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WO2013015644A1 - Procédé pour la prolifération de cellules souches issues du placenta - Google Patents

Procédé pour la prolifération de cellules souches issues du placenta Download PDF

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Publication number
WO2013015644A1
WO2013015644A1 PCT/KR2012/006000 KR2012006000W WO2013015644A1 WO 2013015644 A1 WO2013015644 A1 WO 2013015644A1 KR 2012006000 W KR2012006000 W KR 2012006000W WO 2013015644 A1 WO2013015644 A1 WO 2013015644A1
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Prior art keywords
cells
stem cells
passage
placenta
conditions
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Korean (ko)
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문지숙
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Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
Chabio and Diostech Co Ltd
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Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
Chabio and Diostech Co Ltd
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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions

Definitions

  • the present invention relates to a method for propagation of placental-derived stem cells, and more specifically, to placenta-derived stem cells comprising subcultured under hypoxia conditions having an oxygen partial pressure of 1 to 8%. It relates to a method of proliferating stem cells.
  • stem cells such as embryonic stem cells and adult stem cells into various cells
  • various studies have been made on the possibility of application as cell therapy.
  • Embryonic stem cells with multipotency have been noted as cell therapy by differentiating into various cells, but due to ethical problems in utilizing embryonic stem cells, they have difficulty in practical use as cell therapy.
  • studies using adult stem cells have been actively conducted.
  • stem cells derived from bone marrow, adipose, umbilical cord blood, etc. are known.
  • Various studies have been conducted on how to differentiate these cells into specific cells.
  • adult stem cells have a low proliferation rate and are easily aged and have a limited number of cells that can be obtained from a tissue.
  • bone marrow-derived adult stem cells are known to have extremely limited differentiation into various tissues and are difficult to obtain a large number of cells (Matikainen T. and Laine J. Placenta-an alternative source of stem cells.Toxicology and Applied Pharmacology 2005; 207: S544-S549).
  • Other types of adult stem cells, umbilical cord blood or fat stem cells are relatively easy to obtain, but have limited differentiation capacity.
  • Placent-derived stem cells which are of recent interest, have the advantage that they do not require invasive procedures compared to other adult stem cells, but the number of cells from one placenta is also limited, and the number of cells can be expanded. Even if the subculture is carried out for risk, the period of proliferation of the cells is reduced, and thus the number of cells eventually obtained is intact.
  • adult stem cells have different factors on stem cell maintenance, aging mechanism, and proliferation, depending on the source.
  • differentiation may be induced or differentiation may be suppressed depending on the type of stem cells.
  • the types of cells differentiated and produced according to the types of stem cells may appear at all. For example, when preadipocytes are exposed to hormones that induce differentiation into adipocytes, they differentiate into adipocytes.However, in hypoxia conditions, progenitor cells are exposed to hormones that induce differentiation of adipocytes.
  • the present inventors conducted various studies to develop a method capable of proliferating at high proliferation rate while maintaining stem stemity of placental-derived adult stem cells and also inhibiting aging as much as possible.
  • the differentiation of placental-derived adult stem cells is not only suppressed but also high proliferation rate. It was found that can be achieved. This is surprising given the reports that many stem cells disclose that differentiation is induced under hypoxic conditions.
  • an object of the present invention is to provide a method for propagation of placental-derived stem cells comprising culturing placental-derived adult stem cells under hypoxic conditions having an oxygen partial pressure of 1 to 8%.
  • a method of proliferating placental-derived stem cells which comprises subcultured placental-derived stem cells under low oxygen conditions having an oxygen partial pressure of 1 to 8%.
  • the placental-derived stem cells are derived from amniotic mesenchymal stem cells derived from amnion of human placenta or isolated human placenta.
  • Amniotic epithelial cells can be preferably used.
  • the passaging may be preferably performed up to 10 to 15 passages.
  • the subculture is performed in alpha-MEM medium to which fetal bovine serum, antibiotic, heparin, and fibroblast growth factor-4 are added.
  • the subculture is alpha-MEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug / ml heparin, 25 ng / ml fibroblast growth factor-4. It can be carried out in the.
  • the proliferation method according to the present invention can achieve a significantly higher proliferation rate compared to normoxia conditions. Therefore, the proliferation method of placental-derived stem cells according to the present invention can shorten the culture rate of the cells, thereby enabling mass production of placental-derived stem cells.
  • FIG. 1A and 1B show passage of mesenchymal stem cells (hAMSC) derived from amnion of human placenta (FIG. 1A) and amnion epithelial cells (hAEC) of human placenta (FIG. 1B), respectively, under normal oxygen and hypoxic conditions.
  • hAMSC mesenchymal stem cells
  • FIG. 2A to 2C show the cumulative number of cells obtained when subcultured hAMSC under normal oxygen conditions and low oxygen conditions (FIG. 2A), the ratio of cell numbers under low oxygen conditions to normal oxygen conditions of the number of accumulated cells obtained. (FIG. 2B) and the results of measuring the doubling time of the cells (FIG. 2C).
  • FIG. 3A to 3C show the cumulative number of cells obtained when passage of hAEC under normal oxygen conditions and low oxygen conditions (FIG. 3A), the ratio of cell numbers under low oxygen conditions to normal oxygen conditions of the number of accumulated cells obtained. (FIG. 3B), and the result of measuring the Population doubling Level (PDL) (FIG. 3C).
  • 4A to 4F show the results of analyzing surface antigens of proliferated cells using a flow cytometer when the hAMSCs were passaged under normal oxygen conditions and low oxygen conditions.
  • 5A to 5H show the results of analyzing surface antigens of proliferated cells using a flow cytometer when hAEC was passaged under normal oxygen conditions and low oxygen conditions.
  • FIG. 6A and 6B show the results of staining analysis of stem cell markers (FIG. 6A) and proliferation markers (FIG. 6B) of proliferated cells when hAMSCs were passaged under normal oxygen and hypoxic conditions.
  • FIG. 7A and 7B show the results of staining analysis of stem cell markers (FIG. 7A) and proliferation markers (FIG. 7B) of proliferated cells when hAEC was passaged under normal oxygen and hypoxic conditions.
  • FIG. 8 shows the expression of Oct4, Sox2, nanog, c-Myc, KLF4 using reverse transcription polymerase chain reaction (RT-PCR) for harvested cells when hAMSC was passaged under normal oxygen and hypoxic conditions.
  • RT-PCR reverse transcription polymerase chain reaction
  • placenta-derived stem cells includes all stem cells isolated from the placenta, and preferably, four types of stem cells isolated from the human placenta separated in vitro. That is, (1) human amniotic epithelial cells (hAEC), (2) amniotic mesenchymal stromal cells or human amniotic mesenchymal stem cells (hAMSC), and 3) chorionic mesenchymal stem cells. Cells (human chorionic mesenchymal stromal cells or human chorionic mesenchymal stem cells (hCMSC)), and (4) human chorionic trophoblastic cells (hCTC).
  • hAEC human amniotic epithelial cells
  • hAMSC human amniotic mesenchymal stem cells
  • hCMSC human chorionic mesenchymal stem cells
  • hCTC human chorionic trophoblastic cells
  • the placental-derived stem cells may be mesenchymal stem cells (hAMSC) derived from the amnion of the human placenta separated in vitro or amniotic epithelial cells (hAEC) of the human placenta separated in vitro.
  • hAMSC mesenchymal stem cells
  • hAEC amniotic epithelial cells
  • the placental-derived stem cells can be obtained by known methods (eg, Current protocols in Stem Cell Biology 1E.3.1-1E.3.10 and 1E.5.1-1E5.11).
  • the present invention provides a method for propagating placental-derived stem cells, comprising subcultured placenta-derived stem cells under low oxygen conditions having an oxygen partial pressure of 1 to 8%.
  • placenta-derived adult stem cells are passaged under hypoxia conditions as described above, stem cell differentiation can be suppressed to maintain stem cellity and to suppress aging as much as possible.
  • the passage number of the passage is not particularly limited, and the passage number may be appropriately selected according to the number of desired proliferating cells. Typically, at least 5 passages, preferably 10 passages or more, more preferably 10 to 15 passages, can obtain a clinically necessary number of cumulative proliferating cells.
  • the growth media used in the method for propagating placental-derived stem cells according to the present invention are not particularly limited, and preferably, fetal bovine serum, antibiotics, heparin, and fibroblast growth factor-4 -4) can be performed in the added alpha-MEM medium.
  • the subculture may be performed in alpha-MEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug / ml heparin, and 25 ng / ml fibroblast growth factor-4. have.
  • hAMSCs human amniotic mesenchymal stem cells
  • hAMSCs Mesenchymal stem cells obtained from the amniotic membrane of human placenta were subjected to normal oxygen conditions (ie, 37 ° C, 5% CO 2 culture conditions and hypoxia conditions, 37 ° C, 5% CO 2 , 1 to 3). The cells were cultured in a culture condition of 8% O 2 . Expansion media include 10% fetal bovine serum, 1% penicillin-streptomycin, 1ug / ml heparin, 25ng / ml fibroblast growth factor-4 (FGF4). Added alpha-MEM medium was used. hAMSCs were counted and inoculated with 6000-7000 cells per cm 2 and cultured with medium replacement every other day.
  • normal oxygen conditions ie, 37 ° C, 5% CO 2 culture conditions and hypoxia conditions, 37 ° C, 5% CO 2 , 1 to 3
  • the cells were cultured in a culture condition of 8% O 2 .
  • Expansion media include 10% fetal bo
  • More than 80% of the cells were grown (confluent) in the flask for about 3 to 4 days after incubation.
  • the cultured cells were washed with phosphate-buffered saline (PBS) and then washed for 2 minutes using 0.25% trypsin / EDTA.
  • PBS phosphate-buffered saline
  • fetal bovine serum was added to stop the enzyme reaction, centrifuged at 1000 rpm for about 5 minutes, and then the supernatant was removed to harvest the cells.
  • Harvested cells were again counted to 6000-7000 cells per cm 2 and subjected to the same method of subculture.
  • hAECs human amniotic epithelial cells
  • hAECs Human placenta amnion epithelial cells
  • Test Example 1 Comparison of Stem Cells Cultured in Normal Oxygen and Hypoxic Conditions
  • Example 1 (1) the results of observing the passaged hAMSCs (first passage, fifth passage, and tenth passage) under normal oxygen conditions and low oxygen conditions, respectively, with an optical microscope are shown in FIG. 1A. From the results of FIG. 1A, it can be seen that cells cultured under normal oxygen conditions and low oxygen conditions were passaged while maintaining the morphology of mesenchymal cells.
  • FIG. 1B is an optical microscope of hAECs cultured to the 0th passage, the 1st passage, the 5th passage, the 10th passage, and the 15th passage, respectively, in the normal oxygen condition and the low oxygen condition in Example 1 (2). The result is.
  • the results of cumulative counting of the proliferated cells are shown in FIG. 2A.
  • the results showing the number of cells proliferated in each passage (the number of accumulated cells) as the ratio of cell numbers of hypoxic conditions to normal oxygen conditions is shown in Figure 2b.
  • the number of cells proliferated in the normal oxygen condition was only about 1.6x10 13 , but the number of cells proliferated in the low oxygen condition was about 5x10 15 cells.
  • the cumulative number of cells showed a proliferation rate of about 6000 times higher than that in the normal oxygen condition.
  • the level of doubling of cells under each condition was measured, and the sum of doubling levels up to the 11th passage was normal oxygen condition of 20 and low oxygen condition of 29.5. At all times, the doubling levels of hypoxic conditions remained high. Particularly, the third passage showed the highest value of 3.5, the low oxygen condition of the 11th passage was low value of 1.5, and the normal condition rapidly decreased after the 9th passage, and cell growth could be stopped. Particularly, in the eleventh passage, it can be estimated that the cell death starts by recording a value of -0.5 (see FIG. 2C). From the above results, it can be seen that the hypoxic culture conditions increase the proliferation rate of the cells and also delay the aging of the cells.
  • the number of cells proliferated under normal oxygen conditions was only about 6x10 11 , but the number of cells proliferated under low oxygen conditions was about 1.3 x 10 15 , and the cumulative number of cells was
  • the culture rate in the hypoxic condition showed a growth rate of about 2000 times or more than that in the normal oxygen condition.
  • the sum of the doubling levels up to the 10th passage was 10.8 normal oxygen condition, 21.9 hypoxic condition and approximately 2 times higher hypoxic condition.
  • doubling levels of hypoxic conditions remained high.
  • the doubled level (PDL) level was below 1.5, while in low oxygen conditions, it gradually increased to 2 or more after the second pass, and reached the maximum value of 4 in the fourth pass, and 2 to the 9th pass. It stayed above. From the above results, as in hAMSCs, it can be seen that when hAEC is cultured under low oxygen, cell proliferation is increased and cell aging is delayed.
  • Example 1 the surface antigens of cells harvested by subcultured hAMSCs to the first passage, the fifth passage, and the tenth passage under normal oxygen conditions and low oxygen conditions were analyzed. That is, each harvested cells were washed with a mixture of 5% FBS and PBS, and then centrifuged at 1000 rpm for 5 minutes. After the supernatant was discarded, the cells were suspended in FACS buffer and 10000 cells were divided by the number of samples. In each well, add antibodies (FITC-labeled anti-SSEA4, anti-TRA-1-81, anti-CD34 and PE-labeled anti-TRA-1-60, anti-CD9, anti-CD44), respectively.
  • FITC-labeled anti-SSEA4, anti-TRA-1-81, anti-CD34 and PE-labeled anti-TRA-1-60, anti-CD9, anti-CD44 antibodies
  • FIGS. 4A to 4F show that the MSC marker CD44 was maintained up to the 10th passage in cells cultured under normal oxygen conditions and low oxygen conditions, indicating that the characteristics of the mesenchymal cells were not changed.
  • the stem cell marker SSEA4 decreased with passage, but the decrease was lower in the cells cultured in hypoxic conditions than in cells cultured in normal oxygen conditions.
  • Example 1 (2) the surface of cells harvested by subcultured hAEC to 0th passage, 1st passage, 5th passage, 10th passage, and 15th passage under normal oxygen conditions and low oxygen conditions, respectively Antigen was analyzed in the same manner as above.
  • SSEA4, TRA-1-60, and TRA-1-81 as a stem cell marker
  • CD9 as a nontropoblast marker
  • CD34 as a hematopoetic stem cell marker
  • CD15 as a NPC marker
  • CD 133, And CD 184, CD 44 and CD90 as MSC markers
  • Example 1 staining analysis of marker proteins of cells harvested by subcultured hAMSCs to the first passage, the fifth passage, and the tenth passage under normal oxygen conditions and low oxygen conditions, respectively, was performed. That is, each harvested cell was washed three times with PBS and fixed for 10 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, and treated with a blocking buffer (5% goat serum) containing 0.3% Triton-X100 for 1 hour at room temperature, the stem cell markers Oct4 and Sox2 in blocking buffer 1 Dilute to 400 and react overnight at 4 ° C. Washed three times with PBS, and reacted with secondary antibodies Alexa Fluor TM 488 and Alexa Fluor TM 594 in the dark for 1 hour.
  • a blocking buffer 5% goat serum
  • Example 1 (2) the markers of cells harvested by subcultured hAEC to 0th passage, 1st passage, 5th passage, 10th passage, and 15th passage under normal oxygen conditions and low oxygen conditions, respectively was analyzed in the same manner as above. From the results of FIGS. 7A and 7B, as in hAMSCs, Oct4 was not expressed in both normal oxygen and hypoxic conditions, but Sox2 was expressed, and the expression level of Ki-67, a proliferation marker, was also high in cells cultured in hypoxic conditions. It can be seen that the proliferation rate is significantly high.
  • RT-PCR Reverse transcription polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction

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Abstract

La présente invention concerne un procédé pour la prolifération de cellules souches issues du placenta qui comprend la sous-culture de cellules souches issues du placenta dans des conditions faibles en oxygène ayant une pression partielle en oxygène de 1-8 %. Le procédé pour la prolifération des cellules souches issues du placenta selon la présente invention peut maintenir le caractère souche des cellules souches par l'inhibition de la différentiation de celles-ci, peut inhiber le vieillissement de celles-ci autant que possible et, en particulier, peut permettre un taux significativement élevé de prolifération.
PCT/KR2012/006000 2011-07-28 2012-07-27 Procédé pour la prolifération de cellules souches issues du placenta Ceased WO2013015644A1 (fr)

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KR10-2011-0075079 2011-07-28
KR1020110075079A KR20130013435A (ko) 2011-07-28 2011-07-28 태반-유래 줄기세포의 증식방법

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KR20150083439A (ko) * 2014-01-08 2015-07-17 사회복지법인 삼성생명공익재단 영양막 기저층으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제
KR20150083440A (ko) * 2014-01-08 2015-07-17 사회복지법인 삼성생명공익재단 순수 영양막층으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제
WO2020260431A1 (fr) 2019-06-28 2020-12-30 F. Hoffmann-La Roche Ag Procédé de production d'anticorps

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CN104382827A (zh) * 2014-11-28 2015-03-04 广州赛莱拉干细胞科技股份有限公司 人羊膜间充质干细胞外泌体的用途
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Cited By (7)

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KR20150083439A (ko) * 2014-01-08 2015-07-17 사회복지법인 삼성생명공익재단 영양막 기저층으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제
KR20150083440A (ko) * 2014-01-08 2015-07-17 사회복지법인 삼성생명공익재단 순수 영양막층으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제
KR20160036031A (ko) * 2014-01-08 2016-04-01 사회복지법인 삼성생명공익재단 연골 재생용 세포 치료제
KR101669009B1 (ko) * 2014-01-08 2016-10-26 사회복지법인 삼성생명공익재단 순수 영양막층으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제
KR101669038B1 (ko) * 2014-01-08 2016-10-26 사회복지법인 삼성생명공익재단 영양막 기저층으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제
KR101697141B1 (ko) * 2014-01-08 2017-01-17 사회복지법인 삼성생명공익재단 연골 재생용 세포 치료제
WO2020260431A1 (fr) 2019-06-28 2020-12-30 F. Hoffmann-La Roche Ag Procédé de production d'anticorps

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