WO2008056963A1 - Procédé permettant la prolifération de cellules souches au moyen de leptine - Google Patents

Procédé permettant la prolifération de cellules souches au moyen de leptine Download PDF

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Publication number
WO2008056963A1
WO2008056963A1 PCT/KR2007/005670 KR2007005670W WO2008056963A1 WO 2008056963 A1 WO2008056963 A1 WO 2008056963A1 KR 2007005670 W KR2007005670 W KR 2007005670W WO 2008056963 A1 WO2008056963 A1 WO 2008056963A1
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Prior art keywords
stem cells
leptin
cells
cell
culture medium
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PCT/KR2007/005670
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English (en)
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Chanil Moon
Hyunwon Yang
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Chanil Moon
Hyunwon Yang
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Publication of WO2008056963A1 publication Critical patent/WO2008056963A1/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
    • C12N5/0607—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
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/355—Leptin

Definitions

  • the present invention relates, in general, to a method for proliferating stem cells with leptin and, more specifically, to a method for proliferating various bone marrow- or blood-derived stem cells to a great extent in a culture medium supplemented with leptin without an increase in the number of subcultures
  • a stem cell is an undifferentiated somatic cell that is capable either of division to give rise to daughter stem cells, or of differentiating into any specialized cell type given the appropriate signals.
  • In vitro culturing and differentiation of stem cells affords specific cells useful in the treatment of various diseases (Petit-Zeman S., Regenerative medicine. Nat. Biotechnol. 19: 201-206, 2001) .
  • organ transplantation was developed as a predominant promising therapy for replacing recipients' damaged or failing organs with working ones from donors .
  • organ transplantation is problematic in that organs are in very short supply and donors as well as recipients must undergo surgery.
  • stem cell therapy does not suffer from these problems, and can be effectively administered merely by injecting specific cells.
  • stem cells can grow and be transformed into specialized cells having characteristics consistent with cells of various tissues, their use in therapy has been proposed for various incurable diseases.
  • patients suffering from diabetes must be administered with insulin daily or must have the pancreas transplanted from brain-dead patients.
  • Stem cell therapy can be used to treat diabetes simply by injecting stem cells, which then differentiate into insulin-secreting pancreatic cells.
  • Effective medical treatment is also anticipated for Parkinson' s disease by injecting stem cells, which then differentiate into dopamine-secreting cerebral. cells, and for myocardial infarction, merely by injecting stem cells, which differentiate into cardiomyocytes . Further, this medical technology is applicable to cancer treatment.
  • Stem cells can be used for the development of cellular agents involved in apoptosis of cancer.
  • stem cells are administered to reconstruct tissues or organs which to replace the excised tissues or organs in which cancer occurred.
  • tissue engineering which has recently progressed greatly, allows the development of artificial organs (Petit-Zeman S., Regenerative medicine. Nat. Biotechnol. 19: 201-206, 2001, Gussoni E. et al., Nature 401: 390-394, 1999; Kim, Byoung-Soo, Bioindustry. VoI 31, Development of Artificial Organs Stem Cell-Based Tissue Engineering, 2002) .
  • stem cell therapy is a new and promising strategy for the treatment of various incurable diseases. Furthermore, the successful results of stem cell therapy in the recovery of function of damaged organs are noteworthy enough to attract immediate and special attention from patients, scientists and various persons in the medical and pharmaceutical fields. In spite of the positive side thereof, stem cell therapy, however, suffers from many disadvantages, evidenced in some clinical tests using stem cells. Of them, the greatest disadvantages are that it is difficulty to obtain stem cells in an amount sufficient for use in treatment and that cells, after administered, fail to graft after a long engraftment period.
  • Leptin is a cytokine-like protein produced by adipose tissue, which is known to play a key role in regulating energy intake and energy expenditure, including the regulation (decrease) of appetite and (increase) of metabolism.
  • leptin receptors As the expression of leptin receptors (Ob-R) is found in immune cells and hematopoietic stem cells as well as the hypothalamus, which recognizes satiation, leptin is known to be involved in the proliferation and differentiation of hematopoietic stem cells
  • leptin receptors show structural similarity to receptors of gpl30 cytokines
  • leptin is believed to act as a cytokine for hematopoietic stem cells, like Epo, G-CSF and GM-CSF, which play essential roles in the proliferation of stem cells.
  • a method for proliferating stem cells comprising culturing the stem cells in a culture medium supplemented with leptin or an analogue or derivative thereof.
  • the method for proliferating stem cells with leptin in accordance with the present invention has advantages over conventional methods in terms of growth rate, stability and economy. Therefore, it is expected that thanks to the high growth rate and stability thereof, the method featuring the use of leptin in culturing stem cells in accordance with the present invention may be a basic technology for stem cell therapy. Also, the method of the present invention is expected to play an essential role in the development of cell therapeutics for various incurable diseases, thereby making a contribution to the generation of a high value-added industry.
  • FIG. 1 is a graph showing cell counts of the human bone marrow-derived stem cells cultured in serum-free media supplemented or not supplemented with leptin in the presence or absence of a growth factor.
  • FIG. 2 is a graph showing cell counts of the human bone marrow-derived stem cells cultured in serum media supplemented or not supplemented with leptin.
  • FIG. 3 is a graph showing cell counts of the human umbilical cord blood hematopoietic stem cells cultured in serum media supplemented or not supplemented with leptin.
  • FIG. 4 is a graph showing cell counts of the murine bone marrow-derived stem cells cultured in serum-free media supplemented or not supplemented with leptin in the presence or absence of a growth factor.
  • FIG. 5 shows cell counts of the murine bone marrow- derived stem cells cultured in serum-free media not supplemented with any additives ⁇ upper left panel) , and supplemented with leptin (upper right panel) , with G-CSF (lower left panel) and with a combination of leptin and G-CSF (lower right panel) in microscopic photographs.
  • FIG. 6 shows the effect of lejptin on the secretion of cytokines from the murine bone marrow-derived stem cells cultured in culture media in graphs .
  • the present invention pertains to a method for proliferating stem cells, comprising culturing stem cells.
  • Leptin secreted by adipocytes, is known to regulate obesity and appetite. Recent studies indicate that leptin is involved in the proliferation of hematopoietic stem cells. In the present invention, it is revealed that, when supplemented with leptin, cell culture media allow the proliferation of various stem cells in a large quantity without many rounds of sub-culturing. The addition of leptin once to a cell culture medium during the culture of stem cells makes it possible to stably and conveniently increase the number of stem cells without many processes for conventional stem cell proliferation until cell recovery, thereby greatly reducing the production cost.
  • five human hematopoietic stem cells including bone marrow-derived stem cells, bone marrow-derived hematopoietic stem cells, blood stem cells, blood hematopoietic stem cells, and umbilical cord blood hematopoietic stem cells, and murine bone marrow-derived stem cells were isolated and cultured in serum-added or serum-free media supplemented or not supplemented with growth factors in the presence or absence of leptin, followed by counting the cells .
  • Human bone marrow-derived stem cells (bone marrow cells with CD105+, CD166+, CD29+, CD44+, CD14-, CD34- and CD45- markers) were observed to increase four times in counts thereof when cultured in serum-free media in the presence of leptin compared to when cultured in a control, and to a greater extent than when cultured in media supplemented with growth factors (FIG. 1) .
  • FIG. 1 In the case of serum media, far greater effects, e.g., cell counts increased seven times, were observed (FIG. 2) .
  • Human umbilical cord blood hematopoietic stem cells were also observed to increase at least four-fold in number when cultured in the presence of leptin (serum medium) compared to when cultured in the absence of leptin (FIG. 3) .
  • leptin serum medium
  • FIG. 3 Human umbilical cord blood hematopoietic stem cells
  • stem cells useful in the present invention include bone marrow-derived stem cells, bone marrow-derived hematopoietic stem cells, blood stem cells, blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, fetal stem cells and adipocyte-derived stem cells, but are not limited thereto.
  • Leptin may be used alone or in concert with conventional proliferation methods, for example, in combination with other growth factors, for example, erythropoietin, G-CSF, GM-CSF, LIF, IL-3, and the like.
  • leptin analogues or leptin derivatives can afford the proliferation effect, as well.
  • any culture medium whether supplemented with serum or not, can be used in the present invention (e.g., Dulbecco's medium or a special medium containing cellulose (e.g., a medium from Stem Cell Technology Inc.).
  • the method of the present invention has an advantage in that stem cells can proliferate to a large quantity within a short period of time even in the absence of additional growth factors or additives.
  • the method of the present invention guarantees approximately eight times as many cells within the same period compared to conventional proliferation methods (after 2 weeks of culture, the number of cells increased 7.17 times : 58.87 times) .
  • the time necessary to reach the same number of stem cells can be reduced approximately 64% by the method of the present invention, compared to conventional methods.
  • stem cells can be grown to an amount sufficient for use in clinical application, thus obviating the repetitive rounds of stem cell extraction processes for cell therapeutic agents.
  • the method of the present invention which can guarantee a sufficient amount of stem cells, is expected to eliminate the problems of immune suppression or infection due to allo- or xenografts.
  • the reduced culture procedure and time period in accordance with the present invention is believed to result in reduced stress to stem cells as well as in the possibility of differences between the original stem cells and the final stem cells due to self-renewal or apoptosis . Therefore, it is anticipated that the proliferation method of stem cells in accordance with the present invention leads to a first economically favorable process, which can be a solution to the prior art problems .
  • human bone marrow-derived stem cells having CD105+, CD166+, CD29+, CD44+, CD14-, CD34- and CD45- markers were used.
  • an Iscove' s Modified Dulbecco' s Medium Gibco-BRL, USA
  • a special medium containing cellulose Stem Cell Technology Inc.
  • insulin transferrin, glutamine, fetal bovine serum and leptin (all from Sigma, USA)
  • G-CSF and erythropoietin both from Gibco, USA
  • the bone marrow was sampled in an amount of 10 ml from persons who were identified to be free of infection and transferred into vacutainer tubes coated with an anticoagulant agent, followed by rotating the vacutainer tubes two or three times to homogenously mix the bone marrow with the anticoagulant agent.
  • the content was homogeneously mixed by shaking the tubes about 20 times prior to centrifugation on a horizontal rotor (swing-out head, Beckman Coulter Inc, USA) at 3000 rpm at 20 0 C for 20 min (within 2 hours after the sampling of the bone marrow) .
  • the upper plasma layer thus separated was removed from the tube and transferred into cryogenic vials in an amount of 0.5 ml per vial.
  • the cell layer and the remaining plasma were well mixed with a pipette, transferred into 50 mL tubes, and diluted to a final volume of 45 ml with saline. After 10 rounds of inversion, 50 ⁇ l of the mixture was used for cell counting while another 50 ⁇ l was stored for subsequent flow cytometry.
  • the tube containing the remainder was centrifuged at 1,500 rpm at room temperature for 5 min, followed by removing the supernatant and suspending the cells in a culture medium.
  • the cell suspension was treated with antibodies (Sigma, USA) to surface markers, CD105, CD166, CD29, CD44, CD14, CD34 and CD45, and the properties thereof were analyzed using a flow cytometry analyzer (Beckman Coulter Inc. USA) , followed by isolation of the cells having surface markers CD105+, CD166+, CD29+, CD44+, CD14-, ,,CD34- and CD45- only. Of them, 50 ⁇ l was stored separately until the measurement of cell count.
  • a culture medium was aliquoted in an amount of 5 ml into 15 ml culture dishes and then supplemented with leptin, serum and other growth factors in predetermined amounts (EPO 3 IU/ml, G-CSF 10 ng/ml, leptin 100 nM) in accordance with test groups (Table 1) , followed by inoculating the cells at a density of 10 6 cells/ml of medium. After 14 days of incubation at 37°C in a 5% CO 2 atmosphere, the cell cultures were centrifuged at 1,500 rpm at room temperature for 5 min. After the removal of the supernatant, centrifugation was again conducted at 1500 rpm at room temperature for 5 min.
  • the cell pellets thus obtained were washed twice with a Hartman Dex solution (Choongwae Pharma Corporation) or Iscove' s MDM, and suspended in 20 ml of a Hartman Dex solution.
  • the cell suspension was filtered through a cell strainer, followed by 10 rounds of inversion 100 ⁇ l of the cell suspension was taken using a micropipette. 50 ⁇ l was used to count the cells while the other 50 ⁇ l was subjected to flow cytometry.
  • EXAMPLE 2 Proliferation of Human Bone Marrow-Derived Hematopoietic Stem cell
  • CD34 (+) cells were isolated from 10 ml of the bone marrow in the same manner as in Example 1 after the pre-blood test (CD34+ progenitor cell isolation kit, MACS, Miltenyi Biotec, Germany) .
  • a blood sample was taken from a suitable site in the arm tied with a tourniquet using a vacuum needle and a scalp needle, both connected to a holder, after which the vacutainer tube was rotated two or three times in order to mix the blood with an anticoagulant agent therein. Centrifugation was conducted within 2 hours after blood sampling. The vacutainer tube was turned upside down about 20 times to homogenously mix the content thereof before centrifugation at 3000 rpm at 20°C for 20 min on a horizontal rotor (swing-out head). The plasma in the upper layer (0.5 cm above the buffy coat layer) was removed using a 15 ml tube and transferred into cryogenic vials at an amount of 0.5 ml per vial.
  • lymphocytes and the remaining plasma in the buffy coat layer were well mixed with a pipette, transferred into 50 iriL tubes and diluted to a final volume of 45 ml with saline. After 10 rounds of inversion, 50 ⁇ l of the mixture was used for cell counting while another 50 ⁇ l was stored for subsequent flow cytometry.
  • the tube containing the remainder was centrifuged at 1,500 rpm at room temperature for 5 min. Following the removal of the supernatant, the cells were suspended in a culture medium and well mixed with Ficoll-Hypaque. Following flotation on Ficoll-Hypaque gradients, stem cells were carefully removed from a layer located 1/3 of the total length from the top thereof?.
  • CD34 (+) cells were isolated from 10 ml of the bone marrow in the same manner as in Example 1 after the pre-blood test (CD34+ progenitor cell isolation kit, MACS, Miltenyi Biotec, Germany) .
  • mice (Charles River, USA) under general anesthesia.
  • the hind legs were dissected, and the tibia were separated from the surrounding soft tissues and cut at both the fore end and the rear end, followed by the injection of a 23G syringe needle thereinto.
  • a culture medium was passed through the 23G syringe needle, the bone marrow was effused.
  • the bone marrow mass thus obtained was loosened into single cells by being passed back and forth in a Pasteur pipette.
  • the culture medium containing the bone marrow was transferred into a 15 ml culture tube and centrifuged at 1300 rpm for 5 min.
  • the culture tube was swung to loosen the cell pellet, which was then suspended with 1 ml of a culture medium. Thereafter, the same procedure as in Example 3 was repeated to isolate and culture the stem cells.
  • the culture tube was centrifuged at 1300 rpm for 5 min when using a general culture medium.
  • the culture tube was allowed to stand for 30 min at 4 0 C after the addition of 5 ml of chilled Iscove' s MDM thereinto.
  • the culture medium was observed to turn from a semi-solid state to a liquid state, it was centrifuged at 1300 rpm for 5 min.
  • Example 3 The supernatant was removed and the cell pellet was dissolved in 5 ml of the culture medium and again centrifuged at 1300 rpm for 5 min, followed by washing the cells twice with a Hartman Dex solution. The same procedure as in Example 3 was repeated to isolate and count the cells .
  • the experimental results after the cell isolation and proliferation are shown in FIGS. 4 and 5.
  • the culture medium was measured for cytokine levels 24 hours after treatment with leptin. The results are shown in FIG. 6.

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Abstract

L'invention porte sur un procédé qui permet la prolifération de cellules souches. La présence de leptine dans un milieu de culture cellulaire permet d'obtenir une prolifération considérable de diverses cellules souches sanguines ou de moëlle osseuse sans augmentation du nombre de sous-cultures. Le procédé précité permet de réduire le temps nécessaire à l'obtention d'une quantité prédéterminée de cellules et, par conséquent, de réaliser des avantages économiques. L'invention permet également la prolifération stable de cellules souches non modifiées.
PCT/KR2007/005670 2006-11-10 2007-11-12 Procédé permettant la prolifération de cellules souches au moyen de leptine WO2008056963A1 (fr)

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Cited By (8)

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WO2009155041A2 (fr) * 2008-05-28 2009-12-23 Children's Medical Center Corporation Méthode de modulation de la croissance de cellule souche hématopoïétiquie
US8551782B2 (en) 2006-03-24 2013-10-08 Children's Medical Center Corporation Methods for promoting HSC engraftment
US9051548B2 (en) 2009-02-03 2015-06-09 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9056085B2 (en) 2009-02-03 2015-06-16 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9402852B2 (en) 2006-10-20 2016-08-02 Children's Medical Center Corporation Method to enhance tissue regeneration
US9452186B2 (en) 2011-12-02 2016-09-27 Fate Therapeutics, Inc. Enhanced stem cell composition
US10111907B2 (en) 2011-12-02 2018-10-30 Fate Therapeutics, Inc. Methods of treating ischemia
US10851412B2 (en) 2013-03-15 2020-12-01 Fate Therapeutics, Inc. Cell potency assay for therapeutic potential

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KR101350144B1 (ko) * 2013-05-03 2014-01-10 재단법인 통합의료진흥원 팔물탕 추출물을 포함하는 골수유래 줄기세포 증식 촉진용 조성물
WO2018226051A2 (fr) 2017-06-07 2018-12-13 주식회사 엑소스템텍 Composition de milieu sans sérum pour la culture de cellules comprenant un exosome dérivé d'une cellule souche humaine

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US8551782B2 (en) 2006-03-24 2013-10-08 Children's Medical Center Corporation Methods for promoting HSC engraftment
US8563310B2 (en) 2006-03-24 2013-10-22 Children's Medical Center Corporation Methods for promoting hematopoietic reconstitution
US9028811B2 (en) 2006-03-24 2015-05-12 Children's Medical Center Corporation Methods for promoting HSC self-renewal
US10278990B2 (en) 2006-03-24 2019-05-07 Children's Medical Center Corporation Methods for promoting hematopoietic reconstitution
US10736906B2 (en) 2006-10-20 2020-08-11 Children's Medical Center Corporation Method to enhance tissue regeneration
US9402852B2 (en) 2006-10-20 2016-08-02 Children's Medical Center Corporation Method to enhance tissue regeneration
WO2009155041A3 (fr) * 2008-05-28 2010-03-11 Children's Medical Center Corporation Méthode de modulation de la croissance de cellule souche hématopoïétiquie
WO2009155041A2 (fr) * 2008-05-28 2009-12-23 Children's Medical Center Corporation Méthode de modulation de la croissance de cellule souche hématopoïétiquie
US9051548B2 (en) 2009-02-03 2015-06-09 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US10092599B2 (en) 2009-02-03 2018-10-09 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US10159697B2 (en) 2009-02-03 2018-12-25 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9737567B2 (en) 2009-02-03 2017-08-22 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9056085B2 (en) 2009-02-03 2015-06-16 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US10111907B2 (en) 2011-12-02 2018-10-30 Fate Therapeutics, Inc. Methods of treating ischemia
US10172888B2 (en) 2011-12-02 2019-01-08 Fate Therapeutics, Inc. Enhanced stem cell composition
US9452186B2 (en) 2011-12-02 2016-09-27 Fate Therapeutics, Inc. Enhanced stem cell composition
US10980838B2 (en) 2011-12-02 2021-04-20 Fate Therapeutics, Inc. Methods of treating ischemia
US11052118B2 (en) 2011-12-02 2021-07-06 Fate Therapeutics, Inc. Enhanced stem cell composition
US10851412B2 (en) 2013-03-15 2020-12-01 Fate Therapeutics, Inc. Cell potency assay for therapeutic potential

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