477.1, ACOO2480.3, ACOO2480.5, ACOO2539.1, AC003090.1, AC003102.3, AC003664.1, ACOO3988.1, AC004053.1, AC004057.1, AC004152.5, AC004158.2, AC004158.3, AC0041604, AC004166.7, ACOO4221.2, AC004231.2, AC004381.6, AC004386.4, AC004448.5, ACOO4449.6, AC004475.1, AC004538.3, ACOO4540.5, AC004543.2, ACOO4593.2, ACOO4623.2, ACOO4799.3, AC004837.4, ACOO4840.9, AC004862.6, AC004893.10, AC004893.11, ACOO48954, AC004906.3, AC004947.2, AC004957.5, AC004967.11, AC004967.7, ACOO4980.10, AC004980.7, AC004980.9, AC004985.12, AC004987.8, AC004987.9, ACOO5000.1, ACOO5003.1, ACOO5009.1, AC005009.2, AC005O13.5, AC005019.3, ACOO5029.1, AC005034.3, AC005042.4, AC005071.1, AC005086.4, ACOO5154.5, ACOO5154.8, AC005204.2, ACOO5215.1, AC005256.1, ACOO5262.3, ACOO5300.5, ACOO5306.3, AC005324.6, AC005324.8-001, AC005326.2, AC005329.1, ACOO5329.7, AC00533.0.2, AC005391.3, AC005488.11, ACOO5513.1, ACOO5519.4, ACOO5534.6, AC005540.3, AC005562.2, ACOO5592.1, ACOO5592.3, ACOO5609.1, ACOO5625.1, AC005682.5, AC005682.6, AC005702.3, AC005702.4, ACOO5754.1, ACOO5757.6, ACOO5775.2, AC005841.1, ACOO5943-4, ACOO5943.5, ACOO5944.2, ACOO5971.3, ACOO60.00.5, ACOO6003.3, ACOO6014.8, AC006023.8, ACOO6028.10, ACO06042.7, AC006111.1, AC006116.1, AC006116.20, AC006132.1, AC006156.1, AC006160.5, ACOO6370.2, ACOO6372.1, ACOO6372.4, ACOO6372.5, ACOO6445.7, ACOO6465.4, ACOO6509.6, ACOO6547.14, AC006547.8, ACOO6946.15, ACOO7000.10, AC007000.12, AC007256.5, ACOO7285.7, ACOO7308.6, ACOO7383.3, ACOO7387.2, ACOO7391.2, ACOO7395.4, AC0074.01.1, AC007403.3, ACOO7464.1, ACOO7551.2, ACOO7557.1, ACOO7557.3, ACOO7563.3, ACOO7563.4, ACOO7563.5, ACOO7875.2, ACOO78814, ACOO7969.4, ACO08063.2, ACO08073.7, ACO08073.9, ACO08088.4, AC008103.5, ACOO8278.3, ACOO8440.10, ACOO8674.1, ACOO8676.1, ACOO8687.1, ACOO8697.1, ACOO8746.3, AC008993.2, AC008993.5, ACOO9065.1, ACOO9110.1, AC009120.5, ACOO9133.12, ACOO9133.15, ACOO9133.20, ACOO9227.2, ACOO9264.1, ACOO9336.24, ACOO948.0.3, ACOO9492.1, ACOO9505.2, US 2014/03297.04 A1 Nov. 6, 2014 38

TABLE 4B-continued Genes having higher expression levels in vitro-differentiated insulin-positive-like cells compared to mature f-cells ACOO9542.2, AC009945.4, AC009955.8, AC009963.5, AC009965.2, AC010.095.7, ACO10127.3, ACO10132.11, ACO10148.1, ACO 10325.1, AC010336.1, ACO10492.2, ACO104924, ACO10507.5, ACO 10525.6, ACO 10536.2, ACO10615.1, AC010620.1, ACO10649.1, ACO10677.5, ACO10729.1, ACO10731.2, ACO10746.4, ACO10761.10, ACO10761.13, AC010761.9, ACO10894.3, ACO10894.4, ACO10904.1, AC010976.2, ACO10982.2, ACO10997.1, ACO11290.4, ACO 11306.2, ACO11343.1, ACO 11385.2, ACO11443.1, ACO11450.1, ACO11475.1, ACO11484.1, ACO11491.1, ACO11497.1, ACO11551.1, ACO11558.5, ACO 11718.1, ACO 11742.3, ACO11899.9, ACO12123.1, ACO12358.4, ACO12358.7, ACO12363.7, ACO12368.1, ACO12445.1, ACO12485.2, ACO12499.1, ACO125064, ACO 12668.2, ACO 13283.1, ACO13400.2, ACO13402.2, ACO13439.4, ACO13460.1, ACO13472.3, ACO13472.4, ACO15691.13, ACO15815.2, ACO15815.6, ACO15818.11, ACO15818.6, ACO15842.1, ACO15849.2, ACO15884.1, ACO16405.1, ACO16670.1, ACO16683.5, ACO16700.5, ACO16708.2, ACO16712.2, ACO16716.2, ACO16722.1, ACO16722.2, ACO167234, ACO16725.4, ACO16757.3, ACO16773.1, ACO16894.1, ACO1711.6.8, ACO18359.1, AC018638.1, AC018648.5, ACO18693.6, ACO18705.5, ACO18737.6, AC018738.2, ACO18799.1, ACO18804.1, ACO18804.6, AC018865.5, ACO18865.8, ACO18865.9, AC018892.9, ACO19068.2, ACO19070.1, ACO19100.3, ACO19118.2, ACO191854, ACO19205.1, AC020550.7, AC020743.3, ACO20907.3, ACO20915.1, ACO21860.1, ACO22007.4, ACO22007.5, ACO22182.1, ACO22182.2, ACO22210.2, ACO22400.1, ACO22498.1, ACO22532.1, ACO22819.2, ACO23480.1, ACO23490.1, ACO23490.2, ACO23590.1, ACO24162.2, ACO24475.1, ACO24580.1, ACO24896.1, ACO24937.2, ACO24937.4, ACO24937.6, ACO24940.1, ACO25165.1, ACO25165.8, ACO25171.1, ACO26956.1, ACO27119.1, ACO27307.1, AC027601.1, ACO27612.1, ACO37445.1, AC0483824, AC051649.6, ACO55764.1, ACO55811.5, ACO60834.2, ACO60834.3, ACO61961.2, ACO61975. 1, ACO61992.2, AC062016.1, ACO63976.7, ACO64836.3, ACO64852.5, ACO66593.1, ACO68O14.1, ACO68020.1, ACO68129.2, ACO68134.10, ACO68137.1, ACO68499.6, ACO68522.4, ACO68535.3, ACO68580.6, ACO68587.2, AC068641.1, ACO68718.1, ACO68831.10, ACO68831.3, ACO68831.6, ACO69277.2, ACO69278.4, AC069294.1, ACO69513.4, ACO72052.7, AC073052.1, AC073130.3, AC073133.2, AC073236.3, AC073283.4, AC073342.12, AC073415.2, AC073850.6, AC073869.2, AC073869.20, AC073957.15, ACO74019.2, AC074212.5, ACO74212.6, ACO74289.1, AC074363.1, ACO74389.6, ACO78842.3, ACO78883.3, ACO7894.1.1, ACO78942.1, ACO79233.1, ACO79354.5, ACO79586.1, ACO7961.0.2, ACO79753.1, ACO79776.1, ACO79776.3, ACO79780.3, ACO79781.5, ACO79781.8, ACO79790.2, ACO798.07.2, ACO80091.1, ACO8.0125.1, ACO83855.4, ACO83862.6, ACO83949.1, ACO84018.1, ACO84117.3, ACO84125.4, ACO87289.1, ACO87650.1, AC090103.1, AC090519.5, ACO90559.1, ACO90587.5, AC090602.2, ACO90627.1, ACO91038.1, ACO91153.4, ACO91633.3, ACO91878.1, ACO92106.1, ACO92159.2, ACO92171.1, ACO921714, ACO922.91.1, ACO923.29.1, ACO92338.5, ACO92535.1, ACO92535.3, ACO92619.1, ACO92620.3, ACO92638.2, ACO92641.2, ACO92675.3, ACO92798.1, ACO92839.1, ACO92839.4, ACO92902.1, AC093106.5, AC093107.7, ACO93159.1, ACO93375.1, ACO934.15.3, ACO93415.5, ACO93609.1, ACO93616.4, ACO93627.10, ACO93627.6, ACO93668.3, ACO.93690.1, ACO93724.2, ACO93734.11, ACO93734.13, ACO93818.1, ACO93838.4, ACO93838.7, AC096574.5, ACO96669.2, ACO97374.2, ACO97468.4, ACO97499.2, ACO97635.4, ACO97721.1, ACO98474.1, ACO98784.2, ACO98826.5, ACO99489.1, AC099668.5, AC10O830.3, AC10O830.5, AC10O852.2, AC103828.1, AC103965. 1, AC104113.3, AC104131.1, AC104532.2, AC1045324, AC104655.3, AC104809.3, AC105052.1, AC105339.1, AC106782. 18, AC106782.20, AC106801.1, AC106869.2, AC107057.2, AC108025.2, AC108039.2, AC108142.1, AC108479.2, AC109333.10, AC109642.1, AC109828.1, AC110079.1, AC110373.1, AC110619.1, AC110619.2, AC110781.3, AC110781.5, AC110926.4, AC112211.2, AC112211.3, AC112229.1, AC114273.1, AC114730.11, AC114730.8,AC115618.1, AC116609.2, AC116614.1, AC117372.1, AC118138.2, AC123023.1, AC124057.5, AC125232.1, AC125238.2, AC125634.1, AC126365.4, AC127496.1, AC130888.1, AC131180.3, AC131180.4, AC131971.3, AC132186.1, AC132216.1, AC133633.1, AC133919.6, AC135048.13, AC136704.1, AC137723.5, AC137934.1, AC138035.2, AC138623.1, AC138649. 1, AC139100.3, AC139530.1, AC1398874, AC140061.2, AC140061.8, AC140542.2, AC141586.5, AC141928.1, AC142528.1, AC145124.1, AC147651.2, AC15954.0.2, AC226118.1, ACAA1, ACAP3, ACBD4, ACBD7, ACCN2, ACCN4, ACE, ACER1, ACHE, ACIN1, ACOT11, ACOT7, ACOX2, ACOX3, ACP2, ACPT, ACR, ACSF2, ACSS3, ACTG1P1, ACTL6B, ACTL7B, ACTN3, ACTN4P2, ACTR1A, ACTR1B, ACTR3P2, ACVR1C, ACVR2A, ACVR2B, ACVR2B-AS1, ADOOOO90.2, ADOOO864.1, ADOOO864.5, ADAD2, ADAM11, ADAM12, ADAM19, ADAM2, ADAM23, ADAM33, ADAM7, ADAM8, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS9-AS1, ADAMTS9-AS2, ADAMTSL1, ADAMTSL3, ADAMTSL4, ADAMTSL5, ADAP1, ADARB2, ADCK1, ADCY2, ADCY3, ADCY5, ADCY6, ADCY8, ADCYAP1R1, ADD1, ADD2, ADH1A, ADH1B, ADH5P3, ADH6, ADORA2A, ADRA2B, ADRA2C, ADRB1, ADRBK1, ADSL, AEOOO658.27, AEOOO659.23, AEBP1, AES, AFO13593.1, AFO38458.5, AFO64858.6, AFO64863.1, AF121898.1, AF131217.1, AF1461914, AF165138.7, AF196970.3, AF235103.1, AF238380.3, AF238380.5, AFAP1, AFAP1L1, AFAP1L2, AFF2, AFF3, AFG3L2P1, AFMID, AFP, AGAP11,

US 2014/03297.04 A1 Nov. 6, 2014 56

0108. It is to be understood that the levels of expression of genes listed in Table 3 or Table 4 in the B-cell or the popula one or more of the genes listed in Table 4 are depicted in FIG. tion of B-cells compared to the levels of expression of the 5C as a fold change in expression of in vitro-differentiated same one or more genes in mature B-cells. insulin-positive B-like cells compared to mature B-cells. 0.115. In some aspects, the present invention provides a 0109. It should also be appreciated that any gene listed in method of identifying fetal B-cells. An exemplary method of Table 4 can be used as a marker for detecting immature B-cells identifying fetal B-cells comprises (a) obtaining a putative or in vitro-differentiated insulin-positive f3-like cells by mea B-cellor a population of putative f3-cells; and (b) detecting an Suring the level of expression of the gene in a cell, culture, cell expression level in the f-cell or the population of B-cells of line, tissue, or population of cells (e.g., Suspected of being one or more genes listed in Table 3, wherein: (i) an elevated B-cells), wherein if the level of expression of the gene in the level of expression of one or more genes listed in Table 3 in cell, culture, cell line, tissue, or population of cells is elevated the f-cell or the population of B-cells indicates that the B-cell (for example, as depicted in FIG.5C), the cell, culture, cell or the population of B-cells are fetal B-cells. line, tissue, or population of cells comprises immature B-cells 0116. In some embodiments of this and other aspects of or in vitro-differentiated insulin-positive B-like cells. the invention, the presence of elevated levels expression com 0110 Those skilled in the art will also appreciate that any prises at least a 2 fold increase, a 3 fold increase, a 4 fold two or more of the genes listed in Table 4 can be used in increase, a 5 fold increase, or N-fold increase (where N is a combinations of up to N genes (where N is a positive integer positive integer) in the levels of expression of the one or more greater than or equal to 2) as markers for detecting immature genes listed in Table 3 in the f-cell or the population of B-cells B-cells or in vitro-differentiated insulin-positive f3-like cells compared to the levels of expression of the same one or more by measuring the levels of expression of the combination of genes in mature B-cells. genes in a cell, culture, cell line, tissue, or population of cells 0117. In some aspects, the present invention provides a (e.g., Suspected of being B-cells), wherein if the levels of method of identifying in vitro-differentiated insulin-positive expression of the combination of genes in the cell, culture, (3-like cells. An exemplary method of identifying in vitro cell line, tissue, or population of cells is elevated (for differentiated insulin-positive B-like cells, comprises: (a) example, as depicted in FIG.5C), the cell, culture, cell line, obtaining a putative B-cell or the population of putative tissue, or population of cells comprises immature B-cells or in B-cells; and (b) detecting an expression level in the B-cell or vitro-differentiated insulin-positive B-like cells. the population of B-cells of one or more genes listed in Table 0111. In some aspects, the present invention provides a 4, wherein: (i) an elevated level of expression of one or more method of identifying the functional maturity of B-cells. An genes listed in Table 4 in the B-cellor the population of B-cells exemplary method of identifying the functional maturity of indicates that the f-cell or the population of B-cells are in B-cells comprises (a) obtaining a putative B-cell or a popula vitro-differentiated insulin-positive B-like cells. tion of putative B-cells; and (b) detecting an expression level 0118. In some embodiments of this and other aspects of in the B-cell or the population off-cells of one or more genes the invention, the presence of elevated levels expression com listed in Table 1, Table 2, Table 3 or Table 4, wherein: (i) an prises at least a 2 fold increase, a 3 fold increase, a 4 fold elevated level of expression of one or more genes listed in increase, a 5 fold increase, or N-fold increase (where N is a Table 1 or Table 2 in the B-cell or the population of B-cells positive integer) in the levels of expression of the one or more indicates that the B-cell or the population of B-cells are func genes listed in Table 4 in the f-cell or the population of B-cells tionally mature f3-cells; and (ii) an elevated level of expres compared to the levels of expression of the same one or more sion of one or more genes listed in Table 3 or Table 4 in the genes in mature B-cells. B-cell or the population of B-cells indicates that the B-cell or 0119. In some embodiments of this and other aspects of the population of B-cells are functionally immature B-cells. the invention, the elevated levels expression of the one or 0112 In some aspects, the present invention provides a more genes listed in Table 3 and Table 4 are depicted in FIG. method of identifying mature B-cells. An exemplary method 5C. Those skilled in the art will appreciate how to interpret the of identifying mature B-cells comprises (a) obtaining a puta negative relative expression levels depicted in FIG. 5C as tive B-cell or a population of putative B-cells; and (b) detect positive levels of elevated expression. ing an expression level in the B-cell or the population of I0120 In some embodiments of this and other aspects of B-cells of one or more genes listed in Table 1 or Table 2, the invention, the elevated levels expression of the one or wherein: (i) an elevated level of expression of one or more more genes listed in Tables 2 and 4 are depicted in FIG. 5C. genes listed in Table 1 or Table 2 in the B-cell or the popula Those skilled in the art will appreciate how to interpret the tion of B-cells indicates that the B-cell or the population of negative relative expression levels depicted in FIG. 5C as B-cells are mature B-cells. positive levels of elevated expression. 0113. In some embodiments of this and other aspects of 0121. In some aspects, an exemplary method of distin the invention, the presence of elevated levels expression com guishing mature and immature B-cells comprises: (a) obtain prises at least a 2 fold increase, a 3 fold increase, a 4 fold ing a putative B-cell or a population of putative B-cells; (b) increase, a 5 fold increase, or N-fold increase (where N is a measuring expression of a group of genes in the B-cell or the positive integer) in the levels of expression of the one or more population of B-cells to produce an expression profile of the genes listed in Table 1 or Table 2 in the (3-cell or the popu B-cell or the population of B-cells; (c) comparing the expres lation of B-cells compared to the levels of expression of the sion profile of the f-cell or the population of B-cells to any or same one or more genes in immature B-cells. all of: (i) a reference mature B-cell expression profile selected 0114. In some embodiments of this and other aspects of from the group consisting of a first group of genes having the invention, the presence of elevated levels expression com higher expression levels in mature B-cells compared to fetal prises at least a 2 fold increase, a 3 fold increase, a 4 fold B-cells, wherein the first group of genes is selected from the increase, a 5 fold increase, or N-fold increase (where N is a group consisting of STAT4, NPAS2, STAT3, NPAS2, STAT3, positive integer) in the levels of expression of the one or more PBX3, NR3C2, DDIT3, SIX4, ETV5, SIX2, TP53, BCL6,