Use of SP0CK2 as a regulator of differentiation of human pluripotent stem cells towards pancreatic β-cells

Abstract

The subject of the present invention is a use of SPARC / osteonectin, CWCV and a Kazal-like domain proteoglycan-2 (SPOCK2) as a regulator of differentiation of human pluripotent stem cells (hPSCs) towards pancreatic β-cells. SPOCK2 influences the formation of β-cells in a process of differentiation from human stem cells (SC-β, stem cell-derived β-cells), regulating the proliferation and functionality thereof. SPOCK2 is a negative regulator of these processes. Decreasing the level of SPOCK2 contributes to a substantial increase in the proliferation of the SC-β-cells, while activation of SP0CK2 inhibits their expansion. Additionally, the lack of SPOCK2 during the differentiation of hPSCs towards β-cells already at their progenitor step accelerates this process, which results in a more efficient differentiation thereof. The SC-p-cells formed in this manner are characterized by an increased glucose-stimulated in vitro and in vivo insulin (INS) secretion, comparable to human pancreatic islet β-cells. Use of SPOCK2 as a regulator of differentiation of human pluripotent stem cells towards pancreatic β-cells.

Description

[0001] Use of SPOCK2 as a regulator of differentiation of human pluripotent stem cells towards pancreatic p-cells

[0002] The subject of the present invention is a use of SP0CK2 as a regulator of differentiation of human pluripotent stem cells towards pancreatic p-cells.

[0003] The present invention discloses a use of SP0CK2 (full name: SPARC / osteonectin, CWCV and a Kazal-like domain proteoglycan-2) as a regulator of differentiation of human pluripotent stem cells (hPSCs) towards pancreatic p-cells. SPOCK2 influences the formation of p-cells in a process of differentiation from human stem cells, regulating the proliferation and functionality thereof. SPOCK2 is a negative regulator of these processes. Decreasing the level of SPOCK2 contributes to a substantial increase in the proliferation of the stem cell derived p-cells (SC-p-cells), while activation of SPOCK2 inhibits their expansion. Additionally, the lack of SPOCK2 during the differentiation of hPSCs towards p-cells already at their progenitor step accelerates this process, which results in a more efficient differentiation thereof. The SC-p-cells formed in this manner are characterized by an increased glucose- stimulated in vitro and in vivo insulin (INS) secretion, comparable to human pancreatic islet P-cells.

[0004] Current protocols of in vitro differentiation of hPSC towards p-cells are based on mimicking the consecutive developmental steps of the pancreas by modulating the signaling pathways involved in this process. The modulation of the individual signaling pathways occurs with the employment of low-molecular-weight compounds and growth factors. Currently, the highest differentiation efficiency is achieved when employing 3D protocols in which the cells form organoids suspended in a differentiating medium. The first step is the differentiation of the hPSCs into the definitive endoderm (DE) by activating the canonical pathway of WNT and TGFp. CHIR99021, by inhibiting GSK3P, activates the canonical pathway of WNT. Activation of the TGFp pathway typically occurs through the activin A protein. The differentiation efficiency at this step amounts to about 90-95% (D'Amour et al., 2005, Nat Biotechnol, 23(12), 1534-1541), (Borowiak & Melton, 2009, Curr Opin Cell Biol, 21(6), 727-732), (Takenaga, Fukumoto, & Hori, 2007, J Cell Sci, 120(Pt 12), 2078-2090).

[0005] After reaching the DE stage, activation of the FGF pathway takes place by adding the keratinocyte growth factor (KGF). The activation of the FGF pathway leads to further specification into the primitive gut-tube (PGT) endoderm cells, that is, the precursors for the esophagus, stomach, Liver and pancreas (D‘ Amour et al., 2006, Nat Biotechnol, 24(11), 1392- 1401), (Kroon et al., 2008, Nat Biotechnol, 26(4), 443-452), (Russ et al., 2015, EMBOJ, 34(13), 1759-1772 The next stage reached by the cells during the differentiation are the pancreatic progenitor cells (PPs). The addition of retinoic acid (RA) to the differentiating medium induces expression of the marker gene PDX1 , a transcription factor necessary for pancreatic formation (Johannesson et al., 2009, PLoS One, 4(3), e4794). Additionally, a combination of the embryonic growth factor (EGF) and nicotinamide increases the in vitro differentiation efficiency of the human PSCs into the [3-cells by activating the expression of the specific NKX6-1 transcription factor already at the PP cell step. NKX6-1 is necessary for the proper development and functioning of the 3-cells (McGaugh & Nostro, 2017, J Vis Exp(l2l)). S0X9 is also a distinguishing marker of the PP cells. At this step of the differentiation, an efficiency of about 70% is achieved. A further step of the in vitro differentiation is acquisition of the features of endocrine progenitors (EPs) by the cells. Inhibition of the Notch pathway is necessary to activate NGN3 and initiate the formation of the EP cells (Murtaugh, Stanger, Kwan, & Melton, 2003, Proc Natl Acad Sci U S A, 100(25), 14920-14925), (Zhang et al., 2021, Cell Death Dis, 12(10), 867). Furthermore, the inhibition of the TGF3 pathway by the addition of the ALK5 receptor inhibitor (a type-l TGF£ receptor) promotes the differentiation of the endocrine pancreatic cells (Nostro et al., 2011, Development, 138(5), 861-871), (Rezania et al., 2012, Diabetes, 61(8), 2016-2029). The EP cell stage is characterized by the expression of genes such as CHGA and NGN3, and the differentiation efficiency amounts to about 60%. The final stage is maturation of the EP cells and specialization towards functional p-cells. However, the mechanisms guiding the processes of maturation of the EPs and their differentiation towards the [3-cells are still insufficiently recognized. During the in vitro differentiation, the low and variable efficiency of obtaining functional [3-cells, which is about 20-50% of cells, continues to pose a problem. After reaching the stage of the early immature [3-cells, maturation thereof ensues, i.e., they acquire features characteristic of mature functional [3-cells, such as the expression of specific genes (INS, NKX6-1, PDX1, CHGA, PCSK1) and the ability to secrete INS in response to an increased level of glucose.

[0006] Patents concerning protocols aimed at yielding [3-cells formed in the process of differentiation from hPSCs.

[0007] D'Amouret et al. describe the production of enriched cultures of definitive endoderm (DE) derived from human embryonic stem cells (ESs) in the presence of a high concentration of activin and a low serum level (D'Amour et al., 2005, Nat Biotechnol, 23(12), 1534-1541), which has been illustrated in the U.S. Patent No. 7704738. Transplantation of these cells under the Bowman's capsule of mice caused differentiation into more mature cells with features of the endodermal tissue, which is discussed in the U.S. Patent No. 7704738.

[0008] The endoderm cells derived from human embryonic stem cells may be further differentiated into pancreatic precursor cells (PPs - pancreatic progenitors) with expression of the PDX1 transcription factor after the addition of FGF-10 and retinoic acid (U.S. No. 2005 / 0266554A1). Subsequent transplantation of the PP cells to the fat cushion of immunodeficient mice caused the formation of functional endocrine pancreatic cells after a 3- 4-month maturation phase. The problem has been illustrated in the U.S. Patent No. 7993920 and the U.S. Patent No. 7534608.

[0009] Fisk et al. describe a system for producing pancreatic islet cells from human embryonic stem cells, presented in the U.S. Patent No. 7033831. In this case, the differentiation pathway was divided into three steps. Human embryonic stem cells were first differentiated into the endoderm by employing a combination of sodium butyrate and activin A, discussed in the U.S. Patent No. 7326572. The cells were then cultured with BMP antagonists, such as Noggin, combined with EGF or betacellulin in order to produce PDX1 -positive cells. The final differentiation was induced by nicotinamide.

[0010] A use of ephrin and sphingosine-1 -phosphate ligands as regulators of differentiation of pluripotent stem cells into endocrine cells has been illustrated in the patent US10066210B2.

[0011] The latest papers presenting protocols for differentiation of the hPSCs towards the pancreatic P-cells:

[0012] • Barsby T. et al., Differentiating functional human islet-like aggregates from pluripotent stem cells. STAR Protoc. 2022 Dec 16;3(4): 101711. Epub 2022 Sep 21. PM ID: 36136756; PMCID: PMC9508476. https: / / doi: 10.1016 / j.xpro.2022.101711.

[0013] • Nair, G. G. et al. Author Correction: Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived beta cells. Nat Cell Biol 21 , 792 (2019). https: / / doi.Org / 10.1038 / S41556-019-0316-3

[0014] • Velazco-Cruz, L., et al., Acquisition of Dynamic Function in Human Stem Cell-Derived P Cells. Stem Cell Reports, 12(2), 351-365 (2019). https: / / d0i.0rg / l 0.1016 / j.stemcr.2018.12.012

[0015] • Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133 (2014). https: / / d0i.0rg / l 0.1038 / nbt.3033 • Pagliuca, F. W. et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428-439 (2014). https: / / doi.Org / 10.1016 / j.cell.2014.09.040

[0016] State of art concerning the SPOCK2 protein:

[0017] SPOCK2 is a secreted multi-domain proteoglycan which belongs to the BM-40 / SPARC protein family and is a part of the extracellular matrix (Viloria et al., 2016, Sci Rep, 6, 37839.)' (Hartmann & Maurer, 2001, Matrix Biol, 20(1), 23-35). SPOCK2 isexpressed in brain endothelial cells and neurons (Vannahme et al., 1999, J Neurochem, 73(1), 12-20) as well as in pancreatic p-cells and their endocrine progenitors (EPs) (Single-cell RNA sequencing atlases: https: / / eolaniru.shinvapps.io / shinyapp / : http: / / singlecell.charite.de / cellbrowser / pancreas / ). The SP0CK2 gene encodes a full-length protein with a length of 424 amino acids, containing thyroglobulin-1 , a vesicle-like domain and a calcium-binding domain (Viloria et al., 2016, Sci Rep, 6, 37839). Currently, few functional studies focus on SPOCK2. As demonstrated so far, SPOCK2 has been associated with pancreatic ductal adenocarcinoma (Aghamaliyev et al., 2023, J Cancer Res Clin Oncol, 149(11), 9191-9200), lung adenocarcinoma (Zhao et al., 2020, Front Genet, 11, 588499), prostate cancer (Liu, Ren, & Song, 2019, ), ovarian cancer (Lou et al., 2019, PeerJ, 7, e7163.) , and bronchopulmonary dysplasia (Hadchouel et al., 2011, Am J Respir Crit Care Med, 184(10), 1164-1170) and may become a potential candidate for a prognostic marker. In a recent genome-wide association study (GWAS) on pain, it has been demonstrated that the SPOCK2 gene may contribute to a chronic, but not acute back pain (Freidin et al., 2019, Pain, 160(6), 1361-1373)- (Bortsov et al., 2022, Pain Rep, 7(5), e1018.). SPOCK2 has been shown to modulate neuropathic pain by interacting with the MT1-MMP metalloproteinase (MMP14) in order to regulate the activation of matrix metalloproteinase-2 (MMP2) in rat astrocytes (Wang et al., 2024, J Neuroinflammation, 21(1), 57). So far, the role of SPOCK2 in the process of differentiation and maturation of p-cells has not been recognized.

[0018] The essence of the present invention is a use of SPOCK2 as a regulator of differentiation of human pluripotent stem cells towards pancreatic p-cells.

[0019] The implementation of the solution according to the invention resulted in the following technical and ractical effects:

[0020] The p-cells obtained from human pluripotent stem cells, hPSCs, constitute a source of cells for transplantations in patients with diabetes. Current protocols for generation of these cells still leave much to be desired in terms of the amount of functional SC-p- cells. Despite the fact that certain mitogens may be utilized to multiply the p-cells, adult p-cells replicate extremely rarely. In the present invention, the p-cells derived from hPSCs were used and an extracellular matrix protein - SPOCK2 was identified as an inhibitor of proliferation of the early SC-p-cells. Decreasing the level of SP0CK2 contributed to a substantial increase in the proliferation, while activation of SP0CK2 inhibited the expansion of the SC-p-cells. Additionally, the lack of SP0CK2 during the differentiation of hPSCs towards the SC-p-cells already at their progenitor step accelerated this process, which resulted in a more efficient differentiation thereof. The human SC-p-cells with an increased proliferation following a decrease in the level of SPOCK2 were characterized by an increased glucose-stimulated in vitro and in vivo insulin secretion, comparable to human pancreatic islet p-cells.

[0021] • Improving the functioning of the p-cells in diabetes.

[0022] • Regulation, differentiation and proliferation of the SC-p-cells.

[0023] • Possibilities for development of drugs and targeted therapies aimed at increasing the mass and functionality of the SC-p-cells.

[0024] • In vivo functionality and transplantation success:

[0025] • Effective transplantation of the SC-p-cells with a deletion of SPOCK2 under the Bowman's capsule of mice, leading to improved glucose-stimulated insulin secretion and maintenance of euglycemia, highlights the in vivo application and potential success of regeneration strategies.

[0026] • Potential therapeutic application in diabetes:

[0027] • Possibility of increasing the mass and functionality of the p-cells in patients with diabetes.

[0028] • Improving the effectiveness of diabetes treatment.

[0029] Thus, the SPOCK2 inhibition-controlled method for cell culture offers a viable approach to increase the amount and quality of the p-cells derived from hPSCs, which may constitute a robust source of functional cells for transplantation.

[0030] Examples:

[0031] The method for culture and the process of differentiation of all the hPSC lines employed in the examples towards the pancreatic p-cells were performed in the following manner: The hPSC cells were cultured in a StemFlex medium (Thermo Fisher Scientific) on vitronectin- coated plates (rh VTN; Thermo Fisher Scientific) at a temperature of 37°C, in an atmosphere containing 5% CO2. The cells were passaged every 3-4 days by means of PBS-EDTA, when their confluence reached -80%. Before the differentiation, the cells were dissociated by means of TrypLE (Thermo Fisher Scientific) in order to obtain a single-cell suspension. The differentiation towards the pancreatic p-cells was performed in the form of 3D organoids, the plate was on an orbital shaker throughout the whole duration of the differentiation. Before initiating the differentiation, the organoids were washed with a DMEM / F12 medium. The differentiating medium was changed daily until day 20 and, after the day 20, every two days according to the protocol of Nair et al. (Nair et al., 2019, Nat Cell Biol, 21(6), 792) with minor changes. The detailed composition of the basal media is shown in Table 1 below.

[0032] Table 1.

[0033] Low-molecular-weight compounds and differentiation-directing proteins were added each day to an appropriate volume of the basal differentiating medium. The detailed composition of the differentiating medium employed during the differentiation is given below. The final concentration of the low-molecular-weight compound or the protein is written in a bracket.

[0034] • Day 1 : basal medium S1 , CHIR99021 (3 pM), activin A (100 ng / ml), vitamin C (250 pM).

[0035] • Day 2-3: basal medium S1 , activin A (100 ng / ml), vitamin C (250 pM).

[0036] • Day 4-6: basal medium S2, KGF (50 ng / ml), vitamin C (250 pM), IWP2 (1.25 pM). • Day 7: basal medium S3, KGF (50 ng / ml), retinoic acid (2 pM), PdBu (500 nM), SANT-1 (250 nM), LDN193189 (200 nM), vitamin C (250 pM), Y-27632 (10 pM).

[0037] • Day 8-12: basal medium S3, activin A (5 ng / mL), KGF (50 ng / ml), retinoic acid (100 nM), SANT-1 (250 nM), vitamin C (250 pM), Y-27632 (10 pM), IWP2 (1.25 pM), EGF (100 ng / mL), nicotinamide (10 mM). 216

[0038] • Day 13-19: basal medium S5, retinoic acid (100 nM), SANT-1 (250 nM), LY-411575 (1 pM), T3 hormone (1 pM), Alk5 inhibitor (10 pM), betacellulin (20 ng / mL), vitamin C (250 pM).

[0039] • Day 20-26: ESFM basal medium.

[0040] The protocol has been shown in Fig. 1 which illustrates the steps of in vitro differentiation from human PSCs towards pancreatic p-cells. Marked in the diagram are the individual stages (hPSC - SC-P), the day of the differentiation on which a given stage is reached, and the low-molecular-weight compounds (e.g., CHIR, IWP2, T3), the proteins (e.g., activin A - AA, KGF, EGF, betacellulin) influencing the reaching of a specific stage by the differentiated cells.

[0041] The subject of the present invention is illustrated by the three examples below.

[0042] Example 1: Identification and validation of the inhibitory role of SPOCK2 in the differentiation of human pluripotent stem cells towards pancreatic p-cells

[0043] In order to identify and verify the role of SPOCK2 as the inhibitor of the process of differentiation of the pancreatic p-cells from hPSCs, two hPSC lines: a wild type (WT) and an hPSC line with deletion of the SPOCK2 protein (SPOCK2 KO or KO), were utilized. At the pluripotent cell step, the CRISPR / Cas9 technology was used to inactivate the SPOCK2 gene, which in effect resulted in the lack of formation of the SPOCK2 protein in these cells. In the course of the differentiation, the first considerable change was observed already at the early PP step of day 10. The SPOCK2 KO PP cells exhibited higher expression of NKX6-1 and PDX1 than in the WT PP cells (Fig. 2). Fig. 2 shows representative confocal microscope images of the WT and SPOCK2 KO cells from day 10 (the early PP stage) and a quantification of fluorescence signals of the cells stained with antibodies against CHGA (green) and NKX6-1 (red), and PDX1 (white). DAPI (blue) denotes the cellular nuclei. Scale = 100 pm. A Student's t-test was used. The data are shown as means ± SD. N = 3 biological replicates.

[0044] On day 13, at the late PP stage, the expression of NKX6-1 and PDX1 still persisted at a higher level in the SPOCK2 KO PPs. Moreover, the expression of CHGA - a marker of endocrine cells, was also considerably elevated in the SPOCK2 KO cells, unlike in the WT PP cells (Fig. 3 and Fig. 4), as demonstrated by the immunofluorescence staining analysis. Fig. 3 shows representative confocal microscope images of the WT and SPOCK2 KO cells from day 13 (the late PP stage) and a quantification of fluorescence signals of the cells stained with antibodies against NKX6-1 (green) and PDX1 (red), and SOX9 (white). Similarly, Fig. 4 shows the WT and SPOCK2 KO cells from day 13 (the late PP stage) and a quantification of fluorescence signals of the cells stained with antibodies against PDX1 (green) and CHGA (red). In both cases, DAPI (blue) denotes the cellular nuclei. Scale = 100 pm. A Student's t-test was used. The data are shown as means ± SD. N = 3 biological replicates.

[0045] Single-cell RNA sequencing (scRNA-seq) as well as immunofluorescence staining analyses demonstrated that the early SPOCK2 KO SC-p-cells from day 21 were characterized by higher expression of p-cell markers, such as C-PEP, NKX6-1 and PCSK1, than in the WT (Figures 5 - 8), suggesting that the SPOCK2 KO SC-p-cells are more mature at this step of differentiation. Fig. 5 shows representative confocal microscope images of the WT and SPOCK2 KO cells from day 21 (the p-cell stage) and a quantification of fluorescence signals of the cells stained with antibodies against C-PEP (green) and NKX6-1 (red). Similarly, Fig. 6 shows the WT and SPOCK2 KO cells from day 21 (the p-cell stage) and a quantification of fluorescence signals of the cells stained with antibodies against C-PEP (green) and PCSK1 (red). In both cases, DAPI (blue) denotes the cellular nuclei. Scale = 100 pm. A Student's t- test was used. The data are shown as means ± SD. N = 3 biological replicates. Fig. 7 shows a graph of relative expression of selected genes, EP markers, whereas Fig. 8 shows a graph of relative expression of p-cell markers, in the SPOCK2 KO and WT cells from day 21 of the differentiation. The dot size represents the percentage of cells exhibiting expression of a gene. Red denotes up-regulation and blue denotes down-regulation of the expression of genes. Additionally, the scRNA-seq analysis rendered evident significant differences in the proportions between the WT and SPOCK2 KO SC-p-cells. The populations of the EP and endocrine cells were significantly elevated in the SPOCK2 KO SC-ps (Fig. 9). Fig. 9 shows a percentage estimate of the proportion of the PP, EP and endocrine cells among the cells differentiated up to day 21 (the SC-p stage) by employing the data concerning scRNA-seq for WT and SPOCK2 KO.

[0046] Conclusions:

[0047] In summary, the differences between SPOCK2 KO and WT during the differentiation indicate that the lack of SPOCK2 accelerates the differentiation process already at the PP stage, and as a result, in the eventual pool of the SC-p-cells, we have more INS-producing cells, characterized by a higher expression of the p-cell markers compared to the WT cells, which suggests that the SPOCK2 KO SC-p-cells are more functional (Example 3).

[0048] Example 2: Identification and verification of the role of SPOCK2 as an inhibitor of the proliferation of the pancreatic p-cells formed in the process of differentiation from hPSCs.

[0049] In order to identify and verify the role of SPOCK2 as the inhibitor of the proliferation of the pancreatic p-cells formed in the process of differentiation from hPSCs, the KO and WT hPSC lines already mentioned in Example 1 were utilized. Additionally, a line with 70% decreased expression of SPOCK2 (SPOCK2 KD or KD) was employed formed by employing a lentiviral (shRNA) system at the EP step. Furthermore, in order to increase the amount of the SPOCK2 protein (SPOCK2 GoF), a recombinant human SPOCK2 (rh SPOCK2) (R&D Systems) was used, rh SPOCK2 in the concentration range of 0 - 250 ng / ml was directly added to the differentiating medium at the EP step for 2 - 7 days. All the hPSCs were cultured and differentiated into the SC-p-cells as in the above-described Example 1 and as shown in Figure 1. Then, the cells were subjected to a varied analysis for their proliferative abilities.

[0050] The rh SPOCK2 protein was demonstrated to inhibit proliferation of the SC-p-cells. The SC- P-cells were enriched by employing antibodies against the CD49a and TM4SF4 antigen which are surface markers of human p-cells and a-cells, respectively. The sorted-out SC-p- cells (CD49a+ / TM4SF4-) were treated with 100 ng / ml or 250 ng / ml rh SPOCK2, causing a 44% drop in expression of the pHH3 proliferation marker at the highest concentration of rh SPOCK2, as demonstrated by a flow cytometry analysis (Fig. 10). Fig. 10 shows a FACS analysis of the p-cells derived from hPSCs, stained with antibodies against a surface marker of p-cells, a GFP-A-conjugated CD49a (x-axis), and a surface marker of a-cells - an APC-A- conjugated TM4SF4 (y-axis), showing the percentage of p-cells (gate P4) and a-cells (gate P5). Quantification of proliferating cells (pHH3 +) among the p-cells (CD49a+ and TM4SF4-) treated with the rh SPOCK2 protein for seven days. The data were normalized relative to a non-treated control. In order to determine the p-values, an ANOVA test was used. The data are shown as means ± SD. N = 5 biological replicates.

[0051] A lentiviral system was used to deliver an anti-SPOCK2 shRNA or a hollow GIPZ plasmid, which lead to a 70% decrease in the expression level of SPOCK2 and, simultaneously, to a 55% and 77% rise of the SC-p-cells with expression of pHH3 and C-PEP (a C-peptide which is a by-product of INS maturation and a p-cell marker) for SPOCK2 KD, relative to WT or GIPZ, respectively, as demonstrated by an immunofluorescence staining analysis (Fig. 11). Fig. 11 shows a quantification of the proliferating SC-p-cells. The C-PEP+ / pHH3+ cells were compared to the C-PEP+ WT, GIPZ or SPOCK2 KD cells. A higher number of the SPOCK2 KD SC-p-cells with expression of pHH3 compared to the control cells is shown in %. In order to determine the p-values, an ANOVA test was used. The data are shown as means ± SD. N = 5 biological replicates.

[0052] To study the proliferation of the SPOCK2 KO SC-p-cells, -6400 WT and -5500 KO cells were transcriptionally profiled by employing the scRNA-seq technology. After performing appropriate normalizations and identification of cell clusters and genes with altered expression, it was demonstrated that twice as many cells were formed with the expression of INS and NKX6-1 in SPOCK2 KO than in WT SC-p (Fig. 12). Fig. 12 presents a UMAP graph showing the distribution and percentage of cells with co-expression of INS and NKX6-1 for WT (left) and SPOCK2 KO (right). Cells with the expression of NKX6-1 were marked in blue, whereas those positive for INS were marked in red.

[0053] Then, a cell cycle evaluation was performed by employing the Seurat package in order to identify the proliferating SC-p-cells. 19% of the SC-p-cells with SPOCK2 KO, compared to 12% of the WT SC-p-cells, with the expression of INS or INS / NKX6-1 , were in active phases of the cell cycle: S and G2 / M (Fig. 13). Fig. 13 shows the proportion (%) of the SPOCK2 KO and WT SC-p-cells (referred to as INS+ / NKX6.1+) in the G1, S, or G2M phase of the cell cycle.

[0054] Conclusions:

[0055] In summary, SPOCK2 was consistently observed to inhibit the proliferation of immature human p-cells. Several approaches were used to manipulate the expression of SPOCK2 and two independent hPSC lines - HLIES8 and H1 were used in order to obtain the SC-p-cells, indicating that the influence of SPOCK2 on the proliferation of human p-cells is not limited to a particular hPSC line.

[0056] Example 3: Identification and verification of the role of SPOCK2 as an inhibitor of glucose- dependent insulin secretion by the pancreatic p-cells formed in the process of differentiation from hPSCs.

[0057] In order to identify and verify the role of SPOCK2 as the inhibitor of the glucose-dependent insulin (INS) secretion by the pancreatic p-cells formed in the process of differentiation from hPSCs, WT hPSC lines and a (KD) line with transient expression of SPOCK2, formed by employing the lentiviral technology (shRNA) at the EP step, were utilized. The WT and KD hPSCs were cultured and differentiated into the SC-p-cells as in the above-described Example 1 and as shown in Figure 1. Then, the cells were subjected to an analysis of glucose-stimulated insulin secretion (GSIS) in order to evaluate the function of SPOCK2- deficient SC-ps.

[0058] The cells were incubated for 1 hour in a Krebs buffer: 128 mM NaCI, 5 mM KCI, 2.7 mM CaCI2, 1.2 mM MgCI2, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHC03, 10 mM HEPES, 0.1% BSA, then incubated in a Krebs buffer with a low glucose content of 2.8 mM D-(+)- glucose and a high glucose content of 16.7 mM D-(+)-glucose for 30 minutes in each step. After each incubation step, supernatants were collected. The cells were then dissociated by means of TrypLE in order to obtain a single-cell suspension and counted (Countess, Invitrogen). Human insulin was measured from the supernatants by employing an ELISA (Mercodia) test, in accordance with the manufacturer's instructions.

[0059] The WT control cells and the SPOCK2 KD cells exhibited minimal secretion of INS in response to 2.8 mM of glucose, but after exposure to 16.7 mM of glucose, the SPOCK2 KD SC-p-cells secreted an average of 5.5 plll / ml of INS, which constitutes a rise by 85% (Fig. 14). An INS stimulation index (SI), defined as the ratio of high-glucose-level- to low-glucose- level-induced insulin secretion, was significantly higher for the SPOCK2 KD SC-p-cells than for WT (Fig. 14). Fig. 14 shows a graph from the measurement of insulin secretion by employing an ELISA test in the SC-p WT or SPOCK2 KD cells after treatment with low glucose (2.8 mM) or high glucose (16.7 mM) normalized to total cells. The insulin stimulation index of the WT and SPOCK2 KD SC-p-cells, compared to human primary islets, is calculated as the ratio of insulin secretion in response to 16.7 mM vs. 2.8 mM of glucose. To determine the p-values shown in the graph, a one-way ANOVA analysis was used for multiple comparisons. The data are shown as means ± SD. N = 4 biological replicates. It is worthwhile to note that the INS secretion in response to 16.7 mM of glucose and the INS SI for the SPOCK2 KD SC-p-cells were comparable to the levels for human primary pancreatic islets.

[0060] Conclusions:

[0061] Decreasing the level of SPOCK2 expression in the cells differentiating towards p-cells caused a pronounced in vitro expansion of the SC-p-cells and significantly increased their glucose-stimulated insulin secretion.

Claims

Claim1. Use of SP0CK2 as a regulator of differentiation of human pluripotent stem cells towards pancreatic p-cells.

Images