WO2013137567A1 - Méthode de différenciation de cellules souches pluripotentes d'origine humaine en progéniteurs sanguins, en progéniteurs de l'endothélium vasculaire, en cellules endothéliales et en cellules musculaires lisses avec du sélénium - Google Patents

Méthode de différenciation de cellules souches pluripotentes d'origine humaine en progéniteurs sanguins, en progéniteurs de l'endothélium vasculaire, en cellules endothéliales et en cellules musculaires lisses avec du sélénium Download PDF

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WO2013137567A1
WO2013137567A1 PCT/KR2013/001403 KR2013001403W WO2013137567A1 WO 2013137567 A1 WO2013137567 A1 WO 2013137567A1 KR 2013001403 W KR2013001403 W KR 2013001403W WO 2013137567 A1 WO2013137567 A1 WO 2013137567A1
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cells
stem cells
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서원희
송선화
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아주대학교 산학협력단
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    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to a method for differentiating human pluripotent stem cells using selenium, and specifically, to a method for effectively differentiating human pluripotent stem cells into blood progenitor cells, vascular progenitor cells, vascular endothelial cells, and vascular smooth muscle cells.
  • Stem cells are cells that can be differentiated into various cells constituting biological tissues, which collectively refer to undifferentiated cells obtained from each tissue of embryo, fetus and adult. Stem cells are differentiated into specific cells by differentiation stimulation (environment), and unlike the cells in which cell division is stopped due to differentiation, proliferation is possible due to cell division (self-renewal). (Proliferation; expansion) is characterized by being able to differentiate into other cells by different environments or different differentiation stimulation, which is characterized by having plasticity (differentiation).
  • stem cells can be classified in various ways. One of the most commonly used methods is according to an individual in which stem cells are separated. Embryonic stem cells (ES cells) isolated from embryos and adult stem cells isolated from adults Can be divided into Another common classification is according to the differentiation capacity of stem cells, which can be divided into pluripotency, multipotency and unipotency stem cells. Pluripotent stem cells are pluripotency cells that have the potential to differentiate into all cells. Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPSCs). ), Embryonic germ cells (EGC), embryonic tumor cells (embryonic carcinoma cells, ECC) and the like. Multipotent and / or unipotent stem cells include adult stem cells.
  • ES cells Embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • ECC embryonic tumor cells
  • ECC embryonic tumor cells
  • Multipotent and / or unipotent stem cells include adult stem cells.
  • Embryonic stem cells are formed from the inner cell mass of the blastocytes, which are the early stages of embryonic development, and have the potential to differentiate into all cells, which can differentiate into any tissue cell and also do not die. It can be cultured in undifferentiated state, and unlike adult stem cells, it is also possible to produce germ cells, so it can be inherited to the next generation (Thomson et al., Science, 282: 1145-1147, 1998; Reubinoff et al., Nat. Biotechnol., 18: 399-404, 2000). Human embryonic stem cells are prepared by separating and culturing only inner cell mass when forming human embryos. Currently, human embryonic stem cells made worldwide are obtained from frozen embryos remaining after infertility.
  • iPSCs are included as a concept of pluripotent stem cells.
  • Induced pluripotent stem cells are cells which have redifferentiated differentiated adult cells in various ways and returned to embryonic stem cells, which are the early stages of differentiation.
  • dedifferentiated cells have been reported to exhibit almost the same characteristics as embryonic stem cells, pluripotent stem cells, in gene expression and differentiation capacity.
  • human pluripotent stem cells have a pluripotency capable of differentiating into almost all somatic cells with self-renewal to constantly proliferate and maintain a constant number.
  • the research is expected to bring radical advances in various academic fields such as embryology, regenerative medicine, and new drug development, and differentiates human pluripotent stem cells into desired cells, which can be used in various intractable patients such as diabetes, neurological diseases, and cardiovascular diseases. It is very useful in that it can be used as a therapeutic agent.
  • an increasing number of people with ischemic cardiovascular disease and cell therapy using adult stem cells have been studied for patients who are inadequate to existing surgery or drug treatment, but have difficulty in securing a sufficient number of adult stem cells. Therefore, efforts are being actively made to differentiate human pluripotent stem cells, which have superior self-proliferation and differentiation ability to adult stem cells, into vascular endothelial cells and utilize them for treatment.
  • human pluripotent stem cells In order to use human pluripotent stem cells as a cardiovascular cell therapy, it is necessary to secure a sufficient number of differentiated cells necessary for transplantation.In order to do this, human pluripotent stem cells can be effectively induced into vascular progenitor cells or vascular endothelial cells and smooth muscle cells. Skill is essential.
  • human embryonic stem cells can be differentiated into vascular endothelial cells using coculture with mouse-derived stromal cells, or using fetal bovine serum (FBS) or human recombinant growth factor / cytokines. It has been reported how. Among them, differentiation using co-culture with mouse bone marrow stromal matrix was performed by co-culture with human embryonic stem cells and 10% FBS culture using mouse bone marrow-derived stromal cells as a feeder layer. As a separate method, isolated KDR-positive cells can be further differentiated in culture containing 10% FBS and vascular endothelial growth factor A (VEGF) to obtain vascular endothelial cells (Pathway for differentiation of human embryonic stem cells to vascular cell components).
  • FBS fetal bovine serum
  • VEGF vascular endothelial growth factor A
  • MIT's Robert Langer team reported how to induce differentiation of human embryonic stem cells into vascular cells using bovine serum.
  • the method produces a three-dimensional embryonic body (EB) of human embryonic stem cells, incubates the embryonic body in a culture medium containing 20% FBS for 12 days, and isolates CD34 positive cells using a flow cytometer.
  • EB embryonic body
  • CD31 positive vascular endothelial cells were obtained by incubating for another 10 to 15 days in EGM-2 (endothelial growth medium-2) to which 5% FBS was added (Vascular Progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle-like cells and form vascular networks in vivo.Ferreira LS, Gerecht S, Shieh HF, Watson N, Rupnick MA, Dallabrida SM, Vunjak-Novakovic G, Langer R. Circulation Research 2007 101: 286-94).
  • the conventional method of differentiating human embryonic stem cells into vascular cells uses co-culture with bovine serum or animal-derived cells, and thus in the clinical application of vascular endothelial cells differentiated from human embryonic stem cells.
  • human recombinant growth factor / cytokines required for differentiation into vascular cells in particular human recombinant vascular endothelial growth factor A, human recombinant bone morphogenic protein-4, human Using serum-free and xeno-free media containing recombinant recombinant fibroblast growth factor (bFGF), or human recombinant Activin A
  • bFGF recombinant fibroblast growth factor
  • the present inventors conducted various studies to develop a technique for improving the efficiency of vascular endothelial cell differentiation method that does not use serum and animal-derived materials or animal-derived cells, and as a result selenium Using human recombinant growth factor / cytokine medium comprising the human pluripotent stem cells were found to be able to differentiate into high efficiency blood progenitors, vascular progenitors, vascular endothelial and vascular smooth muscle cells and came to complete the present invention.
  • the present invention selectively differentiates human pluripotent stem cells into mesoderm using selenium-added human recombinant growth factor / cytokine differentiation medium to increase the differentiation efficiency into blood and vascular progenitor cells or vascular endothelial and smooth muscle cells. It is an object to provide a method.
  • the present invention provides a method for differentiating human pluripotent stem cells into mesodermal stem cells using a medium containing selenium (selenium).
  • Human pluripotent stem cells used in the present invention include human embryonic stem cells, human induced pluripotent stem cells, embryonic germ cells, and embryonic tumor cells.
  • the mesoderm stem cells may be differentiated into blood progenitor cells, vascular progenitor cells, vascular endothelial cells, and vascular smooth muscle cells.
  • the medium may further contain human recombinant growth factor or cytokine.
  • the human recombinant growth factor or cytokine used in the present invention is not limited, but may include human recombinant BMP-4, human recombinant bFGF, and / or human recombinant VEGF.
  • the differentiation medium containing selenium When using the differentiation medium containing selenium according to the method of the present invention, there is an effect that can selectively differentiate human pluripotent stem cells into mesodermal stem cells, ultimately blood precursor cells, vascular precursor cells, vascular endothelial cells with high efficiency Cells, vascular smooth muscle cells and the like.
  • FIG. 1 is a graph illustrating the gene expression patterns of cells in the selenium-treated group (white) and the treated group (black: selenium 20 ng / ml, gray: selenium 50 ng / ml) during differentiation of human embryonic stem cells.
  • A undifferentiated human embryonic stem cell marker gene
  • B mesoderm specific marker gene
  • C endoderm specific marker gene
  • D ectoderm specific marker gene
  • Figure 2 shows the results of immunofluorescence staining of the cell differentiation of the selenium-treated group and the treated group (50 ng / ml) during the differentiation of human embryonic stem cells, 9 and 15 days after differentiation, respectively.
  • Cell differentiation into mesoderm, endoderm, and ectoderm are shown (CD34: mesoderm marker, AFP (a-fetoprotein): endoderm marker, b III-tubulin: ectoderm marker).
  • Figure 3 shows the gene expression patterns of cells in the selenium-free group (white) and the treated group (black: selenium 20 ng / ml, gray: selenium 50 ng / ml) during the differentiation of human embryonic stem cells
  • EC endothelial cell
  • SMC smooth muscle cell
  • Figure 4 is an immunofluorescence staining showing the differentiation of vascular endothelial cells and vascular smooth muscle cells in the group treated with selenium (50 ng / ml) during the differentiation process of human embryonic stem cells (vascular endothelial cell specific markers : CD31, VE-cad, vascular smooth muscle cell specific marker: SMA).
  • FIG. 5 is a flow cytometry analysis of the differentiation of human embryonic stem cells into vascular precursors and vascular endothelial cells in the selenium-treated group and the treated group (20 or 50 ng / ml) during the differentiation of human embryonic stem cells.
  • CD31 vascular endothelial cell specific marker
  • CD34 vascular progenitor cell specific marker
  • the term 'stem cells' refers to master cells that can be regenerated without limitation to form specialized cells of tissues and organs. Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter cells, or one daughter cell and one derived ('transit') cell, and then proliferate into mature, fully formed cells of the tissue.
  • the term 'pluripotent stem cell' refers to a stem having pluripotency capable of differentiating into three germ layers constituting a living body, that is, endoderm, mesoderm, and ectoderm. It refers to cells, and generally corresponds to embryonic stem cells, induced pluripotent stem cells, embryonic germ cells, embryonic tumor cells.
  • the term 'embryonic stem cell' is a cell cultured by separating and cultured from an inner cell mass (inner cell mass) of the blastocyst, which is an early development after fertilization.
  • the term 'adult cell' refers to a cell derived from an adult that is born and alive as opposed to an embryonic cell.
  • the term 'induced pluripotent stem cell (iPSC)' is a cell induced by artificially dedifferentiating (reprogramming) the already differentiated adult cells to the pluripotency (pluripotency)
  • iPSC induced pluripotent stem cell
  • the term 'embryonic germ cell' used in the present invention refers to a cell derived from primordial germ cells occurring in the gonadal ridge region of the fetus and has characteristics similar to embryonic stem cells but embryonic stem. It has weaker differentiation and division ability than cells.
  • the term 'embryonic tumor cell' used in the present invention is an undifferentiated stem cell established from teratocarcinomas formed by the tumorization of primordial germ cells, and the main material for multipotent research before embryonic stem cells are established. Was used. Embryonic tumor cells, although having problems such as chromosomal abnormalities, have pluripotency.
  • the term "differentiation” refers to a phenomenon in which structures or functions are specialized to each other during cell division and proliferation. It means to change.
  • a relatively simple system is divided into two or more qualitatively different sub systems. For example, qualitatively between parts of a living organism that were almost homogeneous in the first place, such as head or torso distinctions between eggs that were initially homogenous in the development, or cells such as muscle cells or neurons.
  • Phosphorus difference or as a result, is a state divided into subclasses or subclasses that can be distinguished qualitatively.
  • the term 'cell therapeutic agent' is a cell and tissue prepared by separating, culturing and special manipulation from a human being and used as a medicine used for the purpose of treatment, diagnosis, and prevention, and to restore the function of the cell or tissue. Or a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise altering a cell's biological properties in vitro.
  • Cell therapy agents are classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells.
  • Human embryonic stem cells (H9, WiCell, USA) in undifferentiated state were removed using a cell detachment solution (ReproCell, Japan) according to a known method (Takahashi K et al. Cell (2007) 131 , 861-872). Formed.
  • the embryoid body obtained from the above was human recombinant BMP-4 (10 ng / ml, Prospec), human recombinant bFGF (5 ng / ml, Prospec), human recombinant VEGF (10 ng / ml, R & D), sodium selenite (20-50).
  • DMEM / F12 Gibco, composition: http://www.atcc.org/Portals/1/Pdf/30-
  • 10% serum replacement Knockout TM Serum Replacement xenofree, Gibco
  • the embryonic body differentiated in step I was transferred to a culture plate using a pipette, attached to a plate bottom, and then human recombinant BMP-4 (20 ng / ml, Prospec) and human recombinant bFGF (5 ng / ml , Prospec), endothelial cell Basal Medium-2 (Lonza) supplemented with human recombinant VEGF (50 ng / ml, R & D), sodium selenite (20-50 ng / ml, Sigma) Ie, 20% oxygen concentration) for an additional 7 days.
  • human recombinant BMP-4 (20 ng / ml, Prospec
  • human recombinant bFGF (5 ng / ml , Prospec)
  • endothelial cell Basal Medium-2 Longza
  • human recombinant VEGF 50 ng / ml, R & D
  • sodium selenite (20-50 ng / ml, Sigma
  • Cells were harvested after 8 and 15 days of differentiation from Example 1, and the differentiation patterns into ectoderm, endoderm, and mesoderm were examined by real-time RT-PCR.
  • RNA of the cell was extracted using Trizol (Invitrogen), and expression of genes was observed using Real-time PCR.
  • 1 ⁇ g of RNA was first synthesized into cDNA using a Superscript first-strand synthesis system (Invitrogen), and the amplified gene expression was amplified using the primers and real time PCR SYBR-Green PCR master mix (Applied Biosystems) of each gene. Measured by Step One PlusTM Real-time PCR system (Applied Biosystems), the results are shown in FIG.
  • Oct4 and Nanog genes which are markers of undifferentiated stem cells, was significantly reduced during differentiation compared to undifferentiated human embryonic stem cells (H9), whereas ectoderm (Pax6, Nestin), Mesoderm (Brachyury, Mesp1, CD34), endoderm (Sox17, Gata6) It can be seen that the expression of various genes are increased. In particular, it can be seen that the expression of mesoderm marker genes is significantly increased when using a medium containing selenium during differentiation compared to ectoderm and endoderm.
  • the differentiated cells obtained from Example 1 were fixed in 4% paraformaldehyde for 10 minutes and reacted for 10 minutes at 0.5% Triton X-100.
  • PBS phosphate buffered saline
  • the antibody was reacted at 4 ° C. for 24 hours.
  • the resultant was washed with PBS, and reacted with fluorescent labeled secondary antibody at room temperature for about 2 hours, followed by washing with PBS.
  • the nuclei were stained with 4,6-diamino-2-phenylindole (DAPI) and observed using a Nikon ECLIPSE Ti-U inverted microscope (Nikon Instruments Inc.).
  • DAPI 4,6-diamino-2-phenylindole
  • CD34 blood and vascular precursor cell-specific marker
  • AFP endodermal cell marker
  • bIII-tubulin ectoderm cell marker
  • Example 4 Confirmation of gene expression promotion of pluripotent endothelial and vascular smooth muscle cells of human pluripotent stem cells by selenium treatment
  • Example 2 Cells after 8 and 15 days of differentiation obtained from Example 1 were collected and examined for differentiation into vascular endothelial cells and vascular smooth muscle cells through real-time RT-PCR. RT-PCR method was performed in the same manner as in Example 2.
  • PECAM CD31
  • VE-cadherin genes which are vascular endothelial cell-specific markers, as well as specific markers of vascular smooth muscle cells during differentiation compared to undifferentiated human embryonic stem cells (H9) It can be seen that the expression of SMA and Myocardin genes are also increased.
  • Example 5 Confirmation of the selective differentiation of human pluripotent stem cells into vascular endothelial and vascular smooth muscle cells by selenium treatment
  • CD31 and VE-cadherin vascular endothelial specific marker
  • SMA vascular smooth muscle cell specific marker
  • selenium promotes the selective differentiation of human pluripotent stem cells into vascular endothelial and vascular smooth muscle cells.
  • the cells were separated by treatment with Accutase (Innovative cell technologies, Inc.) for 15 minutes, and then reacted with an antibody (anti-human CD31, CD34 antibody) having a complex of fluorescent materials (anti-human CD31, CD34 antibody) at 4 ° C. for 1 hour, followed by PBS (phosphate buffered). saline).
  • the number of CD34 positive cells, blood and vascular progenitor cell-specific markers in the cells on the 15th day of differentiation of 3.6% of the total increases by about 5% by selenium treatment.
  • the excellent efficient differentiation induction technology of the present invention is essential for practical application of human pluripotent stem cells as a cardiovascular disease cell therapy, and the present invention is expected to contribute greatly to the practical use of cardiovascular cell therapy.
  • selenium promotes the differentiation of hematopoietic progenitor cells, CD34-positive cells, into the treatment of malignant diseases such as immunodeficiency diseases, aplastic anemia, hemoglobin disease, myeloid leukemia, and malignant lymphoma that can be treated by hematopoietic stem cell transplantation. It is expected to contribute greatly.

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Abstract

Cette invention concerne une méthode permettant d'augmenter l'efficacité de la différenciation de cellules souches pluripotentes d'origine humaine en progéniteurs sanguins, en progéniteurs de l'endothélium vasculaire, en cellules vasculaires endothéliales et en cellules vasculaires de muscle lisse. Plus particulièrement, l'invention concerne une méthode de différenciation de cellules souches pluripotentes d'origine humaine utilisant un milieu contenant du sélénium. Cette invention peut permettre à des cellules souches pluripotentes d'origine humaine de se différencier de manière sélective en cellules souches du mésenchyme, et l'invention présente l'effet remarquable de différenciation de cellules souches pluripotentes d'origine humaine en progéniteurs sanguins, en progéniteurs de l'endothélium vasculaire, en cellules vasculaires endothéliales et en cellules vasculaires de muscle lisse avec une grande efficacité. L'invention peut donc contribuer au développement d'une thérapie cellulaire pour les maladies cardiovasculaires ou d'une thérapie cellulaire pour la dyscrasie sanguine utilisant des cellules souches pluripotentes d'origine humaine.
PCT/KR2013/001403 2012-03-16 2013-02-21 Méthode de différenciation de cellules souches pluripotentes d'origine humaine en progéniteurs sanguins, en progéniteurs de l'endothélium vasculaire, en cellules endothéliales et en cellules musculaires lisses avec du sélénium WO2013137567A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014200340A1 (fr) * 2013-06-10 2014-12-18 Academisch Ziekenhuis Leiden Différentiation et expansion de cellules endothéliales à partir de cellules souches pluripotentes et formation in vitro de structures de type vasculaire

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101656396B1 (ko) * 2014-04-21 2016-09-09 한국생명공학연구원 Oct4 및 Sox2를 이용한 인간 치수강세포의 리프로그래밍 방법 및 이의 용도
KR102676444B1 (ko) 2022-01-28 2024-06-18 중앙대학교 산학협력단 Rec8 유전자의 과발현을 통한 배아줄기세포 분화 효율 증진 기술

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011100286A2 (fr) 2010-02-09 2011-08-18 The Johns Hopkins University Compositions et méthodes de production d'une cellule mésodermique différenciée

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CONLEY, B.J. ET AL.: "Derivation propagation and differentiation of human embryonic stem cells", THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, vol. 36, 2004, pages 555 - 567 *
GUAN, K. ET AL.: "Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro", CYTOTECHNOLOGY, vol. 30, 1999, pages 211 - 226, XP019236607, DOI: doi:10.1023/A:1008041420166 *
PEIFFER, 1. ET AL.: "Simultaneous differentiation of endothelial and trophoblastic cells derived from human embryonic stem cells", STEM CELLS AND DEVELOPMENT, vol. 16, 2007, pages 393 - 401 *
ZAMBIDIS, E.T. ET AL.: "Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development", BLOOD, vol. 106, 1 August 2005 (2005-08-01), pages 860 - 870 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014200340A1 (fr) * 2013-06-10 2014-12-18 Academisch Ziekenhuis Leiden Différentiation et expansion de cellules endothéliales à partir de cellules souches pluripotentes et formation in vitro de structures de type vasculaire
US10119122B2 (en) 2013-06-10 2018-11-06 Academisch Ziekenhuis Leiden Differentiation and expansion of endothelial cells from pluripotent stem cells and the in vitro formation of vasculature like structures

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