WO2020035480A1 - Structures cellulaires de type îlots pancréatiques viables et leur procédé de préparation - Google Patents

Structures cellulaires de type îlots pancréatiques viables et leur procédé de préparation Download PDF

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WO2020035480A1
WO2020035480A1 PCT/EP2019/071680 EP2019071680W WO2020035480A1 WO 2020035480 A1 WO2020035480 A1 WO 2020035480A1 EP 2019071680 W EP2019071680 W EP 2019071680W WO 2020035480 A1 WO2020035480 A1 WO 2020035480A1
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hlsc
cell
cells
pancreatic islet
ils
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Victor Manuel NAVARRO TABLEROS
Giovanni Camussi
Yonatan GOMEZ
Chiara GAI
Chiara PASQUINO
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Unicyte Islet Ag
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/14—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from hepatocytes

Definitions

  • Type 1 diabetes is a disease with an important socio-economic impact, caused by autoimmune destruction of pancreatic insulin producing cells (b-cells) and associated with development of debilitating micro vascular and macro vascular complications. Treatment with exogenous insulin to achieve a glycemic control does not completely prevent long term complications. Both pancreas and islets transplantation restore islets function and potentially reduce long-term diabetic complications, but are limited by both donor shortage and need for immunosuppression. Beta-cell replacement is an attractive therapy to achieve normo- glycemia in patients with diabetes mellitus and several studies focused on generation of human b-cells. Pluripotent stem cells for their unlimited capacity of self-renewal may represent a scalable source of insulin producing cells.
  • Beta-like cells have been differentiated from human induced pluripotent stem cells (hiPCS) and human embryonic stem cells (hESC). Nevertheless, most current approaches are complex and require virus-induced transfection or methods of genetic engineering. The challenge of producing b-cells from pluripotent stem cells faces several inefficiencies including rate of differentiation, generation of immature insulin producing cells, ethical concern, tumors formation or failure to maintain a sustained correction of hyperglycemia. Recently, many efforts have been focused on identifying alternative and abundant cell sources of functional b-cells.
  • pancreatic islets contain different endocrine cells: alpha, beta, gamma, epsilon and PP cells that secrete glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide, respectively.
  • the characteristic conformation of pancreatic islets is important for a fine regulation of the islet function.
  • paracrine cell interactions among the insular cells help to modulate the hormone secretion and the tuning of normal b-cell function.
  • Several multistep protocols have been described to differentiate human embryonic or inducible pluripotent stem cells into pancreatic progenitors able to undergo in vivo maturation with generation of glucose sensitive insulin secreting cells able to reverse diabetes in mice.
  • in vitro mature endocrine cells would advantage reversal of diabetes.
  • progenitors generated in vitro are immature and the protocols mainly produce poly-hormonal cells concomitantly expressing insulin, glucagon and somatostatin.
  • Rezania, A., et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. (2012) Diabetes, 61(8):2016-2029 have recently described a seven-stage protocol that generate glucose responsive insulin secreting cells from human embryonic stem cells. These cells, even if not fully mature, were able to rapidly reverse diabetes. Insulin producing cells may also be generated from pancreatic progenitors.
  • Insulin producing cells have been obtained also by trans-differentiation of adult stem cells, for instance islet-derived mesenchymal stem cells cultured in absence of serum and with an appropriate mix of factors, as well as bone marrow mesenchymal stem cells in conditions mimicking diabetic niche.
  • Other approaches focused on reprogramming pancreatic cell types such as ductal, acinar or alpha cells by inducing epigenetic changes.
  • liver and pancreas share common embryonic origins and the developing liver has been shown to exhibit transcriptional features similar to the adult pancreas.
  • Insulin producing cells were also generated by reprogramming hepatocytes inducing overexpression of PDX1 plus exendin-4 to force proliferation and differentiation.
  • a limitation in using human liver hepatocytes is their low proliferative potential in vitro.
  • Herrera, M. B. et al. Isolation and characterization of a stem cell population from adult human liver. (2006) Stem Cells. 24, 2840-2850 describes the isolation from the adult human liver by a culture strategy leading death of mature hepatocyte of a cell population with high proliferative capacity defined as adult human liver stem-like cells (HLSC).
  • HLSC adult human liver stem-like cells
  • HLSC are clonogenic and express markers of both mesenchymal stem cells and immature hepatocytes. Moreover, HLSC express several stem cells and embryonic markers, such as nanog, Oct4, SOX2 and SSEA4 and are able to undergo multiple differentiations including islet-like phenotype. However, the differentiation protocol was limited by a very low efficiency and the insulin secretion under glucose control was not demonstrated.
  • WO2015091493 discloses in vitro generation of insulin-producing 3D spheroid structures from an isolated adult stem cell by a one-step protocol based on charge-dependent aggregation promoted by a poly-cationic substance added to the cell culture medium.
  • WO2015091493 teaches that an in vitro maturation period of at least 14 days is required to generate glucose-sensitive insulin-secreting pancreatic islet-like cell clusters which are suitable for transplant.
  • pancreatic islet- like cell cultures In contrast to the teaching of the prior art, the present inventors have surprisingly found that shortening the maturation period of pancreatic islet- like cell cultures to less than 14 days is highly beneficial to islet viability without affecting their functional properties. Indeed, viability assessment conducted over multiple days of maturation on cultures of pancreatic islet- like cell clusters grown from an adult stem cell as described in WO2015091493, revealed that the number of viable islets progressively decreased starting from day 7 with a significant viability loss between day 13 and day 14 of post-differentiation.
  • spheroid pancreatic islet-like cell clusters cultured for less than 14 days were capable of responding in vitro to high glucose stimulation by secreting human C-peptide (hC -peptide), starting after day 4 of post-differentiation.
  • C-peptide hormone is an indicator of insulin secretion and is a widely used as a measure of pancreatic b cell function.
  • the secretion of hC- peptide in response to high glucose was higher in less differentiated pancreatic islet-like cell clusters compared to islets at a later stage of in vitro maturation (day 14).
  • pancreatic islet like cell clusters under analysis were capable of undergoing further in vivo maturation following implant under the kidney capsule of streptozotocin-induced diabetic SCID mice and rapidly reversing in vivo hyperglycemia in these mice.
  • the present inventors have therefore found that, compared to WO2015091493, a reduced maturation period of cultures of pancreatic islet-like cell clusters advantageously provides islet structures which are viable and at the same time functional both in vitro and in vivo, making them particularly suitable for clinical applications.
  • the invention provides a method of preparing a population of artificially grown spheroid pancreatic islet-like cell structures, comprising the following culturing steps: (i) maintaining an adult stem cell in a first differentiation liquid cell culture medium comprising a poly-cationic substance;
  • isolated adult stem cells aggregate and differentiate into spheroid pancreatic islet-like cell structures when cultured in a liquid cell culture medium in the presence of a poly-cationic substance.
  • Any poly-cationic substance which is capable of promoting adult stem cell aggregation and differentiation into pancreatic islet cells at a given concentration and which, at that concentration is non-cytotoxic to cells, may be used in the method of the present invention.
  • Illustrative, non-limiting examples of poly-cationic substances suitable for use in the method of the invention are protamine, poly-lysine and cationic dextrans.
  • Protamine preferably in the form of a salt, such as for example protamine sulfate or protamine hydrochloride, is the preferred positive polycation because it is suitable for clinical applications, and is added to the medium at a concentration which preferably ranges between 5 and 20 qg/ml.
  • the most preferred form of protamine is protamine hydrochloride.
  • the adult stem cell used in the method of the invention is preferably an adult mammalian stem cell. Therefore, any liquid cell culture medium capable of sustaining the growth of mammalian cells is suitable for use in the method of the invention.
  • the liquid cell culture medium is a serum-enriched cell culture medium. Serum-enriched DMEM or RPMI are mentioned as non-limiting examples. Serum concentration in the cell culture medium is preferably comprised within the range of from 5 to 20%.
  • the liquid cell culture medium also comprises one or more carbon sources, such as for example glucose and glutamine. Preferred glucose concentrations are within the range of 6-25 mM. Preferred glutamine concentrations are within the range of 0.5-3 mM.
  • the liquid cell culture medium is serum free and serum is replaced by a substitute such as e.g. a protein supplement that provides the beneficial growth-promoting activities of albumin and a and b-globulins.
  • a substitute such as e.g. a protein supplement that provides the beneficial growth-promoting activities of albumin and a and b-globulins.
  • the selection of the most suitable serum substitutes to be used in the method of the invention falls within the knowledge of the person skilled in the art.
  • the expression "adult stem cell” is intended to mean a stem cell that is isolated from an adult tissue, in contrast with an “embryonic stem cell” which is isolated from the inner cell mass of a blastocyst.
  • Adult stem cells are also known as "somatic stem cells”.
  • a post-differentiation period of from 5 to 9 days, preferably of from 5 to 7 days, is the key characteristic of the method according to the invention in that it enables to generate viable pancreatic islet-like cell structures which are at the same time glucose responsive in vitro and capable to restore glycemia in vivo, despite their not fully mature state.
  • the post-differentiation period starts with the first day (day 1) of culture of adult stem cells in liquid cell culture medium in the presence of a poly-cationic substance.
  • the post-differentiation period comprises a first culturing step wherein adult stem cells are maintained in a first differentiation cell culture medium containing a poly-cationic substance, and a second culturing step wherein the first differentiation liquid cell culture medium is replaced with a second differentiation liquid cell culture medium not comprising a poly-cationic substance, and the cells are cultured in said second medium.
  • Any liquid cell culture medium capable of sustaining the growth of mammalian cells is suitable for use as the second differentiation liquid cell culture medium.
  • the same liquid cell culture medium which was used in the first step shall also be employed in the second step, except that it does not contain the poly-cationic substance.
  • the method of the present invention is further characterized in that, following the post- differentiation phase which duration is comprised between 5 to 9 days, preferably between 5 to 7 days, the cultivation of spheroid pancreatic islet-like cell clusters is blocked. Blocking of cultivation may be carried out for example by harvesting the islet-like structures, for example by collecting the supernatant medium containing the islets in suspension and recovering islet-like structures adhering to the culture substrate. It is understood that other methods of blocking islets cultures in vitro may be used within the scope of the invention, as are known in the art.
  • the adult stem cell which is differentiated into pancreatic islet-like cell structures is an adult human liver stem cell (HLSC).
  • a preferred human liver stem cell is the human non-oval liver stem cell (HLSC) expressing both mesenchymal and embryonic stem cell markers disclosed in WO 2006/126219.
  • This cell line is in particular characterized in that it is a non-oval human liver pluripotent progenitor cell line which is isolated from adult tissue, which expresses hepatic cell markers and which has multipotent differentiation abilities and regenerative properties. More in particular, this cell line is capable of differentiating into mature liver cells, insulin producing cells, osteogenic cells and epithelial cells.
  • it expresses one or more markers selected from the group comprising albumin, a- fetoprotein, CK18, CD44, CD29, CD73, CD146, CD105, CD90 and any combination thereof, and it does not express markers selected from the group comprising CD 133, CD117, CK19, CD34, cytochrome P450.
  • the human non-oval liver pluripotent progenitor/stem cells disclosed in WO 2006/126236 were shown to undergo differentiation into a variety of tissue cell types (namely, mature liver cells, epithelial cells, insulin-producing cells and osteogenic cells) and to exert organ regenerating effects.
  • tissue cell types namely, mature liver cells, epithelial cells, insulin-producing cells and osteogenic cells
  • Such cells are derived from a non-oval human liver pluripotent progenitor cell line which expresses hepatic cell markers.
  • Such cells are isolated by a method comprising the steps of: (i) culturing adult liver-derived human mature hepatocytes in a cell culture medium until death of mature hepatocytes and selection of a population of surviving cells having epithelioid morphology;
  • step (ii) expanding the population of surviving cells having epithelioid morphology by culturing in a serum-containing, glucose-containing culture medium supplemented with hEGF (human epithelial growth factor) and bFGF (basic fibroblast growth factor) and comprising the usual inorganic salts, amino acids and vitamins necessary for the growth of mammalian cells and in particular wherein the mature hepatocytes are frozen in a serum- containing culture medium in the presence of a cryoprotecting agent and then thawed prior to culturing according to step (i).
  • hEGF human epithelial growth factor
  • bFGF basic fibroblast growth factor
  • HFSCs human non-oval liver stem/progenitor cells
  • the use of HFSCs in the present invention is preferred for a number of reasons: 1) they are relatively easy to isolate and expand, 2) they are characterized by a degree of proliferation that allows to provide a suitable number of cells for therapeutic use, 3) they can be used autologously and 4) they avoid ethical concerns.
  • the liver and the pancreas share common embryonic origins and the developing liver has been shown to exhibit transcriptional features similar to the adult pancreas (Bose B et al. Cell Biol Int. 2012; 36(11): 1013-20).
  • pancreatic islet-like cell structures obtained by the method of the present invention are characterized by a unique combination of high viability, in vitro functional properties and further in vivo maturation ability, notwithstanding their not complete differentiated state. Accordingly, the scope of the invention also includes a composition comprising a population of artificially grown spheroid pancreatic islet-like cell structures as defined in the appended claims. As further illustrated in the experimental section which follows, viability assessment was conducted by double staining with fluorescein diacetate (FDA) and propidium iodide (PI) on samples of islet-like structures taken at day 4, 5, 7, 10, 11, 13 and 14 during post- differentiation phase.
  • FDA fluorescein diacetate
  • PI propidium iodide
  • the population of spheroid pancreatic islet-like structures according to the present invention are advantageously characterized by a viability of at least 75%as measured by the fluorescein diacetate (FDA) and propidium iodide (PI) assay.
  • the population of spheroid pancreatic islet-like structures shows a viability of at least 80 %, more preferably of at least 85%, even more preferably of at least 90%.
  • the spheroid pancreatic islet-like structures according to the present invention are further characterized by enhanced glucose-responsiveness as measured by the ability of secreting C-peptide in response to high glucose (HG) stimulation in an amount which is at least two fold the amount secreted in response to low glucose (LG) stimulation in both static or dynamic conditions.
  • HG high glucose
  • LG low glucose
  • the amount of C-peptide secreted in response to high glucose (HG) stimulation is of at least 50 pg/ml/400 IEQ preferably at least 80 pg/ml/400 IEQ, more preferably at least 95 pg/ml/400 IEQ.
  • the term“high glucose” is intended to refer to a stimulus of at least 28 mM glucose
  • the term“low glucose” is intended to refer to a stimulus of up to 2.8 mM glucose, excluding 0 value, for example from 0.1 mM up to 2.8 mM glucose.
  • at least 50% of the spheroid pancreatic islet like cell structures in the population have a diameter of less than 150 pm, preferably equal to or less than 100 pm, more preferably of from 50 to 100 pm, as measured during the post- differentiation phase from day 4 to day 13.
  • the mean diameter in the population of spheroid pancreatic islet-like cell structures is less than 100 pm as measured during the post-differentiation phase from day 4 to day 13.
  • the morphometric analysis conducted by the inventors demonstrated that the size distribution in the population of pancreatic islet size changes across in vitro maturation, from day 4 to day 21, with an increase in the average diameter in the islet population above 100 pm from day 14 ( Figure 2B).
  • the size increase appears to correlate with the loss in viability observed in pancreatic islet-like populations during differentiation.
  • islet survival rate both in vitro and in vivo, declines with increasing islet size because of reduced oxygen and nutrient diffusion within larger structures.
  • the pancreatic islet-like cell structures according to the invention are therefore more suitable for in vivo implantation in that they can be better vascularized and/or provided with nutrients from the immediate environments compared to larger islet structures.
  • the spheroid pancreatic islet-like structures express at the protein level the pancreatic hormones which are usually produced by human pancreatic islets (i.e . insulin, glucagon, pancreatic polypeptide, somatostatin and ghrelin).
  • human pancreatic islets i.e . insulin, glucagon, pancreatic polypeptide, somatostatin and ghrelin.
  • the pancreatic islet-like structures according to the invention express one or more additional markers of pancreatic b-cell development and/or function such as, for example, the b-cell transcription factors Nkx6.l, Nkx6.3 and MafB, the endocrine specific markers chromogranin A (CgA) and Ngn3, and the exocrine pancreatic markers PDX1 and synaptophysin, and any combination thereof.
  • the pancreatic islet-like structures according to the invention do no not express or express a reduced level of adult stem cell markers such as, but not limited to, CD90, CD73, CD 105, CD31, as compared to reference undifferentiated adult stem cells.
  • reference undifferentiated adult stem cells refers to corresponding adult stem cells grown in vitro prior to differentiation into spheroid pancreatic islet-like structures.
  • pancreatic islet-like structures according to the invention after in vivo implantation under the renal capsule of streptozotocin-diabetic SCID mice, were able to significantly reduce hyperglycemia in diabetic mice after 1 week of implantation and restore a normal profile of glucose tolerance test.
  • reduction of hyperglycemia was associated with detection of human C-peptide in diabetic mice, which in turn exhibited very low, and in some animal undetectable, levels of murine C-peptide.
  • islet implant was removed, increased glycemia and diabetic profile of glucose tolerance test were restored suggesting that the reversal of diabetes was dependent on implanted pancreatic islet-like structures according to the invention.
  • pancreatic islet-like structures according to the invention By comparing gene expression profiles, a closer association was observed between the explanted pancreatic islet-like structures according to the invention and human islets than to pancreatic-like cell structures cultured in vitro and undifferentiated adult stem cells, respectively.
  • higher expression levels were determined in the explants for several genes involved in insulin secretion and signaling pathway, glucose response, ion channels function, cell differentiation and islets maturation.
  • IEQ/100 ILS is the Islets Equivalent of an average diameter of 150 mih (IEQ) normalized to 100 islets (ILS).
  • IEQ Islets Equivalent of an average diameter of 150 mih
  • ILS islets normalized to 100 islets
  • IEQ of islets isolated from the pancreas and used for transplantation is determined as follows. Suspended islets isolated from the pancreas are evaluated both in number and size (diameter) in order to determine the total islets equivalent (i.e. total islets volume). Islets diameter assessment takes into account 50 pm diameter range increments, from 50 pm to >350 pm, without taking into account diameters ⁇ 50 pm, that do not provide a significant contribution to the total volume.
  • a relative conversion factor is conventionally used to convert the total islets number to Islets Equivalent of an average diameter of 150 pm (IEQ).
  • the pancreatic islet-like structures according to the invention are particularly suitable for use both in clinical application, such as in a method treatment of diabetes by pancreatic islet transplantation, and in in vitro applications, such as screening methods for identifying substances capable of promoting the expression of one or more pancreatic hormones by pancreatic islet cells or for identifying substances capable of exerting a cytotoxic effect on pancreatic islet cells.
  • FIG. 1 in vitro differentiation of HLSC into islet-like structures (HLSC-ILS).
  • A) Representative micrographs of 10 experiments showing l.3xl0 4 /cm 2 non-stimulated cells (HLSC) and cells stimulated with protamine 10 gg/ml (HLSC+P) or with 10 IU/ml heparin -protamine (10 gg/ml) (HLSC+P+Hep). Scale bars 200 pm.
  • B) Representative micrographs of transmission electron microscopy (left; scale bar 100 pm) and of dithizone staining (right) of HLSC-ILS showing their characteristic phenotype.
  • C) Graph showing dependence of HLSC-ILS generation on protamine concentrations (n 6).
  • F Graph showing contribution of glucose and FBS to the generation of HLSC-ILS.
  • Figure 2 graph showing HLSC-ILS viability over in vitro maturation (A), from day 4 to day 14 of post-differentiation, as measured by FDA/PI staining. Percentage of viable islets was calculated as percentage alive (FDA)/percentage dead (PI).
  • B Graphs showing the size distribution of islet-like structures derived from HLSCs during in vitro maturation, from day 7 to day 14. Data are expressed as relative frequency.
  • Figure 3 Bar graph showing the results of flow cytometry analysis of stem cells markers (CD73, CD90, CD105, CD31), albumin and islets specific markers (PDX1, Ngn3, synaptophysin, C-peptide and glucagon) in HLSC and HLSC-ILS.
  • stem cells markers CD73, CD90, CD105, CD31
  • albumin and islets specific markers PDX1, Ngn3, synaptophysin, C-peptide and glucagon
  • Figure 4 phenotypic characterization of non-differentiated HLSC (HLSC) and differentiated HLCS-ILS at different times of culture (7, 14 and 21 days).
  • FIG. 1 Representative micrographs of immunofluorescence and percentage of different cell subpopulations expressing insulin alone, insulin and glucagon and negative for both hormones.
  • Figure 5 analysis of human C-peptide (hC-peptide) in vitro secretion in static and dynamic conditions.
  • A) Graph showing hC-peptide and glucagon in vitro basal secretion by HLSC- ILS (4, 7, 14 and 21 days) as evaluated in static condition, compared with human islets.
  • HLSC-ILS 400 IEQ
  • cadaveric human islets were stimulated with 2.8 mM of glucose.
  • B Graph showing dynamic secretion of hC-peptide by HLSC-ILS at 14 days of differentiation stimulated with high glucose (HG) and high potassium (KC1).
  • Figure 6 in vivo reversal of diabetes in SZT-treated SCID mice by implantation of 800 HLSC-ILS (28,000 IEQ/kg) under the right renal capsule.
  • A) Graph illustrating blood glucose levels in sham non-diabetic (non-DM, n. 12), diabetic (DM, n. 8), DM mice implanted with HLSC-ILS (DM+HLSC-ILS, n. 12) and DM+HLSC-ILS before (Pre-expl) and after (Post-expl) explantation (n. 4).
  • ANOVA with Newman-Keuls multi-comparison tests was performed.
  • HLSC Human Liver Stem Cells
  • HLSCs were isolated from human cryopreserved normal hepatocytes obtained from Lonza Bioscience (Basel, Switzerland) and cultured and characterized as previously described (Herrera, M. B. et al., Isolation and characterization of a stem cell population from adult human liver. (2006) Stem Cells. 24: 2840-2850.). Cadaveric human islets used as control were obtained from Tebu-bio (Magenta, Italy).
  • HLSC-ILS Islet-Like Structures
  • HLSC cultured in a RGFP/RFG medium enriched with protamine chloride resulted in a fast formation of numerous HLSC-ILS structures.
  • Such effect was blunted after the neutralization of protamine with heparin (HLSC+P+Hep).
  • Scanning electron microscopy showed 3D spheroidal structures that were positive for dithizone staining ( Figure 1B).
  • the addition of protamine was followed by cell cluster formation that was dose dependent (Figure 1C) and time-dependent (Figure 1D), with a maximum effect after a culture period of 14 days in the presence of a protamine concentration of 10 pg/ml. Based on these results, in subsequent experiments a protamine concentration of 10 pg/ml was used.
  • HLSCs are cultured in a differentiation medium containing polylysine, preferably at a concentration comprised between 1 and 50 pg/ml.
  • experiments were performed by seeding HLSC in a RFG medium supplemented with protamine chloride at the most efficient concentration (10 pg/ml; RFGP) with the following different cell densities: a) l.3xl0 4 /cm 2 , b) 2.6xl0 4 /cm 2 and c) 3.9xl0 4 /cm 2 .
  • Cells were cultured for a period of 4 days, without changing the RFGP medium, in a humidified 5% CO2 incubator at 37°C. On day 5, the medium was replaced with RFG medium; such medium was subsequently changed every other day over a period of 14 days.
  • HLSC-ILS were then counted as previously described by Molano, R.D., et al. (2001). Prolonged islet graft survival in NOD mice by blockade of the CD40-CD154 pathway of T-cell co-stimulation. Diabetes, 50, 270-276. Viability was assessed by fluorescein diacetate (FDA; Thermofisher Scientific, Milan, Italy) and propidium iodide (PI; Sigma). As shown in Figure 1E, structure formation was cell density-dependent. Indeed, HLSC-ILS number was significantly higher in the presence of a basal cell density of 3.9xl0 4 /cm 2 after a culture period of 14 days.
  • FDA fluorescein diacetate
  • PI propidium iodide
  • HLSCs were cultured at a density of 1.3x10 4 /cm 2 for a period of 14 days in a humidified 5% CO2 incubator at 37°C in a medium consisting of RFGP medium. On day 5, the medium was replaced with the RFG medium in absence or presence of glucose 5.6 mM, 11.6 mM or 28 mM that was subsequently changed every other day. At the end of the experiments, HLSC-ILS were counted. Once the efficient concentration of protamine and cell density was established, the kinetic of HLSC-ILS formation was assessed.
  • HLSCs with a cell density of l.3Exl0 4 /cm 2 were cultured in the RFGP medium for 2 and 4 days and the total number of HLSC-ILS was assessed for each condition. On day 4, the medium was replaced with the RLG medium that was subsequently changed every other day for a period of 4, 5, 7, 10, 11,
  • HLSC-ILS in suspension were harvested by collecting the supernatant medium while islet-like structures adhering to the culture substrate were recovered by carefully and repeatedly pipetting a physiological saline solution over them. The number of HLSC-ILS were counted. As a result of this analysis, neither LBS nor glucose concentration did appear to impact on cell cluster formation, as HLSC-ILS number was similar in the absence or in the presence of different glucose concentrations (5.6 mM, 11.6 mM or 28 mM) or LBS 10 % ( Figure 1F).
  • Transmission electron microscopy was performed on Kamovsky’s-fixed, osmium tetraoxide-postfixed tissues and embedded in epoxy resin, according to standard procedures. Ultra-thin sections were stained with uranyl acetate and lead citrate and were examined with a Jeol JEM 1010 electron microscope (Jeol). By transmission electron microscopy, endocrine granules typical of human islets were observed in the 3D spheroidal structures at day 4, 7 and 14 of differentiation. Three main types of electron-dense granules were observed: granules containing crystalloids, granules with a diffuse gray pale core, and granules with a dense core.
  • HFSC-IFS were centrifuged at 1100 rpm for 5 minutes and the islet pellet was seeded in a 24-well plate with 200 m ⁇ of PBS IX. The islets were subsequently incubated for 30 seconds with 20 m ⁇ diluted FDA and 20 m ⁇ diluted PI solutions, and micrographs taken. Dead cells were stained in RED whereas viable cells were stained in GREEN. HFSC-IFS were then assigned to the following categories:
  • Cat.0 few/n° green cells (non viable)
  • Cat.l 75% red cells (25% viability)
  • Total viable islets were then calculated as follows: (0.25 x n°. Cat.l) + (0.5 x n°. Cat.2) + (0.75 x n°. Cat.3) + n°. Cat.4.
  • Total islets number was calculated as follows: n°. Cat.O + n° . Cat.l + n° . Cat.2 + n° . Cat.3 + n° . Cat.4.
  • N° corresponds to the number of HLSC-ILS in each category. The percentage of viability was determined by applying the formula: Total viable HLSC-ILS x 100 / Total n° of HLSC-ILS.
  • pancreatic markers was assessed by immunofluorescence in HLSC-ILS during post-differentiation culturing in the RFGP and/or RFG medium.
  • HLSC-ILS were fixed in paraformaldehyde 4% (PAF, overnight), then in 30% of sucrose (overnight), included in Tissue -Tek-II (Bio-optica, Milan, Italy) and frozen at -80°C.
  • Serial slides were cut (3-5 pm) by a cryostat and fixed in acetone.
  • some HLSC-ILS were disaggregated in single cells by using Trypsin IX (Sigma) and fixed in PAF 4%.
  • tissue sections or cells were first washed with PBS l x , incubated for 30 minutes at room temperature with a permeable solution containing PBS lx and Triton X-100 (0.25%), washed twice with PBS 1 x for 5 min each one, incubated for 20 min with a blocking solution containing PBS l x , Tween (0.1%), and 0.1% bovine serum albumin (wt/vol) for 30 minutes at room temperature, and then incubated overnight with specific primary antibodies or irrelevant isotype controls.
  • Sections were incubated overnight with the following primary anti-human proteins antibodies: PDX1 (1 :500), Ngn3 (1 :50), MafB (2 pg/ml), Nkx6.l (1 :50), Nkx6.3 (2 pg/ml), chromogranin A (CgA) (2 pg/ml), C- peptide (2pg/ml), glucagon (1 :50), somatostatin (2 pg/ml), pancreatic polypeptide (3 pg/ml) and ghrelin (3 pg/ml) (1 :200) (Abeam, Cambridge, MA); insulin (1 :200, Dako, Copenhagen, Denmark); Glut-2 (1 :200, Santa Cruz, Santa Cruz, TX ).
  • An appropriate isotopic irrelevant antibody (Abeam) was used as control. Following washing with PBS-Tween solution, sections were incubated with an appropriate secondary antibody 1 :1000 (Alexa Fluor 488 Donkey anti-goat IgG for PDX1 and PP; Goat anti-Mouse IgG for CgA, C-peptide, ghrelin; Goat anti-Rabbit IgG for MafB, Nkx6.l, Nkx6.3, Glut-2, glucagon, somatostatin, Goat anti- Guinea pig for insulin; Texas Red Goat anti-Mouse IgG for Ngn3 (Invitrogen, Carlsbad, CA) at room temperature for one hour, washed with PBS-tween solution and incubated 10 minutes with DAPI (Dako).
  • an appropriate secondary antibody 1 :1000 Alexa Fluor 488 Donkey anti-goat IgG for PDX1 and PP
  • HLSC-ILS expressed the b-cell transcription factors Nkx6.l, Nkx6.3, MafB and the endocrine specific markers chromogranin A (CgA) and Ngn3 at 14 days of differentiation. Furthermore, at the same time -point HLSC-ILS expressed the exocrine pancreatic marker PDX1 and the human islet specific hormones, including insulin/C-peptide, glucagon, somatostatin and ghrelin. All isotype controls were negative. Similar but less intense positivity was detectable at 4 and 7 days of differentiation. To characterize cells co-expressing hormones, HLSC-ILS were dissociated into single cells and stained by immunofluorescence.
  • Insulin-expressing cells were 51.9 % ⁇ 8.1, insulin/glucagon co-expressing cells were 31.8 % ⁇ 11.0, and negative cells for both hormones were 16.3% ⁇ 8.9 (Figure 4C). No cells expressing only glucagon were detected. The presence of cells positive for somatostatin, ghrelin and PP was less than 1%. 8. FACS analysis of stem cell, hepatic and pancreatic markers
  • the following antibodies were phycoerythrin (PE) or fluorescein isothiocyanate (FITC) or allophycocyanin (APC)- conjugated: anti-CDl05, -CD29, -CD31, -CD44 (Dako Denmark A/S, Copenhagen, Denmark); -CD73, -CD90 (BD Biosciences Pharmingen, San Jose); and unconjugated antibodies (1 :20) anti-albumin, -synaptophysin (Dako), -PDX1, c-peptide, -glucagon, -Ngn3 (Abeam); Alexa fluor 488-conjugated goat antibodies against mouse/rabbit/pig IgG (Dako) were used as secondary antibodies (1 :1000) when needed.
  • PE phycoerythrin
  • FITC fluorescein isothiocyanate
  • APC allophycocyanin
  • the cells were resuspended in FACS buffer (PBS containing 0.1% bovine serum albumin or PBS containing 0.1% Triton to permeabilize cells and 0.1% bovine serum albumin). All the incubations were performed at 4°C for 1 hour. Cells were washed twice following each incubation. For each sample, 10,000 cells were analyzed on a FACScan cytometer (BD Biosciences Pharmingen, San Diego, CA). Gating was constructed based on negative controls, and compensation controls were included in all the analyses performed. Population percentages and numbers were generated for gated populations from each experiment using Cell Quest software (BD Biosciences Pharmingen). Results were calculated as percentage of positive cells and as geometric mean of fluorescence.
  • FACS buffer PBS containing 0.1% bovine serum albumin or PBS containing 0.1% Triton to permeabilize cells and 0.1% bovine serum albumin.
  • HLSC and HLSC-ILS were lysed at 4°C for 1 hour in RIPA buffer added with phosphatase and protease inhibitors (Sigma) and centrifuged at 15,000 g. After addition of lysis buffer, HLSC structures were subjected to two cycles of 30 seconds of sonication with a pause of 10 seconds on ice. Sonication was necessary to obtain disintegration of the capsule enveloping the structures. Aliquots containing 30 pg of proteins quantified by Bradford method were subjected to 4-15% gradient Tris-HCl gel electrophoresis under reducing conditions and electroblotted onto nitrocellulose membranes.
  • the membranes were blocked with non-fat milk and incubated overnight with primary antibodies: anti-C peptide (Abeam); anti-Glucagon (Abeam) and anti-Actin (Santa Cruz Biotechnology) at the appropriate concentration. After extensive PBS-T washings, the membranes were incubated with the appropriate secondary antibodies (Thermofisher Scientific) for 1 hour at room temperature, washed with PBS-T, developed with ECL detection reagents, and subjected to Chemidoc (Bio-Rad, Hercules, CA). Western Blot analysis confirmed the expression of Ngn3, PDX1, C-peptide, glucagon and somatostatin proteins in HLSC-ILS throughout post-differentiation (Figure 4A). In contrast, non-differentiated HLSC expressed low levels of Ngn3 and PDX1.
  • HLSCs and HLSC-ISL were harvested from flasks and centrifuged at 900 rpm for 10 minutes. Supernatant was then discarded and the pellet was washed with PBS and centrifuged at 900 rpm for 10 minutes. Supernatant was discarded and pellet was resuspended in 200 m ⁇ of RNase free water (Ambion) plus 5 m ⁇ of RNAse Inhibitor (Ambion, Thermo Fisher Scientific). Cell suspension was cooled on ice and then islets, but not HLSC, were sonicated at 30% potency for 30 seconds two times.
  • RNA concentration and quality were measured by Nanodrop ND-1000 (Thermo Fisher Scientific).
  • RNA A total of 500 ng of total RNA were retro-transcribed to cDNA with Superscript IV VILO Master Mix with ezDNase enzyme (Invitrogen, Thermo Fisher Scientific) following kit’s instructions and by using Veriti Thermal Cycler (Applied Biosystems).
  • the specific TaqMan Gene Expression Assay was spotted on a custom TaqMan Array microfluidic cards (Applied Biosystems, Thermo Fisher Scientific).
  • the samples were mixed with TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific), TaqManArray microfluidic cards were prepared according to manufacturer’s instruction and run with the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Each sample was run in triplicate and 3 samples for each experiment were tested. Retro-transcription negative controls (no RT enzyme) were run for 6 TaqMan Gene Expression Assay selected among the 48 spotted on the TaqMan® Array microfluidic cards.
  • HFSC-IFS Gene expression analysis confirmed that HFSC-IFS cultured in vitro undergo a differentiation process into b-cells.
  • HFSC-IFS showed decreased expression of HFSC-specifie markers, such as e.g. cytokeratin 18 (KRT18), cytokeratin 8 (KRT8) and vimentin (VIM), along with an increased expression of NANOG and nestin (NES), two stem cell markers.
  • KRT18 cytokeratin 18
  • KRT8 cytokeratin 8
  • VIM vimentin
  • NES NANOG and nestin
  • HFSC-IFS insulin maturation enzymes and several markers involved in b- cell differentiation were upregulated in HFSC-IFS compared to HFSC. Increased expression levels were also detected for several transcripts encoding proteins involved in insulin granules release, such as PFKFB2, STX1A, STXBP1, VAMP2, and the calcium (CACNA1C) and potassium (KCNJ11) voltage gated calcium, which trigger insulin secretion.
  • qRT-PCR showed an enhanced expression in HFSC-IFS of several transcription factors specifically involved in b-cell differentiation, such as FOXA2, MAFA, MAFB, NEUROD1, NGN3, NKX6.1, and PDX1.
  • the present inventors carried out incubations of the islet-like structures under static conditions as described below. After post-differentiation culturing for 4, 6, 7, 10 and 14 days in the RFGP and/or RFG medium, the HLSC-ILS were cultured overnight in a medium consisting in DMEM (5.6 mM) supplemented with FBS (10%), L-glutamine (2 mM) and penicillin 100 Ul/ml/streptomycin 100 pg/rnl.
  • IEQ HLSC-ILS were placed in 12 well-culture plates and cultured in Krebs buffer (25 mM HEPES, 115 mM NaCl, 24 mM NaHCOs, 5 mM KC1, mM MgCl2-6 H 2 0, 0.1% BSA, 25 mM CaCk -2H 2 0, pH at 7.4) with a low concentration of glucose (2.8 mM) for 1 hour.
  • Krebs buffer 25 mM HEPES, 115 mM NaCl, 24 mM NaHCOs, 5 mM KC1, mM MgCl2-6 H 2 0, 0.1% BSA, 25 mM CaCk -2H 2 0, pH at 7.4
  • the medium was collected and replaced with a Krebs buffer supplemented with high glucose concentration (28 mM). Islet-like structures were then placed in a humidified 5% CO2 incubator at 37°C for 1 hour.
  • the ALPCO KIT ELISA (Alpco Diagnosis, Windham, NH) is designed for the quantitative determination of C-peptide in biological samples by using a dual-monoclonal antibody sandwich ELISA format. Briefly, 25 m ⁇ of each standard, control, and sample were added with 50 m ⁇ of the proprietary Assay Buffer to the 96-well microplate coated with a monoclonal antibody specific for C-peptide. The microplate was then incubated for 1 hour at room temperature on a microplate shaker at 700-900 rpm. After the first incubation was complete, the wells were washed 6 times with 200 m ⁇ of Working Strength Wash Buffer per well and blotted dry.
  • a total of 100 m ⁇ of Working Strength Conjugate were then added to each well, and the microplate was incubated a second time on a microplate shaker at 700- 900 rpm for 1 hour, washed (6 times with 350 m ⁇ of Working Strength Wash Buffer), and blotted dry.
  • TMB Substrate was added to each well (100 m ⁇ ), and the microplate was incubated a third time for 15 minutes at room temperature on a microplate shaker at 700-900 rpm. Once the third incubation was complete, 100 m ⁇ of Stop Solution were added into each well, and the optical density (OD) was measured by a spectrophotometer at 450 nm. The intensity of the color generated was directly proportional to the amount of C-peptide in the sample.
  • HLSC-ILS 400 IEQ
  • HLSC-ILS 400 IEQ
  • FCS 2.8 mM of glucose
  • HLSC-ILS were detached from the culture chip, total protein was extracted (see Western blot analysis methods) and samples were stored at -20°C prior to analysis.
  • the secretion of human C peptide (hC -peptide) was assessed by ELISA (ALPCO Diagnosis, Windham, NH, see the paragraph above) according to the manufacturer’s instructions, while total protein was quantified by BCA colorimetric detection assay (BCA protein assay kit, Pierce).
  • BCA protein assay kit Pierce
  • a basal secretion of human C-peptide was detectable in pancreatic islet-like structures at four days post-differentiation, increased at day 7 and maintained until day 21 (Ligure 5 A).
  • the level of C-peptide basal secretion was markedly inferior to that of human islets and the secretion of glucagon was not detected (Ligure 5A).
  • the glucose-induced secretion was evaluated in the pancreatic islet-like structures according to the present invention at day 4, 6, 7, 10 and 14 of post-differentiation by static incubation with 2.8 mM low glucose (LG) and 28 mM high glucose (HG) and 50 mM KC1 (Ligure 5C).
  • pancreatic islet-like clusters did not respond to either LG or HG at day 4 of post-differentiation. A slight increase in secretion was observed in response to KC1. In contrast, the secretion of hC-peptide significantly increased after HG stimulation between day 4 and day 6 of post-differentiation (at least two-fold the amount secreted in response to LG stimulation) and was reduced again at day 14 (Ligure 5C).
  • the kinetic of secretion evaluated at 14 days of post-differentiation by using the dynamic microfluidic system showed an initial basal secretion to LG, followed by two peaks of secretion in response to HG and KC1 (Ligure 5B). Each peak was reduced when shifted to LG. The ratio of secretion was 4.5 and 5.6 in response to HG and KC1, respectively. 17.
  • Experimental animals Animal studies were approved by the Ethics Committee of Istituto Superiore di Sanita (741/2015-PR) and conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 8-9 weeks old male SCID mice obtained from Charles Rivers (Calco, Italy) were maintained in specific pathogen-free conditions and provided with sterile food and water ad libitum in the animal facility.
  • Blood glucose >250 mg/dl was induced in SCID mice weighing ⁇ 22 g by intraperitoneal injections of streptozotocin (STZ; Sigma)-citrate buffer (55 mg/kg/day) for 5 consecutive days (53-Breyer). Sham mice injected with sodium citrate buffer were used as controls. Blood glucose was measured between 2:00 p.m. and 4:00 p.m. via tail vein sampling on alert, non-fasted animals, using the diagnostic glucose oxidase enzymatic test (Glucometer Accu-Chek, Roche, Mannheim, Germany).
  • mice Groups of diabetic SCID mice with equivalent blood glucose levels and nondiabetic control mice were studied in parallel and divided in the following subgroups: a) nondiabetic control SCID mice; b) STZ-induced diabetic SCID mice (DM); c) DM mice that received HLSC-ILS under the right renal capsule (DM+HLSC-ILS). 19. HLSC-ILS implantation under the kidney capsule
  • HLSC-ILS grown in post-differentiation culturing for periods up to 14 days were counted and scored for size, according to the algorithm previously described for the calculation of the 150 pm diameter islet equivalent (IEQ) number (Ricordi, C., et al. (1990). Islet isolation assessment in man and large animals. Acta Diabetologica Latina, 27: 185-195). Diabetic SCID mice were anesthetized by intramuscular injection of zolazepam (Zoletil; VIRBAC, Milan, Italy) (0.2 ml/Kg) and xilazine (Rompun; Bayer. Milan, Italy) (16 mg/Kg).
  • the right kidney was carefully externalized through a dorsal lumbotomy incision and the HLSC-ILS (800 IEQ) delivered under the renal capsule with a 24 GA catheter.
  • the kidney was then replaced in the retroperitoneal space and the skin incision closed using a 6-0 silk suture.
  • Lollowing islets-like structures implants blood glucose obtained via tail vein sampling between 2:00 p.m. and 4:00 p.m. on alert 4h-fasted animals was measured weekly (Glucometer Accu-Chek, Roche) with a maximum follow-up period of seven weeks.
  • Intraperitoneal injection of streptozotocin was followed by induction of hyperglycemia in SCID mice (Figure 6A).
  • Implant of HLSC-ILS under the renal capsule of diabetic SCID mice was associated with a progressive reduction of hyperglycemia which was already significant after 1 week of implantation and was sustained up to 8 weeks. When the implant was removed after 8 weeks, a significant increase of glycemia was observed, suggesting that euglycemia was dependent on the HLSC-ILS implant.
  • Non-implanted diabetic mice and non-diabetic mice were used respectively as hyperglycemic and normo-glycemic controls. Changes in glycemia directly correlated with serum levels of C-peptide (Figure 6B).
  • the murine C-peptide (mC-peptide) levels were minimal or absent in both diabetic mice and diabetic mice treated with HLSC-ILS ( Figure 6B).
  • the presence of sustained hyperglycemia in diabetic mice supports that this remnant quantity of mC-peptide is not sufficient to maintain euglycemia.
  • human C peptide (hC- peptide) was detected only in the diabetic mice implanted with HLSC-ILS and was undetectable after removing the implants.
  • the histological analysis demonstrated the presence of spheroid structures under the renal capsule in the right kidney of diabetic mice treated with HLSC-ILS.
  • the expression of both protein and mRNA of human insulin in the implant was detected by immunohistochemistry and by in situ hybridization. Human islets were used as positive control and mouse kidney and pancreas as negative controls.
  • Intraperitoneal glucose tolerance test flPGTT Intraperitoneal glucose tolerance test flPGTT
  • IPGTT intraperitoneal glucose tolerance tests
  • non-DM non-diabetic mice
  • DM mice diabetic mice
  • HLSC-ILS HLSC-ILS
  • IPGTT intraperitoneal glucose tolerance tests
  • Blood glucose was measured between 2:00 p.m. and 4:00 p.m. on alert 4h-fasted animals via tail vein sampling and analyzed by glucometer (Accu-Chek, Roche, Mannheim, Germany).
  • IPGTT blood glucose was measured immediately before and at 15, 30, 60 and 120 min after an IP glucose (2g/kg in 0.9% NaCl) injection.
  • IP glucose 2g/kg in 0.9% NaCl
  • diabetic animals showed a high basal glycemia and an altered glucose tolerance test with a sustained hyperglycemia.
  • the HLSC-ILS- implanted diabetic mice showed normalization in basal glycemia and a response to IPGTT comparable to non-diabetic control mice. Glycemia returned to basal levels after 120 min. Following explant of HLSC-ILS, mice were hyperglycemic and showed an altered IPGTT curve comparable to diabetic mice (Figure 6C).
  • HLSC-ILS implants were removed from normoglycemic implanted animals. Briefly, under aseptic conditions, an abdominal lobotomy incision was performed, the right kidney was carefully exposed and the portion of renal capsule containing the structures removed to explant the HLSC-ILS. The kidney was then replaced in the retroperitoneal space and the muscle and skin incisions closed using a 6-0 silk suture. One week after the explant, glycemia was assessed in the blood obtained via tail vein sampling between 2:00 p.m. and 4:00 p.m. on alert 4h-fasted animals (Glucometer Accu-Chek, Roche).
  • mice were sacrificed at week 8 post-implant; blood samples were collected and serum separated.
  • the human C-peptide levels were assessed by using an ultrasensitive human C- peptide enzyme-linked immunosorbent assay kit (Alpco Diagnosis, Windham, NH, see the paragraph titled“Quantitative determination of C peptide”).
  • Sections were dewaxed and hydrated, boiled in citrate buffer (IX) at l00°C for 20 minutes and then incubated with PBS-0.l% Tween- 0.1% bovine serum albumin (wt/vol) for 30 minutes at room temperature to block the unspecific reactivity. Subsequently, sections were incubated overnight with the specific antibodies: anti-human C-peptide and anti-human glucagon (1 :200; Abeam). In the negative controls the primary antibodies were omitted or substituted with non-immune human IgG.
  • FISH Fluorescence in situ hybridization
  • HLSC-ILS human islets
  • HLSC ILS cultured in vitro and HLSC three genes for adaptor proteins involved in the transduction of insulin signal (GAB1, NCK1, SORBS 1) were expressed at similar or higher levels in EXP-ILS compared to human islets, and were upregulated in respect to HLSC-ILS and HLSC, suggesting an increased activation of the insulin signaling pathway.
  • GAB1, NCK1, SORBS 1 three genes for adaptor proteins involved in the transduction of insulin signal (GAB1, NCK1, SORBS 1) were expressed at similar or higher levels in EXP-ILS compared to human islets, and were upregulated in respect to HLSC-ILS and HLSC, suggesting an increased activation of the insulin signaling pathway.

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Abstract

L'invention concerne un procédé de préparation de structures cellulaires de type îlots pancréatiques caractérisées par une combinaison unique de viabilité élevée et de caractéristiques morphologiques et fonctionnelles qui les rendent particulièrement appropriées pour une utilisation dans une application à la fois de dépistage clinique et de drogues, ainsi que les structures cellulaires de type îlots pancréatiques obtenues à partir dudit procédé.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3816276A1 (fr) * 2019-10-31 2021-05-05 Unicyte Islet AG Structures de cellules de type îlots pancréatiques viables et leur procédé de préparation
WO2021228104A1 (fr) * 2020-05-11 2021-11-18 中国科学院分子细胞科学卓越创新中心 Amas de cellules du type îlot, procédé de préparation et application de celui-ci

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126219A1 (fr) 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progeniteurs hepatiques
WO2015091493A1 (fr) 2013-12-16 2015-06-25 Fresenius Medical Care Deutschland G.M.B.H. Structures cellulaires de type îlots pancréatiques et leur procédé de préparation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126219A1 (fr) 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progeniteurs hepatiques
WO2006126236A1 (fr) 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progenitrices du foie
WO2015091493A1 (fr) 2013-12-16 2015-06-25 Fresenius Medical Care Deutschland G.M.B.H. Structures cellulaires de type îlots pancréatiques et leur procédé de préparation

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BOSE B ET AL., CELL BIOL INT., vol. 36, no. 11, 2012, pages 1013 - 20
CABRERA, O. ET AL.: "The unique cytoarchitecture of human pancreatic islets has implications for islet cell function", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., vol. 103, 2006, pages 2334 - 2339
HERRERA, M. B. ET AL.: "Isolation and characterization of a stem cell population from adult human liver", STEM CELLS, vol. 24, 2006, pages 2840 - 2850, XP008146144, doi:10.1634/stemcells.2006-0114
MOHAMMED, J.S. ET AL.: "Microfluidic Device for Multimodal Characterization of Pancreatic Islets", LAB ON A CHIP, vol. 9, no. 1, 2009, pages 97 - 106, XP055021721, doi:10.1039/B809590F
MOLANO, R.D. ET AL.: "Prolonged islet graft survival in NOD mice by blockade of the CD40-CD154 pathway of T-cell co-stimulation", DIABETES, vol. 50, 2001, pages 270 - 276
MOLANO, R.D. ET AL.: "Prolonged islet graft survival in NOD mice by blockade of the CD40-CD154 pathway of T-cell costimulation", DIABETES, vol. 50, 2001, pages 270 - 276
REZANIA, A. ET AL.: "Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice", DIABETES, vol. 61, no. 8, 2012, pages 2016 - 2029, XP008162152, doi:10.2337/db11-1711
RICORDI, C. ET AL.: "Islet isolation assessment in man and large animals", ACTA DIABETOLOGICA LATINA, vol. 27, 1990, pages 185 - 195
SAMAD NADRI ET AL: "Differentiation of conjunctiva mesenchymal stem cells into secreting islet beta cells on plasma treated electrospun nanofibrous scaffold", ARTIFICIAL CELLS, NANOMEDICINE AND BIOTECHNOLOGY, 14 December 2017 (2017-12-14), US, pages 1 - 10, XP055544175, ISSN: 2169-1401, DOI: 10.1080/21691401.2017.1416391 *
SHABARI SARANG ET AL: "Umbilical Cord Derived Mesenchymal Stem Cells Useful in Insulin Production - Another Opportunity in Cell Therapy", INTERNATIONAL JOURNAL OF STEM CELLS, vol. 9, no. 1, 31 May 2016 (2016-05-31), pages 60 - 69, XP055544164, ISSN: 2005-3606, DOI: 10.15283/ijsc.2016.9.1.60 *
WANG HEFEI ET AL: "Morphological characteristics and identification of islet-like cells derived from rat adipose-derived stem cells cocultured with pancreas adult stem cells : Coculture of ADSCs/PASCs", CELL BIOLOGY INTERNATIONAL., vol. 39, no. 3, 26 January 2015 (2015-01-26), GB, pages 253 - 263, XP055544171, ISSN: 1065-6995, DOI: 10.1002/cbin.10387 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3816276A1 (fr) * 2019-10-31 2021-05-05 Unicyte Islet AG Structures de cellules de type îlots pancréatiques viables et leur procédé de préparation
WO2021083962A1 (fr) * 2019-10-31 2021-05-06 Unicyte Islet Ag Structures cellulaires de type îlots pancréatiques viables et leur procédé de préparation
WO2021228104A1 (fr) * 2020-05-11 2021-11-18 中国科学院分子细胞科学卓越创新中心 Amas de cellules du type îlot, procédé de préparation et application de celui-ci

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