WO2011067465A1 - Formulations et procedes de culture de cellules souches - Google Patents

Formulations et procedes de culture de cellules souches Download PDF

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WO2011067465A1
WO2011067465A1 PCT/FI2010/050980 FI2010050980W WO2011067465A1 WO 2011067465 A1 WO2011067465 A1 WO 2011067465A1 FI 2010050980 W FI2010050980 W FI 2010050980W WO 2011067465 A1 WO2011067465 A1 WO 2011067465A1
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serum replacement
culture medium
present
cells
stem cells
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PCT/FI2010/050980
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Kristiina Rajala
Marjo-Riitta Suuronen
Outi Hovatta
Heli Skottman
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Kristiina Rajala
Marjo-Riitta Suuronen
Outi Hovatta
Heli Skottman
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Application filed by Kristiina Rajala, Marjo-Riitta Suuronen, Outi Hovatta, Heli Skottman filed Critical Kristiina Rajala
Priority to CA2782296A priority Critical patent/CA2782296A1/fr
Priority to JP2012541546A priority patent/JP2013512667A/ja
Priority to CN2010800545573A priority patent/CN102906247A/zh
Priority to EP10800970A priority patent/EP2507360A1/fr
Priority to KR1020127017428A priority patent/KR20130009943A/ko
Priority to AU2010326512A priority patent/AU2010326512A1/en
Publication of WO2011067465A1 publication Critical patent/WO2011067465A1/fr

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    • C12N5/0602—Vertebrate cells
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance

Definitions

  • the present invention relates to xeno-free formulations for use in the derivation, maintenance and differentiation of stem cells, such as human embryonic stem cells.
  • Human embryonic stem cells are pluripotent cells that have the potential to differentiate into all cell types of a human body. Human ESCs are of great therapeutic interest because they are capable of indefinite proliferation in culture and are thus capable of supplying cells and tissues for replacement of failing or defective human tissue. There are high expectations that, in the future, human ESCs will be proliferated and directed to differentiate into specific cell types, which can be transplanted into human bodies for therapeutic purposes or used as cell models in drug discovery and toxicology studies.
  • Embryonic stem cells are d ifficult to maintain in culture because they tend to follow their natu ral cel l fate and spontaneously differentiate. Most culture conditions result in some level of unwanted differentiation. Stem cells differentiate as a result of many intrinsic and extrinsic factors, including growth factors, extracellular matrix molecules and components, environmental stressors and direct cell-to-cell interactions. Long- term proliferative capacity, pluripotent developmental potential after prolonged culture and karyotypic stability are the key features with respect to the utility of stem cell cultures.
  • the undifferentiated stage of hESCs can be monitored by judging the morphological characteristics of the cells.
  • Undifferentiated hESCs have a characteristic morphology with very small and compact cells. While some differentiated cells usually appear at the margin of colonies of hESCs, an optimal culture method provides growth support with minimal amount of differentiated cells.
  • biochemical markers that are used to track the status of undifferentiated stage of hESCs such as the transcription factor Oct4 and Nanog as well as cell surface markers TRA-1 -60, TRA-1 -81 , SSEA-3/4. These markers are lost when hESCs begin to differentiate to any cell lineage.
  • Basic techniques to create and culture hESCs have been described .
  • Embryonic stem cells have typically been derived and proliferated in culture medium containing animal serum (especially fetal bovine serum) or other animal derived products to permit the desired proliferation during such culturing.
  • animal serum especially fetal bovine serum
  • animal derived products may contain toxic proteins or immunogens that evoke an im m u n e response in the recipient and thus lead to rejection upon transplantation (Martin et al., Nat Med. 2005 Feb;1 1 (2):228-32).
  • feeder-free culture methods have been developed for hESCs. Many of these feeder-free methods utilize animal derived components. In addition, these methods suffer from inadequate reproducibility and currently are unable for long-term maintenance of undifferentiated hESCs with stable and normal karyotype. Feeder-free cultures with enzymatic passaging may also be so demanding for the hESCs that they become more prone to abnormalities.
  • the present invention provides means and methods for derivation, maintenance and differentiation of clinical-grade stem cells. More specifically, the present invention relates to a serum replacement, a final culture medium comprising said serum replacements, and uses thereof.
  • An object of the present invention is to provide a xeno-free serum replacement comprising at least one fatty acid selected from a group consisting of conjugated linoleic acid and eicosapentaenoic acid.
  • the concentration of conjugated linoleic acid (CLA) is such that a final culture medium, which is a basal medium supplemented with said serum replacement, comprises from about 0.5 mg/l to about 5 mg/l CLA, and the concentration of eicosapentaenoic acid (EPA) is such that the final culture medium comprises from about 1 mg/l to about 10 mg/l EPA.
  • the serum replacement may further comprise Activin A and/or retinol.
  • the concentration of Activin A is such that the final culture medium comprises from about 0.001 mg/l to about 0.02 mg/l Activin A and/or the concentration of retinol is such that the final culture medium comprises from about 0.25 mg/l to about 1 .0 mg/l retinol.
  • the serum replacement may further comprise stearic acid.
  • the concentration of stearic acid is such that a final culture medium comprises from about 0.5 mg/l to about 5 mg/l stearic acid.
  • Another object of the present invention is to provide a xeno- free cell culture medium comprising a basal medium and a serum replacement according to the embodiments of the present invention.
  • Still another object of the present invention relates to the use of the present serum replacement or the cell culture medium for maintenance, proliferation or differentiation of stem cells.
  • an object of the present invention relates to the use of the present serum replacement or the cell culture medium for derivation or isolation of stem cells.
  • a further object of the present invention is to provide a method for initiating a new stem cell line in vitro.
  • Said method comprises a) providing isolated cells of desired origin, b) contacting said cells with the present xeno-free culture med ium, and c) cultivating said cells under conditions suitable for stem cell culture.
  • the cultivation may be performed on a feeder cell layer.
  • said isolated cells are of embryonic, adult somatic, or mesenchymal origin.
  • a still further object of the present invention is to provide a method for culturing stem cells.
  • Said method comprises a) contacting said stem cells with the present xeno-free medium, and b) cultivating said cells under conditions suitable for stem cell culture.
  • the cultivation may be performed on a feeder cell layer.
  • the present culture medium is able to support the maintenance and proliferation of stem cells in a substantially undifferentiated state over numerous in vitro passages.
  • the stem cells cultured in the culture medium according to the present invention are substantially undifferentiated, retain their pluripotency or multipotency and maintain their genomic integrity.
  • an object of the present invention is to provide a method for differentiating stem cells.
  • Said method comprises a) contacting said stem cells with the present xeno-free medium supplemented with a differentiating agent, such as a growth factor or differentiating cells (e.g. END2 cells), and b) cultivating said cells under conditions suitable for differentiation of stem cells.
  • a differentiating agent such as a growth factor or differentiating cells (e.g. END2 cells)
  • Figures 1 A - 1 D are light microscopic images of hESC line HS401 cultured in the culture medium according to the present invention in different osmolarities for 5 passages: 260 mOsm (Fig . 1A), 290 mOsm (Fig. 1 B), 320 mOsm (Fig. 1 C) and 350 mOsm (Fig. 1 D). Scale bar 500 ⁇ .
  • Figures 2A - 2B show the morphology and differentiation stage of hESCs cultured in the culture medium according to the present invention and in the presence of lipids and lipid derivatives.
  • UD, PD and DIFF represent undifferentiated, partly differentiated and differentiated hESC colonies, respectively.
  • Fig. 2A HS401 cell line cultured in the culture medium according to the present invention supplemented with different lipids and lipid derivatives.
  • F ig . 2B HS401 cell line cultured in control hES medium supplemented with different lipids and lipid derivatives.
  • Figures 3A - 3F demonstrate the increase in the proliferation and expression of stem cell markers in response to retinol.
  • Fig 3A Bright-field microscopic image of hESCs (Regea 07/046) at day 3 cultured in the culture medium according to the present invention without retinol for 5 passages.
  • Fig 3B Bright-field microscopic image of hESCs (Regea 07/046) at day 3 cultured in the culture medium according to the present invention containing 2.0 ⁇ retinol for 5 passages. The size of the colonies is larger in the presence of retinol when compared to the colonies cultured without retinol. Scale bar 500 ⁇ .
  • 3C - D Fluorescent microscopic image of hESCs (HS401 ) cultured in the culture medium according to the present invention containing 2.0 ⁇ retinol for 12 passages showing positive expression of Nanog and TRA-1 -81 . Insets represent DAPI staining. Scale bar 200 ⁇ .
  • Fig. 3E Cell proliferation analysis of hESC line Regea 07/046 cultured in the culture medium according to the present invention without and in the presence of 0.5, 2.0 and 3.5 ⁇ retinol for 10 passages. Fig.
  • Figures 4A - 4C demonstrate the increase in the proliferation and expression of stem cell markers in response to Activin A.
  • Fig . 4A Cell proliferation analysis of hESC l ine Regea 07/046 cultured in the culture medium according to the present invention without and in the presence of 5ng/ml and 10 ng/ml Activin A and hES control medium for 10 passages.
  • Fig. 4A Cell proliferation analysis of hESC l ine Regea 07/046 cultured in the culture medium according to the present invention without and in the presence of 5ng/ml and 10 ng/ml Activin A and hES control medium for 10 passages.
  • Fig. 4C FACS analysis of SSEA4 and TRA-1 -60 stem cell markers of hESC line Regea 07/046 cultured in the culture medium accord ing to the present invention without and in the presence of 5ng/ml and 10 ng/ml Activin A and hES control medium for 10 passages.
  • Figures 5A - 5J show characterization of hESC lines derived and cultured for long-term in the culture medium according to the present invention.
  • Fig. 5A A Giemsa band karyogram showing normal karyotypes of hESC lines, Regea 07/046 at passage 36, Regea 08/013 at passage 25, and Regea 06/040 at passage 71 .
  • Fig. 5B Quantitative FACS analyses indicating expression of SSEA-4 and TRA-1 -81 of hESC lines at day 7. Regea 07/046 at passage 45, Regea 08/013 at passage 41 , and Regea 06/040 at passage 26.
  • Fig. 5A A Giemsa band karyogram showing normal karyotypes of hESC lines, Regea 07/046 at passage 36, Regea 08/013 at passage 25, and Regea 06/040 at passage 71 .
  • Fig. 5B Quantitative FACS analyses indicating expression of SSEA-4 and
  • Fig. 5C Cell proliferation analysis of hESC lines Regea 06/040 at passage 29, Regea 07/046 at passage 53 and Regea 08/01 3 at passage 41 .
  • Fig . 5D Quantitative RT-PCR analysis of Nanog, Oct4, GABRB3, GDF3, DNMT3B and TDGF1 expression in hESC lines Regea 07/046 at passage 52, Regea 08/013 at passage 45, and Regea 06/040 at passage 33.
  • Fig. 5E Bright-field (scale bar, ⁇ ) microscopic image showing undifferentiated colony morphology of hESC line 07/046 (p33) 1 after freezing and subsequent thawing in the culture medium according to the present invention at passage 1 .
  • Fig. 5E Bright-field (scale bar, ⁇ ) microscopic image showing undifferentiated colony morphology of hESC line 07/046 (p33) 1 after freezing and subsequent thawing in the culture medium according to the present invention at passage 1 .
  • Fig. 5H Differentiated cardiomyocytes from hESC line Regea 08/013 stain positively with ventricular myosin heavy chain . Scale bar is 1 00 ⁇ .
  • Fig. 5I RT-PCR analysis of neurospheres derived from hESC line Regea 08/013 cultured in the culture medium accord ing to the present invention showed expression of neural precursor markers Musashi, Nestin and PAX6; neuronal markers MAP-2, NF68 and 07X2; and astrocytic marker GFAP. No expression of pluripotent markers Oct4 and Nanog, nor endo- AFP or mesodermal markers T/Brachyury were detected.
  • Fig. 5J Most of the cells migrating out from the plated neurospheres stained positive for neuronal marker MAP-2 and few cells were positive for astrocytic marker GFAP. Scale bar is 100 ⁇ .
  • Figures 6A - 6C show characterization of human induced pluripotent stem cells (iPS cells) cultured in the culture medium according to the present invention .
  • Fi g . 6A Quantitative FACS analyses indicating expression of SSEA-4 and TRA-1 -81 of human iPS cell lines cultured in hES medium and in the culture medium according to the present invention.
  • Cell samples cultured in hES medium are from 6 day old colonies, cell samples from iPS cell line A cultured in culture medium according to the present invention from 7 day old colonies and samples from iPS cell line B from 8 day old colonies.
  • Fig. 6B Quantitative RT-PCR analysis of Nanog, Oct4, GABRB3, GDF3, DNMT3B and TDGF1 expression of day 6 colonies in iPS cell line A in hES medium at passage 1 0, in culture medium according to the present invention at passage 7 and iPS cell line B in hES medium at passage 1 1 , in culture medium according to the present invention at passage 8.
  • Fig. 6B Quantitative RT-PCR analysis of Nanog, Oct4, GABRB3, GDF3, DNMT3B and TDGF1 expression of day 6 colonies in iPS cell line A in hES medium at passage 1 0, in culture medium according to the present invention at passage 7 and iPS cell line B in hES medium at passage 1 1 , in culture medium according to the present invention at passage 8.
  • Fig. 6B Quantitative RT-PCR analysis of Nanog, Oct4, GABRB3, GDF3, DNMT3B and TDGF1 expression of day 6 colonies in iPS cell line A in hES medium at passage 1
  • FIG. 6C RT- PCR analysis of in v/ ' fro-derived EBs showing transcripts for AFP and SOX-17 (endodermal markers), a-cardiac actin and T (Brachyury; mesodermal markers), SOX-1 and PAX6 (ectodermal markers), and ⁇ -actin as a housekeeping control. Lane 1 , 50-bp DNA ladder. Both cell lines at passage 10.
  • Figures 7A - 7E show characterization of human adipose stem cells (ASCs) cultured in the culture medium according to the present invention.
  • Fig . 7A Morphology of ASCs cultured in human serum (HS) containing medium at day 8. (Scale bar 50 ⁇ ).
  • Fig. 7A Morphology of ASCs cultured in human serum (HS) containing medium at day 8. (Scale bar 50 ⁇ ).
  • Fig. 7A Morphology of ASCs cultured in human serum (HS) containing medium at day 8. (Scale bar 50
  • FIG. 7B Morphology of ASCs cultured in culture medium according to the present invention at day 8.
  • FIG. 7C Morphology of ASCs cultured in HS medium at day 1 1 .
  • Fig. 7D Morphology of ASCs cultured in culture medium according to the present invention at day 1 1 .
  • the present invention relates to means and methods for derivation, maintenance and differentiation of clinical-grade stem cells. More specifically, the present invention relates to a xeno-free serum replacement formulation and to a culture medium comprising said serum replacement. Furthermore, the present invention relates to methods for stem cell derivation, culture, maintenance and differentiation.
  • the present invention thus provides a defined xeno-free seru m replacement formu lation or com position that may be used to supplement any suitable basal medium for use in the in vitro derivation, maintenance, proliferation, or differentiation of stem cells.
  • said serum replacement may be used to supplement either serum- free or serum-containing basal mediums, or any combinations thereof.
  • xeno-free basal medium is supplemented with the present xeno-free serum replacement, the final culture medium is xeno-free, too.
  • stem cells include both pluripotent and multipotent stem cells.
  • Embryonic stem cells are pluripotent cells being able to differentiate into a wide variety of different cell types.
  • True embryonic stem cell l ines (i) are capable of indefinite prol iferation in vitro in an undifferentiated state; (ii) are capable of differentiation to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm), even after prolonged culture; and (iii) maintain a normal karyotype throughout the prolonged culture.
  • Embryonic stem cells are, therefore, referred to as being pluri potent.
  • iPS cells are another example of pluripotent stem cells.
  • iPS cells are generated from differentiated cells, typically from adult somatic cells such as fibroblasts by developmental reprogramming. Such cells have been described e.g. in WO 2008/151058 and US 2008/076176.
  • Multipotent stem cel ls incl ude are not l im ited to, hematopoietic stem cells and mesenchymal stem cells (MSCs), which are adult stem cells capable of differentiating into a variety of cell types.
  • MSCs may be isolated from different sources including bone marrow and adipose tissue. MSCs derived from adipose tissue are termed as adipose stem cells (ASCs).
  • ASCs adipose stem cells
  • Stem cells including hESCs, cultured in accordance with the present invention can be obtained from any su itable source using any appropriate technique, including, but not limited to, immunosurgery.
  • procedures for isolating and growing human embryonic stem cells are described in U .S. Pat. No. 6,090,622.
  • Procedures for obtaining Rhesus monkey and other non-human primate embryonic stem cells are described in U.S. Pat. No. 5,843,78 and international patent publication WO 96/22362.
  • methods for isolating Rhesus monkey embryonic stem cells are described by Thomson et al., (1995, Proc. Natl . Acad. Sci. USA, 92:7844- 7848).
  • Blastomere biopsy is an attractive new technology which allows isolation and propagation of embryonic stem cells without damaging the donor embryo. Other methods for obtaining stem cells are readily available in the art.
  • the means and methods provided herein are applicable to stem cells derived from any desired animal, preferably mammals including primates such as humans, mon keys, and apes, as well as non-primate mammals such as mice, rats, horses, sheep, pandas, goats and zebras.
  • xeno-free it is meant herein that the origin of the reagent is not from a foreign source, i.e. does not contain material of non- human animal origin when human stem cells are to be cultured .
  • culturing of, for instance, murine stem cells has to be done in the absence of any mice derived material in order to be xeno-free.
  • Suitable xeno-free sources for culturing human stem cells may include chemical synthesis or synthetic preparations or isolation, preparation or purification of the reagent of interest from bacteria, yeasts, fungi, plants and humans.
  • seru m repl acement a formulation that may be used to replace animal serum in a final cell culture medium.
  • a conventional serum replacement comprises typically vitamins, albumin, lipids, amino acids, transferrin, antioxidants, insulin and trace elements.
  • the final cell culture medium may further comprise growth factors, non-essential amino acids, ⁇ -mercaptoethanol, L-glutamine and/or antibiotics added d irectly to the basal med ium or further comprised in the serum replacement.
  • CLA conjugated linoleic acid
  • EPA eicosapentaenoic acid
  • the serum replacement according to the present invention is a xeno-free formulation comprising at least one fatty acid selected from the group consisting of conjugated linoleic acid and eicosapentaenoic acid.
  • a preferred concentration range of CLA in the serum replacement is such that the final culture medium comprises from about 0.5 mg/l to about 5 mg/l, more specifically about 2.5 mg/l CLA.
  • a preferred concentration range of EPA in the serum replacement is such that the final culture medium comprises from about 1 mg/l to about 10 mg/l, more specifically about 5 mg/l EPA.
  • said serum replacement comprises CLA from about 2.5 mg/l to about 25 mg/l, more specifically about 12.5 mg/l and/or EPA from about 5 mg/l to about 50 mg/l, more specifically 25 mg/l. It is evident to a person skilled in the art that the serum replacement may be provided in a form to be added to the basal medium with different percentages, whereby the concentrations of individual ingredients change accordingly.
  • the next best fatty acid for use in the serum replacement is stearic acid.
  • a preferred concentration range of stearic acid in the serum replacement is such that the final culture medium comprises from about 0.5 mg/l to about 5 mg/l, more specifically about 2.5 mg/l stearic acid.
  • said serum replacement comprises stearic acid from about 2.5 mg/l to about 25 mg/l, more specifically about 12.5 mg/l.
  • Activin A especially in combination with CLA and/or EPA promotes stem cell proliferation and expression of stem cell markers such as Nanog, Oct4, GDF3, DNMT3B, GABRB3 and GDF3.
  • the serum replacement according to the present invention may further comprise Activin A.
  • a preferred concentration range of Activin A in the serum replacement is such that the final culture medium comprises from about 0.001 mg/l to about 0.02 mg/l, more specifically about 0.005 mg/l Activin A.
  • said serum replacement comprises Activin A from about 0.005 mg/l to about 0.1 mg/l, more specifically about 0.025 mg/l .
  • retinol i.e. vitamin A
  • the effective concentration rage of retinol is about 10 times lower than that disclosed in WO 2008/148938.
  • Preferred concentration range of retinol in the serum replacement is such that the final culture medium comprises from about 0.25 mg/l to about 1 .0 mg/l, more specifically about 0.57 mg/l retinol.
  • said serum replacement comprises retinol from about 1 .25 mg/l to about 5.0 mg/l, more specifically about 2.85 mg/l.
  • the serum replacement according to the present invention comprises Activin A and CLA and/or EPA.
  • the serum replacement comprises retinol and CLA and/or EPA.
  • the serum replacement comprises Activin A, retinol, and CLA and/or EPA. Preferred concentrations of these ingredients are given above.
  • Each and every serum replacement according to these embodiments may further comprise stearic acid.
  • the serum replacement comprises in addition to the ingredients given above at least one ingredient, preferably free of endotoxins, selected from the group consisting of lipids or lipid derivatives, vitamins, albumins or albumin substitutes, amino acids, vitamins, transferrins, transferrin substitutes, antioxidants, insulin or insulin substitutes, trace elements, and growth factors.
  • Such ingredients are to be present in the serum replacement formulation in a concentration sufficient to support the proliferation of stems cells in a substantially undifferentiated state, while maintaining both the pluripotency and the karyotype of the cells.
  • the serum replacement according to the present invention may further comprise at least one other lipid or lipid derivative including, but not limited to, lipoproteins such as very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and cholesterol; phospholipids such as phosphatidylcholine, lysophosphatidylcholine, phosphatidylserine, phosphatidylinositol, sphingomyelin, and phosphatidylethanolamine; fatty acids such as linoleic acid, gamma-linoleic acid, linolenic acid, arachidonic acid, oleic acid, docosahexaenoic acid, palmitic acid, palmitoleic acid, myristic acid and their derivatives such as prostaglandins.
  • the serum replacement may comprise at least one other lipid or lipid derivative including, but not limited to, lip
  • the serum replacement may further contain other vitamins than retinol, such as ascorbic acid, biotin, choline chloride, D-Ca Pantothenate, Folic acid, iinositol, niacinamide, Pyridoxal, Pyridoxine, Riboflavin, thiamine, Vitamin B 12, Vitamin D2.
  • vitamins typically several vitamins are included in the basal medium and additional vitamin supplementation can be added to the final medium.
  • thiamine is used in a concentration of about 9 mg/l
  • ascorbic acid is used in a concentration of about 50 g/ml in the cell culture medium according to the present invention.
  • Albumin substitutes suitable for use in the present invention include any compound , which may be used instead of albumin and has essentially similar effects as albumin. Suitable concentration of albumin or albumin substitute in the serum replacement and in the final culture medium according to the present invention, can be readily determined by a skilled person using routine methods well known in the art. Typically, albumins or albumin substitutes are used in the final medium in the range of about 1 mg/ml to about 20 mg/ml, preferably of about 5 mg/ml to about 1 5 mg/ml . In one embodiment, albumin is present at about 10 mg/ml in the cell culture medium according to the present invention.
  • Fetuin, a-fetoprotein, and any combination thereof may be used to replace albumin in the serum replacement. However, due to their high price it may be feasible to use them in combination with albumin .
  • the serum replacement comprises about 0.5 mg/ml fetuin and about 0.25 mg/ml a-fetoprotein.
  • a basal medium is to be supplemented with 20% serum replacement.
  • a typical final cell culture medium comprises from about 0.01 mg/ml to about 1 mg/ml fetuin and/or a-fetoprotein.
  • Amino acids suitable for use in the present invention include, but are not limited to amino acids, such as glycine, L-histidine, L-isoleucine, L- methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and their D-forms and derivatives.
  • amino acids such as glycine, L-histidine, L-isoleucine, L- methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and their D-forms and derivatives.
  • Suitable concentrations of amino acids can be readily determined by a skilled person using routine methods well known in the art. Typical concentration ranges are presented in Table 3.
  • the serum replacement accord ing to the present invention may contain additional non-essential amino acids, such as L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, L-serine, and their D-forms and derivatives.
  • additional non-essential amino acids may be included in the serum replacement or added directly to the final cell culture medium according to the present invention.
  • Non-essential amino acids may be provided as a commercially available mixture, such as MEM non-essential amino acids (NEAA) provided by Invitrogen .
  • NEAA MEM non-essential amino acids
  • concentration of said mixture in the final medium according to the present invention is about 1 %.
  • L-glutamine is preferably added to the cell culture medium accord ing to the present invention as a stabil ized , d ipeptide form of L- glutamine such as Glutamax (Invitrogen, 2 mM).
  • L-glutamine may be included in the serum replacement according to the present invention.
  • Transferrins are involved in iron delivery to cells, controlling free iron concentration in biological fluids and preventing iron-mediated free radical toxicity.
  • Suitable transferrin substitutes for use in the present invention include any compound which may be used instead of transferrin and has essentially similar effects as transferrin.
  • Such substitutes include, but are not limited to, iron salts and chelates (e.g., ferric citrate chelate or ferrous sulfate).
  • Suitable concentrations of transferrin or transferrin substitute in the serum replacement and the final medium according to the present invention can be readily determined by a skilled person using routine methods well known in the art.
  • suitable range of transferrin or transferrin substitute in the final medium according to the present invention is about 1 g/ml to about 1 000 g/ml, preferably about 5 g/ml to about 1 00 pg/ml, and more preferably, about 5 g/ml to about 10 g/ml.
  • transferrin is present at about 8 g/ml in the cell culture medium according to the present invention.
  • Antioxidants suitable for use in the present invention include, but are not limited to glutathione and ascorbic acid. Suitable concentrations of antioxidants in the serum replacement and the final medium according to the present invention can be readily determined by a skilled person using routine methods well known in the art. According to one embodiment, glutathione is present at 1 .5 g/ml and ascorbic acid is present at 50 g/ml in the cell culture medium according to the present invention.
  • Insulin substitutes suitable for use in the present invention include any compound , wh ich may be used instead of insul in and has essentially similar effects as insulin.
  • Suitable concentration of insulin or insulin substitute in the serum replacement and the final medium according to the present invention can be readily determined by a skilled person using routine methods well known in the art.
  • suitable range of insulin in the final medium is about 1 g/ml to about 1000 pg/ml, preferably about 1 g/ml to about 100 pg/ml, more preferably about 50 g/ml to about 15 g/ml. In one embodiment, insulin is present at about 10 g/ml.
  • Trace elements suitable for use in the present invention include, but are not limited to Mn 2+ , Si 4+ , Mo 6+ , V 5+ , Ni 2+ , Sn 2+ , Al 3+ , Ag + , Ba 2+ , Br “ , Cd 2+ , Co 2+ , Cr 3+ , F " , Ge 4+ , ⁇ , Rb + , Zr 4+ and Se 4+ and salts thereof.
  • Suitable concentrations of trace elements or salts thereof can be readily determined by a skilled person using routine methods known in the art.
  • Commercially available trace element compositions such as Trace Elements B and C provided by CellGro Mediatech Inc. may also be used.
  • trace elements Cu 2+ and/or Zn 2+ may be included e.g. in the form of a commercially available Trace Element A composition provided by CellGro Mediatech Inc.
  • the present inventors have shown that lithium chloride may be harmful for embryonic stem cells resulting in differentiation thereof.
  • the serum replacement is devoid lithium chloride.
  • Growth factors su itable for use in the present invention include fibroblast growth factors (FGFs) such as basic FGF (bFGF or FGF-2).
  • FGFs fibroblast growth factors
  • bFGF or FGF-2 basic FGF
  • Suitable range of FGF in final medium according to the present invention is about 1 ng/ml to about 1000 ng/ml, preferably about 2 ng/ml to about 1 00 ng/ml , and more preferably about 4 ng/ml to about 20 ng/ml .
  • FGF is present at about 8 ng/ml.
  • FGF is preferably used, other materials, such as certain synthetic small peptides (e.g. produced by recombinant DNA variants or mutants) designed to activate fibroblast growth factor receptors, may be used instead of FGF.
  • Growth factors may be included in the serum replacement according to the present invention or they may be added separately to the final cell culture medium according to the present invention.
  • Antibiotics can also be used, to avoid contamination of the serum replacement or the medium according to the present invention. Suitable antibiotics or combinations thereof, as well as suitable concentrations are apparent to a person skilled in the art. However, if the medium is to be used in the culture of cells for clinical applications one might want to avoid the use of antibiotics.
  • ⁇ -mercaptoethanol may be included in the serum replacement according to the present invention or it may be added separately into the final culture medium according to the present invention. Typically, the final concentration of ⁇ -mercaptoethanol is about 0.1 mM in the culture medium.
  • any of the components of the serum replacement described above may be added directly into a basal medium to provide a final cell culture medium instead of being provided in the serum replacement according to the present invention.
  • the present invention further provides a defined xeno-free culture medium for the in vitro derivation, maintenance, proliferation and differentiation of stem cells.
  • Said culture medium comprises a basal medium and a serum replacement composition set forth herein.
  • Suitable basal media for use in the present invention include, but are not limited to KnockOut Dulbecco's Mod ified Eagle's Med ium (KO-DMEM), Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Med ium and HyQ ADCF-MAb (HyClone) and any combinations thereof.
  • the basal medium is KO-DMEM.
  • basal medium refers to any medium which is capable of supporting growth of stem cells, and in general supplies standard inorganic salts, vitamins, glucose, a buffer system and essential amino acids.
  • the basal medium can be supplemented with about 1 g/L to about 3.7 g/L sodium bicarbonate.
  • the basal medium is supplemented with about 2.2 g/L sodium bicarbonate.
  • the basal medium to be supplemented with the serum replacement may itself be xeno-free or it may be further supplemented with e.g. serum.
  • the osmolarity of the culture affects to the success and vitality of stem cell cultures.
  • Osmolarity measured in mill i-osmoles, is a measure of the number of dissolved particles in a solution, which is a measurement of the osmotic pressure that a solution will generate.
  • Normal human serum has an osmolarity of about 290 milli-osmoles.
  • Media for in vitro culture of other mammalian cells vary in osmolarity, but some media have an osmolarity as high as 330 nnill i-osnnoles.
  • the osmolarity of the medium according to the present invention is between about 280 and about 330 mOsmol.
  • osmolarity of the medium can be as low as about 260 mOsmol and as high as about 340 mOsmol.
  • hESCs are grown in an osmolarity of about 320-330 nnilli-osnnoles.
  • lipids, albumin, amino acids, vitamins, transferrin, antioxidants, insulin, and trace elements are included in the serum replacement, while growth factors, non-essential amino acids, ⁇ - mercaptoethanol, L-glutamine and antibiotics are added directly to the cell culture medium .
  • Final composition of one preferred culture med ium is exemplified in Table 2.
  • the serum replacement or the culture medium according to the present invention may be provided in a liquid or a dry form. Furthermore, they may be provided as any su itable concentrated formulation. As an example, basal medium may be supplemented with 10%, 15% or 20% (vol/vol) serum replacement so as to result in final concentrations of ingredients as given above. When desired, ingredients of the serum replacement or the medium may be divided into compatible subformulations.
  • the serum replacement and the culture medium according to the present invention are useful in a plethora of applications.
  • the serum replacement and the culture medium according to the present invention may also be used for initiation, i.e. derivation or isolation, of new stem cell lines such as ESCs, ASCs and iPS cell lines.
  • the present invention thus provides a method for such purposes.
  • the method comprises the steps of providing isolated cells of desired origin, contacting said cells with a xeno-free medium according to the embodiments of the present invention, and cultivating said cells under conditions suitable for cell culture.
  • the medium is supplemented with lam in ine, such as human placental lamin ine, and fibronectin, such as human plasma fibronectin .
  • laminine and fibronectin are used in a concentration of about 5 g/ml.
  • This aspect of the present invention may also be formulated such that the invention relates to the use of the serum replacement or the culture medium for initiating new stem cell lines.
  • all embodiments of the present method apply for said use of the serum replacement or culture medium.
  • the serum replacement and the final culture medium are suitable for supporting the maintenance and proliferation of stem cells in a substantially undifferentiated state, preferably over numerous in vitro passages. More specifically, said formulations may be used for maintaining and proliferating stem cells for at least about 20, preferably at least about 30, and more preferably at least about 50 passages.
  • stem cel l s have been successfu l ly ma i nta i ned i n a su bstantia l ly undifferentiated state for even over 80 passages.
  • Said stem cells retain their pluripotency, or multipotency. For instance, embryonic stem cells maintain their potential to differentiate into derivatives of endoderm, mesoderm and ectoderm tissues. Furthermore, said stem cells retain their genomic integrity as judged e.g. by their unchanged karyotypes.
  • the culture medium according to the invention comprises no components, such as feeder cells, conditioned medium, serum or other medium components, purified from a non-human animal source. More preferably, the culture medium comprises components that are synthesized using recombinant or chemical methods.
  • the present invention thus provides a method for culturing and maintaining stem cells in a xeno-free culture as described above.
  • Said method comprises contacting stem cells with the culture medium according to the present invention, and cultivating said cells under conditions suitable for stem cell culture. Such conditions are apparent to a person skilled in the art.
  • This aspect of the present invention may also be formulated such that the invention relates to the use of the serum replacement or the culture med ium for culturing and maintaining stem cell l ines .
  • the invention relates to the use of the serum replacement or the culture med ium for culturing and maintaining stem cell l ines .
  • all embodiments of th e present method apply for sa id use of the seru m replacement or culture medium.
  • the present culture medium may also be used for studying cell proliferation and differentiation, including studying, identifying and/or screening molecules, such as drug candidates, which i) affect the proliferation of undifferentiated stem cells, ii) affect the differentiation of stem cells, and iii) regulate tissue regeneration .
  • the culture medium may also be used for producing various agents, such as therapeutic proteins, in genetically modified stem cells or differentiated cells obtained therefrom.
  • the serum replacement and the final culture medium may further be used for differentiating stem cells into a desired linage, especially for therapeutic purposes. Th is may be ach ieved by adding appropriate and sufficient concentrations of differentiating agents into the present culture medium.
  • Non-limiting examples of differentiating agents include Noggin, which may be used to differentiate oligodentrocytes; sonig hedgehog and retinoic acid, which may be used to differentiate motor neurons; bFGF, which may be used to differentiate retinal cell lineages; and BMP2, which may be used to differentiate cardiomyocytes; Activin A and IGF2, which may be used to differentiate insulin-producing cells; and Activin A, BMP2 and BMP4, which may be used to differentiate hepatic cells.
  • the differentiating agent may be a differentiating cell, such as END2, which may be used to differentiate cardiomyocytes.
  • Other differentiating agents are well known in the art.
  • This aspect of the present invention may also be formulated such that the invention relates to a method for differentiating stem cells into a desired linage.
  • the method comprises contacting stem cel ls with the cu ltu re med ium according to the present invention supplemented with a differentiating agent, and cultivating said cells under conditions suitable for stem cell culture.
  • Current differentiation protocols utilize a variety of undefined products and culture media that may have unknown effects to the cell characteristics and differentiation.
  • the present formulations, differentiation methods and uses do not share these disadvantages.
  • Suitable feeder cells include but are not l imited to fibroblasts, such as human foreskin fibroblasts, e.g . CRL-2429 (ATCC, Mananas, USA).
  • the formulation, methods, and uses according to the present invention are used for feeder cell-free culture of stem cells.
  • Example 1 Effect of culture medium osmolarity on hESCs.
  • Example 2 Specific lipids and lipid derivatives enhance the undifferentiated growth of hESCs.
  • the hESC colonies were classified into three categories; undifferentiated, partly differentiated and differentiated. Number of each colony type was calculated before each passaging. Later, a percentage value for each colony type of the total amount of colonies was calculated ( Figure 2).
  • the number of undifferentiated colonies increased and the number of differentiated col o n i es d ecrea sed i n th e presen ce of co nj u g ated l i no l e i c a cid , eicosapentaenoic acid, stearic acid, retinol, linoleic-oleic-arachidonic acid mix, DL-isoproterenol, palmitoleic acid and linoleic acid when compared to the colonies cultured in the control hES or Albumax-RegES medium containing Albumax (Invitrogen) instead of human serum albumin.
  • Albumax-RegES medium containing Albumax (Invitrogen) instead of human serum albumin.
  • hES culture medium In hES culture medium the number of undifferentiated colonies increased and the number of differentiated colonies decreased in the presence of cholesterol, arachidonic acid, conjugated linoleic acid, retinol and phosphatidylcholine when compared to the colonies cultured in the control hES medium.
  • retinol and conjugated linoleic acid overall improved the colony morphology and the number of undifferentiated colonies in both culture media.
  • eicosapentaenoic acid resulted in excellent performance by increasing the number of undifferentiated colonies in the culture medium according to the present invention.
  • conjugated linoleic acid and eicosapentaenoic acid are the most preferred fatty acids to be included in a xeno-free serum replacement. The third best performance was observed with stearic acid in the culture medium according to the present invention.
  • Linoleic acid LA 2.5 ⁇ g ml ++/++
  • Phospholipids Phosphatidylcholine, PC 2.5 ⁇ g ml ++/-
  • Lysophosphatidylcholine LPC 5 g/ml -/++
  • Retinol was selected to be further evaluated in the maintenance of undifferentiated hESCs. Initial studies showed that retinol at a concentration of 0.1 -0.5 ⁇ was not effective and no improvement in the morphology or in the number of undifferentiated colonies was seen. Further evaluation, however, showed that retinol at a concentration of 2.0 ⁇ or above improved the proliferation of hESCs as well as induced the expression of hESC specific markers ( Figure 3). In the presence of 2.0 ⁇ retinol, the growth of the colonies started earlier and already at day 3 the size of the colonies was bigger ( Figure 3A - 3B).
  • Proliferation assay demonstrated that hESCs cultured in the presence of 2.0 ⁇ or 3.5 ⁇ retinol had almost two-fold proliferation rate when compared to hESCs cultured without retinol or in the presence of 0.5 ⁇ retinol (Figure 3E).
  • Immunocytochemical staining of hESCs cultured in the presence of retinol showed expression of stem cell markers Nanog and TRA-1 - 81 ( Figure 3C - 3D).
  • retinol increased the expression of pluripotency supporting genes, especially Nanog, which relative expression level was over twentyfold in the presence of 2.0 ⁇ and 3.5 ⁇ retinol ( Figure 3F).
  • Example 4 Activin A further enhances the performance of the culture medium of the present invention.
  • Proliferation assay demonstrated that hESCs cultured in the presence of 5 or 10 ng/ml Activin A in the culture medium according to the present invention had almost two-fold proliferation rate when compared to hESCs cultured without Activin A and the proliferation rate was comparable to h ESCs cultured in the control h ES med ium ( Figure 4A).
  • Fluorescence- activated cell sorting (FACS) and quantitative reverse transcription PCR (qRT- PCR) analysis demonstrated that Activin A increased the expression of pluripotency supporting markers at both transcriptional and translational level ( Figures 4B - 4C).
  • Example 5 Derivation, long-term culture and characterization of hESCs in the culture medium of the present invention.
  • hESC lines (07/046 and 08/013) have been successfully derived from surplus bad quality human embryo donated for stem cell research.
  • Human ESC lines have been continuously cultured for over 80 passages. These cell lines have been karyotyped regularly and exhibit a normal diploid karyotype (Figure 5A).
  • Fluorescence-activated cell sorting (FACS) and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that these cell lines express stem cell markers at levels comparable to the hESC line Regea 06/040 derived and cu ltu red using h ES med iu m ( Figure 5B, D).
  • EB embryoid body
  • hESCs derived and cultured for long-term in xeno-free cond itions can differentiate to card iomyocytes and neural cell lineages. Spontaneously beating areas were observed after 12-16 days after the initiation of the cardiac differentiation.
  • hESC colonies cultured in the culture medium according to the present invention and hES media were dissected into small clusters and cultured in suspension for up to 20 weeks.
  • the differentiated cells expressed neural precursor markers, neuronal markers and astrocytic marker in RT-PCR ( Figure 5I).
  • Immunocytochemical staining verified the neuronal and glial fate of the cells ( Figure 5J).
  • Example 6 Culture and characterization of iPS cells in the culture medium according to the present invention.
  • Example 7 Culture and characterization of ASCs in the culture medium according to the present invention.
  • ASCs isolated from adipose tissue samples were used to assess the performance of the culture medium according to the present invention for the culture of mesenchymal stem cel ls.
  • the proliferation analysis showed already at day 4 that cultures with the culture medium according to the present invention exhibited a higher proliferation rates of ASCs as compared to HS medium ( Figure 7E).
  • ASCs continued to proliferate at a higher rate in the culture medium according to the present invention compared to HS medium at day 7 and 1 1 .
  • CD90 Thy-1 T-cell surface glycoprotein 93,1 ⁇ 11,2 99,8 ⁇ 0,1
  • H LA-ABC * Major histocompatibility class I 0,6 ⁇ 0,4 10,0 ⁇ 11 ,4 antigen
  • HLA-DR Major histocompatibility class II 0,8 ⁇ 0,6 0,4 ⁇ 0,1
  • Cell lines 5/08, 19/08, 24/08 and 25/08 cultured in HS medium were at passage 2-3 and cell lines 9/08, 11/08, 25/08 and 31/08 cultured in the culture medium according to the present invention were at passage 3-4.
  • Data are presented as mean ⁇ standard deviation from the number of donors/samples indicated in parentheses. * p ⁇ 0.05.

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Abstract

L'invention concerne une formulation de remplacement de sérum et un milieu de culture approprié pour dériver, préserver et différencier des cellules souches, comprenant un acide linoléique conjugué, un acide éicosapentaénoïque, une activine A et/ou un acide stéarique.
PCT/FI2010/050980 2009-12-04 2010-11-30 Formulations et procedes de culture de cellules souches WO2011067465A1 (fr)

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JP2012541546A JP2013512667A (ja) 2009-12-04 2010-11-30 幹細胞を培養するための処方物および方法
CN2010800545573A CN102906247A (zh) 2009-12-04 2010-11-30 用于培养干细胞的制剂和方法
EP10800970A EP2507360A1 (fr) 2009-12-04 2010-11-30 Formulations et procedes de culture de cellules souches
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US10138465B2 (en) 2012-12-31 2018-11-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
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FI20096288A0 (fi) 2009-12-04
CN102906247A (zh) 2013-01-30
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