WO2019198962A1 - Procédé pour favoriser la prolifération et la différenciation de cellules souches pluripotentes induites préparées à partir de cellules souches issues d'urine - Google Patents

Procédé pour favoriser la prolifération et la différenciation de cellules souches pluripotentes induites préparées à partir de cellules souches issues d'urine Download PDF

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WO2019198962A1
WO2019198962A1 PCT/KR2019/003834 KR2019003834W WO2019198962A1 WO 2019198962 A1 WO2019198962 A1 WO 2019198962A1 KR 2019003834 W KR2019003834 W KR 2019003834W WO 2019198962 A1 WO2019198962 A1 WO 2019198962A1
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stem cells
urine
cells
cell
induced pluripotent
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PCT/KR2019/003834
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English (en)
Korean (ko)
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조쌍구
김경석
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건국대학교 산학협력단
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Priority claimed from KR1020180041144A external-priority patent/KR102142254B1/ko
Priority claimed from KR1020180041134A external-priority patent/KR102016257B1/ko
Application filed by 건국대학교 산학협력단 filed Critical 건국대학교 산학협력단
Publication of WO2019198962A1 publication Critical patent/WO2019198962A1/fr

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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • C12N5/0684—Cells of the urinary tract or kidneys
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources

Definitions

  • the present invention is a method for isolation and proliferation of urine-derived stem cells; Methods for promoting proliferation or differentiation of stem cells; And it relates to a method of increasing the formation rate of EB (Embryoid Bodies) from induced pluripotent stem cells.
  • EB Embryoid Bodies
  • Stem cell research is an explosive and exciting study with the potential to improve therapies for humans. Such stem cell research is being carried out by mesenchymal stem cells and pluripotent stem cells.
  • mesenchymal stem cells bone marrow derived stem cells, adipose derived stem cells, and umbilical cord derived stem cells have been studied for a long time. Attempts have been made to experimental studies and preclinical studies. In addition, autologous stem cells are clinically useful because they do not induce immune and rejection responses.
  • stem cells which can be separated from tissues and organs, make up a very small cell population in the body and have difficulty in separating cells because a special tissue separation technique is required.
  • Urine derived stem cells used in the present invention can be easily separated from human urine, and has the ability to differentiate into various tissues and organs like other somatic stem cells. In recent decades, urine-derived stem cells have emerged as promising cellular resources for cell therapy and tissue engineering, and play an important role in stem cell biology based on these applications.
  • Pluripotent stem cells are included in embryonic stem cells and induced pluripotent stem cells.
  • Embryonic stem cells are derived from the inner cell mass of early embryos and can differentiate into three types of germ layer cells in vitro and in vivo.
  • induced pluripotent stem cells were reprogrammed similar to embryonic stem cells by introducing the Oct3 / 4, Sox2, c-Myc, and Klf4 genes into mouse somatic cells using a retrovirus in 2006 by Yamanaka's team at Kyoto University. Reprogrammed cells were generated, which were termed induced pluripotent stem cells.
  • Induced pluripotent stem cells can generate cell lines of various lines in vitro by circumventing the ethical problems of embryonic stem cells resulting from the destruction of early embryos.
  • Induced pluripotent stem cells are cells that can solve the ethical issues of embryonic stem cells, and have a pluripotency that can be differentiated into all the cells of the human body. Is attracting attention.
  • An object of the present invention is to provide a method for separating and propagating urine-derived stem cells comprising the step of culturing in a cell culture dish coated with a coating material by separating the cells from the urine collected from the individual.
  • Still another object of the present invention is a stem comprising treating 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone. It is to provide a method for promoting the proliferation or differentiation of cells.
  • Another object of the present invention is an induction comprising treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones. It is to provide a method for increasing the formation rate of EB (Embryoid Bodies) from pluripotent stem cells.
  • EB Embryoid Bodies
  • the present invention provides a method for separating and propagating urine-derived stem cells comprising the step of culturing in a cell culture dish coated with a coating material by separating the cells from the urine collected from the individual.
  • the urine may be collected from the middle urine of the urine.
  • the cells may be isolated within 2 hours by collecting from the urine of the individual.
  • the separation may be performed by centrifugation for 5 to 15 minutes at 300g to 500g, preferably at 400g may be performed for 10 minutes.
  • the method may further comprise the step of washing with PBS (phosphate buffered saline) after separating the cells.
  • PBS phosphate buffered saline
  • the coating material may be Matrigel.
  • the culturing may be to culture by adding Y-27632.
  • the present invention is a stem cell comprising the step of treating 3,2'-dihydroxyflavones (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavones (3,4'-dihydroxyflavone) Provided are methods for promoting proliferation or differentiation.
  • the stem cells may be stem cells differentiated from induced pluripotent stem cells, the induced pluripotent stem cells may be reprogrammed from urine-derived stem cells.
  • the stem cells may be hematopoietic stem cells.
  • the 3,2'- dihydroxy flavone or 3,4'- dihydroxy flavone may be a concentration of 1 to 20 ⁇ M, preferably a concentration of 5 to 15 ⁇ M It may be, and more preferably may be a concentration of 10 ⁇ M.
  • the present invention is the induced pluripotent stem comprising the step of treating 3,2'-dihydroxyflavones (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavones (3,4'-dihydroxyflavone)
  • EB Embryoid Bodies
  • the induced pluripotent stem cells may be reprogrammed from urine-derived stem cells.
  • the 3,2'- dihydroxy flavone or 3,4'- dihydroxy flavone may be a concentration of 1 to 20 ⁇ M, preferably a concentration of 5 to 15 ⁇ M It may be, and more preferably may be a concentration of 10 ⁇ M.
  • the method for isolating and propagating urine-derived stem cells according to the present invention can efficiently separate and proliferate stem cells from urine, and can be utilized in various fields using stem cells.
  • induced pluripotent stem cells can be prepared from urine-derived stem cells, and treated with 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones to increase the EB formation rate in induced pluripotent stem cells, Proliferation and differentiation of differentiated stem cells can be further promoted, and can be utilized in various fields using stem cells.
  • FIG. 1 is a schematic diagram showing a method for separating urine-derived stem cells.
  • Figure 2 shows the results of analyzing the characteristics of the urine-derived stem cells.
  • 3 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And the number of colony-forming cells on the 5th day of isolation and culture of urine-derived stem cells according to Matrigel coating + Y-27632 treatment group.
  • 5 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And Matrigel-coated + Y-27632 treatment group to compare the proliferation efficiency of urine-derived stem cells.
  • Figure 11 shows a schematic diagram (top) showing the production of induced pluripotent stem cells from urine-derived stem cells (top) and produced induced pluripotent stem cells (bottom).
  • Figure 13 shows a schematic diagram (top) and the prepared kidney organoid (bottom) showing the production process of kidney organoid in the induced pluripotent stem cells.
  • Figure 14 shows the EB formation rate in induced pluripotent stem cells following treatment with 3,2'-dihydroxyflavone (3,2'-DHF) or 3,4'-dihydroxyflavone (3,4'-DHF). The result is a comparison.
  • FIG. 15 is a schematic diagram showing the differentiation process of the induced pluripotent stem cells into hematopoietic stem cells (top) and hematopoietic stems according to treatment of 3,2'-DHF or 3,4'-DHF through markers of hematopoietic stem cells This is a result of comparing the differentiation efficiency of the cells.
  • Figure 16 is a result of comparing the total cell number and CD34 + CD45 + cell number after treatment with 3,2'-DHF or 3,4'-DHF in hematopoietic stem cells differentiated from induced pluripotent stem cells.
  • stem cell refers to a cell having pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of an individual, Embryonic stem cells, induced pluripotent stem cells and adult stem cells.
  • embryonic stem cell refers to a cell cultured in vitro by extracting an inner cell mass from an blastocyst embryo just before the fertilized egg implants in the mother's uterus, which can differentiate into cells of all tissues of the individual. It refers to a stem cell having a pluripotent or totipotent self-renewal.
  • iPSC induced pluripotent stem cells
  • iPSC pluripotent pluripotent stem cells
  • Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically showing similar cell shapes, gene and protein expression patterns are similar, and have the characteristics of differentiation ability in and outside the body. It is not derived from female eggs, such as human embryonic stem cells, but is derived from already differentiated somatic cells, so it does not involve ethical problems such as human embryonic stem cells, and can be converted from a patient's somatic cells to stem cells. Therefore, the problem of immune rejection is less. However, despite such usefulness, there is a disadvantage in that a sufficient number of induced pluripotent stem cells cannot be obtained due to low dedifferentiation efficiency.
  • reprogramming refers to a process by which differentiated cells can be restored or converted into a pluripotent state.
  • the reprogramming includes any process of returning differentiated cells having a differentiation capacity of 0% to less than 100% to an undifferentiated state, preferably differentiated cells having a differentiation capacity of 0% or more than 0%. It includes both restoring or converting partially differentiated cells having a differentiation capacity of less than 100% into cells having 100% differentiation ability.
  • Such reprogramming may be performed by a reprogramming reprogramming factor.
  • reprogramming inducer refers to a substance that, when processed or expressed in differentiated cells, induces iPSC-specific gene expression and induces induced pluripotent stem cells having pluripotency.
  • the reprogramming inducer may be included without limitation in the present invention as long as it is a substance that induces reprogramming of differentiated cells, and examples thereof include Oct3 / 4, sox-2, c-Myc, and Klf-4. It can select according to the kind of cell to do, and is not limited to the said example.
  • adult stem cell refers to an undifferentiated cell capable of self-reproduction as a stem cell generated in differentiated tissue and capable of differentiating into all cell types of the derived tissue.
  • the adult stem cells are cord blood (umbilical cord blood), adult bone marrow, spleen, ovary, testis, peripheral blood, amniotic fluid, brain, blood vessels, skeletal muscle, skin or gastrointestinal epithelium, cornea, tooth dimensions, retina, liver or It can be extracted from the pancreas, and refers to primitive cells just before differentiation into cells of specific organs such as bone, liver, and blood.
  • the spinal cord has been reported to have hematopoietic stem cells (HSCs) and mesenchymal stem cells, and neural stem cells, stem cells derived from the brain, have subventricular zones. It is known to separate from the subventricular zone, ventricular zone and hippocampus of the central nervous system (CNS).
  • HSCs hematopoietic stem cells
  • mesenchymal stem cells stem cells derived from the brain
  • CNS central nervous system
  • adult stem cells are known to be difficult to proliferate, but have a strong tendency to differentiate.
  • adult stem cells can be used not only to regenerate organs required by actual medicine but also to characterize each organ after transplantation. It has characteristics that can be differentiated accordingly.
  • hematopoietic stem cell refers to a variety of blood cells and parental cells of lymphocytes that form the immune system, for example stems that can develop into lymphocytes, granulocytes, platelets, and red blood cells, such as B cells and T cells. Says a cell. It is an essential cell for bone marrow transplantation. In normal bone marrow blood, about 1% of cells (CD34 positive cells) that have the ability to produce all blood cells are called hematopoietic stem cells. Found throughout the body, including cord blood and umbilical cord blood, but is produced in large quantities in the bone marrow, and also has the ability to replicate itself. Peripheral blood hematopoietic stem cells are derived from bone marrow and have the property of self-replicating and matured cells as hematopoietic stem cells circulating in the bloodstream.
  • proliferation of the stem cell in the present invention is a concept that is differentiated from the differentiation (differentiation) of stem cells, the stem cells are not differentiated into specific cells, the cells are divided while maintaining the characteristics of the stem cells intact It means that the total number of cells is increased.
  • stem cells differentiate into specific cells when multicellular organisms occur during the formation of complex tissues in a single zygote, or when they repair damaged tissues as adults.
  • the term “medium” refers to a composition comprising nutrients necessary to maintain the growth and survival of cells in vitro .
  • the term "passage culture” means a method of continually culturing the cell band while transferring a portion of the cell to a new culture vessel periodically to continuously culture the cell in a healthy state for a long time. do. As the number of cells increases in a culture vessel with a limited space, proliferation of nutrients is consumed or contaminants accumulate, causing the cells to die naturally, and thus used as a method for increasing the number of healthy cells. Replacing a culture vessel or culturing a cell group is called one passage.
  • the active ingredient of the present invention is 3,2'-dihydroxyflavone (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavone (3,4'-dihydroxyflavone), each structure is represented by the following formula .
  • Urine-derived stem cells were obtained from six or more different sexes and ages. The urine-derived stem cells were collected from urine intermediate urine and separated within 2 hours at room temperature. A total of 100-200 mL of urine was put in a urine container, divided into four 50 mL tubes, and then centrifuged at 400 g for 10 minutes using a centrifuge at room temperature, and then the supernatant was removed. To wash the cells, 15 mL of PBS containing Antibiotic-Antimycotic (Gibco) was placed in a 50 mL tube, centrifuged at 400 g for 10 minutes, seeded in a cell culture dish coated with Gelatin, Matrigel, and Y-27632 was added to the cell culture medium. It was.
  • PBS containing Antibiotic-Antimycotic Gibco
  • RNA was extracted using an Easy-Blue total RNA extraction kit (iNtRON Biotechnology, Republic of Korea). The concentration of total RNA was measured with a Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington DE, USA). CDNA was synthesized using 2 ⁇ g total RNA and M-MLV reverse transcriptase, and analyzed after completion of the PCR reaction in 1% or 1.5% agarose gel. qPCR was analyzed using SYBR Green master mix and mRNA expression was calculated using House keeping gene (GAPDH) as a reference value.
  • GPDH House keeping gene
  • Cell lysis buffer [1% Triton X-100 (Sigma-Aldrich), 100 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10% glycerol (Amresco, Solon OH, USA), 50 mM sodium fluoride (Sigma-Aldrich ), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), 1 mM p-nitrophenyl phosphate (Sigma-Aldrich), and 1 mM sodium orthovanadate (Sigma-Aldrich)] at 13,000 rpm for 15 minutes at 4 ° C. Centrifuged as.
  • Protein supernatants were quantified with Bradford protein reagents and proteins were subjected to SDS-PAGE (10% or 12%).
  • the isolated protein was transferred to a nitro-ceulocellulose membrane and blocked for 1 hour with 5% skim milk powder dissolved in a tris-buffered saline (TBS) solution, followed by anti-OCT4, anti-SOX2, anti-NANOG and anti Primary antibodies of -ACTIN (Santa Cruz Biotechnology, Dallas, TX, USA) were reacted at 4 ° C. for 12-18 hours. Then, HRP-tagged anti-mouse, anti-goat or anti-rabbit secondary antibody was reacted, and the protein was identified using an ECL kit (Amersham Bioscience, Piscataway NJ, USA).
  • Urine-derived stem cells, adipose derived stem cells, umbilical stem cells in culture were counted and laid on a cell culture plate by 50 / cm 2 , and cultured for 10-14 days to confirm colony formation of cells. When the colonies were formed after incubation of the cells, the cells were washed with PBS, stained with crystal violet reagent for 20 minutes, and then washed with PBS again to select colonies.
  • Proliferated cells were reprogrammed using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) according to the manufacturer's manual. Reprogramming induced pluripotent stem cell colonies were transferred to Matrigel (BD Biosciences) coated culture plates and incubated with mTeSR TM 1 (STEMCELL Technologies) and 10 ⁇ M of Y-27632 (STEMCELL Technologies).
  • EB formation was induced for 5 days to differentiate into hematopoietic stem cells using proliferated cultured induced pluripotent stem cells. Then, in IMDM medium containing 15% FBS, SCF (40ng / mL), FLT-3L (20ng / mL), IL-3 (10ng / mL), IL-6 (10ng / mL), BMP4 (20ng / mL) Differentiation was induced by the addition of the medium, and half of the culture medium was changed every day for a total of 21 days of differentiation.
  • EB formation rate was measured as an average of three times by counting the number of EB through the microscope at the end of EB formation.
  • EB was treated with StemPro Accutase (Thermo Fisher Scientific) at 37 ° C. for 10 minutes, the cells were slowly pipetted to separate into single cells, and the total cell number was measured.
  • the inventors performed RT-PCR to characterize the isolated urine-derived stem cells.
  • epithelial markers E-cadherin, Claudin 1, Occludin in urine-derived stem cells was confirmed, and the expression of Vimentin, Twist1, Fibronetin, fibroblast markers, and specific expression of Renal epithelial markers SLC2A1 and L1CAM. (Fig. 2 top panel).
  • the cell culture plate was divided into Gelatin-coated groups, Matrigel-coated groups, and Y-27632-treated and untreated groups on cell culture plates, respectively, to confirm cell adhesion efficiency (top panel of FIG. 3).
  • the cell separation efficiency was about 10 times higher than that of the group coated with Gelatin alone.
  • the cell separation efficiency was about 6 times, and in the group treated with Matrigel and Y-27632 (Matrigel + Y-27632), about 60 times higher than the group coated with Gelatin alone. Showed cell separation efficiency (FIG. 4).
  • the proliferation rate of urine-derived stem cells is higher than that of using a conventionally commercialized medium, the time to mass cultivation can be shortened.
  • the early cultured urine-derived stem cells can be used.
  • chondrocytes and adipocytes can be used as a cell therapy can have many advantages.
  • stem cells were cultured by adding Y-27632 and Matrigel in addition to Gelatin.
  • the cell proliferation rate was increased in the group treated with Y-27632 together with Gelatin alone, and the cell proliferation rate was increased in the group treated with Matrigel rather than Gelatin, and treated with Matrigel and Y-27632 together.
  • the proliferation rate of the cells increased about 4 times compared to the group treated with Gelatin alone (FIG. 5).
  • Matrigel coated on the plate, and treated with Y-27632 was confirmed that can increase the proliferation of urine-derived stem cells.
  • the present inventors performed cell migration experiments to confirm the effective regeneration of damaged urine-derived stem cells into damaged tissues after transplantation.
  • the urine-derived stem cells isolated from the general gelatin showed a cell migration capacity of 50% at 24 h, 60% cell migration capacity at 48 h.
  • Urine-derived stem cells isolated from the Y-27632 group showed 60% cell migration at 24 h and about 80% cell migration at 48 h.
  • urine-derived stem cells isolated from Matrigel-coated petri dishes showed 40% cell migration at 24 h and 70% at 48 h, and approximately 65%, 48 at 24 h in the Matrigel-coated and Y27632 treated groups. It showed cell migration capacity of about 90% at h.
  • Matrigel coated on the plate, and treated with Y-27632 was confirmed that can increase the cell migration capacity of the urine-derived stem cells.
  • the inventors performed CFU-F (colony-forming unit fibroblast) analysis to compare the self-renewal ability which is one of the characteristics of adult stem cells.
  • the size of colonies is different depending on the self-proliferative capacity of the colony, that is, small colony shows low self-proliferative power, and large colony shows high self-proliferative power.
  • Matrigel coated on the plate, and treated with Y-27632 was confirmed that can increase the proliferation of urine-derived stem cells.
  • the present inventors performed experiments comparing the time-dependent proliferation rate of the isolated adipose derived stem cells, umbilical cord derived stem cells and urine derived stem cells. As a result, the urine-derived stem cells showed a faster proliferation rate than the adipose derived stem cells or umbilical stem-derived stem cells (FIG. 8). In addition, it was confirmed that the urine-derived stem cells can secure a larger amount of cells than adipose derived stem cells or umbilical cord stem cells (FIG. 9).
  • the urine-derived stem cells have a similar level of colony forming ability as the adipose derived stem cells or the umbilical cord derived stem cells (FIG. 10).
  • the present inventors have produced induced pluripotent stem cells through the reprogramming process shown in the upper panel of FIG. 11 to reprogram the urine-derived stem cells (reprogramming) to produce induced pluripotent stem cells. As a result, it was confirmed that colonies were formed from urine-derived stem cells to be seen as induced pluripotent stem cells (Fig. 11 lower panel).
  • the inventors carried out experiments to determine whether the cells of the formed colonies are induced pluripotent stem cells.
  • experiments were performed to determine whether Oct4, Sox2, and Nanog were expressed as markers of pluripotent stem cells. As a result, it was confirmed that Oct4, Sox2 and Nanog, which were not expressed in urine derived cells, were expressed in the induced pluripotent stem cells (FIG. 12 left panel).
  • the present inventors attempted to differentiate into several cells from induced pluripotent stem cells (USC-iPSCs) produced from urine-derived stem cells.
  • USC-iPSCs induced pluripotent stem cells
  • FIG. 13 differentiation into kidney organoid was induced through known differentiation methods. As a result, it was confirmed that differentiation into kidney organoid was well induced on the induced pluripotent stem cells at 16 days (Fig. 13 lower panel).
  • EB formation is a step that should be formed upon differentiation into not only hematopoietic stem cells but also various cells, and a relatively large and large number of EB formation within the same time may be advantageous for further differentiation.
  • the present inventors performed experiments to form EB in induced pluripotent stem cells (USC-iPSCs) prepared as described above and induce differentiation into hematopoietic stem cells (Fig. 15 top panel).
  • USC-iPSCs induced pluripotent stem cells
  • experiments were conducted to determine whether the efficiency of differentiation can be enhanced by adding 3,2'-DHF and 3,4'-DHF as in EB formation.
  • the present inventors confirmed that differentiation of hematopoietic stem cells from induced pluripotent stem cells can be induced well.
  • addition of 3,2'-DHF and 3,4'-DHF did not affect the differentiation of hematopoietic stem cells (Fig. 15 lower panel).
  • CD34 is a positive marker for hematopoietic stem cells isolated from peripheral blood or bone marrow, and when 3,2'-DHF and 3,4'-DHF were added, the number of CD34-expressing cells was higher than that of the control group.
  • CFU Coldy forming unit

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Abstract

La présente invention concerne : un procédé d'isolement et de prolifération de cellules souches issues d'urine ; un procédé permettant de favoriser la prolifération ou la différenciation de cellules souches différenciées à partir de cellules souches pluripotentes induites qui sont reprogrammées à partir des cellules souches issues d'urine ; et un procédé permettant d'augmenter le taux de formation d'EB à partir des cellules souches pluripotentes induites.
PCT/KR2019/003834 2018-04-09 2019-04-02 Procédé pour favoriser la prolifération et la différenciation de cellules souches pluripotentes induites préparées à partir de cellules souches issues d'urine WO2019198962A1 (fr)

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KR1020180041144A KR102142254B1 (ko) 2018-04-09 2018-04-09 3,4''-다이하이드록시플라본을 이용한 소변줄기세포의 분리 효율 향상 및 소변줄기세포유래 만능줄기세포의 조혈줄기세포 분화 효율을 촉진시키는 방법
KR10-2018-0041144 2018-04-09
KR10-2018-0041134 2018-04-09
KR1020180041134A KR102016257B1 (ko) 2018-04-09 2018-04-09 소변 유래 줄기세포의 분리 효율 및 자가 증식을 증가시키는 방법

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CN112410286A (zh) * 2020-11-30 2021-02-26 河南省生殖健康科学技术研究院(河南省出生缺陷干预工程技术研究中心) 一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法及应用
CN114958730A (zh) * 2022-04-22 2022-08-30 南京农业大学 一种肌肉干细胞增殖培养基、分化培养基及其应用
WO2023085743A1 (fr) * 2021-11-09 2023-05-19 연세대학교 산학협력단 Procédé d'obtention de cellules pour la création de nft sur la base d'une image cellulaire

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CN112410286A (zh) * 2020-11-30 2021-02-26 河南省生殖健康科学技术研究院(河南省出生缺陷干预工程技术研究中心) 一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法及应用
CN112410286B (zh) * 2020-11-30 2023-10-27 河南省生殖健康科学技术研究院(河南省出生缺陷干预工程技术研究中心) 一种利用诱导多能干细胞构建妊娠期致流产药物筛查模型的方法及应用
WO2023085743A1 (fr) * 2021-11-09 2023-05-19 연세대학교 산학협력단 Procédé d'obtention de cellules pour la création de nft sur la base d'une image cellulaire
CN114958730A (zh) * 2022-04-22 2022-08-30 南京农业大学 一种肌肉干细胞增殖培养基、分化培养基及其应用

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