WO2014053709A1 - Procédé de culture de cellules ouches - Google Patents

Procédé de culture de cellules ouches Download PDF

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Publication number
WO2014053709A1
WO2014053709A1 PCT/FI2013/050966 FI2013050966W WO2014053709A1 WO 2014053709 A1 WO2014053709 A1 WO 2014053709A1 FI 2013050966 W FI2013050966 W FI 2013050966W WO 2014053709 A1 WO2014053709 A1 WO 2014053709A1
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Prior art keywords
stem cell
cells
carbohydrate
binding protein
rho
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PCT/FI2013/050966
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English (en)
Inventor
Milla Mikkola
Tero Satomaa
Jari Natunen
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Glykos Finland Oy
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Publication of WO2014053709A1 publication Critical patent/WO2014053709A1/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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the invention relates to a method for cultur- ing stem cells.
  • the invention also relates to stem cells obtained by the method, to a composition, to a culture system and to a use.
  • Stem cells have great potential in various lines of developmental and genetic research and regenerative medicine. However, they are highly sensitive to culture conditions, and there are several technical issues involved in their cultivation.
  • matrices on which the stem cells can adhere to such as feeder cell layers comprising mouse embryonic fibroblasts.
  • a medium for a feeder-free culture of stem cells including an extracellular matrix extracted from a mouse sarcoma is fre- quently also used; it is sold under the trademark Mat- rigelTM (BD Biosciences, US) .
  • Mat- rigelTM BD Biosciences, US
  • This matrix herein referred to as Matrigel, comprises laminin and collagen in particular, but also a number of other undefined components, and introduces significant lot-to-lot var- iation.
  • the purpose of the present invention is to provide methods for culturing stem cells.
  • the method for culturing stem cells according to the present invention is characterized by what is presented in claim 1.
  • the stem cell or stem cell population according to the present invention is characterized by what is presented in claim 26.
  • the culture system according to the present invention is characterized by what is presented in claim 29.
  • the stem cell population according to the present invention is characterized by what is present ⁇ ed in claim 33.
  • Figure 1 demonstrates analyses of basic char ⁇ acteristics of stem cells cultivated according to the invention ;
  • Figure 2 shows results of clonogenicity and cell growth assays of stem cells
  • Figure 3 demonstrates validation of binding specificity of stem cells
  • Figure 4 shows results of differentiation as ⁇ says of stem cells
  • Figure 5 demonstrates human pluripotent stem cells growing as a cell layer and without colony for ⁇ mation on culture surface coated with ECA lectin.
  • the present invention provides a method and a culturing system suitable for culturing and/or expanding stem cells.
  • At least one stem cell or a stem cell population is seeded or passaged to a cultivation system, which may include, for in- stance, a cultivation vessel, a matrix or substrate and a culture medium.
  • the starting point of the culti ⁇ vation may thus be defined as the time point at which at least one stem cell is contacted with the cultiva ⁇ tion system.
  • the stem cell may be obtained in a number of ways known in the art, for instance isolated from tis ⁇ sue or from ex vivo culture. Alternatively, various stem cell lines may be obtained commercially.
  • Culture medium may be added to culture (cul- tivation system) or replaced as required. Further, additional substances may be added to culture medium or cultivation system as desired.
  • Stem cells are grown and maintained at an appropriate temperature and gas mixture. Typically, 37°C and 5% CO 2 are used for mam ⁇ malian cells. Atmospheric O 2 pressure can be used, or alternatively lowered O 2 pressure (hypoxia) for opti- mized stem cell growth conditions. However, the exact conditions depend on the type of cell and the desired outcome of the cultivation.
  • At least some of the seeded or passaged stem cells typically adhere to a matrix or a substrate in- eluded in the cultivation system, such as a feeder cell layer or Matrigel. This adherence depends on the interaction of the stem cells with the components of the matrix or substrate.
  • stem cells proliferate and form a stem cell population that expands spatially. Frequent ⁇ ly, cells proliferate exponentially during expansion; this growth is often also called the log phase.
  • stem cell proliferation is greatly re- prised or ceases, and the stem cells enter the station ⁇ ary phase.
  • the log phase of stem cells that grow attached to the surface can be lengthened if the cells can be seeded to the culture at a lower density.
  • the cultivation ends.
  • the stem cells may be collected, for in ⁇ stance by detaching from the matrix or substrate by enzymatic and/or mechanical means. Collected stem cells may be seeded or passaged further, or they may be used e.g. for clinical applications. Alternatively, the stem cells and/or the culture system may be dis ⁇ carded .
  • pluripotent stem cells When cultured in commonly used culture sys ⁇ tems, pluripotent stem cells typically exhibit colony growth, i.e. they grow as colonies of up to about a few hundred stem cells. The colonies thus comprise cells growing within the colony with ample cell-to- cell contacts and cells growing on colony borders with less cell-to-cell contacts. The process of colony for ⁇ mation after seeding or passaging pluripotent stem cells is thought to consist of the steps of attachment of individual cells, migration, and proliferation as a colony. Without cell-to-cell contacts, attached cells may detach from commonly used culture substrates and are lost from the culture.
  • Pluripotent stem cells typically display mor- phological characteristics of small and round cell size and shape, high nucleus-to-cytoplasm ratio and growth as colonies and colony morphology, all of which are used in their identification and characterization (Thomson, J. A., et al . Science 282:1145-7, 1998; Takahashi, K., and Yamanaka, S., Cell 126:663-76, 2006) .
  • pluripotent stem cells are often passaged as picked colonies or partly dissociated col ⁇ onies.
  • the common process of gen ⁇ erating new iPS cell lines includes a step of identi- fying the formed iPS cells by their colony morphology and collecting the formed colonies as starting materi- al for cell line generation (Takahashi, K., and Yama- naka, S., Cell 126:663-76, 2006).
  • Pluripotent stem cell colonies may be for ex ⁇ ample elliptical or circular in shape: a typical colo- ny morphology is shown in Figure 1A.
  • Colony size is often restricted in commonly used culture systems be ⁇ cause of low ability of the cells on the colony bor ⁇ ders to expand the colony. Therefore the cells can utilize only a fraction of the available growth sur- face and the growth rate of pluripotent stem cells in culture is significantly hampered.
  • the cells on the colony periphery are also often phenotypically different from the cells inside the colony by for example a different molecular marker expression profile (Sus- tackova, G., et al . Stem Cells Dev. 21(5) :710- 20,2012) .
  • a culture system that would support growth of pluripo ⁇ tent stem cells over the whole available surface and minimize or avoid formation of colony borders with slower growing and phenotypically distinct cells.
  • the present inventors have surprisingly found that the present invention provides for rapid, undif ⁇ ferentiated growth of stem cells.
  • the present inven ⁇ tion thus provides for very high growth and expansion rates of stem cells, when compared with traditional cultivation methods involving e.g. the use of Matrigel or a feeder cell layer.
  • the growth rate of stem cells such as pluripotent stem cells is at least from 20% to 200%, from 30% to 180%, from 40% to 150% or from 50% to 100% higher calculated based on cell number than the growth rate on a complex matrix such as Matrigel or a feeder cell layer.
  • the growth rate of stem cells such as pluripotent stem cells is from 20% to 200%, from 30% to 180%, from 40% to 150% or from 50% to 100% higher based on cell num- ber than on a complex matrix such as Matrigel or a feeder cell layer.
  • the present invention also significantly in ⁇ creases the plating, passaging and cloning efficiency of stem cells.
  • the present invention further allows for maintenance of pluripotency, robust proliferation with a normal karyotype, and the ability to differen ⁇ tiate both in vitro and in vivo.
  • the present invention allows for non-colony growth of stem cells and in particular plu- ripotent stem cells.
  • non- colony growth should be understood as referring to growth as an essentially continuous layer without col ⁇ ony boundaries, in other words limitless growth, with an ability to essentially fill the available surface.
  • the present inventors have surprisingly found that the present invention provides for such limitless non- colony growth of pluripotent stem cells. Since the standard characteristics of both embryonic and induced pluripotent stem cells include a typical colony mor ⁇ phology (Thomson, J. A., et al .
  • the present inventors have sur ⁇ prisingly found that a Rho-associated kinase inhibitor and a carbohydrate-binding protein according to the invention act synergistically to promote significantly higher cell viability after dissociation compared to for example Matrigel, as described in Example 5 and Figure 2: it was found that stem cells cultured on a surface coated with a carbohydrate-binding protein according to the invention become primed for higher viability after another passaging or transfer in presence of a Rho-associated kinase inhibitor to a cell culture surface coated with a carbohydrate-binding protein according to the invention.
  • the stem cells cultured according to the invention have viabil ⁇ ity after dissociation of at least 85%, 87%, 89%, 90%, or 91%.
  • the present invention relates to a method for culturing stem cells, which method comprises the step of contacting at least one stem cell with a carbohydrate-binding protein and a Rho-associated kinase in ⁇ hibitor simultaneously or sequentially at one or more time intervals during the cultivation, wherein the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosaccharide structure ac ⁇ cording to the formula
  • n 0 or 1.
  • the present invention relates to a method for culturing stem cells, which method comprises the step of contacting at least one stem cell with a carbohydrate-binding protein and a Rho-associated kinase in- hibitor simultaneously at one or more time intervals during the cultivation, wherein the carbohydrate- binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to the formula
  • n 0 or 1.
  • the present invention relates to a method for culturing stem cells, which method comprises the step of contacting at least one stem cell with a carbohy- drate-binding protein and a Rho-associated kinase in ⁇ hibitor sequentially at one or more time intervals during the cultivation, wherein the carbohydrate- binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to the formula
  • n 0 or 1.
  • carbohy ⁇ drate-binding protein should be understood as referring to any protein, provided it is capable of binding the non-reducing terminal oligosaccharide structure according to the formula
  • n 0 or 1.
  • a carbohydrate- binding protein should be understood as referring to at least one carbohydrate-binding protein.
  • the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to the formula Fucal-2Ga ⁇ l-4GlcNAc .
  • the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to the formula Ga ⁇ l-4GlcNAc .
  • the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to both the formulas Ga ⁇ l-4GlcNAc and Fucal-2Ga ⁇ l-4GlcNAc.
  • oligosaccharide structure refers to a glycan structure or portion thereof which comprises sugar residues according to the above formula. Such sugar residues may comprise fucose, galactose, or W-acetylglucosamine linked to each other through gly- cosidic bonds in a particular configuration.
  • Glycolipid and carbohydrate nomenclature is essentially according to recommendations by the IUPAC- IUB Commission on Biochemical Nomenclature (e.g. Carbohydrate Res. 1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998, 257, 29) .
  • the oligosaccharide structure is a common structure in gly- cans of undifferentiated stem cells.
  • Fuc should be understood as L-fucose
  • Gal should be un ⁇ derstood as D-galactose
  • Glc should be understood as D-glucose
  • Neuro5Ac should be understood as N- acetylneuraminic acid
  • Neuro5Gc should be understood as N-glycolylneuraminic acid
  • GlcNAc and "N- acetylglucosamine” should be understood as 2- acetamido-2-deoxy-D-glucose; and all monosaccharides are in pyranose form.
  • the non-reducing end terminal structure is not substi- tuted by any other monosaccharide residue or any other substituent on any other positions than at the reduc ⁇ ing end of the oligosaccharide structure.
  • the term "capable of bind ⁇ ing" should be understood as referring to the ability of the carbohydrate-binding protein to bind to the oligosaccharide structure. Binding to the oligosaccha ⁇ ride structure may be assessed by methods known in the art and/or methods described in the examples. As fur ⁇ ther examples only, binding, significant binding and/or kinetic measurements may be assayed e.g. by utilizing surface plasmon resonance-based methods on a Biacore apparatus, by immunological methods such as ELISA or by e.g. carbohydrate microarrays.
  • the initial attachment of stem cells to a ma ⁇ trix or substrate is dependent on the interaction of the stem cells with the matrix or substrate.
  • the car ⁇ bohydrate-binding protein of the invention ensures efficient attachment and is thus a potent simple defined matrix for stem cells.
  • the binding sites of car ⁇ bohydrate-binding proteins comprise a three- dimensional structure compatible to a relatively rigid carbohydrate structure. Therefore the carbohydrate- binding proteins according to the present invention form a group of structurally defined molecules.
  • the carbohydrate-binding protein is a lectin, an antibody or a carbohydrate-modifying protein, or a modification or a fragment thereof.
  • the carbohydrate-binding protein is a lectin.
  • the term "lectin” should be understood as referring to a sugar-binding protein capable of binding to a specific oligosaccharide struc ⁇ ture.
  • Lectins occur ubiquitously in nature and typi- cally are non-enzymatic in action and non-immune in origin. They may bind to a soluble oligosaccharide structure or to an oligosaccharide structure that is a part of a more complex structure, such as a glycopro ⁇ tein or a glycolipid.
  • Lectins may be derived from plants, but may also have an animal origin.
  • Known lec ⁇ tins isolated from plants are, for example, Con A, LCA, PSA, PCA, GNA, HPA, WGA, PWM, TPA, ECA, DSA, UEA- 1, PNA, SNA and MAA.
  • a number of lectins are readily obtainable using methods known in the art or are com- briefly available. Some lectins however, although capable of binding the oligosaccharide structure, can be mitogenic or toxic.
  • the carbohydrate-binding protein is ECA, UEA-1, DSA, RCA, galectin or a fragment thereof. This embodiment has the added utility that individual lectins, depend- ing to some extent on the lectin, are well tolerated and/or non-toxic to cells.
  • the carbohydrate-binding protein has essentially the binding specificity of ECA, UEA-I, DSA, RCA, galectin, an antibody or a carbohydrate-modifying protein, or a modification or a fragment thereof, the binding speci ⁇ ficity being for the non-reducing terminal oligosac ⁇ charide structure Ga ⁇ l-4GlcNAc and/or Fuc l-2Ga ⁇ l- 4GlcNAc.
  • ECA Erythrina cristagalli
  • lectin aggluti ⁇ nin
  • ESL Erythrina cristagalli
  • homologous lectins from Erythrina species e.g. as de ⁇ fined in Bhattacharyya, L., et al . (1989, Glycoconj . J. 6:141-50), WO2010004096, and WO2008087257, such as lectins of E. corallodendron, E. flabelliformis, and E. indica.
  • ECA is capable of binding to N- acetyllactosamine (type 2 chain) glycoconj ugates , a common structure in glycans of undifferentiated stem cells.
  • ECA should be understood as also referring to a carbohydrate-binding protein that has essentially the binding specificity of ECA, the bind- ing specificity being for the non-reducing terminal oligosaccharide structure Ga ⁇ l-4GlcNAc and/or Fuc l- 2Ga ⁇ l-4GlcNAc.
  • the abbreviation "UEA-1” should be understood as referring to a lectin (agglu- tinin-I) from Ulex europeaus .
  • the abbreviation “DSA” should be understood as referring to a lectin from Da ⁇ tura stramonium.
  • RCA should be understood as referring to a non-toxic lec ⁇ tin domain of an agglutinin from Ricinus communis .
  • galectin should be understood as referring to a family of animal lec ⁇ tins capable of binding beta-galactoside, preferably selected from the group of galectins 1-15 encoded by genes named LGALS .
  • galectin is mammalian or human galectin-1.
  • This embodiment has the added utility that mammalian or human galectin-1 has good binding specificity and can sup ⁇ port stem cell attachment and undifferentiated prolif ⁇ eration .
  • the carbohydrate-binding protein is an antibody or any modification or a fragment thereof.
  • the carbohydrate-binding protein may be an scFv, a single domain antibody, an Fv, a VHH antibody, a di- abody, a tandem diabody, a Fab, a Fab', or a F(ab)2, provided it binds said oligosaccharide sequence.
  • Fur- thermore, the antibody, modification or a fragment thereof may be present in monovalent monospecific, multivalent monospecific, bivalent monospecific, or multivalent multispecific forms. Methods for producing and screening antibodies are well known in the art.
  • the antibody or a modification or a fragment thereof is, or comprises the complementary-determining regions of, an antibody specific for Fuc l-2Ga ⁇ l-4GlcNAc (H type II) or Ga ⁇ l-4GlcNAc as defined in WO2008087259.
  • the carbohydrate-binding protein is a carbohydrate- modifying protein.
  • the carbohydrate-modifying protein is selected from the group of glycosyltransferases and gly- cosidases specific for Fuc l-2Ga ⁇ l-4GlcNAc or ⁇ - 4GlcNAc including ST3GalII, ST3GalIV, ST6Gal, ST6GalII, FucT-IV, FucT-IX, FucT-VI, blood group A GalNAc-transferase, blood group B Gal-transferase, l , 2-fucosidase and ⁇ , 4-galactosidase .
  • fragment should be understood as referring to a portion of the carbo- hydrate-binding protein that is capable of binding the oligosaccharide structure.
  • the carbohydrate-binding protein is ECA.
  • This embodiment has the added utility that ECA binds specifically the oligosaccharide structure as defined above.
  • ECA is also a small-sized protein that can easily be produced recombinantly and thus is suitable for GMP use. Fur ⁇ ther, ECA exhibits low mitogenicity and toxicity.
  • the carbohydrate-binding protein exhibits selective binding to the non-reducing terminal oligosaccharide structures Fuc l-2Ga ⁇ l-4GlcNAc and/or Ga ⁇ l-4GlcNAc over other common structures selected from the group of Ga ⁇ l-4 (Fuc l-3) GlcNAc, Fuc l-2Ga ⁇ l-4 (Fuc l- 3) GlcNAc, Neu5Ac 2-3Ga ⁇ l-4GlcNAc and Neu5Ac 2-6Ga ⁇ l- 4GlcNAc.
  • a galectin according to the invention may exhibit additional binding to Neu5Ac 2- 3Ga ⁇ l-4GlcNAc or Neu5Ac 2-6Ga ⁇ l-4GlcNAc .
  • the selective binding means at least 10, 20, 25, 50, 100, or 1000-fold binding affinity compared to the other common structures.
  • the carbohydrate-binding protein may be contacted with at least one stem cell e.g. as a coating on the sur- face of a culture vessel, such as a culture dish or plate.
  • the carbohydrate-binding protein may be contacted with at least one stem cell in immobilized form immobilized to a three-dimensional structure, such as a gel.
  • the carbohydrate-binding protein may be contacted with at least one stem cell e.g. as immobilized on the surface of particles such as microcarriers con ⁇ tained in a culture system or vessel.
  • the carbohydrate-binding protein is contacted with at least one stem cell as a matrix or comprised in a ma- trix.
  • the term "matrix" should be un ⁇ derstood as referring to a substrate to which stem cells can adhere.
  • the matrix may be provided e.g. as a coating on the surface of a culture vessel, such as a culture dish or plate.
  • the matrix may be provided in immobilized form immobilized to a three-dimensional structure, such as a gel.
  • the matrix may be provided e.g. as a coating on the surface of particles, such as microcarriers, contained in a culture system or vessel.
  • the carbohydrate-binding protein may be provided in covalently conjugated form immobilized to the ma ⁇ trix, vessel, three-dimensional structure or particle.
  • the carbo ⁇ hydrate-binding protein may be provided in immobilized or covalently conjugated form as defined in the published publications ⁇ cations WO2010004096 and WO2008087257.
  • the carbohydrate-binding protein according to the invention should be present in an effective amount.
  • the effective amount may depend on, amongst other things, the particular carbohydrate-binding protein, the stem cell, the culture system used and whether the carbohydrate-binding protein is immobilized or provided as a coating.
  • the amount of the carbohydrate- binding protein used in a solution is about 0.1-500 yg/ml, or about 5-200 yg/ml, or about 10-150 yg/ml .
  • the amount of the carbohydrate-binding protein for im- mobilization is about 0.001-50 yg/cm , or about 0.01- 50 yg/cm 2 , or about 0.1-30 yg/cm 2 for a carbohydrate- binding protein with a molecular weight of about 50 kDa, or a corresponding molar density per surface ar- ea.
  • about 1-50 yg/cm 2 , or about 5-40 yg/cm 2 , or about 10-40 yg/cm 2 of the carbohydrate-binding protein is used in a solution to coat a plastic cell culture surface.
  • the concen- tration of the coating solution is about 50-200 yg/ml for a carbohydrate-binding protein with a molecular weight of about 50 kDa, or in a corresponding molar concentration.
  • the colony is split into aggregated cells or even single cells by any method suitable for dissociation, after which the cells are then placed into new culture containers for passaging.
  • Cell passaging is a technique that enables to keep cells alive and growing under cultured condi ⁇ tions for extended periods of time.
  • Single-cell dissociation of stem cells fol ⁇ lowed by single cell passaging may be used in the pre ⁇ sent methods with several advantages, like facilitat- ing cell expansion, cell sorting, defined seeding for differentiation, enabling automatization of culture procedures, cloning and clonal expansion.
  • stem cells may be dissociated into single individual cells, or a combi- nation of single individual cells and small cell clus ⁇ ters comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 cells or more.
  • the dissociation may be achieved by mechanical force, or by a cell dissociation agent, such as cit ⁇ rate, or an enzyme, for example, trypsin, collagenase, or the like.
  • the stem cell (s) can be contacted with the Rho-associated kinase inhibitor before and/or af ⁇ ter dissociation.
  • the stem cell(s) can be contacted with the Rho-associated kinase inhibitor treated only after dissociation.
  • the dissociated cells may be transferred individually or in small clusters to new culture containers in a splitting ratio such as at least or about 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20, 1:40, 1:50, 1:100, 1:150, 1:200, or any range deriva ⁇ ble therein.
  • Suspension cell line split ratios may be done on volume of culture cell suspension.
  • the passage interval may be at least or about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or any range derivable therein.
  • the achievable split ratios for the different enzymatic passaging protocols may be 1:2 every 3-7 days, 1:3 every 4-7 days, and 1:5 to 1:10 approximately every 7 days, 1:50 to 1:100 every 7 days.
  • Passage is defined herein as the growth of stem cells from an initial seed culture in a culture vessel to confluence in the same culture vessel.
  • the stem cell is con ⁇ tacted with a Rho-associated kinase inhibitor during the cultivation.
  • the medium used in the methods of the present invention may already contain the Rho-associated kinase inhibitor or the methods of the present invention may involve a step of adding the Rho-associated kinase inhibitor to the medium.
  • the concentration of the Rho-associated kinase inhibitor in the medium is particularly not limited as far as it can achieve the desired effects such as the improved survival rate of stem cells.
  • a Rho-associated kinase inhibitor e.g.
  • pinacidil may be used at an effective concentration of at least or about 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 500 to about 1000 ⁇ , or any range derivable therein. These amounts may refer to an amount of a Rho-associated kinase inhibitor individu- ally or in combination with one or more Rho-associated kinase inhibitors.
  • the time for contacting stem cell with the Rho-associated kinase inhibitor is particularly not limited as long as it is a time duration for which the desired effects such as the improved survival rate of stem cells can be achieved.
  • the time for treating is at least or about 1, 2, 5, 10, 15, 20, 25, 30 minutes to several hours (e.g., at least or about one hour, two hours, three hours, four hours, five hours, six hours, eight hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 hours, or any range derivable therein) before dissociation.
  • the pluripotent stem cell can be contacted with the Rho- associated kinase inhibitor for, for example, at least or about 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 24, 48 hours or more to achieve the de ⁇ sired effects.
  • the stem cells are contacted with a Rho-associated kinase inhibitor for at most or at least about 0.5, 1, 2, 4, 8, 12 hours, about 2, about 4, or about 6 days, or any range deriv ⁇ able therein.
  • the stem cells are con ⁇ tacted with a Rho-associated kinase inhibitor for at least one to five passages.
  • the Rho-associated kinase inhibitor is subsequently withdrawn from the culture medium, for example after about 0.5, 1, 2, 4, 8, 12 hours or after about 2, about 4, or about 6 days, or any range derivable therein.
  • the Rho-associated kinase inhibitor is withdrawn after at least one, two, three, four, five passages or more, or any range derivable therein.
  • the concentration of the Rho-associated kinase inhibitor is reduced after at least one, two, three, four, five passages or more, or any range derivable therein, and the reduction is at least 2, 5, 10, 100, 1 000, 10 000, 100 000 or 1 000 000 fold compared to the original concentration.
  • the concentration of the Rho-associated kinase inhibi ⁇ tor is reduced after one passage. In one embodiment of the present invention, the reduction is at least 2, 5, 10, 100, 1 000, 10 000, 100 000 or 1 000 000 fold com ⁇ pared to the original concentration. In some embodi- ments, the concentration of the Rho-associated kinase inhibitor is reduced after about 0.1, 0.2, 0.5, 1, 2,
  • This embodiment has the added benefit that the inhibitory activity to cell growth and proliferation is substantially reduced or essentially removed.
  • a culture vessel used for culturing the stem cell (s) can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, mi ⁇ cro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein.
  • the stem cells may be cultured in a volume of at least or about 0.1, 0.2, 0.5, 1, 2,
  • the culture vessel may be a bio- reactor, which may refer to any device or system that supports a biologically active environment.
  • the biore- actor may have a volume of at least or about 2, 4, 5,
  • the culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose.
  • the cellular adhesive culture vessel can be coated with any of carbohydrate binding protein of the pre- sent invention.
  • the substrate for differentiated cell adhesion can be any ma ⁇ terial intended to attach cells.
  • the substrate for cell adhesion includes any of carbohydrate binding protein of the present invention, collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, vitronectin, collagen and fibronectin and mixtures thereof, for ex ⁇ ample MatrigelTM.
  • Rho-associated ki ⁇ nase inhibitor should be understood as referring to any molecule capable of selectively inhibiting Rho- associated protein kinase.
  • Rho-associated protein ki ⁇ nase is a kinase belonging to the AGC ( PKA/PKG/PKC) family of serine-threonine kinases. It is mainly in ⁇ volved in regulating the shape and movement of cells by acting on the cytoskeleton .
  • Rho-associated kinases occur in a number of species, including human, rat, mouse, cow, and zebrafish, only to mention a few.
  • Rho-associated kinase inhibitors include pinacidil, Y-27632, Fasudil (also known as HA1077), Thiazovivin, N-hydroxyfasudil , (S)-(+)-2- methyl-1- [ (4-methyl-5-isoquinolinyl) sulfonyl] homo- piperazine, N- (4-pyridyl) -N' - (2, 4, 6-trichlorophenyl ) urea, 3- (4-pyridyl) -lH-indole, glycyl (S) - (+) -2-methyl- 4-glycyl-l- (4-methylisoquinolinyl-5-sulfonyl) homo- piperazine, azabenzimidazoleaminofurazan, 4- (1-amino- alkyl) -N- (4-pyridyl) cyclo-hexane-carboxamide, and Rho-
  • the Rho-associated kinase inhibitor is selected from the group consisting of pinacidil, Y-27632, Fasudil, Thiazovivin, N-hydroxyfasudil , (S) - (+) -2-methyl-l- [ (4- methyl-5-isoquinolinyl ) sulfonyl] homopiperazine, N- (4- pyridyl) -N' - (2, 4, 6-trichlorophenyl ) urea, 3- (4- pyridyl) -lH-indole, glycyl (S) - (+) -2-methyl-4-glycyl-l- ( 4-methylisoquinolinyl-5-sulfonyl ) homopiperazine, azabenzimidazoleaminofurazan, 4- (1-amino-alkyl) -N- (4- pyridyl) cyclohexane-carboxamide
  • the Rho-associated kinase inhibitor is pinacidil or Y- 27632.
  • the concentration of pinaci ⁇ dil is between 1-1000 ⁇ , between 10-500 ⁇ , between 50-200 ⁇ , or about 100 ⁇ .
  • the concentration of Y-27632 is between 0.1-100 ⁇ , between 1-50 ⁇ , between 5-20 ⁇ , or about 10 ⁇ .
  • the Rho-associated kinase inhibitor may be contacted with at least one stem cell in a solubilized form, e.g. included in a culture medium or added in a cul ⁇ ture medium.
  • the Rho-associated kinase inhibitor may be contacted with at least one stem cell in an immobi ⁇ lized form, e.g. as a component of the coating on the surface of a culture vessel, such as a culture dish or plate, or of matrix such as a three-dimensional struc ⁇ ture, e.g. a gel.
  • the carbohydrate-binding protein is ECA and the Rho- associated kinase inhibitor is pinacidil.
  • This embodi ⁇ ment has the added utility that ECA together with pi- nacidil promotes a highly efficient expansion of stem cells without any impairment of quality.
  • the carbohydrate-binding protein is ECA and the Rho- associated kinase inhibitor is Y-27632. This embodi ⁇ ment has the added utility that ECA together with Y- 27632 promotes a highly efficient expansion of stem cells without any impairment of quality.
  • the carbohydrate-binding protein is a galectin such as mammalian or human galectin-1 and the Rho-associated kinase inhibitor is pinacidil.
  • the carbohydrate-binding protein is a galectin such as mammalian or human galectin-1 and the Rho-associated kinase inhibitor is Y-27632.
  • the stem cell contacted with the carbohydrate-binding protein and the Rho-associated kinase inhibitor has in a preceding step been contacted with or cultured with the carbohydrate-binding protein.
  • the present inven- tors surprisingly detected that cells grown in a pre ⁇ ceding step on the carbohydrate-binding protein according to the invention and then transferred to clonal density into a culture system in contact with both the carbohydrate-binding protein and the Rho- associated kinase inhibitor had higher clonogenicity than the other systems tested (Example 5) ; in other words, synergistic effect was observed between the carbohydrate-binding protein and the Rho-associated kinase inhibitor according to the invention.
  • the stem cell is contacted with or cultured with the carbohydrate-binding protein continuously or at least 2, 5, 10 or 20 times.
  • the stem cell is a human embryonic stem cell.
  • human embryonic stem cell should be understood as referring to a cell derived from 3-5 day old blastocysts, capable of pro ⁇ liferating on a continuous basis when maintained in an appropriate culture environment and of differentiat ⁇ ing.
  • the differentiation of human embryonic stem cells can be detected by the formation of embryoid bodies in vitro and of teratoma in vivo.
  • the embryo is ei ⁇ ther destroyed or not, i.e. it remains alive.
  • the at least one human embryonic stem cell is harvested by a method that does not include the destruction of a human embryo.
  • the stem cell is other than a human embryonic stem cell .
  • the stem cell is a non-human embryonic stem cell.
  • the stem cell is a mammal (non-human) embryonic stem cell. In one embodiment of the present invention, the stem cell is a primate or a mouse or a rat embryonic stem cell.
  • the stem cell is a pluripotent stem cell.
  • the at least one pluripotent stem cell is an induced pluripotent stem cell (iPS) .
  • iPS induced pluripotent stem cell
  • induced pluripo ⁇ tent stem cell should be understood as referring to a pluripotent stem cell derived from any non-pluripotent or differentiated cell, such as an adult somatic cell (e.g. a fibroblast or a blood cell), that has been in ⁇ quizmatic to have all essential features of embryonic stem cells .
  • the at least one stem cell is a stem cell population. In one embodiment of the present invention, the stem cell or stem cell population remains in an essentially undifferentiated state.
  • the stem cell or stem cell population remains in an essentially undifferentiated state through multiple successive culture passages. In one embodiment of the invention, the stem cell or stem cell population remains in an essentially undifferentiated state for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 passages.
  • the term "essentially undif ⁇ ferentiated state" should be understood as referring to a state wherein the stem cell or stem cell popula- tion has not assumed the morphologic and/or functional characteristics of differentiated cells, and cultures of stem cells are described to be in an essentially undifferentiated state when a substantial proportion of stem cells and their progeny in the population dis- play morphological characteristics of undifferentiated cells, clearly distinguishing them from differentiated cells of embryo or adult origin.
  • Stem cells are recog ⁇ nized by those skilled in the art, and pluripotent stem cells typically appear in the two dimensions of a microscopic view as cells with e.g. high nucle- ar/cytoplasmic ratios and prominent nucleoli. It is understood that colonies of undifferentiated cells or stem cells can have neighboring cells that are differentiated .
  • the term "differentiation” should be understood as referring to the cellular de ⁇ velopment, in which a more undifferentiated cell, or a stem cell, undergoes progressive physiological changes to become a more differentiated cell type having a characteristic function. Differentiation can be assayed by measuring an increase in one or more cell- differentiation specific markers relative to the ex- pression of the undifferentiated cell or stem cell markers. The state of differentiation of a stem cell, stem cell culture, or stem cell population can be assessed by morphological characteristics. Undifferenti- ated stem cells have a characteristic morphology, i.e., small and compact cells with clearly defined cell borders, a morphology which can be easily seen by examination of a stem cell culture under a microscope.
  • stem cell cultures By contrast, cells which have differentiated appear larger and more diffuse with indistinct cell borders. While some differentiated cells can, and normally do, appear at the margin of colonies of undifferentiated cells, the optimal stem cell culture is one that pro ⁇ liferates in the culture vessel with only minimal num- bers of cells at the periphery of the culture appear ⁇ ing to be differentiated. With experience, stem cell researchers can judge the status of differentiation and health of human pluripotent stem cell cultures visually with good accuracy.
  • biochemical cell markers are routinely used to track the status of stem cells.
  • transcription factor Oct-4 is regarded as the most reliable marker of undifferentiated status for ES cells.
  • Other bio- chemical markers used to track the status of undiffer ⁇ entiated stem cells include: SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, alkaline phospha ⁇ tase, H-typel, BMP, Sox2, CXCR4, SSEA-1, Nanog, anti- human alpha-l-fetoprotein, and FOXA2.
  • cardiomyocytes should be understood as referring generally to any cardiomyocytes lineage cells, and can be taken to ap ⁇ ply to cells at any stage of cardiomyocytes ontogeny without any restriction, unless otherwise specified.
  • cardiomyocytes may include both cardiomy- ocyte precursor cells and mature cardiomyocytes.
  • hepatocytes should be understood as referring generally to any hepatocyte lineage cells, and can be taken to apply to cells at any stage of hepatocyte ontogeny without any restriction, unless otherwise specified.
  • hepatocytes may include both cells differentiated into definitive endoderm, hepatocyte precursor cells, oste ⁇ oblasts and mature hepatocytes.
  • neurons should be understood as referring generally to any neuron line ⁇ age cells, and can be taken to apply to cells at any stage of neurons ontogeny without any restriction, unless otherwise specified.
  • neurons may in ⁇ clude both neuron precursor cells and mature neurons.
  • the cultured stem cells of the present inven ⁇ tion may be differentiated into any suitable cell type by using differentiation techniques known to those of skill in the art.
  • the stem cells are cultured in conditions to support differentiation to cells such as retinal pigment epithelium, definitive endoderm, pancreatic beta cells and precursors to pancreatic be ⁇ ta cells, hematopoietic precursors and hemangioblastic progenitors, neurons, respiratory cells, muscle pro- genitors, cartilage and bone-forming cells, gastroin ⁇ testinal cells, liver cells, kidney cells, cardiac muscle cells, as well as many other useful cell types of the endoderm, mesoderm, and endoderm.
  • cells such as retinal pigment epithelium, definitive endoderm, pancreatic beta cells and precursors to pancreatic be ⁇ ta cells, hematopoietic precursors and hemangioblastic progenitors, neurons, respiratory cells, muscle pro- genitors, cartilage and bone-forming cells, gastroin ⁇ testinal cells, liver cells, kidney cells, cardiac muscle cells, as well as many other useful cell types of the endoderm, mesoderm, and endoderm.
  • cytokines such as interleukins , interferons, leukemia inhibitory factor, macrophage colony- stimulating factor, monocyte chemotactic proteins, ac- tivins, amphiregulins , angiogenins, endothelial cell growth factors, neurotrophic growth factors, epidermal growth factors, estrogen receptors, fibroblast growth factors, heparin, granulocyte colony stimulating fac- tor, granulocyte macrophage colony stimulating factor, hepatocyte growth factor, insulin, insulin growth factor binding proteins, insulin-like growth factor bind ⁇ ing proteins, insulin-like growth factors, transform- ing growth factors, tumor necrosis factors, vascular endothelial growth factors, bone morphogenic proteins, enzymes that alter the expression of hormones and hor ⁇ mone antagonists, and extracellular matrix components such as fibronectin, proteolytic fragments of fibron-
  • Pluripotent stem cells may be induced to form embryoid bodies, for example using the methods de ⁇ scribed in Itskovitz-Eldor (2000, Mol Med. 6:88-95).
  • the embryoid bodies contain cells of all three embry- onic germ layers.
  • the embryoid bodies may be further induced to differentiate into different lineages for example by exposure to the appropriate induction fac ⁇ tor or an environmental change.
  • Stem cells can be induced to differentiate to the neuroectodermal and neural lineages by culture in media containing appropriate differentiation factors.
  • Such factors may include one or more of activin A, retinoic acid, basic fibroblast growth factor (bFGF) , and antagonists of bone morphogenetic protein (BMP) , such as noggin (Niknejad et al . European Cells and Ma ⁇ terials Vol. 19 2010 pages 22-29).
  • activin A activin A
  • bFGF basic fibroblast growth factor
  • BMP bone morphogenetic protein
  • Cells differentiating towards the neural lin ⁇ eage may be identified by expression of neural mark ⁇ ers, such as Pax6, Nestin, Map2, beta-tubulin III and GFAP .
  • Cells of the neural lineage may cluster to form neurospheres (which may be nestin-positive cell aggre ⁇ gates) , and these may be expanded by application of selected growth factors such as EGF and/or FGF1 and/or FGF2.
  • Cardiomyocytes may be prepared by inducing differentiation of stem cells by modulation of the MAP kinase pathway as described in Graichen et al (2008, Differentiation, 76:357-370).
  • a medium that contains serum or serum equivalent promotes foci of con ⁇ tracting cells of the cardiomyocyte lineage: for exam ⁇ ple, about 20% fetal bovine serum, or a serum supple- ment such as B27 or N2 in a suitable growth medium such as RPMI .
  • More exemplary methods of cardiac dif ⁇ ferentiation may include methods described by Zhang, et al. (2009, Circ Res. 104:e30-41) or U.S. Patent Publication Nos. 20080038820, 20080226558, 20080254003 and 20090047739.
  • Retinal cell differentiation can be induced by methods described in the International Patent Ap ⁇ plication PCT/FI2011/051142.
  • Hepatocyte differentiation and differentia ⁇ tion to the hepatocyte lineage can be induced by meth ⁇ ods known in the art, for example in EP184905 and EP2256187 for induction of human pluripotent stem cells toward the hepatocyte lineage or by using a car ⁇ bohydrate-binding protein as described in Example 7.
  • the present invention also relates to a meth ⁇ od for culturing and/or expanding undifferentiated stem cells, which method comprises the step of con ⁇ tacting at least one stem cell with a carbohydrate- binding protein and a Rho-associated kinase inhibitor simultaneously at one or more time intervals during the cultivation, wherein the carbohydrate-binding protein is capable of binding the oligosaccharide struc ⁇ ture according to the formula
  • n 0 or 1
  • stem cell remains in an es ⁇ sentially undifferentiated state.
  • At least one stem cell may be contacted with a carbohydrate- binding protein of the invention and with a Rho-kinase inhibitor simultaneously at one or more time intervals during the cultivation.
  • the stem cell is contacted with the carbohydrate- binding protein and the Rho-associated kinase inhibi ⁇ tor simultaneously when the stem cell is seeded or passaged to the cultivation.
  • the stem cell is contacted with the carbohydrate-binding protein and the Rho-associated kinase inhibitor at a time interval starting from the starting point of the cul ⁇ tivation. The end point of the time interval may be soon thereafter, e.g. when the stem cell has effectively adhered.
  • the end point of the time interval may be e.g. at the beginning of, during or after the expansion phase.
  • This embodiment has the added utility that contacting the stem cell with the carbohydrate-binding protein and the Rho-associated kinase inhibitor increases and improves cell survival and adherence. This is especially important e.g. dur ⁇ ing normal passaging, recovery from frozen stock samples, and cloning or subcloning of stem cells.
  • At least one stem cell is contacted with a carbohydrate-binding protein and a Rho-associated kinase inhibitor simulta ⁇ neously at one or more time intervals during the cul ⁇ tivation, and in a further step the stem cell is cul- tured in conditions for supporting differentiation of the stem cell.
  • At least one stem cell is contacted with a carbohydrate-binding protein and a Rho-associated kinase inhibitor simulta- neously at one or more time intervals during the cul ⁇ tivation, and in a further step the stem cell is cultured in conditions for supporting differentiation of the stem cell and contacted with a substrate or ma ⁇ trix .
  • At least one stem cell is contacted with a carbohydrate-binding protein and a Rho-associated kinase inhibitor simulta- neously at one or more time intervals during the cul ⁇ tivation, and in a further step the stem cell is cultured in conditions for supporting differentiation of the stem cell, contacted with a substrate or matrix, and the conditions support differentiation of stem cell into a hepatocyte, a retinal cell, a cardiomyo- cytes, or a neuron.
  • At least one stem cell is contacted with a carbohydrate-binding protein and a Rho-associated kinase inhibitor simulta ⁇ neously at one or more time intervals during the cul ⁇ tivation, and in a further step the stem cell is cultured in conditions for supporting differentiation of the stem cell and contacted with a carbohydrate- binding protein.
  • At least one stem cell is contacted with a carbohydrate-binding protein and a Rho-associated kinase inhibitor simulta ⁇ neously at one or more time intervals during the cul- tivation, and in a further step the stem cell is cul ⁇ tured in conditions for supporting differentiation of the stem cell, contacted with a carbohydrate-binding protein, and the conditions support differentiation of stem cell into a hepatocyte, a retinal cell, a cardio- myocytes, or a neuron.
  • the carbohydrate-binding protein is ECA.
  • a stem cell population derived from at least one stem cell is contacted with the carbohydrate-binding pro ⁇ tein and the Rho-associated kinase inhibitor simulta ⁇ neously during the expansion of the stem cell popula ⁇ tion.
  • the term “during the expansion” should be understood as referring to one or more time intervals during which the cells proliferate and ex ⁇ pand spatially.
  • at least one stem cell is contacted with a carbohy ⁇ drate-binding protein and a Rho-associated kinase in ⁇ hibitor simultaneously essentially during the entire duration of the cultivation.
  • the term “during the entire duration of the cultivation” should be understood as referring to all or essentially all time points and time intervals between the starting point of the cultivation and the end of the cultiva- tion.
  • At least one stem cell may be contacted with the carbohydrate-binding protein during the entire duration of the cultivation, while the Rho-associated kinase inhibitor is contacted with the stem cell only at one or more time intervals dur ⁇ ing the cultivation.
  • the stem cell is contacted with the carbohydrate- binding protein essentially during the entire duration of the cultivation and the Rho-associated kinase in ⁇ hibitor when the stem cell is seeded or passaged to the cultivation.
  • a stem cell population derived from at least one stem cell is contacted with the carbohydrate-binding pro- tein essentially during the entire duration of the cultivation and with the Rho-associated kinase inhibi ⁇ tor during the expansion of the stem cell population.
  • the stem cell is contacted with the carbohydrate- binding protein essentially during the entire duration of the cultivation and with the Rho-associated kinase inhibitor when the stem cell is seeded or passaged to the cultivation, and during the expansion of the stem cell population the amount of the Rho-associated ki ⁇ nase inhibitor is decreased according to the invention.
  • the method further comprises the step of providing a carbohydrate-binding protein of the invention as a matrix or comprised in a matrix.
  • the method further comprises the step of seeding or passaging at least one stem cell of the invention to cultivation.
  • the stem cell is seeded or passaged to the matrix.
  • the method further comprises the step of bringing the stem cell of the invention in contact with culture me- dium.
  • the cul ⁇ ture medium is a defined medium.
  • the cul ⁇ ture medium is a serum- and/or feeder-free medium. In one embodiment of the invention, the culture medium is never having been exposed to feeder cells.
  • the cul ⁇ ture medium can be supplemented, for example, with a single or a plurality of growth factors selected from, for example, a WNT signaling agonist, TGF- ⁇ , bFGF, IL- 6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11, IL- 5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG, an- giopoietin-like 2 and 3, G-CSF, GM-CSF, po, Shh, Wnt- 3a, Kirre, or a mixture thereof.
  • a WNT signaling agonist TGF- ⁇ , bFGF, IL- 6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11, IL- 5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG, an- giopoiet
  • a culture medium may also contain additional components such as nutrients, amino acids, antibiotics, buffering agents, and the like.
  • a culture medium of the present invention may contain non-essential amino ac ⁇ ids, L-glutamine, Pen-strep, and monothioglycerol .
  • the cul ⁇ ture medium is supplemented with higher concentrations of FGF (10 to 1000 ng/ml) together with GABA (gamma- aminobutyric acid) , PA (pipecolic acid) , Li and TGF- ⁇ to support long term, for example at least for 20 pas ⁇ sages or more, undifferentiated stem cell growth.
  • FGF gamma- aminobutyric acid
  • PA pipecolic acid
  • the cul- ture medium is supplemented with bFGF.
  • FGF may be in ⁇ cluded in a culture medium at a concentration of from about 5 to about 100 ng/mL, 5 to about 50 ng/mL, from about 5 to about 25 ng/mL, from about 25 to about 50 ng/mL, or any range derivable therein.
  • bFGF is included in the defined culture me ⁇ dia at a concentration of about 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or about 75 ng/mL. These concentrations may be particularly useful for media used for the maintenance of stem cells or pluripotent stem cells in an undifferentiated or sub ⁇ stantially undifferentiated state.
  • the preferred FGF is bFGF, also referred to as FGF2, but other FGFs, including at least FGF4, FGF9, FGF17, and FGF18, will suffice for this purpose as well. Other FGFs may also work, even if at higher concentrations, which can be empirically determined by researchers .
  • the cul ⁇ ture medium is a conditioned medium.
  • the method further comprises the step of culturing the at least one stem cell for a desired duration. It has been reported that some pluripotent stem cells and cell lines can be adapted to non-colony growth as monolayers when seeded to the culture sur ⁇ face in high density, for example at about 200000 cells/cm2 or from 20000 cells/cm 2 to 200000 cells/cm 2 , in presence of a Rho-associated kinase inhibitor. In these methods, cells become confluent in a short time after which they need be harvested or seeded again to another surface.
  • the present invention allows for more efficient plating or seeding of the cells in lower density, even down to clonal density, to achieve non- colony monolayer growth during culture.
  • a stem cell population derived from at least one stem cell is seeded or passaged to cultivation at a low seeding density .
  • the low seeding density is a seeding density in the range of 0.1 to 20000 cells/cm 2 , or 0.1 to 15000, or 0.1 to 10000, or 0.1 to 7500, or 0.1 to 5000, or 0.1 to 2500, or 0.1 to 1000, or 0.1 to 500, or 0.1 to 100, or 1 to 50 cells/cm 2 , or about 10 cells/cm 2 , or 5 to 15, or 7 to 13, or 8 to 12 cells/cm 2 , or about 35 cells/cm 2 , or to 16 to 45, or 25 to 45, or 30 to 40, or 33 to 37 cells/cm 2 .
  • a single passage of pluripotent stem cell culture is a long time passage lasting more than 4 days, or more than 5, 6, 7, 8, 9, 10, 12, 14, 17 or 21 days or more.
  • the cells are viable, attached to the surface and not apoptotic at the end of the long time passage .
  • the passage is a single passage lasting more than 4 days, or more than 5, 6, 7, 8, 9, 10, 12, 14, 17 or 21 days or more.
  • the cells are viable, attached to the surface and not apoptotic at the end of the passage.
  • the stem cells undergo a large fold expansion of over 5-fold, or over 7-, 8-, 9-, 10-, 12-, 15,- 20-, 25-, 30-, 35-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 125-, 150, 200-, 300-, 500-, 700-, 1000-, 1500-, 2000-, 5000-, 7000-, 10000-, 20000-, 40000-, 70000- or over 100000-fold, or even over 200000-, 500000-, 700000-, 1000000 or 10000000-fold during a single passage.
  • the stem cells are seeded onto the culture surface with the low seeding density of the invention and the passage is the long time passage according to the in ⁇ vention .
  • a stem cell population derived from at least one stem cell is seeded or passaged to cultivation at a low seeding density and the passage is a single passage lasting more than 4 days, or more than 5, 6, 7, 8, 9, 10, 12, 14, 17 or 21 days or more.
  • the stem cell population derived from at least one stem cell is dissociated into single cells during pas ⁇ saging .
  • pluripotent stem cells or cell lines have been reported to adapt to non-colony growth as monolayers when seeded to the culture at a high seed- ing density in the presence of Rho-associated kinase inhibitor even on uncoated plastic surface or regard ⁇ less of the type of the surface coating matrix. Howev ⁇ er, most pluripotent stem cell lines require a suita ⁇ ble matrix for attaching to the culture surface and for efficient proliferation in the pluripotent state even on the presence of a Rho-associated kinase inhib ⁇ itor .
  • the method for culturing stem cells produces stem cells that are homogeneous and borderless.
  • the method for culturing stem cells produces stem cell colonies or populations that are homogeneous and bor ⁇ derless .
  • the stem cells have homogeneous expression of stem cell markers such as BMP and low expression of markers indicating differentiation such as SOX.
  • the steps defined above may be performed in any order.
  • the order of the steps defined above may depend e.g. on the culture system, the culture vessel, the form in which the matrix is provided and the stem cell.
  • the present invention also relates to the use of a carbohydrate-binding protein and a Rho-associated kinase inhibitor for increasing the efficiency of ex- pansion of stem cells.
  • the efficiency of expansion of stem cells is increased in comparison to the efficiency of expansion of stem cells cultured without the use of the carbohydrate- binding protein and the Rho-associated kinase inhibi ⁇ tor .
  • the efficiency of expansion of stem cells may be assayed by methods known in the art or by methods described in the examples. In one embodiment of the present invention, the efficiency of expansion of stem cells is assayed by cell counting. In one embodiment of the present invention, the efficiency of expansion of stem cells is assayed by live cell imaging.
  • the present invention also relates to the use of a carbohydrate-binding protein and a Rho-associated kinase inhibitor for culturing stem cells as non- colony cells.
  • non-colony cells should be understood as referring to stem cells exhib ⁇ iting non-colony growth. In other words, non-colony cells grow as an essentially continuous layer without colony boundaries, essentially filling the available surface, without restriction to colonies.
  • the present invention also relates to a stem cell or stem cell population obtained by the method of the invention.
  • the stem cell or stem cell population obtained by the method of the invention is an isolated stem cell or stem cell population.
  • the present invention also relates to a com- position comprising the stem cell or stem cell population obtained by the method of the invention.
  • the present invention also relates to a com ⁇ position comprising the stem cell or stem cell population obtained by the method of the invention for use as a medicament.
  • the present invention also relates to a cul ⁇ ture system for culturing and/or expanding stem cells, which culture system comprises a culture vessel, at least one stem cell, a carbohydrate-binding protein, a Rho-associated kinase inhibitor, culture medium and optionally a matrix.
  • the culture medium and matrix do not comprise inhibi ⁇ tory amounts of non-reducing terminal oligosaccharide structures according to the formula
  • inhibitory amounts should be understood as referring to amounts that do not significantly inhibit stem cell adherence.
  • Glycoproteins used in cell culture media and matrices such as transferrin, serum proteins, and extracellular matrix components such as fibronectin and laminin may comprise such inhibitory oligosaccharide structures depending on the source and manufacturing process of the glycoprotein.
  • the amount of the inhibitory non-reducing terminal ol ⁇ igosaccharide structures is limited so that the method of the invention is not inhibited.
  • the amounts of the inhibitory non-reducing terminal oligosaccharide structures may be determined by inhibition experiments (described in e.g. Example 6 and Figure 3)
  • the amount of free oligosaccharide containing the non- reducing terminal oligosaccharide structures is less than 100 mM. This embodiment has the added benefit that the inhibition of the method is significantly re ⁇ cuted .
  • the present invention revealed that the inhibition factor was similar with or without addition of a Rho-associated kinase inhibitor, even it has been demonstrated that Rho-associated kinase in ⁇ hibitor strengthens stem cell adhesion to culture sur ⁇ face (Meng, G., et al. 2012. Stem Cells Dev 21:2036- 48) .
  • the present invention thus revealed that control of the inhibitory structure concentration in the medi- urn can be highly beneficial.
  • the culture medium comprises transferrin.
  • the amounts of non-reducing terminal oligosaccharide and other inhibitory structures are defined so as not to inhibit the method of the invention.
  • the present invention also revealed by analysis of culture medium supplements (Example 9) that acceptable of amounts of the inhibitory non-reducing terminal oligosaccharide structures may be defined so as not to inhibit the method of the invention.
  • Transferrin is often added to stem cell culture media and it is an essential compo ⁇ nent of validated pluripotent stem cell culture sys ⁇ tems. Transferrin that is available in industrial scale is often isolated from animal serum, most often bovine serum, or from human serum.
  • Examples of such products used as stem cell medium supplements were an ⁇ alysed in Example 9 and found to contain variable amounts of the inhibitory structures according to the invention.
  • the specific amount of the inhibitory structures was found to depend on the level of sialylation and vary from product to product. Since the product specifications of cell cul ⁇ ture media often do not disclose amounts of specific glycoprotein components or amounts of the inhibitory structures according to the present invention, it is impossible to know without analysing or testing a me ⁇ dium whether it will inhibit the method of the present invention. Since amounts of the inhibitory structures may also vary significantly from lot to lot, e.g. due to variable source of material, animal age or condi- tion, or manufacturing process, it is also impossible to know by testing one lot of a supplement whether a next lot will be similar with regard to amount of the inhibitory structures.
  • the amounts of the inhibitory non-reducing terminal oligo ⁇ saccharide structures in the form of non-sialylated serum transferrin N-linked glycan antennae are within the range of 1.5 nM to 5 ⁇ . In one embodiment of the invention, the amount of the inhibitory non-reducing terminal oligosaccharide structures depends on the source and glycoform of the supplement and on the for- mulation of the medium.
  • the culture medium and matrix comprise less than lOOmM, lOmM, ImM, ⁇ , ⁇ , 5 ⁇ , ⁇ , or 0.3 ⁇ of the inhibitory non-reducing terminal oligosaccharide structures.
  • the culture medium comprising transferrin comprises between 1.5nM and 50 ⁇ , between 5nM and 5 ⁇ , between 5nM and ⁇ , or between 25nM and 0.3 ⁇ of the inhibitory non-reducing terminal oligosaccharide structures.
  • the culture medium and matrix does not comprise the inhibitory non-reducing terminal oligosaccharide structures .
  • the culture system contains only non-glycoprotein components such as recombinant proteins produced in bacteria.
  • the carbohydrate-binding protein according to the invention is a non-glycoprotein.
  • the carbohydrate-binding protein according to the invention is non-glycosylated ECA such as recombinantly produced ECA e.g. as described by Stancombe et al . (2003, Pro ⁇ tein Expr. Purif. 30:283-92).
  • the culture system contains selected or produced gly- coprotein components with controlled glycosylation with regard to the inhibitory non-reducing terminal oligosaccharide structures.
  • the culture system comprises culture medium comprising transferrin.
  • the culture medium comprises transferrin in concentrations between 0.5 mg/1 and lmg/1, between 0.5mg/l and lOOmg/1, between 1 mg/1 and 50mg/l, or between 5 mg/1 and 20mg/l.
  • the transferrin is serum transferrin.
  • the transferrin is human serum transferrin.
  • the transferrin is sialylated with nLN between 0 (fully sialylated) and 0.5 (50 % si alylated) as defined for nLN in Example 9; in pre ⁇ ferred embodiments the transferrin is sialylated with nLN between 0.1 and 0.5, between 0.2 and 0.4 or between 0.2 and 0.35. In one embodiment, the transferrin is sialylated with Neu5Ac. In some embodiments, the Neu5Gc content of the transferrin is below 5%, below 1%, below 0.1%, below 0.01% or below 0.001% of the to ⁇ tal amount of Neu5Ac and Neu5Gc.
  • the inhibitory non-reducing terminal oligosaccharide structure is in the form of non-sialylated glycopro ⁇ tein, a non-sialylated N-glycoprotein, a non- sialylated O-glycoprotein or a non-sialylated N- and O-glycoprotein, or a mixture thereof.
  • its concentration is equivalent to or less than that of the transferrin N-linked glycan antennae with ⁇ in the range of 1.5 nM to 5 ⁇ .
  • the culture medium has pH controlled between 6.8 and 7.8.
  • the culture system comprises cell culture medium with controlled pH.
  • the formation of the inhibitory non-reducing terminal oligosaccharide structures from sialylated glycoproteins such as transferrin may be avoided.
  • Human lysosomal sialidase and cytosolic sialidase have pH optima at about 4.5 and 6.5, respectively.
  • medium pH is lowered to from about 6.5 to about 7.0, their release into the culture medium during stem cell culture can generate the inhibitory oligosaccharide structures from si ⁇ alylated glycoproteins such as transferrin.
  • sialylated glycoproteins such as transferrin are known to undergo acid-catalyzed desialylation to asialogly- coprotein comprising the inhibitory oligosaccharide structures in acidic pH.
  • stem cell cultures are often controlled only by phenol red colour indica ⁇ tor comprised in the medium. Phenol red changes colour to yellow in the pH range from about 6.5 to about 6.8.
  • the present invention revealed that precise con ⁇ trol of the medium pH may be highly advantageous when using sialylated glycoprotein supplements such as transferrin.
  • the medium pH is controlled to pH range not lower than from about 6.8 to about 7.0.
  • the pH of the medium comprised in the culture system is controlled above the pH optima of sialidases or to or above neutral pH 7.0.
  • the gen ⁇ eration of the inhibitory non-reducing terminal oligosaccharide structures may be avoided.
  • pH ranges from 6.8 to 7.6 are generally acceptable for mammalian cell culture and from 6.6 to 7.8 are such that mammalian cells can survive in cell culture, while pH ranges from 7.2 to 7.4 are optimal for mammalian cell culture.
  • Methods to control the medium pH in cell culture are known in the art and include e.g. choosing medium with higher buffer capacity, timely medium changes, pH measurement during culture, and changing the medium pH by addition of e.g. carbon dioxide to lower pH or carbonate salt to increase pH.
  • the medium comprised in the culture system comprising sialylated glycoprotein such as transferrin has pH from 6.8 to 7.8, from 7.0 to 7.8, from 7.0 to 7.6, from 7.1 to 7.5, or from 7.2 to 7.4.
  • the present in- vention also relates to a stem cell population cul ⁇ tured and/or expanded according to the method of the invention, which stem cell population exhibits at least two of the features i-v as compared to a second stem cell population grown on a complex matrix:
  • the stem cell population cultured and/or expanded according to the method of the inven- tion exhibits a faster proliferation rate
  • the stem cell population cultured and/or expanded according to the method of the inven ⁇ tion exhibits a higher proportion of cells in essentially undifferentiated state
  • the stem cell population cultured and/or expanded according to the method of the inven- tion exhibits a morphology as a continuous cell layer
  • the stem cell population cultured and/or expanded according to the method of the inven ⁇ tion is substantially free or over 99%, over 99.5%, over 99.7%, over 99.8%, or over 99.9% free of cells growing on colony borders.
  • second stem cell population should be understood as referring to a comparable stem cell population undergoing cultivation in comparable conditions, but grown on a complex ma ⁇ trix .
  • the complex matrix is Matrigel or a feeder cell layer.
  • the feeder cell layer may comprise e.g. mouse embryon- ic fibroblasts.
  • the present invention also relates to the use of a carbohydrate-binding protein and a Rho-associated kinase inhibitor for culturing stem cells essentially in a monolayer.
  • the present invention also relates to the use of a carbohydrate-binding protein and a Rho-associated kinase inhibitor for rapid culturing of stem cells.
  • Stem cells and differentiated cells of the present invention may also be used to screen for fac ⁇ tors (such as solvents, small molecule drugs, pep ⁇ tides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipula ⁇ tion) that affect the characteristics of stem cells or differentiated cells.
  • Stem cells and differentiated cells of the present invention may also be used to screen for fac- tors that promote pluripotency, or differentiation.
  • differentiated cells are used to screen factors that promote maturation, or promote proliferation and maintenance of such cells in long- term culture. For example, candidate maturation fac- tors or growth factors are tested by adding them to cells in different vessels, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.
  • Particular screening applications relate to the testing of pharmaceutical compounds in drug re ⁇ search ("In vitro Methods in Pharmaceutical Research", Academic Press, 1997) .
  • Assessment of the activity of candidate pharmaceutical compounds generally involves combining the stem cells or differentiated cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (com ⁇ pared with untreated cells or cells treated with an inert compound) , and then correlating the effect of the compound with the observed change.
  • a product, or a use, or a method to which the inven ⁇ tion is related, may comprise at least one of the em ⁇ bodiments of the invention described hereinbefore.
  • hESC lines FES 29, FES 30 and H9
  • hiPS induced pluripotent stem cell line
  • Matrigel as previously described (Vuoristo S, Virtanen I, Tak- kunen M, Palgi J, Kikkawa Y, et al . (2009) Laminin isoforms in human embryonic stem cells: synthesis, re ⁇ ceptor usage and growth support. J Cell Mol Med 13: 2622-2633; Mikkola M, Olsson C, Palgi J, Ustinov J, Palomaki T, et al . (2006) Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Dev Biol 6: 40).
  • HEL11.4 was generated from adult fibroblasts (male, 84 years old) using ret- rovirus-induced overexpression of four genes: Oct-4, Sox2, Klf4, and c-Myc.
  • Cells were infected with equal parts of hES medium and virus-containing supernatant twice at 24-h intervals. Cells were harvested and re- seeded on mitotically inactivated treated mouse embry ⁇ onic fibroblast (mEF) layer three days after infec- tion. Twenty-four days post-transduction, ES-like colonies were picked, expanded, and characterized.
  • mEF mitotically inactivated treated mouse embry ⁇ onic fibroblast
  • Cells were passaged by using O.lmg/ml colla- genase IV (Invitrogen) for 5 min at +37°C and harvest ⁇ ed onto ECA ( Sigma-Aldrich) and Matrigel (Becton Dickinson) coated plates and cultured either in StemPro® or in mEF conditioned-medium (CM) (KO-DMEM supplemented with 20% KO-SR, 2 mM Glutamax, 0.1 mM ⁇ - mercaptoethanol , 0.1 mM non-essential amino acids (NE- AA) , all from Invitrogen, Carlsbad, CA, USA) and supplemented with 8 ng/ml recombinant human bFGF (Invi- trogen, Carlsbad, CA, USA) .
  • CM mEF conditioned-medium
  • Pinacidil (Sigma-Aldrich, ⁇ ) was added to culture media during passaging. In some experiments, Y-27632 (10 ⁇ ) was added to culture media during passaging. In all experiments MatrigelTM (BD Biosciences) was used as control matrix. The Mat- rigel plates were prepared as recommended by the manu ⁇ facturer .
  • ECA lectin (Sigma-Aldrich) solution (lmg/ml in PBS) was let to passively adsorb onto surface of the cell culture plates (5yg/cm2) (Nunc, Roskilde, Denmark or Costar, Corning Life Sciences, MA, USA) o/n at +4°C followed by washing twice with PBS.
  • the coated plates were stored at +4°C and used within four weeks.
  • ECA Erythrina cristagalli agglutinin, bind- ing specificity in type 2 N-acetyllactosamine struc ⁇ tures
  • MAA Melthrina amurensis agglutinin, specific for 2,3-linked sialic acid
  • WFA ⁇ Wisteria floribunda agglutinin binding preferentially to N- acetylgalactosamine in a- or ⁇ -linkage
  • PWA Phy- tolacca americana agglutinin, with N-acetylglucosamine specificity, binding also to polylactosamine struc ⁇ tures
  • FES 29 cells were cultured for 9 passages on ECA or Matrigel in CM media.
  • Total RNA was isolated from three separate plates using RNeasy Mini kit (Qiagen, Valencia, CA) and complementary DNA was synthesized from lyg of total RNA using RT 2 First Strand Kit and RT 2 qPCR Master Mixes ( SABiosciences ) according to manufacturer's instruction.
  • the RT 2 qPCR primer Assays were used to study the gene expression profile of genes related to the identification, growth and differentiation of stem cells (array PAHS-081).
  • Karyotype analysis was used to study the gene expression profile of genes related to the identification, growth and differentiation of stem cells.
  • Karyotype was detected by G-banding technique in cyto ⁇ genetics laboratory of the Yhtyneet Medix Laboratories Inc, Helsinki, Finland. Twenty metaphases were exam ⁇ ined from each sample.
  • Figure 1 shows A. Immunohistochemistry of hESC (FES29) and hiPSC (HEL11.4) lines cultured on ECA for 20 passages.
  • B FACS analysis of pluripotency- associated cell surface markers after 20 passages on ECA.
  • C-D Relative expression level of pluripotency associated genes (Oct4, Nanog, Sox2) and early differ ⁇ entiation associated genes (Brachyuru, Goosecoid) by qPCR at passage 1-20. The data were normalized against the level in cells cultured on Matrigel for the same time.
  • Panel C hESC (FES29)
  • Panel D hiPSC (HEL11.4). Error bars indicate SEM.
  • E Error bars indicate SEM.
  • PCR array anal ⁇ ysis of pluripotency and early differentiation gene expression of cells growing either on Matrigel or on ECA at passage 9.
  • Gene profiles were compared between FES 29 on Matrigel and on ECA.
  • Y-axis is the intensi ⁇ ty ratio and
  • X-axis is the average intensity for a given gene measured on two similar HTqPCR arrays. All differences were less than two-fold.
  • Cells were dissociated with TrypLE for 5 minutes and passed through an 80-ym cell strainer (Becton Dickinson) .
  • Dissociated single cells from either ECA or Mat ⁇ rigel were seeded onto both ECA and Matrigel (35 cells/cm 2 ) and cultured in mouse embryonic fibroblast- conditioned medium (CM) supplemented with 8 ng/ml basic fibroblast growth factor (bFGF) .
  • CM mouse embryonic fibroblast- conditioned medium
  • bFGF basic fibroblast growth factor
  • Pinacidil ( ⁇ ) was used during passaging.
  • To evaluate clono- genic capacity cells were alkaline phosphatase stained and colony numbers were counted 10 days after plating .
  • Cell viability analysis Cells were plated and cultured on ECA and Matrigel for 6 days. Cell viability was analyzed in the beginning, on day 3 and on day 6 using Trypan Blue staining of dissociated cells. The results represent eight sepa- rate experiments, each performed in duplicate. Cell viability was tested also on plate without dissocia ⁇ tion using Live/Dead Viability/Cytotoxity Kit (Invi- trogen) according to manufacturer's instructions. Determination of cell growth rate
  • FES 29 and HEL11.4 cells were passaged by collagenase IV to small clumps from ECA and Matrigel and plated on 12 well plates, approximately 6000 cells/well on both matrices. Cells were counted at two time points, day 3 and day 6.
  • Live cell imaging was used as an alternative method.
  • FES 29 and HEL11.4 cells were dissociated by collagenase IV to small aggregates of 10-20 cells from ECA and Matrigel and plated on 12 well plates, approximately 1000 cells/well on both ma ⁇ trices. Cells were let to adhere in the cell culture incubator for 24 hours and the plates were then trans ⁇ ferred into Cell IQ culture platform (CM- Technologies ) . All wells were imaged every second hour for five days. The images were analyzed using Cell IQ Analyzer .
  • C-D Analysis of cell growth rate.
  • Lactose (composed of galactose ⁇ 1,4- linked to glucose) inhibited cell attachment effec ⁇ tively at 100 mM concentration, while the same concen ⁇ tration of saccharose (fructose ⁇ , ⁇ -linked to glu ⁇ cose) had no inhibitory activity. Further, lacto-N- neotetraose oligosaccharide, which contains the ⁇ 1,4- linked galactose epitope, was as effective as lactose (p ⁇ .001) ( Figure 3) .
  • Figure 3 demonstrates the validation of the binding specificity of hESC and hiPSC to ECA using specific competitive inhibitors either in the presence (A) or absence (B) of Pinacidil.
  • the attached cells were counted 20 h after plating. Data represent the mean ( ⁇ SEM) of both cell lines , FES 29 and HEL 11.4. The total number of attached cells was 2-4 fold higher when Pinacidil was used (p ⁇ 0.001).
  • the disaccharides lactose monohydrate (LacH20) and lacto-N-neoteraose (LnNT) inhibited significantly cell attachment onto ECA (***; p ⁇ 0.001) .
  • Hepatic differentiation was done on FES 29, H9 and HEL11.4 cells, which had been cultured either on ECA or Matrigel for at least 10 passages.
  • the differentia ⁇ tion protocol is described in Table 2.
  • HSCs hepatocyte-like cells
  • Matrigel® Matrigel® as a control.
  • HSCs hepatocyte-like cells
  • the DE cells were further differentiated into hepatocyte progenitors with five days DMSO treatment.
  • the cells formed hepatic endoderm with - fetoprotein (AFP) positive progenitors on both matrices (data not shown) .
  • AFP - fetoprotein
  • hPSCs were successful ⁇ ly differentiated into HLCs on ECA matrix and no sig ⁇ nificant difference was detected when compared to cells differentiating on Matrigel.
  • Figure 4 shows hepatic differentiation of cells cultured on either ECA or Matrigel (MG) .
  • A QPCR analysis for pluripotency and endoderm marker gene ex ⁇ pression. Pluripotency gene OCT4, anterior definitive endoderm (DE) marker FOXA2 and CER1. Error bars indicate SEM.
  • B Immunocytochemical characteristics of the DE- cells differentiated on ECA and MG. FOXA2 (green) and OCT4 (red) .
  • C FACS analysis of DE cells express ⁇ ing the endoderm marker CXCR4 differentiated on ECA and on MG.
  • D QPCR results for hepatic markers AFP and Albumin gene expression.
  • E Cell morphology and immunocytochemical characteristics of HLC differentiated on ECA or on MG. Scale bar ⁇ .
  • StemPro XF supplement, KO serum replacement and ITS supplement were purchased from Invitrogen (Life Tech ⁇ nologies) and subjected to N-glycosidase F digestion (Nyman, T.A., et al . , 1998. Eur. J. Biochem. 253:485- 93) .
  • Sialylated N-glycans were isolated by graphitized carbon microcolumn solid-phase extraction (Hemmoranta, H., et al . , 2007. Exp. Hematol. 35:1279-92) and analyzed by MALDI-TOF mass spectrometry in negative ion linear mode (Heiskanen, A., et al . , 2009. Glycoconj . J. 26:367-84). Neutral N-glycan fraction was similarly analyzed in positive ion reflector mode.
  • Stem Cells 25:197-202 together comprising over 90% of the total detected glycan signals.
  • the major acidic N-glycan com- ponents in KO supplement were S2H5N4, S1G1H5N4, G2H5N4, S1H5N4, and G1H5N4, together comprising over 90% of the total detected glycan signals.
  • nLN ⁇ [%Gi (N-2-S) ]
  • x is the total number of detected glycan signals
  • %Gi is the proportion of glycan Gi of the total detected glycans
  • N is the number of N- acetylhexosamine residues in the proposed glycan com- position
  • S is the number of Neu5Ac and Neu5Gc resi ⁇ dues in the proposed glycan composition
  • N-2 is the number of antennae in glycan Gi
  • N-2-S is the max ⁇ imal number of non-reducing terminal Ga ⁇ l-4GlcNAc an- tennae in glycan Gi .
  • nLNmax can be up to 2 in case the bian- tennary N-glycans are essentially completely desialylated (asialotransferrin) .
  • Transferrin is the major glycoprotein added to the Invitrogen' s serum-free medium supplements XF, KO and ITS. According to the manufacturer's specifica ⁇ tions, final transferrin amounts used in cell culture media can vary between 0.5 mg/1 to 100 mg/1 in promot- ing the growth of both adherent and suspension cell cultures (reference cited: Barnes, D., and Sato, G., 1980. Anal. Biochem. 102:255-70). From these figures effective inhibiting oligosaccharide structure concen ⁇ trations originating from transferrin-containing sup- plements can be calculated.
  • nLNmax 2 in the case of asialotransferrin, the concentration is from about 13 nM (0.5 mg/1) up to about 2.5 ⁇ (100 mg/1) .
  • these final medium concentrations of an inhibiting oligosaccharide struc ⁇ ture are not significantly inhibitory to stem cell culture with a carbohydrate-binding protein in the presence of a Rho-associated kinase inhibitor accord ⁇ ing to the invention.

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Abstract

L'invention concerne un procédé de culture de cellules souches, lequel procédé comprend une étape consistant à mettre en contact une cellule souche avec une protéine liant un hydrate de carbone et un inhibiteur de kinase associé à Rho simultanément et à un ou plusieurs intervalles de temps pendant la culture, la protéine liant un hydrate de carbone pouvant lier la structure d'oligosaccharide non réduisante selon la formule (Fucα1-2)nGalβ1-4GlcNAc, où n = 0 ou 1. L'invention concerne également les cellules souches obtenues par ce procédé, ainsi qu'une composition, un système de culture et leur utilisation.
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