CD99r as marker for alpha and beta cells

Abstract

A method for enriching glucagon producing alpha cells and / or insulin producing beta cells in a mixture of cells, comprising labelling the cell surface marker CD99R in a starting mixture of cells with a label and separating the cells that are positive for the cell surface marker from cells that are negative for the cell surface marker to obtain a final mixture of cells that is more enriched in alpha cells and / or beta cells compared to the starting mixture. A use of an antibody specific for CD99R for detecting glucagon producing alpha cells and / or insulin producing beta cells.

Description

[0001]CD99R AS MARKER FOR ALPHA AND BETA CELLS TECHNICAL FIELDThe present invention relates to the field of cell culture and cell therapy, more particularly tomethods of enriching and analyzing cultures of glucagon producing alpha cells and insulin producing beta cells. BACKGROUND TO THE INVENTION Type 1 diabetes is an autoimmune disease which leads to the destruction of insulin producing beta cells. Beta cells play a crucial role in maintaining glucose homeostasis by producing and secreting hormone insulin in response to blood glucose levels. Loss of beta cells in type 1 diabetes causes insulin deficiency which results in hyperglycemia. Patients with type 1 diabetes are primarily dependent on exogenous insulin injections to manage their blood glucose levels. However, exogenous insulin injections do not provide a solution for physiological glucose homeostasis. Previous studies have shown that pancreatic islet transplantations provide better glycemic regulation and insulin independence in type 1 diabetic patients. However, the lack of donors, procurement of pancreas, yield of islets andimmune rejection are major limitations with the islet transplantation (Shapiro et al., 2017).An alternative approach to overcome these limitations is the use of human embryonic stemcell (hESC) derived or induced pluripotent stem cell (iPSC) derived islet cells including insulinsecreting beta cells and glucagon secreting alpha cells. Although differentiation protocols have been developed by several labs to generate stem cell derived islet cells, the compositionand purity of stem cell derived islet cells remains a challenge in all differentiation protocolsand, as an additional complication, is cell line dependent. Stem cell derived islets, from mostof the protocols, consists of 30% to 50% unwanted / unknown cells apart from the beta andalpha cells (Balboa et al., 2022; Pagliuca et al., 2014; Rezania et al., 2014; Velazco-Cruz et al.,2019). In a cell therapy product, it is important to not only know which cell types are present but also to reach as high purity of the therapeutically beneficial cell type as possible for the best therapeutic potential. Unwanted cells may be detrimental to the intended islet function and at worst may have the potential risk of teratoma formation after transplantation.Therefore, strategies to get rid of these unwanted cells are important for clinical applications.The Xg blood group protein CD99 has been described as human beta cell surface marker andused to purify human pancreas fractions with magnetic beads (Martens et al., 2018). In thecontext of human embryonic stem cell differentiation, CD99 has been used as a marker fordefinitive endoderm stage (EP4039798 A1). These studies relate to the more broadlyexpressed CD99 isoform. Previous studies have demonstrated that CD99R isoform expression is very specific and restricted to subsets of T cells, NK cells and monocytes. In contrast, the CD99 isoform isexpressed broadly in several cell types (Bernard et al., 1984; Gelin et al., 1991; Lanza et al.,1994).CD49a is a well-known cell surface marker for cell stem derived beta cells. A previous studyhas demonstrated that stem cell derived beta cells can be enriched by CD49a positive cellsorting. CD49a sorted islets consist of up to 80% beta cells and around 5% enterochromaffincells. However, the limitation with CD49a enriched islets is that they are deficient in glucagonproducing alpha cells (Veres et al., 2019).An object of the present invention is the provision of alternative and / or improved methodsand / or means for enriching glucagon producing alpha cells and / or insulin producing beta cellsand analyzing populations of such cells.DEFINITIONSThe term glucagon producing alpha cells refers to a type of cells normally found in the isletsof Langerhans, responsible for the production of glucagon, a peptide hormone regulatingglucose homeostasis. Glucagon production starts with preproglucagon, which contains asignal peptide removed by signal peptidase, thus forming proglucagon (160 aa in humans).Proglucagon is converted by proprotein convertase 2 to glucagon (amino acids 33-61 ofpreproglucagon). Further products derived from proglucagon produced in intestinal L cellsinclude proglucagon is cleaved to the alternate products glicentin (amino acids 1–69 ofproglucagon), glicentin-related pancreatic polypeptide (1–30), oxyntomodulin (33–69), glucagon-like peptide 1 (72–107 or 108), and glucagon-like peptide 2 (126–158). In the present context, the term preferably encompasses all cells producing glucagon, including mature and immature forms of glucagon-producing cells.The term insulin producing beta cells refers to refers to a type of cells normally found in theislets of Langerhans, responsible for the production of insulin. Insulin is produced as pre-proinsulin, which is processed post-translationally to proinsulin and then to insulin. Mature insulin contains disulfide-linked A and B chain fragments of proinsulin. In the processing from proinsulin to insulin, insulin C-peptide, which is not hormonal but can be useful as marker of insulin secretion or production, is released. In the present context, the term preferably encompasses all cells producing mature insulin. In preferred embodiments, the term may alsoencompass cells that are immature forms of beta-cells that produce insulin although they donot yet process it to mature insulin and may or may not be glucose-responsive. In most preferred embodiments, the term encompasses cells that are glucose-responsive in the sense of being capable of secreting insulin in response to challenge with 20 mM glucose.The term CD99 refers to the Xg blood group protein encoded by the CD99 gene with theGenBank Gene ID 4267 (as of 2024-01-19). According to the GenBank RefSeq entry (Mar 2016)associated with this gene, the protein is a cell surface glycoprotein involved in leukocytemigration, T-cell adhesion, ganglioside GM1 and transmembrane protein transport, and T-cell death by a caspase-independent pathway. In addition, the encoded protein may have the ability to rearrange the actin cytoskeleton and may also act as an oncosuppressor inosteosarcoma. The human gene is found in the pseudoautosomal region of chromosomes Xand Y and escapes X-chromosome inactivation. There is a related pseudogene locatedimmediately adjacent to this locus. A total of 7 transcripts have been identified originatingfrom the CD99 gene. The protein has been alternatively designated as MIC2, HBA71, MIC2X,MIC2Y and MSK5X in the literature. According to the Human Protein Atlas (online as of 2024-01-19), CD99 the tissue RNA expression has low tissue specificity, and protein expression tissue profile is described as being “Membranous expression most abundant in islets of Langerhans, cells in seminiferous ducts and subsets of lymphoid cells.”The term CD99R refers to a particular isoform of CD99 characterized by a particular epitopehaving a restricted tissue distribution. The unique tissue-restricted epitope of CD99R wasdiscovered when it was noticed that different monoclonal antibodies to CD99 detecteddifferent CD99 isoforms having different tissue distributions (Gelin et al., 1991). CD99R maybe characterized by that it is a CD99 protein comprising an epitope expressed on hematopoietic progenitors, thymocytes, a subset of T cells (such as approximately 30-42% ofCD4+ cells and 15-23% of CD8+ cells) and NK cells, but not on B cells, erythrocytes or platelets.The term “comprising” is to be interpreted as including, but not being limited to. Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value. All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 to 10” is inclusive of the endpoints, 2 and 10, and all the intermediate values). The term “about” can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, “about” also discloses the range defined by the absolute values of the two endpoints, e.g. “about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Schematic diagram showing stepwise differentiation of human pluripotent stemcells into pancreatic islets (A). Flow cytometry plot demonstrating percentage of beta (INS+),alpha (GCG+) and non-alpha / non-beta (INS- / GCG-) cells in stem cell derived islets (B).Figure 2. Graph demonstrating percentages of insulin and glucagon positive cells in positive fraction of different cell surface markers (A). Graph showing percentage of non-alpha / non-beta cells in positive and negation fraction of different cell surface markers (B). CD49a andCD99 are indicated for reference.Figure 3. Flow cytometry plot showing beta, alpha and non-alpha / non-beta (INS- / GCG-) cellsin stem cell derived islets (A), Isotype control for CD99R (B), CD99R positive cell population (C), CD99R expression in insulin positive cells (D) and glucagon positive cells (E). Flowjo analysis showing different predicted cell populations in CD99R positive fraction (F). CD99R antibody used here was from BD Biosciences (Clone HIT4).Figure 4. Reference flow cytometry plot showing beta (insulin+), alpha (glucagon+) and non-alpha / non-beta cells in stem cell derived islets (A), Reference Flowjo analysis showing different predicted cell populations in CD99 positive fraction (B).Figure 5. Flow cytometry plot showing beta (insulin+), alpha (glucagon+) and non-alpha / non-beta cells in stem cell derived islets (A), CD99R positive cell population (B). Flowjo analysisshowing different predicted cell populations in CD99R positive fraction (C). Figure 6. Flow cytometry plot showing beta (insulin+), alpha (glucagon+) and non-alpha / non- beta cells in stem cell derived islets (A), CD99R expression in insulin positive cells (B) and glucagon positive cells (C). Reference flow cytometry plot showing beta, alpha and non-alpha / non-beta cells in stem cell derived islets (D), CD49a expression in insulin positive cells(E) and glucagon positive cells (F). Figure 7. Flow cytometry plot showing CD99R positive fraction before sorting in stem cell derived islets (A). CD99R positive fraction after sorting (B). CD99R negative fraction after sorting (C). CD99R sorted and reaggregated islets at the end of stage 7 (D). Bar graph summarizing CD99R positive and negative fraction before and after sorting (E). Figure 8. Flow cytometry plot showing percentage of beta, alpha and non-alpha / non-beta cells in stem cell derived unsorted islets (A). Percentage of beta, alpha and non-alpha / non-beta cells in CD99R sorted islets (B). Bar graph summarizing percentage of beta, alpha andnon-alpha / non-beta cells in unsorted and CD99R sorted islets (C). Bar graph showing data ofIn-vitro glucose stimulated insulin secretion assay for unsorted and CD99R sorted islets (D).Figure 9. Single cell transcriptome analysis of sorted stem cell derived islets. Uniform ManifoldApproximation and Projection (UMAP) demonstrating Seurat clusters of sorted stem cellderived islets. Figure 10. Bar graph summarizing data for 5 independent experiments (including experiments in Fig 8C, D but with additional replicates) for the percentage of beta, alpha and non-alpha / non-beta cells in unsorted and CD99R sorted islets (A). Bar graph summarizing data for5 independent experiments for In-vitro glucose stimulated insulin secretion assay forunsorted and CD99R sorted islets (B). Error bars denote standard error of the mean (SEM).Figure 11. Flow cytometry plot showing beta (insulin+), alpha (glucagon+) and non-alpha / non-beta cells (A), CD99R expression in insulin positive cells (B) and glucagon positive cells (C) instem cell derived islets generated using the Velazco-Cruz et al 2019 protocol. SUMMARY OF THE INVENTION The present invention relates to the use of CD99R as marker for identification and enrichment of glucagon producing alpha cells and / or insulin producing beta cells in a mixture of cells. The inventors discovered that a cell surface marker CD99R is selectively expressed in stem cell derived beta and alpha cells, but not in unwanted non-alpha / non-beta cells (see Example 2). The CD99R as a marker is a particularly useful tool in the generation of alpha or beta cellsfrom pluripotent stem cells for e.g. cell therapy, in vivo studies or in vitro applications such asdrug screening. Magnetic bead sorting with CD99R is effective in depleting unwanted cells from the stem cell derived islets and was able to generate stem cell derived islets up to 90%purity (see Example 3). The novel marker CD99R resulted in markedly lower percentage ofnon-alpha / non-beta cells in the enriched fraction compared to the known marker CD49a orthe major CD99 isoform (Fig 2). The CD99R-enriched islets have both better purity and glucoseresponsiveness compared to non-sorted cells (see Example 4). Example 5 demonstrates utilityof the CD99R marker irrespective of differentiation method (see Fig 11).The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims. 1. A method for enriching glucagon producing alpha cells and / or insulin producing betacells in a mixture of cells, comprising: a. labeling a cell surface marker in a starting mixture of cells with a label; andb. separating the cells that are positive for the cell surface marker from cells thatare negative for the cell surface marker to obtain a final mixture of cells that is more enriched in alpha cells and / or beta cells compared to the starting mixture; wherein said cell surface marker is CD99R. 2. The method of item 1, wherein the label is an antibody specific to CD99R, optionallycomprising an affinity tag.3. The method of any one of the preceding items, wherein the separation step isperformed using beads having affinity to the label to separate the cells positive for the cell surface marker, preferably magnetic beads.4. The method of item 3, wherein the separation step involves separating the beads fromthe unbound cells by gravity, centrifugation, filtration or magnetism, preferably magnetism.5. The method according to any one of items 1-2, wherein the separating step isperformed using a fluorescence-activated cell sorter.6. The method of any one of the preceding items, wherein the mixture of cells is a culturederived in vitro from human pluripotent stem cells.7. The method of any one of the preceding items, wherein the enrichment results in atleast 3-fold reduction in the fraction of cells other than alpha cells and / or beta cells, preferably at least 5-fold reduction, more preferably at least 7-fold reduction, most preferably at least 10-fold reduction.8. A method for obtaining glucagon producing alpha cells and / or insulin producing betacells comprising: a. Providing human pluripotent stem cells;b. Differentiating the pluripotent stem cells in vitro to obtain a population of cellscomprising alpha cells and / or beta cells; c. Enriching the alpha cells and / or beta cells using the method of item 1 or anyitem dependent thereon.9. The method of item 8, wherein the enriched cells are cultured further in vitro after theenrichment step.10. A method of treatment of a subject in need thereof, comprising:a. Obtaining alpha islet cells and / or beta islet cells with the method of item 8 orany item dependent thereon; b. Transplanting the cells to a subject in need thereof.11. The method according to item 10, wherein the subject suffers from type I diabetes.12. A cell population obtainable by the method of any one of the preceding items.13. A method for analyzing an in vitro cell culture comprising glucagon producing alphacells, insulin producing beta cells and / or other cells, comprising quantitating the number or fraction of cells expressing CD99R, wherein expression of CD99R is indicative of the presence of alpha islet cells and / or beta islet cells. 14. The method of item 13, wherein the cell culture is a culture derived in vitro fromhuman pluripotent stem cells. 15. The method of any one of items 13-14, wherein the quantitation is performed by flowcytometry using an antibody specific for CD99R. 16. A use of an antibody specific for CD99R for detecting glucagon producing alpha cellsand / or insulin producing beta cells. The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other. All references are hereby incorporated by reference. DETAILED DESCRIPTION Methods for enriching alpha or beta cells In a first aspect, the present invention provides a method for enriching glucagon producing alpha cells and / or insulin producing beta cells in a mixture of cells, comprising: a. labelling a cell surface marker in a starting mixture of cells with a label; andb. separating the cells that are positive for the cell surface marker (i.e. positivefor the label) from cells that are negative for the cell surface marker (i.e. negative for the label) to obtain a final mixture of cells that is more enriched in glucagon producing alpha cells and / or insulin producing beta cells (preferably both) compared to thestarting mixture; wherein said cell surface marker is CD99R. Preferably, both glucagon producing alpha cells and insulin producing beta cells are enriched simultaneously. The label may be an antibody specific to CD99R. The label can also be an affibody, or otheraffinity reagent, such as affimer, aptamer or monobody, specific to CD99R. The label maycomprise an affinity tag and / or a fluorescent tag which may be suitable for Fluorescence-activated cell sorting (FACS) or analysis. In some cases, the same tag may have both affinityand fluorescent label functions. Suitable tags include phycoerythrin (PE), biotin, FITC, greenfluorescent protein (GFP), Alexa-set of fluorescent dyes and the like.When an unlabeled antibody (or other affinity reagent) specific to CD99R is used as the label,it may be bound by a secondary antibody (or other affinity reagent) able specifically bind tothe CD99R label.Monoclonal CD99R-specific antibodies have been described in academic literature (Bernardet al., 1984; Gelin et al., 1991; Lanza et al., 1994; Shen et al., 1986) and are also availablecommercially (e.g. clone REA323, cat. no. 130-104-852, Miltenyi Biotech, clone HIT4, cat. no.251093, Abbiotech, and clone 5F57, cat. No. USBIC2444-17A, avantor VWR). It should benoted that many antibodies to CD99 are likely to detect both the more broadly expressedisoform (CD99) and the isoform comprising the restricted epitope (CD99R), since the twoisoforms share many if not most epitopes. In contrast, the CD99R antibodies by definition only bind to the restricted isoform having the unique CD99R epitope. Additional monoclonal antibodies can be generated with standard techniques including but not limited to: providing and optionally isolating CD99 antigen from a source where CD99R is expressed (such as HPB-ALL human peripheral blood leukemia T-cell line (DSMZ no. ACC483) or PBMCs), immunizingmice with the CD99 antigen, generating monoclonal antibodies from the immunized mice andthen screening the antibodies with respect to antigen reactivity and tissue reactivitydistribution (see CD99 and CD99R in the Definitions section) to select a CD99R-specific clone.The separation step may be performed using beads having affinity to the label to separatethe cells positive for the cell surface marker, preferably magnetic beads. To be clear, thismeans that cells positive for the marker bind to the beads, which are then separated fromunbound cells. The separation step may involve separating the beads from any unbound cellsby gravity, centrifugation, filtration, acoustics or magnetism, preferably magnetism. Use of fluorescence activated cell sorting is also possible.The mixture of cells may be a culture derived in vitro from human pluripotent stem cells, suchas embryonic stem cells or induced pluripotent stem cells. However, the CD99R as marker isenvisioned to be useful in other contexts as well. The enrichment results in at least 2-fold reduction in the fraction of cells other than alpha cells and / or beta cells, preferably at least 3-fold reduction, more preferably at least 5-foldreduction, even more preferably at least 7-fold reduction, most preferably at least 10-foldreduction. In general, any delta-cells present in the mixture of cells are not significantly depleted in theenrichment utilizing CD99R, apart from unavoidable losses from cell handling. It is preferredthat the enrichment step results in retention of at least 50%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the delta-cells. Methods for obtaining alpha or beta cells In a second aspect, the present invention provides a method for obtaining glucagon producing alpha cells and / or insulin producing beta cells comprising: a. Providing human pluripotent stem cells such as embryonic stem cells orinduced pluripotent stem cells; b. Differentiating the pluripotent stem cells in vitro to obtain a population of cellscomprising glucagon producing alpha cells and / or insulin producing beta cells, preferably both; c. Enriching the glucagon producing alpha cells and / or insulin producing betacells (preferably both) using the method of the first aspect.The enriched cells may be cultured further in vitro after the enrichment step. Preferably, bothglucagon producing alpha cells and insulin producing beta cells are obtained simultaneously.The enrichment step may be performed when the cells are still at an immature stage, i.e. before the differentiation is complete. Thus, the differentiation and enrichment steps mayoverlap in time. The cells may be stored frozen at any stage, so the steps (or parts of the steps)may also be separated in time.The present invention is not particularly limited to specific steps of differentiating the cells.Indeed, several suitable differentiation methods have been described in the literature(Velazco-Cruz et al. 2019, Balboa et al. 2022, Rezania et al. 2014, Pagliuca et al. 2014, etc),and analysis / detection / enrichment using CD99R according to the present invention can beapplied regardless of the specific method.For example, the differentiating may include the following substeps:Pluripotent stem cells (e.g. hESC) are differentiated into definitive endoderm cells. Preferably,for the first 24±12h, the cells are treated with Activin A (TGF-β superfamily cytokine) and aGSK-3 inhibitor such as CHIR99021. For the next 48±12h), cells may be treated with Activin A(100ng / ml).The definitive endoderm cells are differentiated into primitive gut tube cells. Preferably,keratinocyte growth factor (KGF) / Fibroblast growth factor 7 (FGF-7) is used (72±24 h).The primitive gut tube cells are differentiated into posterior foregut cells. Preferably, the cellsare cultured (24±12h) in media containing the factors KGF, a hedgehog pathway inhibitor suchas SANT-1, a retinoid acid receptor agonist such as retinoic acid, protein kinase C activatorsuch as PDBu and BMP signaling inhibitor such as LDN193189.The posterior foregut cells are differentiated into pancreatic progenitor cells. Preferably, thecells are cultured (for 72±24 h) in media comprising KGF, EGF, Nicotinamide, activin A, ahedgehog pathway inhibitor such as SANT-1, a retinoid acid receptor agonist such as retinoic acid, protein kinase C activator such as PDBu and BMP signaling inhibitor such as LDN193189.The pancreatic progenitor cells are differentiated into endocrine progenitor cells. Preferably,they are cultured in media containing a hedgehog pathway inhibitor such as SANT-1, aretinoid acid receptor agonist such as retinoic acid, BMP signaling inhibitor such asLDN193189, TGF-β type I receptor kinase (ALK5) inhibitor such as ALK5i II, a thyroid hormonereceptor agonist such as GC1, a Notch pathway inhibitor such as a γ-secretase inhibitor (e.g.GSI-XX) and betacellulin for 5±2 days. At the end of day 4 cells may be dissociated and singlecell suspension prepared. These single cells may be seeded into suspension conditions (e.g.ultra-low attachment plates, spinning flasks, bioreactors or the like) and cultured for anadditional 24±12h in the presence of the above-mentioned factors and a ROCK-inhibitor suchas H1152.The endocrine progenitor cells are differentiated into stem cell derived islets comprisingglucagon producing alpha cells and insulin producing beta cells. The cells may be further cultured (the full differentiation may take 3-5 weeks, but the enrichment may be done sooner) in media containing a thyroid hormone receptor agonist such as GC1, a ROCK-inhibitor such as H1152, antioxidants such as N-acetyl cysteine and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. Needless to say, the use of CD99R marker is applicable to other protocols, as well. Methods of treatment In a third aspect, the present invention provides a method of treatment of a subject in need thereof, comprising: a. Obtaining glucagon producing alpha cells and / or insulin producing beta cellswith the method of the second aspect; b. Transplanting the cells to a subject in need thereof.The subject may suffer from type I diabetes. Preferably, both glucagon producing alpha cells and insulin producing beta cells are obtained simultaneously. In a fourth aspect, the present invention provides a cell population obtainable by the method of the first or the second aspects, comprising glucagon producing alpha cells and / or insulinproducing beta cells. The cell population may be for use in therapy, particularly for thetreatment of type I diabetes via cell transplantation. Use of CD99R for analysis and detection of alpha or beta cellsIn a fifth aspect, the present invention provides a method for analyzing an in vitro cell culturecomprising glucagon producing alpha cells, insulin producing beta cells and / or other cells, comprising quantitating the number or fraction of cells expressing CD99R, wherein expression of CD99R is indicative of the presence of glucagon producing alpha cells and / or insulin producing beta cells (preferably both).The cell culture may be a culture derived in vitro from human pluripotent stem cells, such asembryonic stem cells or induced pluripotent stem cells.The quantitation may be performed by flow cytometry using an antibody or other labelspecific for CD99R, such any label described for the first aspect.In a sixth aspect, the present invention provides a use of an antibody specific for CD99R fordetecting glucagon producing alpha cells and / or insulin producing beta cells (preferably both).The detection may be in a mixture of cells, particularly a population of cells obtained bydifferentiating pluripotent stem cells in vitro to obtain a population of cells comprisingglucagon producing alpha cells and / or insulin producing beta cells. The antibody may be asdescribed for the first aspect. EXAMPLES The following examples are not to be regarded as limiting. For further information on the experimental details, the skilled reader is directed to a separate section titled Materials and Methods. Example 1: Stem cell differentiation into pancreatic islet cells For differentiation, human embryonic stem cells (hESCs) were seeded on to laminin 521 (LN521) coated plates at a density of 17000 cells per cm2and cultured in NutriStem media for96 hours. Differentiation was carried out by following a step wise (6 stages) differentiationprotocol (Figure 1A). At the end of stage 6, stem cell derived islets contain approximately 40%insulin producing beta cells, 19% glucagon producing alpha cells and 33% non-alpha / non-beta cells (Figure 1B). Example 2: CD99R expression is specific to stem cell derived beta and alpha cells Cell surface marker screening (BD Biosciences) was performed to identify specific cell surfacemarkers for insulin and glucagon positive beta and alpha cells. We performed Flowjo analysisto check the proportion of alpha (glucagon positive) and beta cells (insulin positive) within positive fraction of different cell surface markers. We found that CD99R positive fraction contains a high percentage of insulin and glucagon positive cells (Fig 2A). We also found a high proportion of insulin positive cells in a previously reported marker CD49a. However, the percentage of glucagon positive cells was significantly low in CD49a positive fraction in compared to CD99R positive fraction (Fig 2A). Moreover, there was a significantly smallernumber of non-alpha / non-beta cells present in CD99R positive fraction in compared to CD49apositive fraction (Figure 2B).Through the screen we found CD99R to be selectively expressed within stem cell derived betaand alpha cells (Figure 3A-3E). Gating for the CD99R positive fraction increased the beta andalpha cell populations to about 59% and about 23% respectively while reducing the fractionof non-alpha / non-beta cells from about 38% to about 8% (Figure 3F). This is in stark contrastto the most common variant CD99 which was broadly expressed on most of the cells. TheCD99 positive fraction also did not eliminate the non-alpha / non-beta cells in a mannercomparable to the selective CD99R (Figure 4B) uncovering an unexpected isotype specificexpression in stem cell derived islets. We next validated CD99R expression in stem cell derivedislets using a different antibody (clone REA323) from Miltenyi Biotec also (Figure 5). These results demonstrate that CD99R is a specific and positive marker for stem cell derived betaand alpha cells. Further, we compared CD99R expression with a known beta cell markerCD49a in stem cell derived islets. It was found that CD99R expression was specific to bothstem cell derived alpha and beta cells (Figure 6A-Figure 6C). While CD49a expression waslimited to only stem cell derived beta cells (Figure 6D-Figure 6F).Example 3: MACS sorting with CD99R to deplete unwanted cells from the stem cell derived islets At the end of stage 6 differentiation (at day 36), stem cell derived islets were dissociated into single cell suspension using accutase enzyme. Anti-human CD99R PE antibody was added to single cell suspension and incubated for 10 minutes at 4°C. Anti-PE microbeads were then added, and incubation was performed at 4°C for 15 minutes. Mini-MACS column preparation and magnetic sorting was carried out as per manufacturer’s protocol. After sorting, CD99Rpositive fraction showed 90% purity (Figure 7B, 7E). The CD99R positive fraction was seededinto ultra-low attachment 6 well plate (3 million cells per well) and cultured for 3 days in stage 6 medium followed by 7 days in stage 7 medium (Figure 7). Example 4: CD99R sorted islets have better purity and glucose responsiveness At the end of stage 7, islets were analyzed by flow cytometry. CD99R sorted islets have better purity and significantly reduced number of unwanted cells in comparison to islets without sorting. CD99R sorted islets consist of approximately 66% insulin producing beta cells, 26% glucagon producing alpha cells and 7% unknown / unwanted cells (Figure 8B, 8C): Figure 10A represents additional replicates (5 independent experiments) including the data in Fig 8. Incontrast, islets without sorting contain approximately 48% beta cells, 13% alpha cells and 36%unknown / unwanted cells (Figure 8A, 8C, 10A). Further, single cell transcriptome analysisshowed that sorted SC-islets are highly pure. They contain approximately 80% beta cells, 17% alpha cells and just 1.4% off-target / unwanted cells (enterochromaffin cells) (Figure 9). Moreover, islets with CD99R sorting and without sorting were compared for glucose responsiveness via in-vitro glucose stimulated insulin secretion assay. In response to 20mM glucose, CD99R sorted islets have significantly increased c-peptide release compared to standard islets without sorting (Figure 8D). Similarly, significantly higher levels of c-peptide release were observed for CD99R sorted islets after KCl challenge (Figure 8D, 10B). Example 5: Utility of CD99R using alternative differentiation protocolTo demonstrate broad utility, we checked the expression of CD99R along with insulin andglucagon in stem cell derived islets generated from using a different protocol (Velazco-Cruz et al 2019). In these stem cell derived islets (Figure 11A) also we observed similar results that CD99R expression was specific to both stem cell derived alpha and beta cells (Figure 11B-Figure 11C). MATERIALS AND METHODS Stem cell differentiation into pancreatic islets: Human pluripotent stem cells (hPSCs) were cultured in NutriStem media. For differentiation, stem cells (17000 cells per cm2) were seeded on to laminin 521 coated dish and cultured for 96 hours. Differentiation was performed in a stepwise manner using following differentiation media.Stage 1 (3 days): S1 media + 100 ng / ml Activin A, 5 µM CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile) for 1 day. S1 media + 100 ng / ml Activin A for 2 days.Stage 2 (3 days): S2 media + 50 ng / mL keratinocyte growth factor (KGF).Stage 3 (1 day): S3 media + 50 ng / mL KGF, 0.25 µM SANT-1 (N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine), 2 µM RA (Retinoic acid), 500nM PDBu (Phorbol 12,13-dibutyrate), and 200 nM LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride).Stage 4 (3 days): S3 media + 50 ng / mL KGF, 100 ng / mL EGF (Epidermal growth factor), 10mM Nicotinamide, 5 ng / mL Activin A, 0.25 µM SANT1, 100 nM RA, 500 nM PDBu, and 200 nM LDN193189.Stage 5 (5 days): S5 media + 20 ng / mL Betacellulin, 0.25 µM SANT1, 100 nM RA, 100 nMGSI-XX (γ-secretase inhibitor-XX, N-[(1S)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide ), 10 µMAlk5i II (ALK5 Inhibitor II, 2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine ), 1µM GC-1 (Sobetirome, 2-[4-[[4-Hydroxy-3-(1-methylethyl)phenyl]methyl]-3,5-dimethylphenoxy]acetic acid), and 100 nM LDN193189 for 4 days. After 4 days, cells weredissociated using accutase enzyme and cultured as suspension (1.5 million cells per ml) infollowing media for 1 day: S5 media + 10 µM H1152 (Rho-kinase inhibitor, (S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride), 20ng / mL Betacellulin, 0.25 µM SANT1, 100 nM RA, 100 nM GSI-XX, 10 µM Alk5i II, 1 µM GC-1, and 100 nM LDN193189.Stage 6 (22 days): S6 media + 10 µM H1152, 1 µM GC-1, 10 µM Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and 1 mM N-acetyl cysteine. Stage 7 (7 to 10 days): S7 media + 1 µM GC-1, 10 µM Trolox and 1 mM N-acetyl cysteine.S1 media: MCDB131 + 2.5 mM D-(+)-Glucose + 25 mM NaHCO3 + 2 mM Glutamax + 0.5%Pen / Strep + 0.2-0.5% FAF-BSA.S2 media: MCDB131 + 2.5 mM D-(+)-Glucose + 25 mM NaHCO3 + 2 mM Glutamax + 0.5%Pen / Strep + 0.2-0.5% FAF-BSA + 0.25 mM Vitamin C.S3 media: MCDB131 + 2.5 mM D-(+)-Glucose + 25 mM NaHCO3 + 2 mM Glutamax + 0.5%Pen / Strep + 0.5% FAF-BSA + 0.25 mM Vitamin C + 1:200 ITS-X.S5 media: MCDB131 + 2.5 mM D-(+)-Glucose + 25 mM NaHCO3 + 2 mM Glutamax + 0.5%Pen / Strep + 0.5% FAF-BSA, 1:200 ITS-X, 20 mM Glucose, 10 µM ZnSO4, and 10 µg / mL Heparin.S6 media: CMRL-1066 + 14 mM NaHCO3 + 2 mM Glutamax + 0.5% Pen / Strep + 1% FAF-BSA +1:200 ITS-X + 1x NEAA + 14.45 mM Glucose + 10 µM ZnSO4 + 10 µg / mL Heparin + 1 mM N- acetyl cysteine + 10 µ M TroloxS7 media: CMRL-1066 + 14 mM NaHCO3 + 2 mM Glutamax + 0.5% Pen / Strep + 1% FAF-BSA +1x NEAA + 14.45 mM Glucose + 10 µM ZnSO4 + 10 µg / mL Heparin + 1 mM N-acetyl cysteine + 10 µM Trolox Cell surface marker screening: Cell surface marker screening was performed by using BD Lyoplate Human cell surface marker screening panel. Staining for cell surface markers was performed as per manufacturer’s instructions. PE conjugated goat anti-mouse Ig and PE conjugated goat anti-rat Ig secondary antibodies were used as detection reagents. After surface marker staining, cells were fixedand permeabilized with cytofix / cytoperm reagent (BD). It was followed by intracellularstaining with Alexa fluor 647 mouse anti Insulin (BD) and BV421 mouse anti Glucagon (BD) antibodies. Samples were analyzed in Cytoflex flow cytometry instrument. Data was analyzed using Flowjo software. CD99R positive cell sorting: SC-islets were dissociated in single cell suspension using accutase. Cells were stained with PE conjugated CD99R antibody (Miltenyi Biotec). Anti-PE microbeads ultrapure was used for magnetic labelling and sorting was performed using MS column as per manufacturer’s instructions. After sorting, CD99R positive fraction was further cultured for 10 days. Static GSIS (glucose stimulated insulin secretion) assay: Twenty to thirty SC-islets were incubated overnight in S7 media with low glucose (5 mM finalconcentration). Next day, SC-islets were washed twice with Krebs buffer and thenequilibrated for 2 hours in Krebs buffer containing 2 mM glucose. After this SC-islets were washed once with Krebs buffer and incubated again in Krebs buffer containing 2 mM glucose for 30 minutes. After 30 minutes incubation supernatant was collected and stored. Next, SC- islets were treated step by step with Krebs buffer containing 20 mM glucose, again Krebsbuffer containing 2 mM glucose and in last with Krebs buffer containing 2 mM glucose and 30mM KCl. After each step (treatment) the supernatant was saved. After KCl challange (Krebs buffer containing 2 mM glucose and 30 mM KCl) islets were dissociated into single cells and number of cells were counted. Human c-peptide ELISA was performed to determine c-peptide levels in supernatants collected from each step. REFERENCES Balboa, D., Barsby, T., Lithovius, V., Saarimäki-Vire, J., Omar-Hmeadi, M., Dyachok, O., Montaser, H., Lund, P. E., Yang, M., Ibrahim, H., Näätänen, A., Chandra, V., Vihinen, H., Jokitalo, E., Kvist, J., Ustinov, J., Nieminen, A. I., Kuuluvainen, E., Hietakangas, V., … Otonkoski, T. (2022). Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nature Biotechnology, 40(7), 1042–1055. https: / / doi.org / 10.1038 / s41587-022-01219-z Bernard, A., Gay-Bellile, V., Amiot, M., Caillou, B., Charbord, P., & Boumsell’, L. (1984). A NOVEL HUMAN LEUKOCYTE DIFFERENTIATION ANTIGEN: MONOCLONAL HEMATOLOGIC CELLS AND A SUBPOPULATION OF MATURE T CELLS ANTIBODY ANTI-D44 DEFINES A 28 KdMOLECULE PRESENT ON IMMATURE. In JOURNAL OF ~MMUNOLOGY (Vol. 132, Issue 5).http: / / journals.aai.org / jimmunol / article- pdf / 132 / 5 / 2338 / 1024320 / 2338.pdf?casa_token=SjBRd_EQjyEAAAAA:S6qO1t22pnyVpdOe_ eQkPRDZaqoT_TSGRKyji2pgg9kQieK7g3Tn3EEx53bsz2LarW7cWXJB EP4039798A1 Gelin, C., Zoccola, D., Valentin, H., Raynal, B., & Bernard, A. (1991). Isoforms of the E2 molecule: D44 monoclonal antibody defines an epitope on E2 and reacts differentially with T cell subsets. European Journal of Immunology, 21(3), 715–719. https: / / doi.org / 10.1002 / eji.1830210326Lanza F, Moretti S, Papa S, Malavasi F, Castoldi G (1994) REPORT ON THE FIFTHINTERNATIONAL WORKSHOP ON HUMAN LEUKOCYTE DIFFERENTIATION ANTIGENS,BOSTON, NOVEMBER 3-7, 1993. Heamatologica 1994: 79_374-386. Martens, G. A., De Punt, V., & Stangé, G. (2018). CD99 as surface anchor for human islet endocrine cell purification. Journal of Tissue Engineering and Regenerative Medicine, 12(1), e171–e176. https: / / doi.org / 10.1002 / term.2329 Pagliuca, F. W., Millman, J. R., Gürtler, M., Segel, M., Van Dervort, A., Ryu, J. H., Peterson, Q. P., Greiner, D., & Melton, D. A. (2014). Generation of functional human pancreatic β cells in vitro. Cell, 159(2), 428–439. https: / / doi.org / 10.1016 / j.cell.2014.09.040 Rezania, A., Bruin, J. E., Arora, P., Rubin, A., Batushansky, I., Asadi, A., O’Dwyer, S., Quiskamp, N., Mojibian, M., Albrecht, T., Yang, Y. H. C., Johnson, J. D., & Kieffer, T. J. (2014). Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nature Biotechnology, 32(11), 1121–1133. https: / / doi.org / 10.1038 / nbt.3033 Shapiro, A. M. J., Pokrywczynska, M., & Ricordi, C. (2017). Clinical pancreatic islettransplantation. In Nature Reviews Endocrinology (Vol. 13, Issue 5, pp.268–277). NaturePublishing Group. https: / / doi.org / 10.1038 / nrendo.2016.178 Shen DC., Chen Z., Yu AX., et al., 1986. A group of monoclonal antibodies reactive with the human thymocyte differentiation antigens–production and specificity analysis. Chinese J. ofImmunology. 6: 331-335Velazco-Cruz, L., Song, J., Maxwell, K. G., Goedegebuure, M. M., Augsornworawat, P., Hogrebe, N. J., & Millman, J. R. (2019). Acquisition of Dynamic Function in Human Stem Cell- Derived β Cells. Stem Cell Reports, 12(2), 351–365. https: / / doi.org / 10.1016 / j.stemcr.2018.12.012 Veres, A., Faust, A. L., Bushnell, H. L., Engquist, E. N., Kenty, J. H. R., Harb, G., Poh, Y. C., Sintov, E., Gürtler, M., Pagliuca, F. W., Peterson, Q. P., & Melton, D. A. (2019). Charting cellular identity during human in vitro β-cell differentiation. Nature, 569(7756), 368–373. https: / / doi.org / 10.1038 / s41586-019-1168-5

Claims

CLAIMS 1. A method for enriching glucagon producing alpha cells and / or insulin producing betacells in a mixture of cells, comprising: a. labeling a cell surface marker in a starting mixture of cells with a label; andb. separating the cells that are positive for the cell surface marker from cells thatare negative for the cell surface marker to obtain a final mixture of cells that is more enriched in glucagon producing alpha cells and / or insulin producing beta cells compared to the starting mixture;wherein said cell surface marker is CD99R.

2. The method of claim 1, wherein the label is an antibody specific to CD99R, optionallycomprising an affinity tag.

3. The method of any one of the preceding claims, wherein the separation step isperformed using beads having affinity to the label to separate the cells positive for thecell surface marker, preferably magnetic beads.

4. The method of claim 3, wherein the separation step involves separating the beadsfrom unbound cells by gravity, centrifugation, filtration or magnetism, preferablymagnetism.

5. The method according to any one of claims 1-2, wherein the separating step isperformed using a fluorescence-activated cell sorter.

6. The method of any one of the preceding claims, wherein the mixture of cells is aculture derived in vitro from human pluripotent stem cells.

7. The method of any one of the preceding claims, wherein the enrichment results in atleast 3-fold reduction in the fraction of cells other than glucagon producing alpha cells and / or insulin producing beta cells.

8. A method for obtaining glucagon producing alpha cells and / or insulin producing betacells comprising: a. Providing human pluripotent stem cells;b. Differentiating the pluripotent stem cells in vitro to obtain a population of cellscomprising glucagon producing alpha cells and / or insulin producing beta cells; c. Enriching the glucagon producing alpha cells and / or insulin producing betacells using the method of any one of claims 1-7.

9. The method of claim 8, wherein the enriched cells are cultured further in vitro afterthe enrichment step.

10. A method of treatment of a subject in need thereof, comprising:a. Obtaining glucagon producing alpha cells and / or insulin producing beta cellswith the method of any one of claims 8-9;b. Transplanting the cells to a subject in need thereof.

11. The method according to claim 10, wherein the subject suffers from type I diabetes.

12. A cell population comprising glucagon producing alpha cells and / or insulin producingbeta cells, obtainable by the method of any one of the preceding claims.

13. A method for analyzing an in vitro cell culture comprising glucagon producing alphacells, insulin producing beta cells and / or other cells, comprising quantitating thenumber or fraction of cells expressing CD99R, wherein expression of CD99R is indicative of the presence of glucagon producing alpha cells and / or insulin producing beta cells.

14. The method of claim 13, wherein the cell culture is a culture derived in vitro fromhuman pluripotent stem cells.

15. The method of any one of claims 13-14, wherein the quantitation is performed by flowcytometry using an antibody specific for CD99R.

16. A use of an antibody specific for CD99R for detecting glucagon producing alpha cellsand / or insulin producing beta cells.

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