WO2011070084A1 - Milieu de culture cellulaire contenant des mogrosides utilisé pour cultiver des cellules souches - Google Patents

Milieu de culture cellulaire contenant des mogrosides utilisé pour cultiver des cellules souches Download PDF

Info

Publication number
WO2011070084A1
WO2011070084A1 PCT/EP2010/069204 EP2010069204W WO2011070084A1 WO 2011070084 A1 WO2011070084 A1 WO 2011070084A1 EP 2010069204 W EP2010069204 W EP 2010069204W WO 2011070084 A1 WO2011070084 A1 WO 2011070084A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
mogroside
culture medium
analogue
cells
Prior art date
Application number
PCT/EP2010/069204
Other languages
English (en)
Inventor
Patrick Prendergast
Original Assignee
Patrick Prendergast
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Patrick Prendergast filed Critical Patrick Prendergast
Publication of WO2011070084A1 publication Critical patent/WO2011070084A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • 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
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants
    • 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
    • C12N2501/90—Polysaccharides

Definitions

  • the present invention relates to a medium for growing stem cells, in particular, but not exclusively, primate stem cells, such as human mesenchymal stem cells.
  • the invention further relates to methods of growing stem cells, to stem cells and differentiated cells provided by the methods of the invention and to use of same, for example, in therapy and drug discovery.
  • Stem cells are characterized by their ability to (i) proliferate for long periods of time and (ii) differentiate into a diverse range of specialized cell types.
  • the two broad types of mammalian stem cells are adult stem cells, which are found in adult tissues, and embryonic stem cells, which are derived from the inner cell mass of an early stage embryo known as a blastocyst.
  • multipotency is differentiation of the cells into osteoblasts, adipocytes and chondrocytes.
  • Pluripotent adult stem cells that is, stem cells that are able to differentiate into cells derived from any of the three primary germ layers, the ectoderm, endoderm and mesoderm, are rare.
  • Adult stem cells have the potential to play an important role in therapy and regenerative medicine.
  • adult stem cells have been successfully used in bone marrow transplants for the treatment of diseases such as leukemia and related bone/blood cancers.
  • the use of adult stem cells is less controversial than the use of embryonic stem cells as the destruction of an embryo is not required.
  • the risk of teratoma formation is greatly reduced when using adult stem cells when compared to the risk with embryonic stem cells.
  • MSCs mesenchymal stem cells
  • MSCs are found in the bone marrow, adipose tissue and the umbilical cord, and can differentiate into a number of cell types including osteoblasts, neuronal cells, chondrocytes, myocytes, adipocytes and beta- pancreatic islets cells.
  • MSCs have potential for use in tissue repair.
  • MSCs can take up and keep introduced genes, a phenomenon that could be exploited for the delivery of beneficial molecules to targeted locations.
  • a further advantage of MSCs is that they can be frozen to preserve them, and upon thawing they appear to function normally.
  • MSCs derived from the umbilical cord have been shown to have a low count per volume of the initial material and very low proliferative activity. It would be advantageous if large quantities of MSCs could be readily made available for use in therapy and other areas.
  • MSCs can be propagated in culture for extended periods of time without losing their multipotency.
  • Embryonic stem cells are pluripotent, that is, they are able to differentiate into cells derived from any of the three primary germ layers, the ectoderm, endoderm and mesoderm. For this reason, they are likely to play an important role in drug screening, functional genomics applications and therapies, such as regenerative medicine and replacement of damaged tissues.
  • Embryonic stem cells were first derived from mouse embryos in 1981 . In 1998, a technique to isolate and grow primate embryonic stem cells derived from human blastocysts was first developed. Specific culture environments are required in order to maintain embryonic stem cells stem cells in an undifferentiated state. These requirements differ for human embryonic stem cells and mouse embryonic stem cells. If these requirements are not met, the embryonic stem cells will rapidly
  • mouse embryonic stem cells are grown on a layer of gelatin in the presence of Leukemia Inhibitory Factor (LIF), which inhibits differentiation of mouse embryonic stem cells.
  • LIF Leukemia Inhibitory Factor
  • differentiation of human embryonic stem cells is inhibited by growing the human embryonic stem cells on a layer of feeder cells, known as a feeder layer.
  • Typical feeder layers include animal cells, such as mouse embryonic fibroblasts, and human cells, such as human embryonic fibroblasts, human foreskin fibroblasts, human adult fallopian tubal epithelial cells, human fetal muscle and human fetal skin cells and adult skin fibroblast cell lines.
  • the feeder cells provide an extracellular matrix and cellular contact for the maintenance of human embryonic stem cells in an undifferentiated state without losing their pluripotency.
  • the feeder cells release factors and nutrients into the culture medium. These factors and nutrients may be used by the stem cells.
  • pre-conditioned medium may be provided wherein the medium has firstly been conditioned by growing the feeder cells in the medium, following which the medium is harvested for subsequent stem cell culture.
  • feeder cells pose a risk of contamination of human embryonic stem cells, in particular, contamination with non-human molecules, pathogens and other unidentified risk factors. This problem was discovered by scientists at the University of California, San Diego when non-human sialic acid in the growth media was found to compromise the potential uses of the embryonic stem cells in humans.
  • bFGF Basic Fibroblast Growth Factor
  • bFGF Basic Fibroblast Growth Factor
  • the inventor has surprisingly developed a culture medium which facilitates the rapid growth of human adult stem cells, for example, mesenchymal stem cells (MSCs), without loss of their ability to differentiate.
  • MSCs mesenchymal stem cells
  • the culture medium of the present invention further facilitates the rapid growth of human embryonic stem cells in a substantially undifferentiated form in the absence of feeder cells or preconditioned medium.
  • the culture medium of the invention may be used to provide improved methods for growing human embryonic stem cells wherein the risk of contamination is reduced.
  • Adult and embryonic stem cells grown using the methods of the invention have potential for use in therapies, such as regenerative medicine, and in drug screening, particularly in cases where the absence of feeder cells is beneficial.
  • a culture medium for growing stem cells comprising at least one mogroside and/or at least one analogue thereof.
  • a method for growing stem cells comprising the step of:
  • a composition comprising a culture medium comprising at least one mogroside and/or at least one analogue thereof and stem cells.
  • a culture medium for growing stem cells comprising an extract of Luo Han Guo fruit.
  • a method for growing stem cells comprising the step of:
  • composition comprising a culture medium comprising an extract of Luo Han Guo fruit and stem cells.
  • stem cells grown using the methods and/or medium of the present invention. The invention further extends to differentiated cells derived from said stem cells, and to the use of said stem cells and/or differentiated cells, for example, in therapy or drug discovery. Description of the Figures
  • FIG 1 shows the growth index for bone marrow derived human Mesenchymal Stem Cells (MSCs) grown in the presence of basic Fibroblast Growth Factor (bFGF) or Luo Han Guo fruit extract (C53a);
  • MSCs bone marrow derived human Mesenchymal Stem Cells
  • bFGF basic Fibroblast Growth Factor
  • C53a Luo Han Guo fruit extract
  • Figure 2 shows the growth index for umbilical cord derived human MSCs grown in the presence of bFGF or Luo Han Guo fruit extract (C53a);
  • Figure 3(a) shows the growth index for umbilical cord derived human MSCs grown in the presence of bFGF;
  • Figure 3(b) shows the growth index for umbilical cord derived human MSCs grown in the presence of Luo Han Guo fruit extract (C53a).
  • the present invention is directed to the use of at least one mogroside, and/or at least one analogue thereof, for culturing stem cells.
  • mogrosides In the course of the inventor's work with mogrosides, he has unexpectedly discovered that the presence of one or more mogrosides in a suitable culture medium is sufficient to proliferate human adult stem cells without loss of their ability to differentiate.
  • the inventor has further identified that the presence of one or more mogrosides in a suitable culture medium is sufficient to proliferate human embryonic stem cells in a substantially undifferentiated state through extended culture, for example, more than six months, in the absence of feeder cells and/or pre-conditioned medium.
  • references to “a mogroside” or “at least one mogroside” includes a single mogroside as well as two or more different mogrosides in combination.
  • reference to “an analogue” or “at least one analogue” includes a single analogue as well as two or more different analogues in combination.
  • Reference to "at least one mogroside and/or at least one analogue thereof includes a single mogroside and two or more different analogues in combination, a single analogue and two or more different mogrosides in combination and two or more mogrosides with two or more analogues in combination.
  • analogue as used herein is intended to refer to compounds having the same functional activity as a mogroside, though not necessarily to the same degree.
  • the term “analogue” is intended to extend to derivatives and solvates of mogrosides, provided said derivative or solvate retains the appropriate functional activity, that is, to support the growth of undifferentiated stem cells, although not necessarily to the same degree as mogrosides.
  • derivative as used herein includes chemical modification of the at least one mogroside.
  • the term “without differentiation” or similar terms as used herein are intended to refer to cells that are substantially undifferentiated, that is cells that contain at least 20%, 40%, 60%, 80%, 85%, 90% or 95%
  • feeder cell as used herein is intended to refer to cells that can be co-cultured with stem cells to inhibit differentiation of the stem cells.
  • essentially free of feeder cells is intended to refer to cells that contain less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 % or 0.01 % feeder cells. In certain embodiments, the term refers to cells that contain less than 2% feeder cells.
  • pre-conditioned refers to a medium which has been contacted with a first population of cells, for example feeder cells, prior to being combined with stem cells.
  • the at least one mogroside and/or the at least one analogue thereof is added to a culture medium.
  • the culture medium is substantially free of feeder cells, even more typically essentially free of feeder cells and preferably free of feeder cells.
  • the presence of at least one mogroside and/or the at least one analogue thereof in the culture medium obviates the requirement for feeder cells as the at least one mogroside and/or the at least one analogue thereof inhibits differentiation of the stem cells whilst maintaining cell division. Accordingly, even after expansion a high proportion of the stem cells retain their ability to differentiate. This can be assessed by analysing morphological characteristics and phenotypic markers of the stem cells, as well as their ability to differentiate into the three germ layers for pluripotent stem cells.
  • the culture medium is not pre-conditioned prior to combining the culture medium with the stem cells.
  • the presence of at least one mogroside and/or the at least one analogue thereof in the culture medium obviates the requirement for the medium to be pre-conditioned. There is therefore no requirement for the medium to be brought into contact with other cell types, such as human or mouse feeder cells, before being combined with the stem cells.
  • the absence of feeder cells in the culture medium and/or the absence of the use of pre-conditioned medium reduces the risk of contamination of the stem cells, for example by other species or other tissue types, thus increasing their potential for use in therapies such as regenerative medicine. It further allows more accurate results to be obtained when carrying out experiments using the stem cells, for example, in drug discovery.
  • the at least one mogroside and/or the at least one analogue thereof acts as a growth stimulator.
  • the addition of the at least one mogroside and/or the at least one analogue thereof to the culture medium has been shown in certain embodiments to increase the speed of growth of the stem cells whilst maintaining the cells' ability to differentiate.
  • the stem cells expand up to one and a half times faster when cultured with at least one mogroside or analogue thereof compared to when cultured in the absence of at least one mogroside or analogue thereof, for example in the presence of feeder cells and/or in pre-conditioned medium.
  • Cucurbitaceae family This fruit grows primarily in the mountains of Guilin, in the South-western Chinese province of Guangxi and is commonly known as Monk fruit.
  • the fruit of Luo Han Guo has been cultivated in China for hundreds of years, primarily because of its sweet taste
  • Extract of the Luo Han Guo plant is currently manufactured and marketed as a natural low-calorie sweetener, for example, PURELOTM.
  • US Patent No. 5,41 1 ,755 describes a process for preparing a sweet juice from Luo Han Guo fruit.
  • mogrosides There are no known toxicity issues relating to the use or consumption of mogrosides. These compounds have been used in contemporary Chinese medicine for centuries and have been in recorded human use since 1813. More recently, mogrosides have been shown to have beneficial effects in the treatment of diabetes, cancer and inflammation.
  • a 90-day toxicity study carried out in canines showed no evidence of toxicity at an impressive dose of 3000 mg/kg bw/day.
  • mogrosides are triterpene glycosides. Mogrosides possessing a triterpene backbone with two to six glucose units attached form mogrosides II to VI.
  • the mogroside and/or the at least one analogue thereof is derived from a plant of the Cucurbitaceae species, in particular the fruit of the Cucurbitaceae species.
  • the plant is Siraitia grosvenori Swingle (formerly Momoridca grosvenori Swingle).
  • the fruit is Luo Han Guo.
  • the at least one mogroside and/or the at least one analogue thereof is a triterpene glycoside or an analogue thereof.
  • the at least one mogroside or analogue is selected from the group consisting of 20-hydroxy-1 1 -oxomogroside IA, 1 1 - oxomogroside IIE, ll-oxomogroside IA, mogroside IIE, mogroside III, mogroside IV, mogroside V, siamenoside I, triterpenoid glycoside V, neogroside, Kaempferol 7-a-i-rhamnopyranoside, Kaempferol 3,7- ⁇ - ⁇ - dirhamnopyranoside, 1 1 -oxomogroside III, ll-Dehydroxymogroside III, II- oxomogroside IV, mogroside II, mogroside VI, ll-oxo-mogroside and siamcroside-l, or an analogue thereof.
  • the at least one mogroside or analogue thereof is selected from the group of compounds having the chemical formula shown in formula 1 below, or an analogue thereof.
  • Formula 1 :
  • the at least one mogroside and/or the at least one analogue thereof is added to the culture medium in an isolated form, for example, wherein the at least one mogroside or the analogue thereof has been isolated and purified from Luo Han Guo.
  • the at least one mogroside and/or the at least one analogue thereof is added to the culture medium in the form of a crude extract of Luo Han Guo.
  • the at least one mogroside and/or the at least one analogue thereof is added to the culture medium in the form of a natural sweetener or juice derived, for example, from Luo Han Guo, for example the juice obtained using the process described in US Patent No.
  • the juice is dried and the at least one mogroside and/or the at least one analogue thereof is provided in a powder form.
  • the at least one mogroside comprises a bacterially-transformed mogroside.
  • the at least one mogroside comprises a mogroside in a synthetic form.
  • the at least one mogroside and/or the at least one analogue thereof may be prepared by chemical synthesis techniques. This may be achieved by conventional techniques, for example as described in "Protective Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons lnc.(1991 ), and by P.J. Kocienski, in “Protecting Groups", Georg Thieme Verlag (1994).
  • a mixture of mogrosides and/or analogues is used.
  • the mogrosides and/or analogues are combined in set proportions by weight to achieve optimal stimulatory growth of the stem cells.
  • the at least one mogroside and/or the at least one analogue thereof is added to the culture medium at a concentration of at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 ng/ml. In alternative embodiments the at least one mogroside and/or at least one analogue thereof is added to the culture medium at a concentration of at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 ng/ml. In alternative embodiments the at least one mogroside and/or at least one analogue thereof is added to the culture medium at a
  • the at least one mogroside and/or the at least one analogue thereof is added to the culture medium at a concentration of at least 10 ng/ml.
  • the at least one mogroside and/or the at least one analogue thereof is added to the culture medium at a concentration of at least 5 ⁇ / ⁇ . This concentration has been shown to be particularly effective in proliferating adult stem cells without differentiation.
  • the culture medium comprises an extracellular matrix.
  • the extracellular matrix supports the stem cells.
  • the extracellular matrix comprises at least one of the group consisting of laminin, entactin, reconstituted basement membrane from Engelbreth-Holm-Swarm tumour cells, vitronectin, fibronectin or proteoglycan.
  • a buffer such as an isotonic buffer
  • proteins such as an isotonic buffer
  • proteins such as an isotonic buffer
  • proteins such as an isotonic buffer
  • proteins such as an isotonic buffer
  • proteins such as an isotonic buffer
  • non-essential and/or essential amino acids such as fetal calf serum
  • serum such as fetal calf serum
  • reducing agent and beta-mercaptoethanol such as fetal calf serum
  • the effective concentrations of such components may be determined by those of skill in the art of culturing cells.
  • one or more additional factors such as fibroblast growth factor, for example, basic fibroblast growth factor (bFGF) may be added to the culture medium. These factors can be added to increase the rate of growth of the stem cells and to further inhibit differentiation of the stem cells.
  • the culture medium is essentially free from fibroblast growth factor, for example, basic fibroblast growth factor.
  • the culture medium is any suitable medium known in the art to which at least one mogroside or an analogue thereof has been added.
  • the medium comprises a growth medium which supports growth of the stem cells.
  • the medium is Minimum Essential Medium (MEM), which is a widely used synthetic cell culture media.
  • MEM alpha is a parameter used synthetic cell culture media.
  • the medium includes a nutrient serum which supports growth of the stem cells.
  • the nutrient serum may replace nutrients that would otherwise be absent when a non pre-conditioned medium is used.
  • fetal calf serum for example 10% fetal calf serum, is used.
  • the medium is free from serum.
  • the medium is pre-conditioned, for example by contact with feeder cells.
  • non-conditioned medium may also be used as the presence of the at least one mogroside or analogue thereof obviates the requirement for a pre-conditioned medium.
  • the stem cells comprise primate stem cells, preferably human stem cells. In alternative embodiments the stem cells comprise non-primate stem cells, for example, non-human stem cells. In certain embodiments the stem cells are embryonic stem cells. In alternative embodiments the stem cells are adult stem cells. In certain embodiments the stem cells comprise pluripotent stem cells, for example, human embryonic stem cells or human pluripotent adult stem cells. Typically the human embryonic stem cells are obtained from established human embryonic stem cells or from human blastocysts. In certain embodiments the stem cells comprise multipotent stem cells, for example, adult stem cells such as mesenchymal stem cells. In certain
  • the stem cells are selected from the group consisting of, but not limited to, adipose derived adult stem cells, induced pluripotent stem cells derived from epithelial cells, haematopoietic stem cells, mammary stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, testicular cells and dental pulp derived stem cells.
  • the stem cells are modified by in-vitro treatment or genetically altered, for example, by transfecting the stem cells with a suitable vector in order to provide a particular characteristic for a specific industrial application.
  • stem cells provided by the method of the present invention are substantially undifferentiated cells.
  • the stem cells comprise mainly undifferentiated pluripotent or multipotent stem cells.
  • the stem cells comprise undifferentiated cells, in addition to differentiated and/or partially differentiated cells.
  • the stem cells consist of undifferentiated cells.
  • the pluripotent stem cells are capable of proliferating in-vitro whilst maintaining the potential to differentiate into derivatives of the three germ layers.
  • the multipotent stem cells are capable of proliferating in-vitro whilst maintaining the potential to differentiate into cells of a closely related family of cells. Typically all the stem cells are derived from the same stem cells and therefore have the same genotype.
  • the stem cells comprise mixed populations of different cell lines.
  • the stem cells are differentiated into different cell types.
  • the method of the present invention therefore further includes the step of differentiating the cells to produce different cells types, for example cells of the three germ layers or cells of a closely related family of cells. Typically the method may be carried out in vivo or in-vitro.
  • differentiated cells can be obtained wherein 95% or more of the cells represent the same tissue type or germ layer, for example, cardiomyocytes, mesenchymal cells, neural cells, hepatocytes or osteoblasts.
  • the present invention further extends to the use of the stem cells proliferated by the method of the present invention, for example, in therapy and tissue replacement, drug screening and development, analysis of mammalian development, analysis of pluripotency or multipotency, differentiation and cell proliferation, analysis of signalling pathways and analysis of genetics.
  • a differentiated or multipotency for example, in therapy and tissue replacement, drug screening and development, analysis of mammalian development, analysis of pluripotency or multipotency, differentiation and cell proliferation, analysis of signalling pathways and analysis of genetics.
  • undifferentiated stem cell of the invention may be transplanted into a human or non-human animal to treat a disease, such as a degenerative neurological disorder.
  • said use includes a method of screening to identify an agent that affects stem cell function.
  • the method includes incubating the agent and at least one stem cell cultured using the method of the present invention under conditions sufficient to allow the agent and the at least one stem cell to interact.
  • the agent is allowed to interact with the at least one stem cell in the presence of the at least one mogroside and/or the at least one analogue thereof.
  • the at least one mogroside and/or the at least one analogue thereof is not present when the agent is allowed to interact with the at least one stem cell.
  • the method includes the step of determining the effect of the agent on the function of the at least one stem cell.
  • the function that may be affected, for example inhibited or stimulated, by the agent includes cell
  • MSCs Mesenchymal Stem Cells
  • MSCs Luo Han Guo fruit extract derived using the process described in US Patent No. 5,41 1 ,755 on human Mesenchymal Stem Cells (MSCs) growth was examined using MSCs derived from bone marrow (BM) and the umbilical cord (UC). One sample was obtained from bone marrow and two samples were obtained from the umbilical cord. MSCs were seeded in 6 well plates at the indicated passage at 250 cells/cm 2 and cultured under the following conditions:
  • Figure 1 shows the results obtained for MSCs derived from bone marrow.
  • Figures 2 and 3 show the results obtained for the two MSCs samples derived from the umbilical cord.
  • Figure 1 shows that Luo Han Guo fruit extract enhanced the growth of MSCs derived from bone marrow. Increased growth was observed for cells cultured with Luo Han Guo fruit extract when compared to the control or to cells cultured with bFGF. The best results were obtained using Luo Han Guo fruit extract at a range of 5 g/ml. However, the proliferative capacity of the MSCs derived from bone marrow was low under all culture conditions. It is hypothesised that the low proliferative capacity was due to the MSCs derived from bone marrow being seeded at the 6 th passage.
  • Figures 2 and 3 demonstrate enhanced support and growth of MSCs derived from the umbilical cord cultured in the presence of Luo Han Guo fruit extract when compared to control cultures (no Luo Han Guo fruit extract). In some cases, the results obtained with Luo Han Guo fruit extract were better than those observed with bFGF (see Figure 2, 7 th passage). The best results were obtained using Luo Han Guo fruit extract at a range of 5 g/ml. These results demonstrate that Luo Han Guo fruit extract supports MSCs growth and expansion.

Abstract

La présente invention concerne un milieu de culture permettant de cultiver des cellules souches, ledit milieu de culture comprenant au moins un mogroside et/ou au moins un de ses analogues. L'invention concerne également des procédés permettant de cultiver des cellules souches, comprenant l'étape consistant à cultiver les cellules souches en présence d'au moins un mogroside et/ou d'au moins un de ses analogues. La présence desdits mogrosides et/ou de leurs analogues permet de s'affranchir du besoin de cellules nourricières et/ou de milieu préconditionné, tout en permettant aux cellules souches de proliférer pendant de longues périodes sans perte de leur aptitude à se différencier. Les cellules cultivées au moyen des procédés de la présente invention présentent un potentiel d'utilisation accru en thérapeutique et en médecine régénérative dans la mesure où le risque de contamination des cellules souches est réduit.
PCT/EP2010/069204 2009-12-11 2010-12-08 Milieu de culture cellulaire contenant des mogrosides utilisé pour cultiver des cellules souches WO2011070084A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IE2009/0931 2009-12-11
IE20090931 2009-12-11

Publications (1)

Publication Number Publication Date
WO2011070084A1 true WO2011070084A1 (fr) 2011-06-16

Family

ID=43662178

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2010/069204 WO2011070084A1 (fr) 2009-12-11 2010-12-08 Milieu de culture cellulaire contenant des mogrosides utilisé pour cultiver des cellules souches

Country Status (1)

Country Link
WO (1) WO2011070084A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019004905A (ja) * 2018-09-10 2019-01-17 アナリティコン ディスカバリー ゲーエムベーハー 甘味料または甘味増強剤として使用される新規なトリペルテングリコシド
CN114774353A (zh) * 2022-03-31 2022-07-22 巴马奥比特生命科技研究有限公司 一种干细胞培养基

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5411755A (en) 1993-02-16 1995-05-02 The Procter & Gamble Company Process and composition for sweet juice from cucurbitaceae fruit
WO2006017370A2 (fr) 2004-07-13 2006-02-16 Geron Corporation Milieu destine a la croissance de cellules souches embryonnaires humaines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5411755A (en) 1993-02-16 1995-05-02 The Procter & Gamble Company Process and composition for sweet juice from cucurbitaceae fruit
WO2006017370A2 (fr) 2004-07-13 2006-02-16 Geron Corporation Milieu destine a la croissance de cellules souches embryonnaires humaines

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Siraitia grosvenorii", WIKIPEDIA THE FREE ENCYCLOPEDIA, 6 February 2006 (2006-02-06), XP002628425, Retrieved from the Internet <URL:http://en.wikipedia.org/wiki/Siraitia_grosvenorii> [retrieved on 20110316] *
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; March 2009 (2009-03-01), SHI AI-WU ET AL: "Ginsenoside Rg1 promotes endothelial progenitor cell migration and proliferation.", XP002628424, Database accession no. NLM19262553 *
P.J. KOCIENSKI: "Protecting Groups", 1994, GEORG THIEME VERLAG
SHI ET AL., ACTA PHARMACOLOGICA SINICA, vol. 30, no. 3, March 2009 (2009-03-01), pages 299 - 306, ISSN: 1745-7254 *
T W GREENE; P G M WUTS: "Protective Groups in Organic Synthesis", 1991, JOHN WILEY AND SONS INC.
TAKASAKI MIDORI ET AL: "Anticarcinogenic activity of natural sweeteners, cucurbitane glycosides, from Momordica grosvenori.", CANCER LETTERS, vol. 198, no. 1, 30 July 2003 (2003-07-30), pages 37 - 42, XP002628426, ISSN: 0304-3835 *
ZHOU YING ET AL: "Insulin secretion stimulating effects of mogroside V and fruit extract of Luo Han Kuo (Siraitia grosvenori Swingle) fruit extract", YAOXUE XUEBAO, 2009, pages 10PP, XP002628423, Retrieved from the Internet <URL:http://www.yxxb.com.cn:8080/yxxb_en/fileup/HTML/article_19215.htm> [retrieved on 20110316] *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019004905A (ja) * 2018-09-10 2019-01-17 アナリティコン ディスカバリー ゲーエムベーハー 甘味料または甘味増強剤として使用される新規なトリペルテングリコシド
CN114774353A (zh) * 2022-03-31 2022-07-22 巴马奥比特生命科技研究有限公司 一种干细胞培养基

Similar Documents

Publication Publication Date Title
Piedrahita et al. Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines
KR101443478B1 (ko) 간엽계 줄기 세포를 포함한 세포 제제 및 그의 제조 방법
LU502505B1 (en) Immortalized yak rumen epithelial cell line and construction method thereof
AU2013221839B2 (en) Feeder-free method for culture of bovine and porcine spermatogonial stem cells
CN102634482B (zh) 一种间充质干细胞的无血清完全培养基
CN101688177A (zh) 来自贴壁胎盘干细胞的肝细胞和软骨细胞；以及cd34+、cd45-胎盘干细胞富集的细胞群
KR102128224B1 (ko) 향상된 산후 부착형 세포 및 그의 제조 방법
KR20120008223A (ko) 양막유래 중간엽 줄기세포 배양을 위한 배지조성물 및 이를 이용한 양막유래 중간엽 줄기세포의 배양방법
AU2013403888B2 (en) Method for differentiating pluripotent stem cell induced from mesenchymal stem cell into chondrocyte
CN112920991B (zh) 一种外泌体分泌诱导剂及诱导培养基和应用其的外泌体的生产方法及应用
CN104830763B (zh) Y-27632在间充质干细胞培养中的应用及间充质干细胞的培养方法
CN108504625A (zh) 一种小鼠成纤维细胞及其用途
CN114480273A (zh) 用于获得间充质干细胞及其外泌体的培养基及其制备方法
Mathis et al. Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured “on top” and “inside” different extracellular matrix substitutes
CN106929470A (zh) 一种用于间充质干细胞体外培养和扩增的无血清培养基
US20120052577A1 (en) Culture system for stem cell propagation and neural and oligodendrocyte specification
JP2003535585A (ja) 哺乳動物の配偶子および胚の培養基サプリメント、およびその使用法
CN102899288A (zh) 一种人胰岛来源的胰腺干细胞系的构建及向胰岛素分泌细胞分化的方法
CN111484970A (zh) 一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基
KR20060032958A (ko) 전구 세포의 분리 및 증식
WO2011070084A1 (fr) Milieu de culture cellulaire contenant des mogrosides utilisé pour cultiver des cellules souches
RU2012107674A (ru) Биотрансплантат для восстановления объема костной ткани при дегенеративных заболеваниях и травматических повреждениях костей и способ его получения
KR101656761B1 (ko) 다능성 줄기 세포 및 심근 세포 이외의 분화성 세포에 대해 세포사멸을 유도하는 방법
JP2007000077A (ja) 付着性動物細胞の無血清培養方法及び付着性動物細胞の無血清培養用培地
CN102206609A (zh) 一种卵巢来源雌性生殖干细胞的分离培养方法

Legal Events

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

Ref document number: 10790417

Country of ref document: EP

Kind code of ref document: A1

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

Ref document number: 10790417

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10790417

Country of ref document: EP

Kind code of ref document: A1