Stem cell differentiation

Abstract

A method for the production of pancreatic islet β cells from at least one progenitor cell capable of lineage specific differentiation comprising the use of one of more cell media factors to promote ma

Description

Field of the Invention The invention concerns a method for the production of pancreatic islet [3 cells from at least one progenitor cell capable of lineage specific differentiation comprising the use of one of more cell media factors to promote mature beta cell formation; a heterogenous population of cells obtained from said method; use of said cells in the treatment of a disorder; and a kit of parts when used in said method of differentiation. Background of the invention Stem cells are cells found in all multicellular organisms with the ability to divide and differentiate into diverse specialised cell types. In mammals, there are two naturally occurring broad types of stem cells: embryonic stem cells (ESCs), which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. There is also a third class of stem cells which are man-made and known as induced pluripotent stem cells (iPSCs). Stem cells might have the ability to differentiate into a wide variety of cell types, and to repopulate and revitalize the damaged cells with healthy tissue, and improve its performance. The potential to regulate the differentiation of stem cells to a specific cell type, potentially allows for the regeneration and repair of tissues that have been damaged or affected by disease. Stem-cell therapy uses stem cells to treat or prevent a disease or condition. Research is underway to develop various sources for stem cells as well as to apply stem-cell treatments for neurodegenerative diseases and conditions such as diabetes and heart disease. When glucose enters the bloodstream after a meal, it is absorbed by [3 cells in the pancreas through glucose transporters in the presence of insulin. Type 1 diabetes is an autoimmune disease resulting to the destruction of pancreatic islet insulin producing beta ([3) cells. The most common form of treatment, insulin injected subcutaneously, does not prevent long-term complications of the disease as the optimal dosage is difficult to adjust. Successful pancreas transplantation does not cure the disease, however, can slow down the effect of long-term complications caused to the eyes, nerves and blood vessels. Development of islet isolation techniques and immunosuppressant treatment has shown significant results as an effective treatment method. However, islet transplantation is severely limited by shortage of donors as requires 2-3 donors per recipient. There are various protocols for differentiating pancreatic progenitor cells from ESCs or iPSCs, all of which simulate the differentiation in a defined sequence through a combination of compounds and differentiate iPSCs into definitive cells; definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitors, endocrine progenitors and mature pancreatic islets. Induction of the definitive endoderm, by regulating Wnt and TGF[3 through the small molecule compound CHIR99021 and Activin A protein, is the primary step towards the formation of endoderm derived tissues. Further differentiation into primitive gut tubes is regulated by the growth factor, keratinocyte growth factor (KGF). Further use of KGF in combination of retinoic acid, SANT1, noggin and other molecules to regulate Shh and Bone Morphogenetic Proteins (BMP) and other signalling pathways, stimulates the differentiation of primitive gut tubes into posterior foregut tissue cells prior to pancreatic progenitor cells. As multipotent stem cells, pancreatic progenitor cells can differentiate to endocrine or exocrine cells. Differentiation into endocrine precursor cells is regulated by compounds modulating signalling pathways, such as Notch and BMP, and are characterised by the co-expression of NGN3 and Nkx6.1, the latter of which plays a critical role in pancreatic islet [3 cell function and proliferation. Prolonged culture time leads to maturation of the endocrine progenitors to mature pancreatic islets resulting in a cells and [3 cells, a cells may be distinguished by the secretion of glucagon, typically as a response to low blood glucose. [3 cells may be distinguished by the secretion of insulin, regulated by the transcription factor, MafA, under glucose stimulation to regulate blood sugar levels. Due to the complex development of iPSCs differentiation to pancreatic islet [3 cells, requiring several compounds to modulate signalling pathways and gene expression, there are many limitations. For example, current techniques require lengthy differentiation stages, resulting in greater costs and poor differentiation efficiency; [3 cells within an iPSC-derived islet body accounts for only 20% of total cells compared with 60% from natural human islets. Importantly, neither can generate fully mature [3 islet cells in vitro, requiring further maturation in the body for several months after transplantation for successful glucose stimulation response. Further, current methods often give rise to heterogeneous populations and of low efficiency due to the generation of undefined cellular identities such as and undifferentiated intermediates. From a cell therapy stand point, these ‘contaminated’ cells greatly compromise efficacy and represent a potential risk of tumour formation if utilised in vivo. The present invention relates to the field of stem cell biology, particularly an improved process for differentiation of iPSCs to pancreatic islet [3 cells to improve the yield and maturity of [3 cells. We have shown [3 cells generated using the disclosed method, participate in the function of secreting insulin to regulate blood sugar; in vitro glucose-stimulated insulin secretion (GSIS) assays are used to test the function of iPSCs-derived [3 cells. This approach generates pancreatic islet [3 cells that can be used to reduce the risk of being unable to regulate blood sugar stability, thereby reducing complications of diabetes. This is of great significance for prolonging human lifespan, improving the quality of life of the wounded and sick, reducing the burden on society and families, and promoting the country's social and economic development. Statements of the Invention According to a first aspect of the invention there is provided a method for the production of pancreatic islet [3 cells from at least one progenitor cell capable of lineage specific differentiation, wherein said method comprises, or consists, of: i) Culturing at least one precursor cell derived from the at least one progenitor cell with a culture medium comprising a Rho-kinase inhibitor, wherein said precursor cell is a primitive gut cell; and / or ii) Culturing at least one precursor cell derived from the at least one progenitor cell with culture medium comprising a ErbB receptor tyrosine kinase agonist, wherein said precursor cell is a pancreatic endoderm cell; and / or iii) Culturing at least one least one precursor cell derived from the at least one progenitor cell with a culture medium comprising a canonical Wnt pathway agonist, wherein said precursor cell is a pancreatic endodermal cell. We have herein surprisingly found that culturing cells with defined media factors, in isolation or combination, during the differentiation of pancreatic cells from pancreatic precursor cells, leads to improved differentiation and / or maturation, and overall improved efficiency of the process for generating pancreatic islet [3 cells when compared to other methods wherein the factors are not used, or not utilised at the specified developmental time point. Advantageously, we have found that the specific timing of the culture of precursor cells with Rho-kinase inhibitor, even in a narrow window, can greatly influence the yield of mature beta cells obtained at the end of the differentiation process. Similarly, the precise concentration of Wnt pathway agonist and ErbB receptor tyrosine kinase inhibitor, when provided at specific time point of differentiation, can positively influence mature beta cell formation. Accordingly, through fine tuning any one or more of these supplements, one can significantly promote and improve efficiency of generating the functional maturation of stem cell derived beta cells in vitro. Efficacious and accelerated controlled differentiation will reduce culture times and thereby costs, whilst also generating cells that more accurately reflect those existing in vivo. However, notwithstanding the above and also encompassed in the present disclosure, we have also find this individual effect on supplementation to be further enhanced when used in any one or more combination. Accordingly, in a preferred embodiment, there is provided a method for the production of pancreatic islet [3 cells from at least one progenitor cell capable of lineage specific differentiation, wherein said method comprises, or consists, of: i) Obtaining at least one progenitor cell and culturing in conditions to differentiate into at least one primitive gut cell; ii) Culturing at least one primitive gut cell obtained in step i) with a culture medium comprising a Rho-kinase inhibitor until said gut cell(s) differentiate into at least one pancreatic progenitor cell; iii) Culturing the pancreatic progenitor cell(s) obtained in step ii) in conditions to differentiate into at least one pancreatic endocrine cell; and iv) Culturing at least one pancreatic endocrine cell obtained in step iii) with a culture medium comprising a ErbB receptor tyrosine kinase agonist until said pancreatic endocrine cell differentiate into at least one immature pancreatic beta cell; and v) Culturing at least one immature pancreatic beta cell obtained in step iv) with a culture medium comprising a Wnt pathway agonist until said pancreatic endocrine cell differentiate into at least one mature beta cell. Preferably, said method is in vitro. In a preferred method, progenitor cell refers to a pluripotent or multipotent stem cells such as but not limited to, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), adult stem cells, somatic stem cells, cancer stem cells, progenitor cells or any other cell capable of lineage specific differentiation into a pancreatic islet [3 cell. In a further preferred method of the first aspect of the invention, said progenitor cell comprises a heterogeneous population of cells. Alternatively, said sample comprises or commences as a homogeneous population of cells such as, but not limited to, stem cells or the like that undergo differentiation into other cell types including mixtures thereof. Most preferably, the pancreatic islet [3 cell is a mammalian [3 cell, and more preferably still a human [3 cell. Numerous differential protocols for differentiation of pancreatic beta cells from precursor cells exist are known in the art. For example, but not limited to, for differentiating pancreatic progenitor cells from ESCs or iPSCs, a defined sequence of developmental stages occurs to differentiate iPSCs into definitive pancreatic precursor cells; definitive endoderm (stage 1), primitive gut tube / cell and posterior foregut (stage 2), early and late pancreatic progenitors (stage 3 and 4, respectively), endocrine progenitors (stage 5) and immature and mature pancreatic islets (stage 6 and 7, respectively). We have found that, at defined such developmental stages of precursor differentiation, supplementation of culture media with specific factors can promote differentiation or maturation. As is known to those skilled in the art, determination of a specific cell precursor phenotype can be determined by standard methods, such as but not limited to, the presence or absence of one or more markers that associate with a defined developmental stage. Assay techniques for separating binding agents bound to specific surface markers and isolating cells having positive expression of said cell surface markers are well known to those skilled in the art and, indeed, used every day by workers in the field of clinical diagnostics and cell manufacturing. Such assay techniques include, but are not limited to, immunocytochemical methods, single cell sorting, fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), or any equivalent methods that isolate cells expressing the cell surface marker as well known to those skilled in the art. Flow cytometry, or FACS, is used to separate cell populations usually based on the intensity of binding to a specific binding agent, as well as other parameters such as cell size and light scatter. In such context, fluorescent labels, radioactive labels, or other readily identifiable and quantifiable labels may be used. MACS commonly involves use of magnetic beads coated with binding agents such as antibodies, or fragments thereof, specific for cell surface markers or antigens on the desired cells. When the antibody-magnetic beads are exposed to a mixed cell population, they attach to the cell surface of the desired cells via antibody-antigen interaction. The desired cell subpopulation can then be separated in the presence of a strong magnetic field. In a preferred embodiment, said primitive gut cell can be identified as a precursor cell as positive for any one or more of the following: SOX17 (Gene Card Identifier GB08P054457) and FOXA2 (Gene Card Identifier GC20M022581), both of which are transcription factors. Together, they work in concert to orchestrate the molecular events leading to the formation and maintenance of definitive endoderm. Their collaborative efforts are instrumental in guiding stem cells through the crucial developmental steps toward a specific lineage, setting the stage for subsequent stages of differentiation, such as pancreatic or hepatic lineage specification. Reference herein to a cell being positive or negative for a specified cell marker concerns the ability of said cell to express said marker wherein positive signifies expression of said marker and negative signifies lack of expression of said marker. This encompasses both the mRNAand protein level. Reference herein to a Rho kinase, or Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC (PKA / PKG / PKC) family of serine-threonine specific protein kinases. ROCK plays a role in a wide range of different cellular phenomena, as ROCK is a downstream effector protein of the small GTPase Rho, which is one of the major regulators of the cytoskeleton, and thus has an important role in many cellular processes such as cell migration, cell apoptosis, and cell proliferation. ROCK inhibitors (also known as Rho-kinase inhibitors or Rho-associated kinases inhibitors) are a series of compounds that target rho kinase (ROCK) and inhibit the ROCK pathway. ROCK inhibitors have been used for cell culture practice, in part to limit cellular death and limited dedifferentiation, and therefore widely adopted for induced pluripotent stem cells (iPSC) and embryonic stem cell culture ROCK inhibitors are known in the art, and commercially available, and those suitable for working the invention include, but are not limited to, Y-27632, AT-13148, BA-210, [3-Elemene, Belumosudil, Chroman 1, MRCKa, MRCK[3 kinases, GSK-576371, GSK429286A, H-1152, RKI-1447, TCS-7001, Thiazovivin, Verosudil (AR-12286), Y-30141, Y-33075. Most preferably, said ROCK inhibitor is Y-27632. As is known in the art, ROCK inhibitors such as Y-27632 find use commonly to prevent apoptosis in human iPSCs and ESCs, particularly during the process of passaging, when cells are more susceptible to stress and detachment-induced apoptosis (anoikis). However, unexpectedly we found its further use as defined in Stage 2 of differentiation has further positive effects on the overall production of beta cells. In a further preferred embodiment, said primitive gut cell is cultured with the Rho-kinase inhibitor from 0-48h after observing expression of the markers identifying the precursor cell as a primitive gut cell, or more ideally still a number of hours selected from the group comprising or consisting of: 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48h, and any 0.05h therebetween. Further, and more preferably, said method comprises culturing and / or differentiation of the primitive gut cell, for at least 1-5 days in said Rho-kinase inhibitor, or more ideally 1-4 days, or more ideally still a number of days selected from the group comprising or consisting of: 1, 2, 3, 4 days. Most ideally, said primitive gut cell is cultured until said cell reaches the next developmental stage, ideally until when said cell identifies as a pancreatic progenitor cell as known in the art. In a preferred embodiment, said cells are not cultured in media supplemented with ROCK inhibitor beyond a developmental stage when said cells no longer identify as primitive gut cell. As will be known by those with experience, differentiation of progenitor cells to a selected phenotype can be achieved by changing the culture conditions. Accordingly, said primitive gut cell is cultured with Rho-kinase inhibitor in cell culture media standard in the art for such differentiation stage such as, but not limited to, a media comprising basal media (RPMI 1640), ideally supplemented with one or more additional factors required for this stage of differentiation including one or more of FGF10, b-27 supplement, KAAD-cyclopamine. In yet a further preferred embodiment of the invention, said Rho-kinase inhibitor is provided at a concentration between 1-50pM, and more ideally between 2.5-20pM, including every 0.1 pM concentration there between, and most ideally 5-15pM. Most ideally, inhibitor is provided at a concentration selected from the group comprising: 5pM; 6pM, 7pM, 8pM, 9pM, 10pM, 11 pM, 12pM, 13pM, 14pM, or15pM, oreverO.lpM therebetween. In a preferred embodiment said pancreatic endoderm cell can be identified as a precursor cell as positive for any one or more of the following: Nkx6.1 (Genecard Identifier GC04M084491) or PDX1 (Genecard identifier GC13P028201). PDX1 serves as a master regulator in pancreatic development, playing a crucial role in the specification and maintenance of pancreatic progenitors. Its expression marks the commitment of cells to the pancreatic lineage. NKX6.1, another essential transcription factor, further refines this commitment by contributing to the development of islet [3 cells within the pancreas. Reference herein to a cell being positive or negative for a specified cell marker concerns the ability of said cell to express said marker wherein positive signifies expression of said marker and negative signifies lack of expression of said marker. This encompasses both the mRNAand protein level. Reference herein to ErbB receptor tyrosine kinase refers to a group of receptor tyrosine kinases (RTKs), structurally related to the epidermal growth factor receptor (EGFR), involved in key cellular functions, including cell growth and survival. They signal through phosphorylated tyrosines on their intracellular domains. Activation of these receptors leads to this phosphorylation and to subsequent intracellular signaling. Accordingly, agonists of this pathway are compounds or factors known to activate this signalling pathway. ErbB receptor tyrosine kinase agonists are known in the art, and those suitable for working the invention include, but are not limited to beta-cellulin, EGF, epiregulins, heregulin. Most preferably, said ErbB receptor tyrosine kinase agonists is beta-cellulin. In a further preferred embodiment said pancreatic endoderm cell is cultured with the ErbB receptor tyrosine kinase agonist for at least 1-7 days in said the ErbB receptor tyrosine kinase agonist, or more ideally 1 -4 days, or more ideally still a number of days selected from the group comprising or consisting of: 1, 2, 3, 4 days after observing expression of the markers identifying the precursor cell as a pancreatic endoderm cell, Most ideally, said pancreatic endoderm is cultured until said cell reaches the next developmental stage, ideally until when said cell identifies as an immature pancreatic beta cell as known in the art. In a preferred embodiment, said cells are not cultured in media supplemented with ErbB receptor tyrosine kinase beyond a developmental stage when said cells no longer identify as pancreatic endoderm cell. As will be known by those with experience, differentiation of progenitor cells to a selected phenotype can be achieved by changing the culture conditions. Accordingly, said pancreatic endocrine cell is cultured with ErbB receptor tyrosine kinase agonist in cell culture media standard in the art for such differentiation stage such as, but not limited to, a media comprising basal media (RPMI 1640), ideally supplemented with one or more additional factors required for this stage of differentiation including one or more of b-27 supplement, DAPT. In yet a further preferred embodiment of the invention, said ErbB receptor tyrosine kinase agonist is provided at a concentration between 1-40pM, and more ideally between 5-30pM, including every 0.1 pM concentration there between, and most ideally 5-15pM. Most ideally, inhibitor is provided at a concentration selected from the group comprising: 5pM; 6pM, 7pM, 8pM, 9pM, 10pM, 11 pM, 12pM, 13pM, 14pM, or 15pM, or ever 0.1 pM therebetween. In a preferred embodiment, said immature pancreatic beta cell is identified as a precursor cell positive for any one or more of the following: PDX1, NKX6.1, NEUROG3, MafAand MafB. Reference herein to a cell being positive or negative for a specified cell marker concerns the ability of said cell to express said marker wherein positive signifies expression of said marker and negative signifies lack of expression of said marker. This encompasses both the mRNAand protein level. Reference herein to Wnt pathway agonist is any compound that promotes or activates the canonical Wnt signaling pathway. As is known by those skilled in the art, the canonical Wnt pathway (or Wnt / [3-catenin pathway) is the Wnt pathway that causes an accumulation of [3-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional co-activator of transcription factors that belong to the TCF / LEF family. In a preferred embodiment of the invention, said canonical Wnt pathway agonist is a Glycogen synthase kinase 3 beta (GSK3[3) inhibitor. As is known by those skilled in the art, GSK-3 has been shown to phosphorylate Beta-catenin, thus targeting it for degradation and inhibitors of this protein are known to specifically promote non-canonical Wnt signaling. Such compounds include, but are not limited to, CHIR99021, Wnt3a, WAY-316606, ISX9 and SKL200 Most ideally, said canonical Wnt pathway agonist is ISX9. In a further preferred embodiment, said immature pancreatic beta cell is cultured with the Wnt pathway agonist for at least 1-10 days in said the Wnt pathway agonist, or more ideally 1-6 days, or more ideally still a number of days selected from the group comprising or consisting of: 1,2,3, 4, 5, 6 days after observing expression of the markers identifying the precursor cell as a pancreatic beta cell, Most ideally, said precursor cells is cultured until said cell reaches the next developmental stage, ideally until when said cell identifies as a mature pancreatic beta cell as known in the art. In a preferred embodiment, mature beta cell differentiation is characterised by the expression of maturing and / or mature beta cell markers, such as NKX6-1, C-peptide, MafA, PDX1, and / or insulin, and / or the ability of the insulin-secreting cell to respond acutely to glucose by processing pro-insulin into insulin and C-peptide and by secreting insulin and C-peptide. Yet more preferably, said method comprises continuing culture in said Wnt pathway agonist until at least 20%, more ideally at least 25%, and more ideally still at least 30%, 31%, 32% 33% 34%, 35%, 36%, 37%, 38%, 39%, 40%, of culture cells are mature beta cells, and so preferably express NKX6-1, C-peptide, MafA, PDX1, and / or insulin, and / or the ability of the insulin-secreting cell to respond acutely to glucose by processing pro-insulin into insulin and C-peptide and by secreting insulin and C-peptide. As is known in the art proinsulin is a precursor molecule that consists of insulin and C-peptide linked together. In the pancreatic beta cells, proinsulin undergoes enzymatic cleavage, resulting in the formation of insulin (which has two chains: A and B) and C-peptide. Since C-peptide has a longer half-life (around 20-30 minutes) compared with insulin (which has a half-life of about 5-10 minutes), measuring C-peptide levels provides a more reliable indication of endogenous insulin production over time. As will be known by those with experience, differentiation of progenitor cells to a selected phenotype can be achieved by changing the culture conditions. Accordingly, said immature pancreatic beta cell is cultured with Wnt pathway agonist in cell culture media standard in the art for such differentiation stage such as, but not limited to, a media comprising basal media (RPMI 1640), ideally supplemented with one or more additional factors required for this stage of differentiation including one or more of b-27 supplement, extendin-4, or nicotinamide. In yet a further preferred embodiment of the invention, said Wnt pathway agonist is provided at a concentration between 1-1 OpM, and more ideally between 2.5-7.5pM, including every 0.1 pM concentration there between, and most ideally 3-7pM. Most ideally, inhibitor is provided at a concentration selected from the group comprising: 3pM; 4pM, 5pM; 6pM, 7pM, or every 0.1 pM therebetween. In a preferred method of this embodiment of the invention, optionally, said method comprises at least one further step that involves, after any one of preceding step and before performing the subsequent step determining the expression of any one or more specified markers using conventional methods such as, but not limited to, immunocytochemical methods or Fluorescence-activated cell sorting (FACS) analysis, to determine the correct differentiation phase has been reached. According to a second aspect of the invention there is provided a heterogenous population of cells, optionally isolated from the method disclosed herein, and comprising mature pancreatic beta cells and wherein said beta cells are provided in at least 20%, more ideally at least 25%, and more ideally still at least 30%, 31%, 32% 33% 34%, 35%, 36%, 37%, 38%, 39%, 40%, of the total cell population, and so preferably express NKX6-1, C-peptide, MafA, PDX1, and / or insulin, and / or the ability of the insulin-secreting cell to respond acutely to glucose by processing pro-insulin into insulin and C-peptide and by secreting insulin and C-peptide. According to a third aspect of the invention there is provided the use of a population of cells disclosed herein wherein said cell is capable of secreting insulin or differentiating into a cell capable of secreting insulin for use as a medicament. 5 According to a fourth aspect of the invention there is provided the use of a population of cells disclosed herein wherein said cell is capable of secreting insulin or differentiating into a cell capable of secreting insulin for use in treatment of a disorder or condition responsive to the secretion of insulin. In a preferred embodiment of this aspect of the invention this includes, but is not limited to, a condition such as Type 1 10 and 2 diabetes, cystic fibrosis-related diabetes, pancreatitis, hypoglycemia-related disorders where destruction of pancreatic tissue, including insulin-producing beta cells, occurs. According to a fifth aspect of the invention there is provided use of a population of cells 15 disclosed herein wherein said cell is capable of secreting insulin or differentiating into CM a cell capable of secreting insulin in the manufacture of a medicament to treat a disorder or condition responsive to the secretion of insulin. i— According to sixth aspect of the invention there is provided a kit for differentiation of £\j 20 pancreatic islet [3 cells from at least one progenitor cell capable of lineage specific differentiation, optionally when used in the method disclosed herein, wherein said kit comprises: i) a Rho-kinase inhibitor; and / or ii) a ErbB receptor tyrosine kinase agonist; and / or 25 iii) a canonical Wnt pathway agonist, wherein said precursor cell is a pancreatic endodermal cell; and optionally reagents and / or instructions for practicing said differentiation. In a preferred embodiment, said kit comprises one or more of the following: a cell culture base media and specific supplementary media for Stage 2 to 6. 30 Reference to a cell culture base media refers to a liquid or gel designed to support the growth of precursor cells, in particular stem cells or progenitor cells. As will be appreciated by those skilled in the art, many different types of chemical medium can be used to support the growth of stem or progenitor cells in culture, such as but not limited to, RPMI 1640 medium. In yet a further preferred embodiment, said kit further comprises one or more binding agents specific for one or markers of precursor cell differentiation, ideally selected from the group comprising: SOX17, FOXA2, Nkx6.1, PDX1, C-peptide, MafA. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds, or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. An embodiment of the present invention will now be described by way of example only with reference to the following wherein: Figure 1. FACS results revealed the treatment of Y-27632 at Stage 2 modulated the differentiation efficiency of [3 cells; A) Control, B) Treatment of Y-27632 0 hr post Stage 2, C) Treatment of Y-27632 12 hr post Stage 2, D) Treatment of Y-27632 24 hr post Stage 2 and, E) Treatment of Y-27632 36 hr post Stage 2. Figure 2. FACS results revealed the treatment of betacellulin at Stage 5 modulated the differentiation efficiency of [3 cells; A) Control, B) Treatment of 5 pM betacellulin, C) Treatment of 10 pM betacellulin, D) Treatment of 20 pM betacellulin and, E) Treatment of 40 pM betacellulin. Figure 3. FACS results revealed the treatment of ISX9 at Stage 6 modulated the differentiation efficiency of [3 cells; A) Control, B) Treatment of 2.5 pM ISX9, C) Treatment of 5 pM ISX9, D) Treatment of 10 pM ISX9, E) Treatment of 20 pM ISX9 and, F) Treatment of 40 pM ISX9. Figure 4. FACS results revealed the combination treatment of Y-27632 (12 hr post Stage 2), betacellulin (10 pM at Stage 5) and ISX9 (5 pM at Stage 6) significantly improved the differentiation efficiency of [3 cells; A) Control, B) Repeat 1 for combination treatment, C) Repeat 2 for combination treatment, D) Repeat 3 for combination treatment and, E) Repeat 4 for combination treatment. Figure 5. Glucose stimulated insulin secretion (GSIS) assays at the end of differentiation. Treatment of combination of Y-27632 (12 hr post Stage 2), betacellulin (10 pM at Stage 5) and ISX9 (5 pM at Stage 6) significantly improved functions of [3 cells in response to the fluctuation of glucose concentration; A) Treatment of Y-27632 at different times post Stage 2, B) Treatment of different concentrations of betacellulin at Stage 5, C) Treatment of different concentrations of ISX9 at Stage 6 and, D) Treatment of combination ofY-27632 (12 hr post Stage 2), betacellulin (10 pM at Stage 5) and ISX9 (5 pM at Stage 6). Table 1. F-statistic of one-way ANOVA analysis for FACS results for NKX6.1+ / C-Peptide+ double positive (%) when Y-27632 (10 pM) was added at different times at Stage 2; Table 2. Tukey HSD procedure tests for FACS results for NKX6.1 + / C-Peptide+ double positive (%) when Y-27632 (10 pM) was added at different times at Stage 2; Table 3. F-statistic of one-way ANOVA analysis for FACS results for NKX6.1+ / C-Peptide+ double positive (%) when different concentrations of betacellulin was added throughout Stage 5; Table 4. Tukey HSD procedure tests for FACS results for NKX6.1 + / C-Peptide+ double positive (%) when different concentrations of betacellulin was added throughout Stage 5; Table 5. F-statistic of one-way ANOVA analysis for FACS results for NKX6.1+ / C-Peptide+ double positive (%) when different concentrations of ISX9 was added throughout Stage 6; Table 6. Tukey HSD procedure tests for FACS results for NKX6.1+ / C-Peptide+ double positive (%) when different concentrations of ISX9 was added throughout Stage 6; Table 7. F-statistic of one-way ANOVA analysis for FACS results for NKX6.1+ / C-Peptide+ double positive (%) when combination ofY-27632 (12 hr post Stage 2), betacellulin (10 pM at Stage 5) and ISX9 (5 pM at Stage 6) was applied; Table 8. Tukey HSD procedure tests for FACS results for NKX6.1+ / C-Peptide+ double positive (%) when combination ofY-27632 (12 hr post Stage 2), betacellulin (10 pM at Stage 5) and ISX9 (5 pM at Stage 6) was applied; Methods Differentiation protocol of iPSCs Standard protocol for pancreatic beta cell differentiation is provided below, with specific factor supplementation discussed in the results section to follow. Culture iPSCs in mTeSRI (STEMCELL Technologies, Cat. 85857) with 10 pM Y27632 (STEMCELL Technolgoies, Cat. 72307) medium. When the cells are over 85% confluent, detach with Accutase (STEMCELL Technolgoies, Cat. 07920) and subculture in Matrigel (Coming, Cat. 354277)-coated dishes at a 1:30 ratio at a density of 55,000 cells / cm2. Differentiation factors The following directed differentiation strategy has been adopted for this study, which goes through 6 main stages. Classification of stages defined by Hogrebe et al. (2021). Stage 1: Definitive Endoderm Day 0: Plate human iPSCs on Matrigel-coated plates in mTeSRI (with 10 pM Y27632 10) Medium. Allow cells to reach 100% confluency next day. Day 1-4: Induce definitive endoderm by changing the medium to RPMI 1640 (Gibco, Cat. 11875093) supplemented with 100 ng / mL Activin A (Peprotech, Cat. 120-14P) and 50 ng / mL Wnt3a (bio-techne, Cat. 5036-WN). Daily medium changes. Confirm the efficiency of differentiation into definitive endoderm on Day 4 by checking for the expression of markers of SOX17 and FOXA2 with FACS. Stage 2: Primitive Gut Tube Day 5-6: Switch to RPMI 1640 medium supplemented with 2% B-27 (minus insulin, Gibco, Cat. A1895601), 50 ng / mL FGF10 (STEMCELL Technologies, Cat. 78037.2), and 25 ng / mL KAAD-cyclopamine (Sigma-Aldrich, Cat. 239804). Daily medium changes. Stage 3: Pancreatic Progenitor (Early) Day 7-11: Transition to RPMI 1640 medium with 2% B-27 (minus insulin), 50 ng / mL Retinoic Acid (Sigma-Aldrich, Cat. R2625), and 50 ng / mL FGF10. Daily medium changes. Stage 4: Pancreatic Progenitor (Late) Day 12-15: Continue differentiation using RPMI 1640 medium with 2% B-27 (minus insulin), 50 ng / mL FGF10, and 50 ng / mL KAAD-cyclopamine. Daily medium changes. On Day 15, confirm the efficiency of differentiation into late pancreatic progenitors by checking for the expression of NKX6.1 and PDX1 with FACS. Stage 5: Pancreatic Endocrine Day 16-19 : Use RPMI 1640 medium with 2% B-27 (minus insulin), 10 pM DAPT (Sigma-Aldrich, Cat. D5942), and 50 ng / mL Nicotinamide (Sigma-Aldrich, Cat. N0636). Daily medium changes. Stage 6: Pancreatic Beta Cells Day 20-26: Final differentiation into pancreatic beta cells using RPMI 1640 medium with 2% B-27 (complete), 10 pM Exendin-4 (Sigma-Aldrich, Cat. E7144), and 10 mM Nicotinamide. Medium changes every other day. On Day 27, confirm efficiency of differentiation into pancreatic beta cells by checking for the expression of NKX6.1 and C-peptide with FACS. Characterisation of cells by fluorescence-activated cell sorting (FACS) Islet dissociation to obtain a single-cell suspension from iPSC-derived islets. 1. Collect differentiated islets and wash them with 1X PBS to remove residual media. 2. Incubate islets in TrypLE (Fisher Scientific, Cat# 11558856) for 5-8 minutes at 37°C. 3. Gently pipette up and down to aid dissociation, being careful not to damage the cells. 4. Neutralise TrypLE by adding Stage 6 medium. 5. Pass the cell suspension through a 40 pm cell strainer (Fisher Scientific, Cat# 15360801) to ensure a single-cell suspension. 6. Centrifuge at 300 x g for 5 minutes and remove the supernatant. Fixation and Permeabilisation 1. Fix the single-cell suspension by resuspending the cell pallet (Step 6) in 4% paraformaldehyde for 15 minutes at room temperature. 2. Centrifuge at 300 x g for 5 minutes and remove the supernatant. 3. Wash cells twice with DPBS (centrifuge at 300 x g for 5 minutes). 4. Permeabilise cells using 0.1 % Triton X-100 and 5% (vol / vol) BSA in PBS for 30 minutes at room temperature to permeabilise and block the cells. 5. Wash cells twice with DPBS (with 1 % vol / vol BSA) Primary Antibody Staining 1. Resuspend cells in DPBS (with 1% vol / vol BSA). Incubate with Rabbit anti-NKX6.1 primary antibody (Abeam, Cat# ab221549) and Mouse anti-PDX1 primary antibody (Abeam, Cat# ab84987) at the recommended dilution and incubate overnight at 4°C. 2. After incubation, wash cells 3 times (centrifuge at 300 x g for 5 minutes) with DPBS (with 1% vol / vol BSA). 3. Resuspend the cells in DPBS (with 1% vol / vol BSA). Incubate with Goat antiRabbit Alexa Fluor 488 secondary antibody (for NKX6.1; Abeam Cat# ab150077) and Goat anti-Mouse Alexa Fluor 597 secondary antibody (for PDX1; Invitrogen Cat# A11005) at the recommended dilution for 1 hour at room temperature in the dark. 4. Wash cells 3 times with DPBS (with 1% vol / vol BSA) after secondary antibody incubation (centrifuge at 300 x g for 5 minutes). FACS Sample Preparation 1. Resuspend the stained cells in DPBS (with 1% vol / vol BSA) and pass through a 40 pm cell strainer to ensure single-cell suspension. 2. Transfer the suspension to FACS tubes. 3. Keep the samples on ice and protected from light until acquisition. FACS Acquisition 1. Run the samples on a flow cytometer equipped with the appropriate lasers for detecting Alexa Fluor 488 (green, for NKX6.1) and Alexa Fluor 597 (red, for PDX1). 2. Set up compensation controls using single-stained samples for each fluorophore. 3. Collect at least 10,000 events per sample to ensure robust analysis. Gating strategy: 1. Gate live, single cells based on forward scatter (FSC) and side scatter (SSC). 2. Use fluorescence channels to analyse the proportion of cells positive for NKX6.1 (488 channel) and PDX1 (597 channel). Analyse the double-positive population (NKX6.1+ / PDX1+). 1. Use appropriate software FlowJo for data analysis. 2. Identify the percentage of cells expressing NKX6.1 only, PDX1 only, and NKX6.1 / PDX1 co-expressing cells. 3. Compare the expression profiles of these markers across different samples or experimental conditions. 4. Perform a One-Way ANOVA to test for overall differences among the groups. If the ANOVA shows significance, use Tukey’s HSD (honestly significant difference) procedure as a post-hoc test to compare the specific treatment of interest to all other treatments. GSIS assay to determine islet secretory function Differentiated iPSC-islet cells in 6-well cell culture plates were equilibrated in Krebs-Ringer buffer (KRB) with 2 mM glucose for 1 h at 37°C before being subjected to sequential 1-h incubation of 2 mM (Low) and then 20 mM (High) glucose with 30 mM KCI in KRB for 60 min. Supernatants were collected, and insulin was measured using human insulin ELISA kits (Mercodia, 10-1113-10). Reagents: • KrB buffer: (glucose free) It contains 128 mM NaCI, 5 mM KCI, 2.7 mM CaCL, 1.2 mM MgSO4, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10mM HEPES, 0.1% (wt / vol) fatty-acid free BSA. pH adjusted to 7.4 • Low glucose KrB buffer: It is KrB containing 2 mM glucose • High glucose KrB buffer: It is KrB containing 20 mM glucose Glucose-stimulated insulin secretion (GSIS) assay: 1. Carefully collect about 30 islet clusters into a cell strainer (Fisher Scientific, Cat# 15360801) for each well of 6-well cell culture plate using a pipette. Three replicates per condition. Gently wash the islets with 2 ml KrB for 3 times. 2. Transfer each cell strainer to new wells containing 2 ml fresh 2mM low glucose KrB, and incubate for 1 hour to equilibrate and remove background insulin secretion. 3. After 1 hour, transfer each cell strainer to new wells containing 2 ml fresh 2mM low glucose KrB, and incubate for 1 hour. 4. Collect supernatants into labelled tubes and store on ice or store them at -80°C until further analysis. This will be used for insulin quantification. 5. After 1 hour, transfer each well to new wells containing 2 ml fresh 20mM high glucose KrB. Incubate for 1 hour. 6. Collect supernatants into separate tubes and store on ice or store them at -80°C until further analysis. 7. Cells are then detached with Accutase (STEMCELL Technologies, Cat# 07922) and counted by Scepter 3.0 Handheld Automated Cell Counter (Merck). Cell counts will be used later to calculate the amount of insulin secreted per cell in each well. Insulin quantification by ELISA i) Use the commercially available insulin ELISA kit (Mercodia Ultrasensitive Insulin ELISA, Cat# 10-1132-01) to measure insulin levels in the supernatants collected from each condition (low and high glucose) ii) Measure absorbance using a microplate reader at the wavelength of 450 nm for ELISA results. Data analysis: 1. Calculate the insulin concentration (pU / mL) in each condition using the ELISA kit's standard curve. 2. Express insulin secretion as plU (international units), where 1 plU equals 0.0348 ng insulin. 3. Normalise each sample by dividing this calculated insulin value by its respective cell count to determine the total insulin. 4. Compare insulin secretion under different conditions to assess the functionality of iPSC-differentiated islets. Statistical analysis: i) Perform One-Way ANOVA test to compare insulin secretion between low and high glucose conditions. Results Treatment of Y-27632 at Stage 2 modulates the differentiation efficiency of 0 cells Pancreatic islet 0 cells can be derived from pluripotent stem cells using a variety of protocols described in the literature and, a greater yield and maturity of 0 cells can be attained using the protocols and reagents described herein. Higher yields of 0 cells can be attained by the addition of Y-27632 during the differentiation of primitive gut tubes. The addition of 10 pM Y-27632 at different times of Stage 2 can affect the yield of p cells at the end of differentiation, characterised by FACS (Figure 1). Untreated control (Figure 1 A) produced 16.6% of NKX6.1 / C-Peptide double positive cells. In contrast to Y-27632 treatment at 0 (Figure 1 B), 12 (Figure 1 C), 24 (Figure 1 D) and 36 (Figure 1 E) hours after entering Stage 2 of the directed differentiation, which respectively gave 25.9%, 27%, 22% and 13.8% of double positive cells. 12-hour post treatment of Y-27632 showed the best results. Specific data is provided in Tables 1-2. Tables 1; he p-value corresponding to the F-statistic of one-way ANOVA is lower than 0.05, suggesting that the one or more treatments are significantly different. Table 2; These post-hoc tests would likely identify which treatment pairs show significant differences. Except for the comparison between T2 (0 hr) and T4 (24 hr), Y-27632 had a statistically significant impact on the abundance of NKX6.1+ / C-peptide doublepositive cells at different time points during Stage 2. The treatment administered 12 hours post-Stage 2 resulted in the highest level of co-expression. Treatment of betacellulin at Stage 5 modulates the differentiation efficiency of 0 cells A critical step in the transition of iPSCs to pancreatic islet 0 cells is the differentiation of pancreatic endocrine cells to 0 cells. The addition of different concentration of betacellulin (BTC, Sigma-Aldrich, Cat. B3670) at Stage 5 can affect the yield of 0 cells at the end of differentiation, characterised by FACs (Figure 2). Untreated control (Figure 2A) produced 15.6% of NKX6.1 / C-Peptide double positive cells, in contrast to BTC treatment concentration 5 (Figure 2 B), 10 (Figure 2 C), 20 (Figure 2 D) and 40 (Figure 2 E) pM throughout Stage 5 of the directed differentiation, which respectively gave 25.9%, 27%, 22% and 13.8% of double positive cells. 10 pM BTC treatment showed the best results. Specific data is provided in Tables 3-4. Tables 3; the p-value corresponding to the F-statistic of one-way ANOVA is lower than 0.05, suggesting that the one or more treatments are significantly different. Table 4; these post-hoc tests would likely identify which treatment pairs show significant differences. Except for the comparison between T2 (5 pM) and T5 (40 pM), betacellulin had a statistically significant impact on the abundance of NKX6.1+ / C-peptide double-positive cells by different concentrations during Stage 5. The treatment administered at 10 pM resulted in the highest level of co-expression. Treatment of ISX9 at Stage 6 modulates the differentiation efficiency of 0 cells To understand how ISX9 may promote a greater yield of 0 cells when cells are treated at the final stage of differentiation was explored. The addition of different concentration of ISX9 (STEMCELL Technologies, Cat. 73202) can affect the yield of 0 cells, characterised by FACs (Figure 3). Untreated control (Figure 3 A) produced 16.4% of NKX6.1 / C-Peptide double positive cells. In contrast to ISX9 treatment concentration 2.5 (Figure 3 B), 5 (Figure 3 C), 10 (Figure 3 D), 20 (Figure 3 E) and 40 (Figure 3 F) pM throughout Stage 6 of the directed differentiation, which respectively gave 27.8%, 14.5%, 11.1% and 8.7% of double positive cells. 5 pM ISX9 treatment showed the best results. Specific data is provided in Tables 5-6. Tables 5; The p-value corresponding to the F-statistic of one-way ANOVA is lower than 0.05, suggesting that the one or more treatments are significantly different. Table 6; These post-hoc tests would likely identify which treatment pairs show significant differences. Except for the comparison between T1 (0 pM as control) and T4 (10 pM), ISX9 had a statistically significant impact on the abundance of NKX6.1+ / C-peptide double-positive cells by different concentrations during Stage 6. The treatment administered at 5 pM resulted in the highest level of co-expression. Combination treatment of Y-27632, betacellulin and ISX9 significantly improves the overall differentiation efficiency of 0 cells As there is a greater yield of 0 cells when differentiating iPSCs are treated with Y-27632 (stage 2), BTC (stage 5) and ISX9 (stage 6) individually, the use of all treatments sequentially and the impact to the yield of 0 cells was to be discovered. We observed the treatment of the combination of 3 small molecules can positively affect the yield of 0 cells at the end of differentiation, characterised by FACs (Figure 4). Untreated control (Figure 4 A) gave 16.8% of NKX6.1 / C-Peptide double positive cells. Similarly, 4 test results showed same improvement when cells were sequentially treated with 10 pM Y-27632 12-hours after entering Stage 2, 10 pM BTC throughout Stage 5, and 5 pM IX9 throughout Stage 6 (Figure 4 B-E). Specific data is provided in Tables 7-8. Tables 7; The p-value corresponding to the F-statistic of one-way AN OVA is lower than 0.05, suggesting that treatment groups are significantly different from control groups. Table 8; These post-hoc tests would likely identify which treatment pairs show significant differences. The combined use of Y-27632 (12 hr post Stage 2), betacellulin (10 pM at Stage 5) and ISX9 (5 pM at Stage 6) brought in a statistically significant improvement on the abundance of NKX6.1+ / C-Peptide double-positive cells at the end of differentiation. Mature [3 cells are crucial to generating a strong glucose response mimicking that of human cells, in the presence of high glucose concentrations. GSIS assays demonstrate iPSC-derived [3 cells by traditional differentiation protocol show a much weaker response to the fluctuation of glucose concentrations, in comparison to the combinational treatment producing the most efficient differentiation (Figure 5). Traditional differentiation protocols include Y-27632 only (Figure 5 A), BTC only (Figure 5 B) and ISX9 only (Figure 5 C). The iPSCs sequentially treated by Y-27632, BTC and ISX9 during their differentiation process demonstrate a stronger response when exposed to the low and high glucose challenges (Figure 5 D). Discussion The entire differentiation process of iPSCs to pancreatic [3 cells was successfully directed through 6 stages, including definitive endoderm, primitive gut tube, early and late pancreatic progenitors, pancreatic endocrine cells, and finally, mature pancreatic [3 cells. Our study demonstrated that addition of certain culture factors, in isolation or more prominently in combination—significantly improved the yield and maturity of [3 cells at the end of differentiation, as evidenced by both FACS analysis and GSIS assays. FACS results clearly indicated that the timing and concentration of ROCK inhibitor addition, exemplified using Y-27632, during Stage 2 positively impacted the differentiation outcome. When Y-27632 (10 pM) was administered at different times during Stage 2, the highest percentage of NKX6.1+ / C-peptide double-positive cells (27%) was achieved 12 hours post-entry into Stage 2, compared to the untreated control group (16.6%). Similarly, adding ErbB receptor tyrosine kinase agonist, exemplified using BTC, at Stage 5 also influenced the differentiation of [3 cells. A dose of 10 pM BTC throughout Stage 5 yielded the highest percentage of double-positive cells (27%), outperforming both lower and higher concentrations. At Stage 6, adding a canonical Wnt pathway agonist, exemplified using ISX9, further optimised [3 cell differentiation. Among the tested concentrations, 5 pM ISX9 throughout Stage 6 gave the best results, increasing the percentage of NKX6.1+ / C-peptide double-positive cells to 27.8%, compared to the untreated control (16.4%). Notably, when the three molecules were used in combination—Y-27632 (10 pM) at 12 hours into Stage 2, BTC (10 pM) throughout Stage 5, and ISX9 (5 pM) during Stage 6—there was a consistent and synergistic improvement in [3 cell yield across multiple tests. The final combination treatment yielded NKX6.1+ / C-peptide double-positive percentages significantly higher than untreated controls, consistently around 27%. Functionality of the differentiated [3 cells was validated using glucose-stimulated insulin secretion (GSIS) assays. iPSC-derived [3 cells treated with the small molecule combination exhibited a robust response to glucose fluctuations, mimicking the behavior of natural human islet [3 cells. In contrast, [3 cells generated via traditional differentiation protocols displayed a much weaker insulin secretion response under the same conditions. These findings underscore the potential of the use of three classes of molecules in enhancing both the quantity and functionality of iPSC-derived [3 cells. This novel method of inducing the differentiation of iPSCs into mature, insulin-secreting [3 cells has significant implications for treating diabetes. By promoting the maturation and functionality of [3 cells, this supports the regulation of blood glucose levels, reduces the risk of diabetes-related complications, and contributes to overall improvements in quality of life, longevity, and societal well-being. Summary We have uncovered a novel approach to enhancing the differentiation of human iPSCs into functional pancreatic [3 cells using a variety of three small molecules in isolation or combination. The differentiation process proceeded through six stages, culminating in mature [3 cells characterised by NKX6.1 and C-peptide coexpression. FACS analysis demonstrated that Y-27632, when added 12 hours into Stage 2, significantly increased the yield of double-positive [3 cells (27%) compared to untreated controls (16.6%). Similarly, the addition of 10 pM BTC during Stage 5 and 5 pM ISX9 during Stage 6 further improved [3 cell differentiation, achieving optimal results with these concentrations. When these molecules were used sequentially, [3 cell yield and functionality were consistently enhanced, as shown by glucose-stimulated insulin 5 secretion (GSIS) assays. The treated iPSC-derived [3 cells responded robustly to low and high glucose challenges, closely mimicking the insulin secretion dynamics of natural human islets. This method significantly improves the efficiency and quality of [3 cell generation, offering promising therapeutic potential for diabetes treatment by supporting better glucose regulation and reducing disease-related complications. .0 References 1. Hogrebe et al. (2021). Generation of insulin-producing pancreatic [3 cells from multiple human stem cell lines. Nat Protoc 16, 4109-4143 (2021). Table 1. Treatment Test 1 Test 2 Test 3 Test 4 Test 5 Mean Std. Dev Control (T1) 16.6 15.4 17.3 14.8 16.1 16.0 0.44 0 hr(T2) 25.9 24.1 22.5 21.4 22.3 0.79 12hr(T3) illiM 26.3 28.1 26.8 iiiiiia 0.33 24 hr(T4) 22 23.2 22.7 20.9 21.7 22.1 0.40 36 hr(T5) 13.8 12.7 14.2 13.1 15.2 13.8 0.44 Source Sum of Squares, SS Degrees of Freedom, DF Mean Square, MS F statistic p-value Treatment 598.6816 4 149.6704 116.6384 1.4588e- 13 Error 25.6640 20 1.2832 Total 624.3456 24 Treatments Pair Tukey HSD Q statistic Tukey HSD p-value Tukey HSD inference T1 vs T2 14.2125 0.0010053 p<0.01 T1 vs T3 22.0294 0.0010053 p<0.01 T1 vs T4 11.9622 0.0010053 p<0.01 T1 vs T5 4.4217 0.0379074 p<0.05 T2 vs T3 7.8169 0.0010053 p<0.01 2 vs 14 Z. Z'OUO 0 hl / 463'1 T2 vs T5 18.6342 0.0010053 p<0.01 T3 vs T4 10.0672 0.0010053 p<0.01 T3 vs T5 26.4510 0.0010053 p<0.01 T4 vs T5 16.3838 0.0010053 p<0.01 10 Treatment Test 1 Test 2 Test 3 Test 4 Test5 Mean Std. Dev Control (T1) 15.6 18.1 14.3 15.9 13.6 liiiiiil 0.77 5 pM (T2) 19.3 21.1 20.5 18.7 20.2 19.9 0.43 10 pM (T3) 31.4 26.8 29.3 20 pM (T4) 24.2 22 6 23.4 24.8 22.0 23.4 0.51 40 pM (T5) 20.7 19.4 21.5 18.8 20.1 20.1 0.47 Source Sum of Squares, SS Degrees of Freedom, DF Mean Square, MS F statistic p-value Treatment 508.1944 4 127.0486 70.4417 1.6904e- 11 Error 36.0720 20 1.8036 Total 544.2664 24 Treatments Pair Tukey HSD Q statistic Tukey HSD p-value Tukey HSD inference T1 vs T2 7.4259 0.0010053 p<0.01 T1 vs T3 22.6440 0.0010053 p<0.01 T1 vs T4 13.1535 0.0010053 p<0.01 T1 vs T5 7.6590 0.0010053 p<0.01 T2 vs T3 15.2181 0.0010053 p<0.01 T2 vs T4 5.7276 0.0050397 p<0.01 T2 vs T8 o 2331 T3 vs T4 9.4905 0.0010053 p<0.01 T3 vs T5 14.9850 0.0010053 p<0.01 T4 vs T5 5.4945 0.0072888 p<0.01 10 15 10 15 Treatment Test 1 Test 2 Test 3 Test 4 Test5 Mean Std. Dev Control (T1) 16.4 18.1 14.3 15.9 13.6 liiiiiil 0.80 2.5 pM (T2) 19.5 18.3 20.7 20.4 21.2 20.0 0.51 5 pM (T3| 29.4 28.2 26.8 27.1 iii®® 10 pM (T4) 14.5 15.4 13.7 15.1 14.8 14.7 0.29 20 pM (T5) 11.1 12.2 10.7 13.2 11.8 11.8 0.44 40 pM (T6) 8.66 10.3 9.54 9.32 7.56 9.08 0.46 Source Sum of Squares, SS Degrees of Freedom, DF Mean Square, MS F statistic p-value Treatment 1,108.4161 5 221.6832 166.8238 1.1102e- 16 Error 31.8923 24 1.3288 Total 1,140.3084 29 treatments pair Tukey HSD Q statistic Tukey HSD p-value Tukey HSD inference T1 vs T2 8.4573 0.0010053 ** p<0.01 T1 vs T3 23.5875 0.0010053 ** p<0.01 1 '1 14 T1 vs T5 7.4875 0.0010053 p<0.01 T1 vs T6 12.7714 0.0010053 p<0.01 T2 vs T3 15.1301 0.0010053 p<0.01 T2 vs T4 10.3195 0.0010053 p<0.01 T2 vs T5 15.9448 0.0010053 p<0.01 T2 vs T6 21.2287 0.0010053 p<0.01 T3 vs T4 25.4496 0.0010053 p<0.01 T3 vs T5 31.0749 0.0010053 p<0.01 T3 vs T6 36.3588 0.0010053 p<0.01 T4 vs T5 5.6253 0.0065398 p<0.01 T4 vs T6 10.9092 0.0010053 p<0.01 T5 vs T6 5.2839 0.0116197 p<0.05 Treatment Test 1 Test 2 Test 3 Test 4 Test5 Mean Std. Dev Control (T1) 16.4 18.1 14.3 15.9 13.6 15.7 0.80 Y-27632+BTC+ISX9 (T2) 37.3 38.6 41.9 43.6 40.7 40.4 ■■■ Source Sum of Squares, SS Degrees of Freedom, DF Mean Square, MS F statistic p-value Treatment 1,532.6440 1 1,532.6440 321.9840 9.5390e-08 Error 38.0800 8 4.7600 Total 1,570.7240 9 5 Table 8. treatments pair Tukey HSD Q statistic Tukey HSD p-value Tukey HSD inference T1 vs T2 25.3765 0.0010053 p<0.01 10