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WO2004104195A1 - Cellules de production d'insuline et procede de constitution de ces cellules - Google Patents

Cellules de production d'insuline et procede de constitution de ces cellules Download PDF

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WO2004104195A1
WO2004104195A1 PCT/JP2003/014681 JP0314681W WO2004104195A1 WO 2004104195 A1 WO2004104195 A1 WO 2004104195A1 JP 0314681 W JP0314681 W JP 0314681W WO 2004104195 A1 WO2004104195 A1 WO 2004104195A1
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cells
insulin
producing
pdx
liver
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Japanese (ja)
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Natsuki Nakazima
Tomonori Sakurai
Yasuhiko Tabata
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Japan Science and Technology Agency
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Japan Science and Technology Agency
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • 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
    • C12N5/067—Hepatocytes
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to an insulin producing cell and a method for producing the same.
  • INDUSTRIAL APPLICABILITY The present invention is particularly useful for the treatment of diabetes, and can be used for the fundamental treatment of diabetes by cell transplantation, and for the field of cell production therefor. Background art
  • liver-derived cells particularly liver-derived undifferent stem cells obtained from healthy liver tissue
  • the liver is an organ that is developed and differentiated from the foregut, which is the same as the knee, and studies on differentiation induction using adult stem cells have shown that insulin production from undifferentiated stem cells derived from adult healthy liver tissue has been reported. This suggests the possibility of inducing differentiation into cells, that is, the possibility of producing insulin-producing cells from liver-derived cells. If a method for producing such insulin-producing cells is established, it will be possible to provide a material that is extremely useful for treating diabetes by transplantation. In addition, for clinical application, it is desirable that the physiological functions related to insulin secretion of transplantation cells be as close to normal islet ⁇ cells as possible. Disclosure of the invention
  • the present invention has been made in view of the above-mentioned problems, and has as its object to provide liver-derived cells obtained from normal liver tissue, which are insulin-producing cells that can be used for the treatment of diabetes or its research. To provide an insulin-producing cell that can be produced using the method, and a method for producing the same.
  • SHC small hepatocytes
  • the present invention includes the following inventions A) to Q) as medically and industrially useful products and methods.
  • liver-derived cell means a cell obtained from a healthy liver tissue. Therefore, cells that appear only when damaged by drugs such as liver round cells and are not suitable for transplantation treatment are not included in the liver-derived cells mentioned here.
  • A) or B) above which is responsive to stimulation by introducing a transcription factor Pdx-1 (Pancreatic duodenal homeobox-l) gene or a gene of a protein that positively regulates its expression. Insulin producing cells with improved extrinsic extracellular secretory function.
  • Pdx-1 Pancreatic duodenal homeobox-l
  • H A method for producing insulin-producing cells as described in G) above, wherein small hepatocytes are cultured as liver-derived cells.
  • K The method for producing insulin-producing cells according to I) or J) above, wherein a mixed medium of DMEM and ham F12K is used in the second culture condition.
  • K Second culture condition The method for producing insulin-producing cells according to any one of the above (I) to (K), wherein the Pdx- ⁇ gene is introduced into the cells during or before the cultivation under the following conditions.
  • N The method for producing an insulin-producing cell according to any of the above G) to M), wherein the method is produced from a cell derived from a rat, a mouse, or a human liver.
  • Insulin producing cells obtained by culturing differentiated stem cells.
  • Ii A method for producing an endocrine hormone-producing cell, comprising inducing glucagon and / or somatostatin production by culturing liver-derived cells and other undifferentiated stem cells.
  • FIG. 1 is a flowchart schematically showing a process for producing insulin-producing cells from small hepatocytes.
  • FIG. 2 is a diagram schematically illustrating the manufacturing process of FIG.
  • FIG. 3 show the results of examining the presence or absence of insulin production in the cells obtained by the above-described production process by immunofluorescent staining.
  • FIG. 4 is a diagram showing the results of examining the expression of genes such as transcription factors involved in development and differentiation by RT-PCR at each stage of the production process.
  • FIG. 5 is a graph showing the results of examining the amount of insulin production in the cells obtained by the above-described production steps.
  • FIG. 6 is a graph showing the results obtained by examining how the amount of insulin secreted by cells changes in response to various stimuli for the cells obtained by the above-described production process.
  • FIG. 7 is a diagram illustrating the mechanism of insulin secretion in knee island ⁇ cells in response to various stimuli.
  • FIG. 8 is a graph showing the results of measuring the amount of residual insulin in cells after the stimulation test.
  • FIG. 9 is a diagram showing the results of gene expression of a protein contributing to insulin secretion measured by RT-PCR.
  • FIG. 10 is a diagram showing, as a model, the interrelationship between Pdx-1, Syt1, NeuroD, Pax6, and HNF-3fi in Tengshima ⁇ cells.
  • FIG. 11 is a diagram showing each primer sequence used in the RT-PCR analysis of FIG. '
  • FIG. 12 is a diagram showing the respective primer sequences used in the RT-PCR analysis of FIG. 4, as in FIG.
  • FIG. 13 shows the cell (+ Pdx-1 and -Pdx-1) cells prepared by the method of Fig. 1.
  • FIG. 4 shows the results of examining the gene expression of glucagon and somatostatin by RT-PCR.
  • the present inventors have focused on small hepatocytes (SHCs), which have the ability to proliferate in vitro for a long time and differentiate into liver parenchymal cells, among liver-derived cells that can be separated from normal liver tissue.
  • SHCs small hepatocytes
  • Various conditions for inducing the function to be produced were examined.
  • they successfully transdifferentiated small hepatocytes into insulin-producing cells by culturing them in a safe manner without using a method that would be toxic to living organisms.
  • transfection and forced expression of the transcription factor Pdx-1 gene in these cells enabled the secretion of insulin hormone in response to various stimuli, similar to the physiological function of knee island ⁇ cells in vivo. .
  • step 1 cells containing small hepatocytes (SHC) were isolated from normal liver tissue of rats using collagenase hepatic perfusion. Specifically, a normal rat (Sague-Dawley breed, male) weighing 100-150 g was laparotomized, and a force urge was introduced from the lower part of the portal vein to the base of the liver, and Hepes was injected according to the usual method. Liver perfusion was performed with -buffered salt solution, and non-real liver cells ( ⁇ -parenchymal cells: NPC) containing SHC were isolated by digestion with collagenase. After further purifying the cells by centrifugation, the cells were divided into three groups.
  • SHC small hepatocytes
  • step 2 the three groups of SHC are each cultured in HGM medium for 5 days. Specifically, inoculate 2-5X10 4 viable isolated cells / cm 2 into Petri dishes for cell culture. Was. At this time, an HGM (hepatocyte growth medium) medium based on DMEM (Dulbecco's Modified Eagle's Medium) to which the following components were added was used as a medium (culture medium).
  • HGM hepatocyte growth medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • EGF epidermal growth factor
  • Antibiotics 100 U / ml penicillin, 100 g / ml streptomycin, 0.25 g / ml Fungizone
  • the cells were cultured for 2 days in an incubator at 37 ° C, 5% C0 2 -95% air, with a new medium
  • the cells were replaced and cultured for another 3 days.
  • the cells were cultured in the HGM medium for 53 times, but the period may be about 3 to 7 days as described later.
  • DMSO dimethylsulfoxide
  • step 4 two groups of SHC cells were cultured for two days under low serum culture conditions different from the medium in step 3.
  • the serum type used was fetal calf serum (FBS), and the concentration in the medium (culture solution) was set to a low concentration of 0.2%.
  • FBS fetal calf serum
  • a medium a medium (DMEM / F12K) in which DMEM and ham F12K (Kaigh's modification of Ham's F12) were mixed at a ratio of about 1: 1 was used, and the following components were added.
  • HGF hepatocytes growth factor
  • the conditions in the incubator were set to the same conditions as in steps 2 and 3.
  • the Pdx-1 IRES-GE gene is used in one of the two groups of SHC cells and the GFP gene is used in the other cell using the adenovirus vector.
  • the former cells are sometimes referred to as “+ Pdx-l” and the latter cells are sometimes called “-Pdx-1”).
  • an expression vector is prepared in which the Pdx-1-IRES-GFP gene or the GFP gene is downstream of the chicken beta-actin (AG) promoter.
  • the Pdx-1-IRES-GFP gene contains the gene sequence encoding the mouse transcription factor Pdx-1 (Pancreatic duodenal homeobox-1), the coding sequence of IRES (internal ribosome entry site), which is a ribosome binding site in mRNA, and And a sequence encoding the green fluorescent protein GFP.
  • GFP is for confirming the presence or absence of gene introduction.
  • the adenovirus expression vector described above was infected (transfection) to the two groups of cells after step 4 under conditions of a duplicate infection rate (MOI) of 30-50. (+ Pdx-1) forcibly expressed Pdx-1 and GFP, and the other cell (-Pdx-) forcibly expressed only GFP.
  • MOI duplicate infection rate
  • the experimental group (+ Pdx-1), the experimental group (-Pdx-1), and the sealed group were prepared: the experimental group (+ Pdx-1), the experimental group (-Pdx-1), and the sealed group. Then, on days 9, 12, and 15, the amount of insulin produced by each cell was measured. The results are shown in the graph of FIG. In the graph, “Group 1” represents the experimental group, and “Group 2” represents the control group. As shown in the graph, on the 15th day, the amount of insulin production in the experimental group (+ Pdx-1 and -Pdx-1) was significantly increased as compared with the control group. In the cells of this experimental group (+ Pdx-1 and -Pdx-1), not only insulin but also production of glucagon and somatostatin were observed (see FIG. 13).
  • the results are shown in the upper graphs of FIG.
  • the graph on the left shows Krebs-Ringer bicarbonate buffer (KRBB) containing 0.2% FBS, ⁇ ⁇ concentration dalcos (25 mM) and 10 iiM glucagon-Iike peptide 1 (GLP-1 [7-37]).
  • the middle graph shows the results of stimulation with 45 mM KCl and 0.2 mM tolbutamide added to a low concentration of darcos (5 mM) KRBB solution, and the right graph shows the control.
  • the measurement results using only the low concentration glucose (5 mM) KRBB solution as the basal buffer.
  • the amount of insulin production was significantly increased as compared with the control group.
  • Producing cells could be induced.
  • the stimulus-responsive insulin secretion function could be improved.
  • insulin-producing secretory cells having physiological functions close to those of knee island ⁇ cells in vivo, which regulate the amount of insulin secreted in response to various stimuli.
  • transplanting these cells into drug-induced diabetic nude mice the effect of reducing the increase in blood glucose level was obtained.
  • the above method uses small hepatocytes (SHC) as a material and Although it is one that induces the surin-producing function, other liver-derived cells can be isolated from normal liver tissue, as long as they have the properties of undifferentiated stem cells. It is considered that insulin production function can be induced by similar culture induction.
  • SHC small hepatocytes
  • undifferentiated stem cells other than liver-derived cells can be induced to differentiate into insulin-producing cells by the production method of the present invention.
  • Such undifferentiated stem cells include: (1) adult-derived undifferentiated stem cells, for example, hematopoietic stem cells, which are isolated from peripheral blood or bone marrow components, and isolated from hepatocyte-like cells. And (2) cells induced to differentiate into ES cells into hepatocyte-like cells.
  • the medium used was changed from a medium with a high serum concentration to a medium with a low serum concentration during the culture. Specifically, in steps 2 and 3, a medium containing 10% fetal calf serum (FBS) was used, and in subsequent steps 4 and 5, the proliferation of small hepatocytes to be differentiated into liver parenchymal cells was performed. To control, a medium with a low serum concentration of 0.2% is used.
  • FBS fetal calf serum
  • the serum concentration in the medium under the second culture condition is preferably about 0.1 to 0.5% in order to suppress the growth, although it depends on the kind of serum used and the like. It is more preferable to set it to about 0.2 to 0.3%.
  • the serum concentration in the medium under the first culture condition is preferably about 5 to 20%, more preferably about 7 to 15%.
  • the type of serum used for the first and second culture conditions is not particularly limited, and examples thereof include serum of mammals other than seaweed and seaweed (such as seaweed chicken).
  • serum include fetal serum (Fetal ca serum: FCS or Fetal bovine serum: FBS), newborn serum (Newborn Bovine Serum: NBS), calf serum (Calf serum: CS), and the like. May include fetal bovine serum (03 or 83). Further, it is preferable to use the same serum type throughout the first and second culture conditions.
  • the medium (culture medium) to be used is not particularly limited. However, in the first culture condition, it is preferable to use an HGM medium based on DMEM from the viewpoint of promoting the growth of hepatocytes. Under the second culture conditions, it is preferable to use a medium other than the HGM medium from the viewpoint of once suppressing the growth of cells.
  • the basal medium component under the second culture condition was higher in DMEM and ham F1 than in a mixed medium (DMEM / F12) of DMEM and Ham's F12, which is widely used. Better results were obtained using a mixed medium (DMEM / F12K) with 2K (Kaigh's modification of Ham's F12). Good results are obtained when the ratio of DMEM to ham F12K is approximately 1: 1, and the glucose (D-glucose) concentration in the medium is around 5 mM (1 to: 1.3 g / L). was gotten.
  • the medium may be prepared from reagents, but since various media including DMEM and ham F12K are currently on the market, these may be prepared using commercially available products.
  • epidermal growth factor EGF
  • nicotinamide Nicotiamide
  • HGF liver growth factor
  • EGF and dycotinamide be added to help the induction of insulin-producing cells
  • HGF be added to help the survival of liver cells that are vulnerable to the severe conditions of low serum concentration.
  • concentration of each added substance is not particularly limited, but for example, the concentration of HGF is in the range of 10-25 ng / mL.
  • Dexamethasone and ITS, cell growth factors (fibroblast growth factor, etc.), hypothalamus and pituitary gland extracts are also preferred medium additives. Other commonly used nutrients, antibiotics, etc. may be added to the medium.
  • the cell culture period is also not particularly limited, and may be appropriately set in relation to other conditions.
  • Step 2 is about 3 to 7 days
  • Step 3 is about 1 to 4 days
  • 4 is preferably set to about 2 to 8 days. If the Pdx-1 gene is not forcibly expressed, step 5 is unnecessary, and step 4 may be extended for an appropriate period.
  • the method for introducing the Pdx-1 gene into cells is not particularly limited, and various gene transfer methods such as a phosphate canolescence method, a lipofection method, a microinjection method, a DEAE dextran method, a virus method, and an electoral poration method can be used. Since a method is known, an appropriate method may be selected from known gene introduction methods. Among them, the virus method using a virus vector is preferable because gene transfer can be performed with high efficiency, and various known virus vectors can be used in addition to a general adenovirus vector.
  • an adenovirus vector in which a coding sequence of IRES is connected downstream of the Pdx-1 gene, which is the target gene, and placed downstream of the chicken betaactin (AG) promoter is used.
  • highly efficient gene transfer was obtained by transfection using this vector.
  • the introduction of the Pdx-1 gene into cells is preferably performed during the culture under the second culture condition, but may be performed before the culture.
  • the mouse Pdx-1 gene was introduced so as to be distinguishable from the expression of endogenous rat Pdx-1, but a gene derived from the same species as the host cell may be introduced.
  • insulin-producing cells can be produced from cells derived from mouse and human liver by similar culture induction.
  • human liver-derived cells When human liver-derived cells are used, a part of the liver may be excised by a known method performed in living liver transplantation or the like, and liver-derived cells may be prepared from the tissue.
  • synaptotagmin 1 which significantly enhances the stimulus-responsive insulin-secreting function of cells. It is thought that it was done. Therefore, in addition to the forced expression of Pdx-1, the expression of membrane proteins for extracellular transport of hormonal substances, in particular, synabtotagmin 1 (or other synaptotagmins), is induced by other methods. The stimulus-responsive insulin secretion function may be improved. Thus synaptotagmin 1 (or other extracellular transport membrane protein SNAP25, GB Y, TGase etc.
  • Another method of inducing expression of, for example, transcription factors Neuro D, the expression of Pdxl upstream For example, a method of forcibly expressing a series of transcription factors and genes that promote extracellular transport of hormones and neurotransmitters, which are controlled by the enzyme, may be considered.
  • the present invention is particularly useful for treating or researching diabetes.
  • the production method of the present invention it is possible to synthesize insulin in the undifferentiated stem cells derived from the liver, and by forcibly expressing the transcription factor Pdx-1, the cells can stimulate the synthesized insulin in response to stimulation. Acquired the ability to secrete efficiently. Even in normal knee ⁇ cells, insulin is always stored in the cells and released into the blood promptly in response to stimulation, enabling strict glycemic control.
  • the insulin-producing cells of the present invention are considered to be cells having physiologically similar functions to knee island ⁇ cells. '
  • the insulin-producing cell and the method for producing the same of the present invention can be used as a donor cell used in cell transplantation therapy, which is expected as a fundamental treatment for diabetes, and in the field of cell production therefor.
  • undifferentiated stem cells can be isolated from the liver of a diabetic patient, and insulin-producing cells produced based on the cells can be used as donor cells for autologous transplantation, or can be produced based on liver-derived cells obtained.
  • Surin producing cells may be used as donor cells for allogeneic transplantation.
  • the method for producing the insulin-producing cells of the present invention can be used not only in direct treatment of diabetes but also in basic research, animal tests, clinical tests, etc. for the development of therapeutic methods as a precedent step. Available.
  • insulin synthesized and secreted by the insulin-producing cells of the present invention can be used as pharmaceuticals (insulin preparations), reagents, experimental materials, and the like. Included in the invention. Insulin isolated and purified from rat and mouse cells may be derived from the insulin 1 gene or the insulin 2 gene. A known method may be used for isolating and purifying insulin.
  • the method of the present invention it is possible to induce production of not only insulin but also glucagon and Z or somatostatin (see FIG. 13).
  • the endocrine hormone-producing cells thus obtained and endocrine hormones isolated and purified from the cells are also industrially useful and included in the present invention.
  • Insulin-producing cells were produced from small hepatocytes by the method shown in FIGS. The manufacturing method is as described above, and the description is omitted here.
  • FIG. 2 “Group 1” indicates the experimental group, and “Group 2” indicates the control group. The following figures (the same applies hereafter).
  • Fig. 3 (a) shows the results of observation of cells on day 7 of culture using a phase contrast microscope
  • Figs. 3 (b) and (c) show the results of detection of insulin using an anti-insulin antibody by immunofluorescence staining. is there.
  • the left panel in (b) shows the results of fluorescent staining of insulin in the cells of the experimental group (+ Pdx-1) on day 15, and the right panel shows the results obtained by DAPI fluorescence of the cell nuclei in these cells. The result is superimposed on the result of staining.
  • D API is a dye substance for nuclear staining of cells.
  • the left panel in (c) shows the results of the fluorescent staining of insulin in the cells of the control group on day 15, and the right panel shows the results and the results of the fluorescent staining of DAPI in the same cells superimposed. .
  • insulin was observed to be produced intracellularly (left panel in (b)). Showed almost no insulin production (left panel in (c)).
  • the amount of insulin production of each cell on day 9, day 12, and day 13 of culture was measured.
  • the cells were collected and immersed in ethanol (containing 0.1N hydrochloric acid) at minus 20 ° C for 12 to 24 hours to extract insulin according to the usual method. .
  • This exudate was measured and quantified by ELISA.
  • the total protein content in the sample was quantified by the BCA method, and the extraction efficiency between samples was corrected.
  • the results are shown in the graph of FIG. As shown in the graph, on the 15th day, the amount of insulin production of the experimental group (+ Pdx-1 and -Pdx-1) was significantly increased as compared with the control group.
  • RNA is extracted from cultured cells according to the conventional method, reverse transcription is performed using Superscript-II Reverse Transcriptase, converted to complementary DNA, and then PCH reaction is performed using Taq polymerase. I got it.
  • the primers used in each PCR reaction were as shown in FIGS. 11 and 12, and nested PCR was performed as necessary. The results were confirmed by agarose gel electrophoresis. Figure 4 shows the results.
  • Insulin 2 expression was observed only in the experimental group (+ Pdx-1), but not in the experimental group (-Pdx-1). Forced expression of mouse mPdx-1 induced expression of endogenous rat rPdx-1 and NeuroD. It is considered that the expression of Pdx-1 and neuron or Neuro D is important for the expression of insulin 2.
  • Mature ⁇ -cell markers Isll and Pax6 were detected after culture 9. In contrast, expression of the exocrine cell marker PTF1 was not observed in any of the cells analyzed.
  • HNF-la hepatocytes nuclear factor-la
  • HNF-lb hepatocytes nuclear factor-la
  • HNF-4a cause diabetes, but there was no change in their expression among the cells examined in this study.
  • the results are shown in the upper graphs of FIG.
  • the graph on the left shows the Krebs-Ringer bicarbonate buffer (KRBB) solution containing 0.2% FBS to which high-concentration glucose (25 mM) and 10 nM glucagon-like peptide 1 (GLP-1 [7-37]) were added.
  • the middle graph shows the results of stimulation with 45 mM KC1 and 0.2 mM tolbutamide added to KRBB solution with low concentration of glucose (5 mM) .
  • the right graph is for control, and the basal buffer This is the result of measurement using only the KRBB solution with a low concentration of dalcose (5 mM).
  • the insulin secretion quantification was performed in the same manner as in the experiment in FIG.
  • glucose promotes insulin secretion in vivo
  • adenosine triphosphate ATP
  • ATP adenosine triphosphate
  • Ca 2+ influx of Ca 2+ is increased, and then the Ca stored in the cell is increased. 2+ is released into the cytoplasm.
  • PKA protein kinase A
  • PKC protein kinase C
  • GLP-1 is an activator of the cAMP-PKA pathway, and K + and tolbutamide enhance Ca2 + entry into cells, and both stimulate insulin secretion.
  • the experimental group in which Pdx-1 was forcibly expressed (+ Pdx-1) had a higher stimulus-responsive insulin secretion function than the experimental group (-Pdx-1).
  • the time change of insulin secretion in the cells of the experimental group (+ Pdx-1) was examined, the time change was similar to the time change of insulin secretion from knee island ⁇ -cells due to glucose stimulation (Fig. 6). See lower graph).
  • RT As a result of PCR analysis, expression of other proteins that contribute to the secretion of insulin hormone, SNAP25, GBy, and TGase2, were determined in the experimental group on day 15 of culture (both + Pdx-1) and -Pdx-1. In the same way. In particular, in the experimental group (+ Pdx-1) in which Pdx-1 was forcibly expressed, induction of synaptotagmin 1 (Sytl) expression was observed. (See Figure 9).
  • synaptotagmin 1 (Syt 1) is considered to be a direct or indirect target substance of the transcription factor Pdx-1, and that Pdx-1 positively regulates the transcription of Syt 1.
  • the model shown in Fig. 10 suggests that the interaction between Pdx-1, Syt1, Neuro D, Pax6, and HNF-3B in knee island ⁇ -cells is considered.
  • Neuro D and the like also positively regulate the transcription of Sytl.
  • the present invention relates to insulin-producing cells obtained by culturing liver-derived cells and a method for producing the same, as described above. It can be used in the field of therapy and cell production therefor, and has various other usefulness.

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Abstract

Cette invention propose des cellules de production d'insuline, que l'on peut constituer en utilisant des cellules d'origine hépatiques obtenues à partir d'un tissu hépatique normal comme matière de départ, ainsi qu'un procédé de constitution de ces cellules. En cultivant des hépatocytes de petite taille formés par des cellules souches non différenciées dans des conditions définies, on parvient à différencier ces cellules en cellules de production d'insuline. On peut en outre améliorer le système de sécrétion d'insuline extra-cellulaire de ces cellules par réponse à un stimulus en transférant un gène Pdx-1 dans ces cellules. Les cellules de production/sécrétion d'insuline ainsi obtenues sont physiologiquement très similaires aux cellules P insulaires et peuvent être utilisées dans le traitement ou l'étude du diabète notamment. On s'attend notamment à ce que ces cellules puissent servir de cellules de donneur dans les thérapies de transplantation cellulaire, dans le cas du diabète.
PCT/JP2003/014681 2003-05-22 2003-11-18 Cellules de production d'insuline et procede de constitution de ces cellules Ceased WO2004104195A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
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