[go: up one dir, main page]

WO2015190522A1 - Procédé de production de cellules souches endodermiques - Google Patents

Procédé de production de cellules souches endodermiques Download PDF

Info

Publication number
WO2015190522A1
WO2015190522A1 PCT/JP2015/066740 JP2015066740W WO2015190522A1 WO 2015190522 A1 WO2015190522 A1 WO 2015190522A1 JP 2015066740 W JP2015066740 W JP 2015066740W WO 2015190522 A1 WO2015190522 A1 WO 2015190522A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
insulin
producing
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2015/066740
Other languages
English (en)
Japanese (ja)
Inventor
誠 土方
阿部　雄一
達哉 山口
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toyobo Co Ltd
Kyoto University NUC
Original Assignee
Toyobo Co Ltd
Kyoto University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toyobo Co Ltd, Kyoto University NUC filed Critical Toyobo Co Ltd
Publication of WO2015190522A1 publication Critical patent/WO2015190522A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for producing endoderm stem cells and a technique using the same.
  • Diabetes is a serious disease that is spreading worldwide.
  • the global diabetic population estimated by WHO is over 150 million in 2000 and is expected to reach about 300 million in 2025.
  • islet (insulin producing cell) transplantation is attracting attention as an effective treatment for patients with type I diabetes.
  • the shortage of donors providing transplantable islets is a serious problem. Yes.
  • HLA antigen suitability is low, it is necessary to use an immunosuppressive agent for the recipient in order to control the rejection reaction, and side effects due to the immunosuppressive agent also become a problem.
  • Non-patent Document 1 a method for isolating pancreatic stem cells from a patient's pancreatic tissue and inducing differentiation into pancreatic islet cells has been reported. It is difficult to obtain islet cells sufficient for transplantation, and it is practically used. The current situation is not.
  • an object of the present invention is to provide a technique capable of stably supplying stem cells that can also be used for islet transplantation.
  • hepatocytes from liver-derived cell groups (primary cultured hepatocytes) but also organs derived from other endoderm (for example, pancreas). It was found that cells that can differentiate into constituent cells (ie, endoderm stem cells) can be obtained.
  • endoderm stem cells ie, endoderm stem cells
  • Exemplary inventions include the following. Item 1.
  • A In a liver cell group, a step of expressing a gene encoding a protein having an activity of passing through the G0 phase or G1 phase and shifting to the S phase; (B) a step of culturing the cells obtained in step (A) in the presence of an extracellular growth factor; and (C) a cell in which expression of the molecular marker AFP is negative from the cells obtained in step (B).
  • a method for producing an endoderm stem cell comprising: Item 2. Item 2. The method according to Item 1, wherein the endoderm stem cell is a stem cell having an ability to differentiate into an insulin-producing cell. Item 3. Item 3.
  • Item 1 or 2 wherein the protein is a cyclin-dependent kinase.
  • Item 4. The method according to Item 3, wherein the cyclin-dependent kinase is cyclin-dependent kinase 4 or cyclin-dependent kinase 6.
  • Item 5. The method according to any one of Items 1 to 4, wherein the steps (A) and (B) are repeated at least twice.
  • Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the extracellular growth factor is a hepatocyte growth factor.
  • Item 7. The method according to any one of Items 1 to 6, which is performed.
  • Item 8. The method according to any one of Items 1 to 7, wherein the culture is continued for 7 days or more.
  • Item 9. The method according to any one of Items 1 to 8, wherein the expression is transient expression.
  • Item 10. An endoderm stem cell obtainable by the method according to any one of Items 1 to 9. Item 11. Item 11. The endoderm stem cell according to Item 10, into which an exogenous gene has been introduced. Item 12.
  • Item 10 A method for producing insulin-producing cells, comprising a step of culturing endoderm stem cells obtained by the method according to any one of Items 1 to 9 in a medium suitable for differentiation into insulin-producing cells. Item 13. Item 14. The method according to Item 12 or 13, wherein the insulin-producing cells have glucose reactivity. Item 14. Item 14. An insulin-producing cell obtainable by the method according to item 12 or 13.
  • Human primary hepatocytes for example, cells derived from liver tissue that have become incompatible with organ transplantation for various reasons
  • the present invention using human primary hepatocytes as a raw material makes it possible to more stably provide cells that constitute an endoderm-derived organ including insulin-producing cells (or islet cells). Therefore, the possibility of constructing a large-scale insulin producing cell bank is expanded by utilizing the present invention.
  • These insulin-producing cell banks not only provide highly tissue-compatible transplant-producing insulin-producing cells to diabetics around the world, but also supply many donor-derived insulin-producing cells for research studies such as regenerative medicine Make it possible.
  • the present invention can also be used for the treatment of diseases relating to organs derived from the endoderm other than the pancreas.
  • FIG. 1 shows the results of examining gene markers expressed in proliferated cells by introducing and expressing the CDK4 gene or CDK6 gene in the primary hepatocyte group.
  • FIG. 2 shows a scheme for inducing differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 3 shows changes in the expression of the Pdx1 gene, which is a pancreatic differentiation marker associated with the induction of differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 4 shows changes in the expression of an insulin gene, which is a pancreatic ⁇ cell marker, with the induction of differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 1 shows the results of examining gene markers expressed in proliferated cells by introducing and expressing the CDK4 gene or CDK6 gene in the primary hepatocyte group.
  • FIG. 2 shows a scheme for inducing differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 3 shows changes in the expression of the Pdx1 gene, which is a pancreatic
  • FIG. 5 shows changes in the expression of a glucokinase gene, which is a pancreatic differentiation marker accompanying the induction of differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 6 shows changes in the expression of the glucagon gene, which is a marker for pancreatic ⁇ cells, with the induction of differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 7 shows changes in the expression of the somastatin gene, which is a pancreatic ⁇ cell marker, with the induction of differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 8 shows changes in expression of the Glut2 gene and the Nkx6-1 gene accompanying the induction of differentiation of endoderm stem cells into insulin-producing cells.
  • FIG. 9 shows changes in the expression of the insulin gene and the somatostatin gene before and after induction of differentiation into insulin-producing cells.
  • FIG. 10 shows the results of a glucose stimulation (glucose responsiveness) test in differentiation-induced insulin-producing cells.
  • FIG. 11 shows changes in expression of various gene markers accompanying the induction of differentiation of endoderm stem cells into hepatocytes.
  • the endoderm stem cells are obtained through the following steps (A) to (C).
  • (A) A step of expressing a gene encoding a protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase in the liver cell group
  • (B) Extracellular growth of the cells obtained in the step (A)
  • (C) A step of selecting a cell in which the expression of the molecular marker AFP is negative from the cells obtained in the step (B)
  • a liver cell group means a cell group prepared from liver tissue.
  • Liver cells are usually composed of a plurality of cells, mainly liver parenchymal cells, but may also include other cells such as sinusoidal endothelial cells, stellate cells, and Kupffer cells.
  • a commercially available product may be used, or a sample collected from a living body may be used.
  • human liver cells when used, commercially available frozen human liver cells sold by XenoTech, In Vitro Technologies, etc. can be used.
  • a cell separation kit or the like to remove dead cells by centrifugation.
  • liver tissue collected using a biopsy needle or the like can be prepared by digesting collagenase according to a conventional method to separate hepatocytes and removing dead cells.
  • the liver cell group preferably has high viability.
  • the viability is 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, particularly preferably. 90% or more.
  • Cell viability can be measured using a commercially available analyzer.
  • the liver cell group preferably has a high adhesion rate (for example, 70% or more) to a plate coated with collagen or the like. Viability can be measured according to a known method, for example, by treating a group of cells with trypan blue dye and measuring the proportion of dead cells stained blue using a microscope or the like. Can do.
  • a protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase passes through the G0 phase or the G1 phase.
  • cell cycle reactivation protein passes through the G0 phase or the G1 phase.
  • Transfer to the S phase by passing the G0 phase or the G1 phase means (1) acting on the cells in the G0 phase and being in a dormant state by deviating (escaping) from the cell cycle. It means to enter the cell cycle again by shifting to (2), or to act on the cells in the G1 phase and shift the cell cycle from the G1 phase to the S phase.
  • the means for expressing the cell cycle reactivation protein gene is not limited as long as the cell cycle reactivation protein can be expressed.
  • transient expression means that a gene is introduced into a cell by a DNA transfection method or the like and expressed transiently. Transient usually refers to a period of hours to days.
  • stable expression means that a gene to be expressed is stably expressed in a chromosome.
  • the gene of the cell cycle reactivation protein is: It is preferably expressed transiently.
  • Transient expression is not particularly limited, and can be performed, for example, by introducing an expression vector having a target gene downstream of an expression promoter into a cell and expressing the gene from this expression vector.
  • an expression promoter for example, CMV promoter, SV40 promoter and the like can be used, but are not limited thereto.
  • expression vectors include, but are not limited to, plasmid vectors and liposomes as non-viral vectors, and adenovirus vectors and retrovirus vectors as virus vectors. It is preferable to use a non-viral vector from the viewpoint of safety when using the cells to be produced for pharmaceutical purposes and ensuring that the gene to be introduced is transiently expressed, and in particular, the origin of replication in the host cell.
  • Non-viral vectors that do not contain are preferred. In order to carry out transient expression more reliably, it is possible to add a step of confirming that the introduced cell is not incorporated into the chromosome.
  • plasmid vectors that can be used from such a viewpoint include pcDNA and pSVL.
  • a method for introducing an expression vector into a cell for example, a lipofection method, an electroporation method, a method in which a gene is incorporated into a viral vector and infected, and the like can be used, but not limited thereto.
  • stable expression is not particularly limited, it can be performed, for example, by the following method.
  • An expression vector having a target gene and a dominant selection marker downstream of the expression promoter is introduced into the cell, and a strain in which the target gene is integrated into the chromosome is established. In this established strain, stable expression is performed.
  • an expression promoter for example, CMV promoter, SV40 promoter and the like can be used, but are not limited thereto.
  • a dominant selection marker for example, various drug resistance genes can be used, but not limited thereto. When a drug resistance gene is used as a dominant selection marker, only cell lines that stably express the drug resistance gene can be selected by continuing cell culture in the presence of a drug exhibiting resistance. .
  • the target gene is considered to be stably expressed as well. Whether or not the target gene is actually stably expressed can be clarified by analyzing the base sequence of the chromosome by a DNA sequence or the like.
  • a lipofection method, an electroporation method, or the like can be used, but is not limited thereto.
  • a viral vector a method of incorporating a gene into a viral vector and infecting it can also be used.
  • the liver cell group can be cultured in advance in a medium capable of maintaining the original function of the cells.
  • a commercially available dedicated medium Human Hepatocyte Serum Free Medium, Toyobo
  • suitable for the characteristics of liver cells can be preferably used.
  • Step (B) is a step of culturing the cells obtained in step (A) while giving growth stimulation with extracellular growth factor.
  • the cell cycle shifts from the G0 phase or the G1 phase to the S phase, and further to the M phase (mitotic phase), and then to the G1 phase again Proceed with
  • the action of the cell cycle reactivation protein is activated by the action of the extracellular growth factor present in the medium.
  • stem cells proliferate predominantly and include endoderm stem cells.
  • the extracellular growth factor used in the step (B) is not particularly limited as long as it has a function of externally supporting the growth of liver-derived somatic stem cells.
  • extracellular growth factors include cell growth factors and hormones that stimulate cell growth.
  • the cell growth factor include epidermal growth factor (EGF), hepatocyte growth factor (Hepatocyte Growth Factor; HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial cells.
  • Examples include growth factors (Vascular Endothelial Growth Factor; VEGF) and fibroblast growth factors (FGF). Of these, EGF and HGF are preferred. These may be used alone or in combination of two or more.
  • a preferred extracellular growth factor is hepatocyte growth factor.
  • the concentration of the extracellular growth factor added in the medium is not particularly limited. For example, it is preferably 0.1 to 200 ng / ml, more preferably 1.0 to 100 ng / ml, and 5 to 50 ng / ml. Further preferred.
  • the medium may be exchanged at an appropriate interval. Although not particularly limited, the medium may be changed once every two or three days.
  • the medium may be replaced with a medium containing the same concentration of extracellular growth factor, or may be replaced with a medium containing a different concentration of extracellular growth factor.
  • the medium may be replaced with a medium containing the same extracellular growth factor, or may be replaced with a medium containing a different extracellular growth factor.
  • the culture in the step (B) is preferably performed using a medium suitable for obtaining embryonic stem cells.
  • a medium in addition to the above-mentioned extracellular growth factor, animal serum, epidermal growth factor, nicotinamide, dimethyl sulfoxide and HuS-E / 2 cells (FERM ABP-10908) culture supernatant It is preferable to include one or more selected, preferably two or more, more preferably three or more, still more preferably four or more, and still more preferably all.
  • Animal serum is not particularly limited, and examples include human serum and fetal bovine serum. These are preferably added to the medium in the range of 0.1 to 20% by volume. When nicotinamide is added to the medium, the amount added is preferably 1 to 100 mM. When dimethyl sulfoxide is added to the medium, the addition amount is preferably 0.1 to 2% by volume. When the culture supernatant of HuS-E / 2 cells is added to the medium, the amount added is preferably 10 to 90% by volume.
  • Steps (A) and (B) may be performed once each, or may be performed a plurality of times as necessary. Alternatively, these may be set as a set and repeated a plurality of times. When it is performed a plurality of times, it can be performed preferably 2 to 10 times, more preferably 3 to 8 times, and even more preferably 3 to 5 times. It is considered that repeating steps (A) and / or (B) in this way is preferable in order to transiently express a gene encoding a cell cycle reactivation protein and shift stem cells to a proliferative state. It is done.
  • Steps (A) and (B) can be continued until a stem cell containing the required amount of endoderm stem cells is obtained.
  • the culture period is not particularly limited and can be, for example, several days to several weeks. In one embodiment, the culture is preferably performed for 7 days or more.
  • the step (B) is performed only once, for example, if the step (B) is continued for a period until the colony formation by the stem cells is sufficiently performed, a group of stem cells including endoderm stem cells can be easily collected.
  • the end point of the step (B) is preferably a time point at which a stem cell colony can be confirmed with a microscope or the naked eye, or a time point at which a stem cell colony consisting of 10 to 10,000 cell groups is formed, and more preferably 100 to 100- This is the time when a stem cell colony consisting of 1000 cell groups is formed.
  • Many cells other than stem cells are killed by the end of the step (B), or even if they are alive, the growth is stopped and the cell morphology is clearly different. For this reason, a highly pure stem cell group can be acquired easily.
  • Process (C) By collecting the colonies obtained by the step (B) and selecting cells that are negative for the cell surface molecular marker AFP, endoderm stem cells can be obtained. Colony recovery can be performed by a conventionally known method, for example, a limiting dilution method or a method using a micropipette under a microscope.
  • the endoderm stem cells are preferably albumin negative, C-Met positive, EpCam positive, Thy1 positive, and CD34 negative.
  • the presence or absence of the expression of the molecular marker or gene marker can be confirmed by any technique, and can be performed using, for example, a commercially available kit.
  • Judgment of being an endoderm stem cell can also be performed by other methods (for example, examining whether or not to differentiate into cells of a plurality of endoderm-derived organs). For example, the differentiation of the obtained cells into pancreatic cells is performed, and the presence or absence of gene markers (Pdx1, insulin, glucokinase, etc.) of the pancreatic cells is confirmed to confirm whether the cells can be divided into pancreatic cells. Can do. In addition, hepatocyte differentiation can be induced on the obtained cells and the presence or absence of hepatocyte gene markers (albumin, drug metabolizing enzyme genes, etc.) can be confirmed. .
  • other methods for example, examining whether or not to differentiate into cells of a plurality of endoderm-derived organs. For example, the differentiation of the obtained cells into pancreatic cells is performed, and the presence or absence of gene markers (Pdx1, insulin, glucokinase, etc.) of the pancreatic cells is confirmed to confirm whether the cells can be divided into pancre
  • a stem cell group preferably containing 50% or more endoderm stem cells in terms of the number of cells, more preferably a cell group containing 80% or more endoderm stem cells can be prepared. According to the present invention, it is possible to produce a group of stem cells, more preferably substantially composed of only endoderm stem cells, and more preferably an isolated endoderm stem cell group.
  • Endoderm stem cells are somatic stem cells that have the ability to differentiate into cells that constitute an organ derived from the endoderm (ie, multipotent) and self-proliferating ability.
  • organs derived from the endoderm include, but are not limited to, the esophagus, stomach, small intestine, large intestine, lung, thyroid gland, pancreas, and liver.
  • organs constituted by cells from which endoderm stem cells can differentiate pancreas and liver are preferable.
  • the endoderm stem cells preferably have the ability to differentiate into insulin-producing cells, and stably supply a high-purity population of insulin-producing cells having properties equivalent to those of insulin-producing cells in vivo. Can be used to The insulin-producing cells thus obtained can be finally used in clinical applications such as cell preparations, or in various research and development such as new drug development or disease research.
  • the endoderm stem cell may further contain an exogenous gene as necessary.
  • Induction of differentiation of endoderm stem cells into insulin-producing cells can be performed by culturing endoderm stem cells in a medium suitable for differentiation into insulin-producing cells and other conditions.
  • a medium and other culture conditions suitable for differentiation can be set by appropriately selecting from known conditions.
  • Zhang D. Can be used to differentiate from ES cells to insulin-producing cells (Cell Research 19 (4), 429-38, 2009). That is, it is a method of inducing differentiation stepwise by changing the medium (culture environment) such as specialization, cell proliferation, and cell maturation from endoderm stem cells toward pancreatic differentiation.
  • the insulin-producing cells thus induced to differentiate preferably have glucose responsiveness.
  • Example 1 Preparation of endoderm stem cells (1) Culture of primary human hepatocytes After thawing frozen primary human hepatocytes (XenoTech), Ficoll isolation using Hepatocyte Isolation Kit (XenoTech) to separate live and dead cells As a result, a high viability hepatocyte suspension was obtained. These cells were suspended in Human Hepatocyte Serum Free Medium (Toyobo Co., Ltd.) supplemented with fetal bovine serum at a rate of 10% by volume, and about 5 ⁇ 10 5 cells / cell-coated 12-well cell culture plate (AGC Techno Glass) / It seed
  • Human Hepatocyte Serum Free Medium Toyobo Co., Ltd.
  • fetal bovine serum at a rate of 10% by
  • RNA purified from HuS-E / 2 cells human hepatocyte-derived cells, deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary: FERM ABP-10908
  • a forward primer consisting of the base sequence shown in SEQ ID NO: 1
  • a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 were used.
  • the base sequence of SEQ ID NO: 1 includes a sequence corresponding to Flag Tag.
  • the plasmid (pcDNA-FLAG-CDK4 or pcDNA-FLAG-CDK6) having Flag Tag introduced into the N-terminal side of the open reading frame of DNA encoding CDK4 or CDK6 obtained in this way was 0.3 ⁇ g per well. It was added to the medium together with Effectene transfection reagent (Qiagen), and the CDK4 or CDK6 gene was introduced into the hepatocyte group. Thereafter, transfection was repeated 5 times at a frequency of once every 5 days. As a negative control, a similar transfection operation was performed using a plasmid not containing the CDK gene.
  • the HuS-E / 2 cells are cultured in a DMEM-based culture solution containing 5% human serum and 5% fetal bovine serum.
  • a DMEM-based culture medium in which the prepared conditioned medium was added and mixed was used.
  • DMEM-based cultures include 35.8 mM sodium bicarbonate, 0.34 ⁇ g / ml insulin, 4.1 ⁇ g / ml transferrin, 4.1 ng / ml EGF (epidermal growth factor), 20 ng / ml.
  • HGF hepatocyte growth factor
  • 8.3 mM nicotinamide 0.81 vol% dimethyl sulfoxide were added.
  • FIG. 1 shows the results of examining the gene expression related to hepatocytes for each of the obtained clones. Although some of them showed the properties of hepatic stem cells, there were clonal cells that were negative for alpha fetoprotein (hereinafter, AFP), which is one of the markers of hepatic stem cells (FIG. 1).
  • AFP alpha fetoprotein
  • the cells having a negative AFP marker can also differentiate into cells constituting an organ (pancreas) derived from the endoderm other than the liver. Therefore, cells that are negative for the AFP marker were obtained as endoderm stem cells (ie, stem cells that can differentiate into endoderm-derived organs). Endodermal stem cells were confirmed to have self-replicating ability because of continued proliferation while maintaining the above-mentioned differentiation ability over one year after establishment.
  • Example 2 Confirmation of differentiation ability into endoderm-derived organ (1) Induction of differentiation into insulin-producing cells A test was conducted to differentiate the endoderm stem cells obtained in Example 1 into insulin-producing cells using a known technique. Specifically, the following three-stage differentiation induction procedure shown in FIG. 2 was performed. First, 0.5 mass / volume% bovine serum albumin, 0.5 volume% ITS solution (Life Technologies), 0.5 times B27 solution (Life Technologies), 2 ⁇ M retinoic acid (Sigma), The culture was performed for 4 days in a 1: 1 mixed medium of F12 medium and IMDM medium supplemented with 20 ng / ml FGF7 (Peprotech) and 50 ng / ml NOGGIN (Peprotech).
  • ITS solution 1 volume% bovine serum albumin
  • 1-fold N2 solution 50 ng / ml EGF (Sigma) were added. Cultivated in DMEM medium for 5 days. Thereafter, 1% by volume ITS solution (Life Technologies), 10 ng / ml bFGF (Peprotech), 10 mM nicotinamide (Sigma), 50 ng / ml Exendin4 (Peprotech), and 10 ng / ml BMP4 ( The cells were cultured for 4 days in DF12 medium supplemented with Peprotech.
  • Insulin and somatostatin are known to be produced in islet ⁇ cells and islet ⁇ cells, respectively.
  • the same cells were stained simultaneously by the indirect fluorescent antibody method using an anti-insulin antibody and an anti-somatostatin antibody before and after differentiation induction, it was confirmed that these two proteins were produced in one cell (FIG. 9). ).
  • Example 3 Induction of differentiation into hepatocytes Human hepatic stem cells having the properties of endoderm stem cells obtained in Example 1 were confirmed to have the ability to differentiate into hepatocytes. That is, human hepatic stem cells having the properties of endoderm stem cells are cultured in a medium in which 30 ng / ml FGF4 and 20 ng / ml BMP2 are added to Hepatic basal medium (Lonza) for 5 days, and then Human Hepatocyte Serum-free Medium. (Toyobo) was cultured for about one month. During this time, as a result of investigating gene markers expressed in the cells over time, AFP turned positive on the 5th day of culture and further showed albumin positive after about 1 month of culture (FIG. 11). This result indicates that human hepatic stem cells having the properties of endoderm stem cells are differentiated into hepatocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention a pour objet : un procédé pour la production efficace de cellules souches endodermiques ; et autres. Le procédé de production de cellules souches endodermiques selon l'invention comprend : (A) une étape d'expression d'un gène dans des cellules hépatiques, le gène étant un gène qui code pour une protéine ayant une activité consistant à permettre à la phase des cellules de subir une transition vers une phase S en passant par une phase G0 ou G1 ; (B) une étape de culture des cellules obtenues dans l'étape (A) en présence d'un facteur de croissance extracellulaire ; et (C) une étape de sélection de cellules dans chacune desquelles l'expression d'un marqueur moléculaire AFP est négative parmi les cellules obtenues dans l'étape (B).
PCT/JP2015/066740 2014-06-12 2015-06-10 Procédé de production de cellules souches endodermiques Ceased WO2015190522A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014-121780 2014-06-12
JP2014121780 2014-06-12

Publications (1)

Publication Number Publication Date
WO2015190522A1 true WO2015190522A1 (fr) 2015-12-17

Family

ID=54833611

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/066740 Ceased WO2015190522A1 (fr) 2014-06-12 2015-06-10 Procédé de production de cellules souches endodermiques

Country Status (1)

Country Link
WO (1) WO2015190522A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126236A1 (fr) * 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progenitrices du foie
WO2009143353A1 (fr) * 2008-05-22 2009-11-26 Vesta Therapeutics, Inc. Procédé de différenciation de cellules progéniteurs de mammifère en cellules d'îlots pancréatiques produisant de l’insuline
WO2012133156A1 (fr) * 2011-03-25 2012-10-04 国立大学法人京都大学 Procédé de production de cellules souches épithéliales

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126236A1 (fr) * 2005-05-26 2006-11-30 Fresenius Medical Care Deutschland G.M.B.H. Cellules progenitrices du foie
WO2009143353A1 (fr) * 2008-05-22 2009-11-26 Vesta Therapeutics, Inc. Procédé de différenciation de cellules progéniteurs de mammifère en cellules d'îlots pancréatiques produisant de l’insuline
WO2012133156A1 (fr) * 2011-03-25 2012-10-04 国立大学法人京都大学 Procédé de production de cellules souches épithéliales

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG D ET AL.: "Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells.", CELL RES., vol. 19, no. 4, 19 April 2009 (2009-04-19), pages 429 - 438, XP055074920, ISSN: 1001-0602 *

Similar Documents

Publication Publication Date Title
JP4336821B2 (ja) 哺乳動物の骨髄細胞または臍帯血由来細胞と脂肪組織を利用した心筋細胞の誘導
US11920160B2 (en) Pancreatic insulin-producing beta-cell lines derived from human pluripotent stem cells
JPWO2008066199A1 (ja) 胚性幹細胞のインスリン分泌細胞への分化誘導方法、該方法により誘導されるインスリン分泌細胞およびその用途
EA035360B1 (ru) Способы трансдифференцировки и способы их использования
CN109689858A (zh) 用于产生具有体内血管形成能力的中胚层和/或内皮集落形成细胞样细胞的方法
Day Epithelial stem cells and tissue engineered intestine
WO2007010858A1 (fr) Cellule souche pluripotente clonée à partir d’une cellule unique dérivée de tissu du muscle squelettique
JP6095272B2 (ja) 上皮系体性幹細胞の製造方法
JP2021512643A (ja) 細胞誘導の方法
US20200291358A1 (en) Stem cell derived from young pig and method for producing same
WO2015190522A1 (fr) Procédé de production de cellules souches endodermiques
JP4913730B2 (ja) 膵臓β細胞再生方法および装置
Narayanan et al. Ductal cell reprograming to insulin-producing cells as a potential beta cell replacement source for islet auto-transplant recipients
KR101587231B1 (ko) 체세포에서 간세포로의 직접교차분화 촉진용 조성물 및 방법
WO2011110722A1 (fr) Procédé pour la prolifération "in vitro" de cellules issues de tissus d'origine endodermique
JP5294041B2 (ja) 膵臓疾患又は糖尿病のための膵臓細胞再生移植用キット
JP6654323B2 (ja) 重層上皮組織形成能を有する細胞、及びその製造方法
US20200095557A1 (en) Cell spheroids containing capillary structures and methods of using same
JPWO2014038655A1 (ja) 腸上皮由来体性幹細胞の製造方法
Li Generation of Human Pancreatic Beta Like Cells from Pluripotent Stem Cells
WO2014038652A1 (fr) Procédé de production de cellules souches somatiques dérivées du pancréas
CN119286927A (zh) 一种将胰岛细胞重编程为诱导多能干细胞的方法
Jahan Multi-stage endothelial differentiation and expansion of human pluripotent stem cells
CN119120581A (zh) 一种双基因修饰的干细胞诱导分化胰岛β细胞的制备方法
WO2014038653A1 (fr) Procédé de production de cellules souches somatiques dérivées du rein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15806119

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15806119

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP