WO2014053709A1 - A method for culturing stem cells - Google Patents

Abstract

The 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 inhibitor simultaneously at one or more time intervals during the cultivation, wherein the carbohydrate-binding protein is capable of binding the non-reducing oligosaccharide structure according to the formula (Fucα1-2)nGalβ1-4GlcNAc, wherein n = 0 or 1. The invention also relates to stem cells obtained by the method, composition, culture system and use.

Description

A METHOD FOR CULTURING STEM CELLS

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

Traditional methods for culturing stem cells require the use of complex 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- rigel™ (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.

One of the major problems of the traditional methods and culture systems is that the use of stem cells or extracts in clinical applications is hampered by the presence of material derived from animals pre- sent in complex matrices in the culture. The current culture methods are also laborious and difficult to scale, and there is always a need to culture cells more efficiently, more cost-effectively and in larger numbers or quantities. Furthermore, it is frequently difficult to maintain the stem cells as undifferentiated and in uniform quality. PURPOSE OF THE INVENTION

The purpose of the present invention is to provide methods for culturing stem cells.

SUMMARY

The method for culturing stem cells according to the present invention is characterized by what is presented in claim 1.

The use of a carbohydrate-binding protein and a Rho-associated kinase inhibitor according to the present invention is characterized by what is present^¬ ed in claim 24.

The use of a carbohydrate-binding protein and a Rho-associated kinase inhibitor according to the present invention is characterized by what is present^¬ ed in claim 25.

The stem cell or stem cell population according to the present invention is characterized by what is presented in claim 26.

The composition according to the present invention is characterized by what is presented in claim

27.

The composition according to the present in- vention is characterized by what is presented in claim

28.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this specification, illus^¬ trate embodiments of the invention and together with the description help to explain the principles of the invention. In the drawings:

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; and

Figure 5 demonstrates human pluripotent stem cells growing as a cell layer and without colony for^¬ mation on culture surface coated with ECA lectin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method and a culturing system suitable for culturing and/or expanding stem cells.

During cell cultivation, 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% CO₂ are used for mam^¬ malian cells. Atmospheric O₂ pressure can be used, or alternatively lowered O₂ 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.

After seeding or passaging, the growth of cell cultures typically proceeds from the initial lag phase to the expansion phase. During the expansion or expansion phase, 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. When stem cells occupy all available substrate or matrix and have no room left for expansion, or when the culture medium no longer has the capacity to support fur^¬ ther growth, stem cell proliferation is greatly re- duced or ceases, and the stem cells enter the station^¬ ary phase.

For efficient growth, proliferation and expansion of stem cells in culture, it is advantageous that they stay in the log phase as long as possible. 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.

When the stem cell culture has been main^¬ tained for the desired duration, the cultivation ends. Optionally, 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 .

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. This colony-restricted growth has been demonstrated for both complex matrices such as feeder cells and Matrigel, defined protein substrates such as vitronectin, fibronectin and collagen, and defined synthetic substrates such as polyly- sine (Meng, G., et al . Stem Cells Dev. 21:2036-48, 2012) .

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) . For example, pluripotent stem cells are often passaged as picked colonies or partly dissociated col^¬ onies. As another example, 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) . Thus it would be highly beneficial to use 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

Furthermore, the present invention allows for non-colony growth of stem cells and in particular plu- ripotent stem cells. In this context, the term "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 . Science 282:1145-7, 1998; Takahashi, K., and Yamanaka, S., Cell 126:663- 76, 2006), thorough characterization of the cells growing as a continuous layer was needed to establish their nature as undifferentiated pluripotent stem cells, including both phenotypical and functional characterization as described in the Examples. This feature has the added utility that a higher surface area is available for cell growth in the cell culture system according to the invention.

Furthermore, 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. In some embodiments 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

(Fuc l-2) _nGa^l-4GlcNAc,

wherein 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

(Fucal-2) _nGa^l-4GlcNAc,

wherein 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

(Fuc l-2) _nGa^l-4GlcNAc,

wherein n = 0 or 1.

In this context, the term "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

(Fucal-2) _nGa^l-4GlcNAc,

wherein n = 0 or 1.

In this context, the term "a carbohydrate- binding protein" should be understood as referring to at least one carbohydrate-binding protein.

In one embodiment of the present invention, the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to the formula Fucal-2Ga^l-4GlcNAc .

In one embodiment of the present invention, the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosaccharide structure according to the formula Ga^l-4GlcNAc .

In one embodiment of the present invention, 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.

The term "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.

In this context, the abbreviation "Fuc" should be understood as L-fucose; "Gal" should be un^¬ derstood as D-galactose ; "Glc" should be understood as D-glucose; "Neu5Ac" should be understood as N- acetylneuraminic acid; "Neu5Gc" should be understood as N-glycolylneuraminic acid; and "GlcNAc" and "N- acetylglucosamine" should be understood as 2- acetamido-2-deoxy-D-glucose; and all monosaccharides are in pyranose form.

The notation of the oligosaccharide structure and the glycosidic bonds between the sugar residues comprised therein follows that commonly used in the art, e.g. "Ga^l-4GlcNAc" should be understood as meaning a galactose residue and an W-acetylglucosamine residue linked by a covalent linkage between the first carbon atom of the galactose residue to the fourth carbon atom of the W-acetylglucosamine residue linked by an oxygen atom in the beta configuration.

In an embodiment of the present invention, 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.

In this context, 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.

It is realized that 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.

In one embodiment of the present invention, the carbohydrate-binding protein is a lectin, an antibody or a carbohydrate-modifying protein, or a modification or a fragment thereof.

In one embodiment of the present invention, the carbohydrate-binding protein is a lectin.

In this context, 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- mercially available. Some lectins however, although capable of binding the oligosaccharide structure, can be mitogenic or toxic. In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In this context, the abbreviation "ECA" should be understood as referring to lectin (aggluti^¬ nin) from Erythrina cristagalli (also called ESL) and 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.

In one embodiment of the present invention, the abbreviation "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.

In this context, the abbreviation "UEA-1" should be understood as referring to a lectin (agglu- tinin-I) from Ulex europeaus . In this context, the abbreviation "DSA" should be understood as referring to a lectin from Da^¬ tura stramonium.

In this context, the abbreviation "RCA" should be understood as referring to a non-toxic lec^¬ tin domain of an agglutinin from Ricinus communis .

In this context, the term "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 .

In one embodiment of the present invention, 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 .

In one embodiment of the present invention, the carbohydrate-binding protein is an antibody or any modification or a fragment thereof. As an example on^¬ ly, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the carbohydrate-binding protein is a carbohydrate- modifying protein. In one embodiment of the present invention, the carbohydrate-modifying protein is selected from the group of glycosyltransferases and gly- cosidases specific for Fuc l-2Ga^l-4GlcNAc or Θβΐβΐ- 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 βΐ , 4-galactosidase .

In this context, the term "fragment" should be understood as referring to a portion of the carbo- hydrate-binding protein that is capable of binding the oligosaccharide structure.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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. In an embodiment a galectin according to the invention may exhibit additional binding to Neu5Ac 2- 3Ga^l-4GlcNAc or Neu5Ac 2-6Ga^l-4GlcNAc . In one em^¬ bodiment, the selective binding means at least 10, 20, 25, 50, 100, or 1000-fold binding affinity compared to the other common structures.

In one embodiment of the present invention, 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. In one embodiment of the present invention, 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. Alterna^¬ tively, 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.

In one embodiment of the present invention, the carbohydrate-binding protein is contacted with at least one stem cell as a matrix or comprised in a ma- trix. In this context, 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. Alternatively, the matrix may be provided in immobilized form immobilized to a three-dimensional structure, such as a gel. Alterna^¬ tively, 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. Alternative- ly, the carbohydrate-binding protein may be provided in covalently conjugated form immobilized to the ma^¬ trix, vessel, three-dimensional structure or particle. In one embodiment of the present invention, the carbo^¬ hydrate-binding protein may be provided in immobilized or covalently conjugated form as defined in the publi^¬ 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. In one embodiment of the present invention, 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/cm², or about 0.1-30 yg/cm² for a carbohydrate- binding protein with a molecular weight of about 50 kDa, or a corresponding molar density per surface ar- ea. In one embodiment of the present invention, about 1-50 yg/cm², or about 5-40 yg/cm², or about 10-40 yg/cm² of the carbohydrate-binding protein is used in a solution to coat a plastic cell culture surface. In one embodiment of the present invention, 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.

In some embodiments of stem cell culturing, once a culture container is full, 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.

In certain embodiments, 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. For example, the stem cell(s) can be contacted with the Rho-associated kinase inhibitor treated only after dissociation.

Based on the source of stem cells and the need for expansion, 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. For example, 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.

In some embodiments, the stem cell is con^¬ tacted with a Rho-associated kinase inhibitor during the cultivation. Thereby, 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 μΜ, 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. For example, when the stem cell is a human pluripotent stem cell, 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. After 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.

In certain embodiments, 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.

In other embodiments, the stem cells are con^¬ tacted with a Rho-associated kinase inhibitor for at least one to five passages. Optionally, 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. In other embodiments, the Rho-associated kinase inhibitor is withdrawn after at least one, two, three, four, five passages or more, or any range derivable therein.

In other embodiments, 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.

In one embodiment of the present invention, 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,

4, 8, 12 hours or after about 1, about 2, or about 4 days, or any range derivable therein. 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,

5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range deriva^¬ ble therein, depending on the needs of the culture. In a certain embodiment, 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,

6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 li- ters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein. 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. For differentiation purposes, 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 Matrigel™.

In this context, the term "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. A number of Rho-associated kinase inhibitors are known, including 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- statin and are also commercially available. In this context, the term "a Rho-associated kinase inhibitor" should be understood as referring to at least one Rho- associated kinase inhibitor.

In one embodiment of the present invention, 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, Rhostatin, and any combination thereof.

In one embodiment of the present invention, the Rho-associated kinase inhibitor is pinacidil or Y- 27632. In one embodiment, the concentration of pinaci^¬ dil is between 1-1000 μΜ, between 10-500 μΜ, between 50-200 μΜ, or about 100 μΜ. In one embodiment, the concentration of Y-27632 is between 0.1-100 μΜ, between 1-50 μΜ, between 5-20 μΜ, or about 10 μΜ.

In one embodiment of the present invention, 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. In another embodiment of the present in^¬ vention, 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.

In one embodiment of the present invention, the carbohydrate-binding protein is ECA and the Rho- associated kinase inhibitor is pinacidil or Y-27632.

In one embodiment of the present invention, 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. In one embodiment of the present invention, 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.

In one embodiment of the present invention, the carbohydrate-binding protein is a galectin such as mammalian or human galectin-1 and the Rho-associated kinase inhibitor is pinacidil.

In one embodiment of the present invention, the carbohydrate-binding protein is a galectin such as mammalian or human galectin-1 and the Rho-associated kinase inhibitor is Y-27632.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the stem cell is contacted with or cultured with the carbohydrate-binding protein continuously or at least 2, 5, 10 or 20 times.

In one embodiment of the present invention, the stem cell is a human embryonic stem cell.

In this context, the term "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. In technologies har^¬ vesting human embryonic stem cells, the embryo is ei^¬ ther destroyed or not, i.e. it remains alive. In one embodiment of the invention, the at least one human embryonic stem cell is harvested by a method that does not include the destruction of a human embryo.

In one embodiment of the present invention, the stem cell is other than a human embryonic stem cell .

In one embodiment of the present invention, the stem cell is a non-human embryonic stem cell.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the stem cell is a pluripotent stem cell.

In one embodiment of the present invention, the at least one pluripotent stem cell is an induced pluripotent stem cell (iPS) .

In this context, the term "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^¬ duced to have all essential features of embryonic stem cells .

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In this context, 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 .

In this context, 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. 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.

In addition to morphological characteristics, biochemical cell markers are routinely used to track the status of stem cells. For example, 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 (depending on species) 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.

In this context, the term "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. For example, cardiomyocytes may include both cardiomy- ocyte precursor cells and mature cardiomyocytes. In this context, the term "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. For example, hepatocytes may include both cells differentiated into definitive endoderm, hepatocyte precursor cells, oste^¬ oblasts and mature hepatocytes.

In this context, the term "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. For example, 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.

In an embodiment, 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.

Compounds added to the conditions to support differentiation of stem cells include but are not lim^¬ ited to: 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- ectin, laminin, tenascin, thrombospondin .

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).

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). Using 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

(Fuc l-2) _nGa^l-4GlcNAc,

wherein n = 0 or 1,

and wherein the stem cell remains in an es^¬ sentially undifferentiated state.

According to the present invention, 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. In one embodiment of the present invention, 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. In other words, 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. Alternatively, 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.

In one embodiment, in one step, 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.

In one embodiment, in one step, 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 .

In one embodiment, in one step, 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.

In one embodiment, in one step, 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.

In one embodiment, in one step, 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.

In one embodiment the carbohydrate-binding protein is ECA.

In one embodiment of the present invention, 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. In this context, 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. In one embodiment of the present invention, 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. In this context, 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.

The carbohydrate-binding protein and the Rho- associated kinase inhibitor need not be contacted with at least one stem cell simultaneously at all time in^¬ tervals at which the carbohydrate-binding protein or the Rho-associated kinase inhibitor is contacted with the at least one stem cell. In other words, at least one stem cell may be contacted with the carbohydrate- binding protein and the Rho-associated kinase inhibi^¬ tor partially at different time intervals, provided that the stem cell is contacted with a carbohydrate- binding protein and a Rho-associated kinase inhibitor simultaneously at least at one time interval during the cultivation. For instance, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the method further comprises the step of providing a carbohydrate-binding protein of the invention as a matrix or comprised in a matrix.

In one embodiment of the present invention, the method further comprises the step of seeding or passaging at least one stem cell of the invention to cultivation. In a further embodiment, the stem cell is seeded or passaged to the matrix.

In one embodiment of the present invention, the method further comprises the step of bringing the stem cell of the invention in contact with culture me- dium.

In one embodiment of the invention, the cul^¬ ture medium is a defined medium.

In one embodiment of the invention, 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.

In one embodiment of the invention, 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-β, 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 culture medium may also contain additional components such as nutrients, amino acids, antibiotics, buffering agents, and the like. In certain embodiments a culture medium of the present invention may contain non-essential amino ac^¬ ids, L-glutamine, Pen-strep, and monothioglycerol .

In one embodiment of the invention, 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-β to support long term, for example at least for 20 pas^¬ sages or more, undifferentiated stem cell growth.

In one embodiment of the invention, 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. In certain em- bodiments, 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 .

In one embodiment of the invention, the cul^¬ ture medium is a conditioned medium.

In one embodiment of the present invention, 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/cm² to 200000 cells/cm², 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. This has the benefit that the cells stay in log phase and grow ex^¬ ponentially for a longer period in each passage, which leads to higher overall growth and expansion rate. In one embodiment of the present invention, a stem cell population derived from at least one stem cell is seeded or passaged to cultivation at a low seeding density .

In one embodiment, the low seeding density is a seeding density in the range of 0.1 to 20000 cells/cm², 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/cm², or about 10 cells/cm², or 5 to 15, or 7 to 13, or 8 to 12 cells/cm², or about 35 cells/cm², or to 16 to 45, or 25 to 45, or 30 to 40, or 33 to 37 cells/cm².

In an embodiment of the present invention, 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. In an embodiment the cells are viable, attached to the surface and not apoptotic at the end of the long time passage .

In one embodiment of the present invention, 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. In one embodiment of the present inven^¬ tion, the cells are viable, attached to the surface and not apoptotic at the end of the passage.

In an embodiment of the present invention, 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.

In an embodiment of the present invention, 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 .

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the stem cell population derived from at least one stem cell is dissociated into single cells during pas^¬ saging .

Some 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 . In an embodiment of the present invention, the method for culturing stem cells produces stem cells that are homogeneous and borderless.

In an embodiment of the present invention, the method for culturing stem cells produces stem cell colonies or populations that are homogeneous and bor^¬ derless .

In another embodiment of the present inven^¬ tion, the stem cells have homogeneous expression of stem cell markers such as BMP and low expression of markers indicating differentiation such as SOX.

In different embodiments of the method, 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.

In one embodiment of the present invention, 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. In this context, the term "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.

In one embodiment of the present 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.

In one embodiment of the present invention, the culture medium and matrix do not comprise inhibi^¬ tory amounts of non-reducing terminal oligosaccharide structures according to the formula

(Neu5R¹ 2-3)_m(Fuc l-2) _nGa^R²,

wherein R¹ is Ac or Gc; m = 0 if the carbohydrate-binding protein is not a galectin; m = 0 or 1 if the carbohydrate-binding protein is a galectin; n = 0 or 1-m; and R² is l-4GlcNAc or l-4Glc or absent.

In one embodiment of the present invention, the culture system does not comprise inhibitory amounts of non-reducing terminal oligosaccharide structures according to the formula (Fuc l-2 ) _nGa^R, wherein n = 0 or 1; and R = l-4GlcNAc or l-4Glc or any glycan structure or absent.

In this context, the term "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.

In one embodiment of the present invention, 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)

In one embodiment of the present invention, 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^¬ duced .

Surprisingly, 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.

In one embodiment of the present invention, the culture medium comprises transferrin. In one embodiment of the present invention, 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. In serum transferrins, 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.

In one embodiment of the invention, 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 μΜ. 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.

In an embodiment of the present invention, the culture medium and matrix comprise less than lOOmM, lOmM, ImM, ΙΟΟμΜ, ΙΟμΜ, 5μΜ, ΙμΜ, or 0.3μΜ of the inhibitory non-reducing terminal oligosaccharide structures. In an embodiment of the invention, the culture medium comprising transferrin comprises between 1.5nM and 50μΜ, between 5nM and 5μΜ, between 5nM and ΙμΜ, or between 25nM and 0.3μΜ of the inhibitory non-reducing terminal oligosaccharide structures.

In an embodiment of the present invention, the culture medium and matrix does not comprise the inhibitory non-reducing terminal oligosaccharide structures .

In an embodiment, the culture system contains only non-glycoprotein components such as recombinant proteins produced in bacteria. In an embodiment, the carbohydrate-binding protein according to the invention is a non-glycoprotein. In an embodiment, 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).

In an embodiment of the present invention, the culture system contains selected or produced gly- coprotein components with controlled glycosylation with regard to the inhibitory non-reducing terminal oligosaccharide structures.

In one embodiment of the present invention, the culture system comprises culture medium comprising transferrin. In some embodiments, 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. In an embodiment the transferrin is serum transferrin. In an embodiment the transferrin is human serum transferrin. In an embodiment 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.

In an embodiment of the present invention, 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. In an embodi- ment, 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 μΜ.

In one embodiment of the present invention, the culture medium has pH controlled between 6.8 and 7.8.

In an embodiment of the present invention, the culture system comprises cell culture medium with controlled pH. In this embodiment, 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. In case 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. Further, sialylated glycoproteins such as transferrin are known to undergo acid-catalyzed desialylation to asialogly- coprotein comprising the inhibitory oligosaccharide structures in acidic pH. However, 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. Thus the present invention revealed that precise con^¬ trol of the medium pH may be highly advantageous when using sialylated glycoprotein supplements such as transferrin. In one embodiment of the invention, the medium pH is controlled to pH range not lower than from about 6.8 to about 7.0.

In an embodiment of the present invention, 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. In this embodiment, 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.

In one embodiment of the present invention, 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:

(i) the stem cell population cultured and/or expanded according to the method of the inven- tion exhibits a faster proliferation rate;

(ii) 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;

(iii) the stem cell population cultured and/or expanded according to the method of the inven^¬ tion exhibits non-colony growth;

(iv) the stem cell population cultured and/or expanded according to the method of the inven- tion exhibits a morphology as a continuous cell layer; and

(v) 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.

In this context, the term "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 .

In one embodiment of the present invention, 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. In some applications, 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.

The embodiments of the invention described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined to^¬ gether to form a further embodiment of the invention. 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. EXAMPLES

In the following, the present invention will be described in more detail. Reference will now be made in detail to the embodiments of the present in^¬ vention, examples of which are illustrated in the ac^¬ companying drawings. The description below discloses some embodiments of the invention in such detail that a person skilled in the art is able to utilize the in- vention based on the disclosure. Not all steps of the embodiments are discussed in detail, as many of the steps will be obvious for the person skilled in the art based on this specification. EXAMPLE 1.

Cell culture and maintenance

Three hESC lines (FES 29, FES 30 and H9) and two induced pluripotent stem (hiPS) cell line (FiPS 5- 7 and HEL11.4) were included and cultured on 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. 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 β- 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) . Pinacidil (Sigma-Aldrich, ΙΟΟμΜ) was added to culture media during passaging. In some experiments, Y-27632 (10μΜ) was added to culture media during passaging. In all experiments Matrigel™ (BD Biosciences) was used as control matrix. The Mat- rigel plates were prepared as recommended by the manu^¬ facturer .

EXAMPLE 2.

Coating of plates with ECA

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.

EXAMPLE 3.

Testing of lectins

ECA (Erythrina cristagalli agglutinin, bind- ing specificity in type 2 N-acetyllactosamine struc^¬ tures) , MAA (Maackia amurensis agglutinin, specific for 2,3-linked sialic acid), WFA {Wisteria floribunda agglutinin, binding preferentially to N- acetylgalactosamine in a- or β-linkage) and PWA (Phy- tolacca americana agglutinin, with N-acetylglucosamine specificity, binding also to polylactosamine struc^¬ tures) were tested for their ability to act as a growth supporting matrix for ESC lines FES 29 and FES 30 in mEF-CM medium. In cell culture conditions, stem cells attached onto ECA and MAA. Furthermore, continu^¬ ous growth was acquired on ECA matrix.

EXAMPLE 4.

Flow Cytometry Analysis of Surface Antigens

Single cell suspensions were generated by incubation with TrypLE (Gibco) for 5 min at +37°C. Cells were stained with specific cell surface antibodies (SSEA-1, SSEA-3, Tra 1-60, H type 1, CXCR4) and fluorescein conjugated secondary antibody prior to analysis by flow cytometry (FACS Calibur, BD Biosciences) . Anti^¬ bodies are listed in Table 1.

Table 1. Antibodies used for analysis.

Immunohistochemistry of the cells on ECA Cells were fixed with 4% paraformaldehyde and permea- bilized with 0.1% Triton X-100 ( Sigma-Aldrich, St. Louis, MO, USA) if needed. The antibodies used are listed in Table 1. The cells were probed with second- ary antibodies for 30min in the dark at RT . Cells were mounted using Vectashield mounting media with 4,6- diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA) . Teratoma formation

Cells were harvested with collagenase IV from ECA and Matrigel plates, and ca. 100,000 cells from each ma^¬ trix were injected into nude mouse testis. After 7-8 weeks, tumors were dissected, fixed with 4% PFA and H&E stained sections histologically examined. The ani^¬ mal experiments were approved by the experimental ani^¬ mal welfare committee of the District Government of Southern Finland. RNA-isolation and Quantitative PCR

Total RNA was isolated using NucleoSpinR RNA II (Macheray-Nagel GmbH & Co. KG, Germany) according to manufacturer's instruction. Complementary DNA was synthesized from 50μg of total RNA using iScript™ cDNASynthesis Kit (Biohit) according to manufacturer's instructions .

Real time SYBR Green quantitative PCR (qPCR) analyses was performed with Corbett Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) using the following conditions: 95°C 7 minutes and 40 cycles of 95°C, 20 s; 56°C, 20s; 72°C, 20s. The data was ana^¬ lyzed according to comparative Ct method (applied Bio- systems, User Bulletin #2) . Cyclophilin gene expression was an internal reference for normalization. All samples and controls were analyzed in duplicates. Pri^¬ mers used for qPCR are shown in Table 3. Table 3. Primers used in qPCR analysis.

PCR Arrays

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 RT² First Strand Kit and RT² qPCR Master Mixes ( SABiosciences ) according to manufacturer's instruction. The RT² qPCR primer Assays (SABiosciences) 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

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.

Basic characteristics of hPSCs cultured on

ECA

Long-term culturing on ECA-coated plates was evaluated with hPSCs lines (FES29 and HEL 11.4) and the results were compared to the same cell lines cul^¬ tured on Matrigel. For most of the experiments the cells were cultured in mEF conditioned media and treated with the Rho-associated kinase inhibitor pina- cidil during passaging. Without pinacidil the cells did not attach as effectively and they also partly changed morphology forming a lot of feeder-like cells. In long-term cultures the analysis of these cell lines by immunocytochemical stainings (Oct4, Nanog, Sox2 and E cadherin) and flow cytometry (Tra 1-60, SSEA-3, H type 1, SSEA-1) demonstrated a profile characteristic for undifferentiated hESCs (Figure 1A and B) . The ex^¬ pression levels of major pluripotency associated genes remained essentially similar throughout 20 passages on both matrices. Minor upregulation of primitive streak/early differentiation markers Brachyuru and Goosecoid occurred at later passages of FES29 cells on ECA (Figure 1C) . With the iPSC line HEL11.4, the plu- ripotency genes tended to remain higher and the dif^¬ ferentiation genes lower on ECA throughout the culture period (Figure ID) . In general, FES29 showed a con^¬ stant gene expression pattern independent of the ma^¬ trix as tested by PAHS-081 qPCR array including 84 genes controlling growth and differentiation of stem cells (Figure IE) . The ECA cultured cells also re^¬ tained their full in vivo differentiation capacity as indicated by highly complex teratoma containing all three germ layer derivatives (Figure IF) . Both cell lines were karyotypically normal after 18 passages on ECA (not shown) . Y-27632 had similar effects on the cells as pinacidil.

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. 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. F. HE staining of a teratoma derived from FES 29 cultured on ECA 9 passages. Derivatives of all germ layers can be de^¬ tected .

Next, the ability of of ECA to support the growth of undifferentiated hPSC in defined cell cul^¬ ture medium StemPro™ was tested. Cells were first adapted to StemPro for one passage using 1:1 mix of StemPro and CM media and then only StemPro was used. The results indicated that also defined media support^¬ ed self-renewal and cells maintained stem cell markers and normal karyotype detected after 9 passages on ECA in StemPro (data not shown) .

EXAMPLE 5.

Clonogenicity assay

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/cm²) and cultured in mouse embryonic fibroblast- conditioned medium (CM) supplemented with 8 ng/ml basic fibroblast growth factor (bFGF) . Pinacidil (ΙΟΟμΜ) 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. For this purpose, 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 .

Clonogenicity and cell growth

The ability of the ECA matrix to support clonogenicity of hPSC cells was studied by plating dispersed cells first adapted to ECA or Matrigel for at least two passages on either of the two matrices at the density of 35 cells/cm². In the presence of pina- cidil the colony forming efficacy was clearly highest (10.3%,) when ECA-adapted cells were plated on ECA, as compared with all other conditions where the efficacy was approximately 6% (p<0.05, one-way ANOVA, Tukey' s post-hoc test) (Figure 2A) . Pinacidil was found to be essential for the development and survival of the sin^¬ gle-cell derived clones in these experiments.

Long-term cell imaging was used to study col- ony area and cell growth. Colonies were imaged every second hour during four days after plating to record colony areas and the number of cells in the colonies. In accordance with the clonogenicity assay, the ini^¬ tial number of colonies was higher on ECA than on Mat- rigel . An explanation to this was provided by cell vi^¬ ability analysis, which showed higher viability for cells grown on ECA than on Matrigel (90.1% vs. 82.9 %, p< 0.01, Figure 2B) . The cells were counted three and six days after plating. The number of cells was sig- nificantly higher on ECA than on Matrigel at both time points (Figure 2C-D) . No difference in speed of cell division was detected and the size of the colonies growing on ECA and Matrigel was similar (Figure 2E) . These results show that culture on ECA generates more cells based on better attachment and survival after dissociation and plating.

Figure 2 shows A. Clonogenicity assay on dis^¬ persed single cells (35 cells/cm2). Cloning efficiency was calculated as the number of clones per the total number of plated cells when transferring the cells be^¬ tween ECA and Matrigel (MG) substrates (p= 0.003, one^¬ way ANOVA) . Error bars indicate SEM. B. Cell viabil^¬ ity during passaging. Cells were counted by trypan blue exclusion (N=8) . On ECA the average cell viabil- ity rate was 92% whereas on Matrigel it was 84 ~6 (p<0.01) . Data represent the mean (± SEM) of eight separate experiments. C-D. Analysis of cell growth rate. Data represent the mean ) of 12 wells of hESC (FES29, C) or hiPSC (HEL11.4, D) . *, p<0.05; **, p<0.01. E. Colony area (2 wells/matrix) for FES 29 cell line. Cells were passaged in small clumps and cultured in live cell imaging system (Cell IQ, CM- technologies) . Areas of the colonies were analyzed in Cell IQ Analyzer program and results are shown as average size. Y-axis is the diameter of colony area counted as pixels.

EXAMPLE 6.

Validation of binding specificity

Cells were passaged and plated as described earlier. The two compounds expected to act as specific binding inhibitors for ECA were lactose monohydrate (Sigma- Aldrich, lOOmM) and lacto-N-neotetraose (Kyowa Hakko Kogyo, lOOmM) . Saccharose ( Sigma-Aldrich, lOOmM) was used as a control. The inhibitors were added to cul^¬ ture media at the time of passaging and the attached cells were counted after 20 hours.

To assess the specificity of the cell-lectin interaction in supporting stem cell attachment to the growth surface, we performed inhibition experiments with specific disaccharide inhibitors and control di- saccharides. Lactose (composed of galactose β1,4- linked to glucose) inhibited cell attachment effec^¬ tively at 100 mM concentration, while the same concen^¬ tration of saccharose (fructose ΐ,ΐ-linked to glu^¬ cose) had no inhibitory activity. Further, lacto-N- neotetraose oligosaccharide, which contains the β1,4- linked galactose epitope, was as effective as lactose (p< .001) (Figure 3) . The inhibition experiments thus demonstrated that initial cell attachment to the ECA matrix was dependent on specific interaction of the surface-bound lectin with stem cell glycan ligands. The experiments were performed either in the presence (Figure 3A) of absence (Figure 3B) of pinacidil. Even if the effect of the inhibitors was similar in both conditions, the total number of attached cells was 4- fold higher with pinacidil.

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) . The control disaccharide, saccha- rose, had no inhibitory activity.

EXAMPLE 7.

In Vitro Differentiation

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.

Table 2. Protocol for hepatic differentiation

Stage 1. 1 Stage 2. 5 days

4 days Stage 3. 10 days day Differentiation

to

The first day

DE differenHepatic progeniHepatocyte matu^¬ of DE induc^¬ tiation tors ration

tion

RPMI1640 RPMI1640 Leibovitz's L-15

KO-DMEM

+Glutamax +Glutamax ( invitrog

B27 2% (v/v) B27 2% (v/v) KO-SR 20% (v/v) FBS 8.2% (v/v)

Tryptose phos^¬

Wnt3a 75 ActA 100

NEAA 1% (v/v) phate broth 8.3% ng/ml ng/ml

(v/v)

Hydrocortisone-

ActA 100

NaB 0.5 mM Glutamine ImM 21-hemisuccinate ng/ml

10μΜ

NaB ImM βΜβΟΗ 0. ImM Insulin ΙμΜ DMSO 1% (v/v) Glutamine 2mM

HGF 10ng/ml

OncM 20ng/ml

Act A= Activin A, NaB= Natrium butyrate, One M= Oncostatin M

Hepatocyte differentiation

The cells were differentiated into hepatocyte-like cells (HLCs) on ECA side by side with Matrigel® as a control. For hepatocyte differentiation we used a three step protocol, modified from the one established by Hay et al . (Hay DC, Fletcher J, Payne C, Terrace JD, Gallagher RC, et al., 2008: Highly efficient dif- ferentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci U S A 105: 12301-12306). During the course of dif^¬ ferentiation the expression of the pluripotency marker gene OCT4 was efficiently downregulated while endoderm marker FOXA2 and anterior DE marker Cerberus (CER1) were strongly upregulated (Figure 3A) . The cells on both coatings changed into morphologically typical de^¬ finitive endoderm (DE) -cells and stained positive for FOXA2 (Figure 3B) . DE- induction yielded on average 68±5 % cells positive for CXCR4 on ECA, while using Matrigel® the average was 78117% (Figure 3C) . However, cells detached easier from ECA than from Matrigel during the DE stage. 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) . When the cells were matured into HLCs with HGF and Oncostatin M treatment (dl0-d20) the more mature hepatocyte marker albumin became strongly expressed as shown by qPCR and immunocytochemistry (Figure 4 D-E) . Taken together, 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 ΙΟΟμιη. EXAMPLE 8.

Continued culture

Long-term cell culture was used to study colony area and cell growth of initially formed colonies described in the previous Examples in continued culture on ECA coated surface. Colonies were imaged with microscopy to evaluate colony areas and the number of cells in the colonies. Unlike on Matrigel, the colonies contin^¬ ued expanding rapidly until they completely fused to^¬ gether and filled in various experiments from 90% to 100% of the available ECA-coated surface. Figure 5 shows an exemplary microscopic image showing unlimited and non-colony restricted growth as a continuous cell layer over the whole available area, effectively form^¬ ing a single colony. In contrast, when any two colo- nies grew together on Matrigel in parallel experiments, they typically did not fuse but instead the former colony border cells retained their different morphology leaving the old colony borders clearly vis^¬ ible in microscopic examination. On ECA, all the cells retained the morphology of pluripotent stem cells growing inside colonies: small and round cell size and shape, and high nucleus-to-cytoplasm ratio (Figure 5) . Stem cell marker expression was analyzed from these cells similarly as in Example 4 and the results were similar (not shown) . These results demonstrated that culture on ECA according to the present invention gen- erated a different non-colony growth morphology for the cells, while the proportion of colony border cells was significantly decreased compared to Matrigel cul^¬ ture. In representative experiments the proportion of colony border cells as measured by microscopy was com- pared on Matrigel and on ECA coated surfaces in stand^¬ ard culture conditions with pinacidil used in passag^¬ ing: after the pluripotent stem cells had essentially completely filled a well in a 6-well plate as a large colony (diameter 3.5cm, surface area 9.5cm2), the ra- tio of cells inside the colony to cells at the colony borders was over about 1000:1 (>99.9%), while in typi^¬ cal colonies such as on Matrigel culture the similar ratio between the phenotypically different cells was depending on the individual colony from about 10:1 (90.9%) to about 100:1 (99.0%) with much variation from experiment to experiment. Thus 10-100 fold reduc^¬ tion in the number of colony border cells and significantly more homogeneous cell population with signifi^¬ cantly less variation could be achieved in pluripotent stem cell culture on ECA coated surface compared to Matrigel .

EXAMPLE 9.

Analysis of stem cell culture medium supplements

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.

In the supplements, neutral N-glycans were not detected and were thus below the detection limit of approximately 1% of the total N-glycans. Glycan signals indicating the presence of Neu5Gc (Heiskanen, A., et al., 2007. Stem Cells 25:197-202) including m/z 1946, 2237, and 2256, were not detected in XF supple- ment, while the major acidic N-glycan components were S2H5N4, S1H5N4, S2H6N5F2, S2H5N4F1 and S2H6N5 (for nomenclature see Heiskanen, A., et al . , 2007. Stem Cells 25:197-202), together comprising over 90% of the total detected glycan signals. This corresponds to a typical human transferrin N-glycan composition of partially sialylated biantennary and triantennary N-glycans that contain non-reducing terminal N-acetyllactosamine at the antennae (Spik, G., et al . , 1988. Biochimie 70:1459-69). Similarly, 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. This corre^¬ sponds to a typical bovine serum transferrin N-glycan profile with presence of Neu5Gc (Heiskanen, A., et al., 2007. Stem Cells 25:197-202). ITS supplement gave essentially similar results as KO serum replacement.

Calculation of approximate number of non- reducing terminal Ga^l-4GlcNAc residues in an N- glycan site (nLN) was performed according to the for- mu1a :

x

nLN = ∑ [%Gi (N-2-S) ] ,

i=l

wherein 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, and N-2-S is the max^¬ imal number of non-reducing terminal Ga^l-4GlcNAc an- tennae in glycan Gi . This calculation yielded that nLN = 0.37 for XF supplement and nLN = 0.24 for KO supple^¬ ment. However, 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. For example, using XF sup^¬ plement containing human serum transferrin with two N- glycosylation sites and a molecular weight of about 80 kg/mol, N-glycan concentration is 25 nmol/mg; and effective inhibiting oligosaccharide structure concen- tration is thus from 5 nM (0.5 mg/1) up to about 1 μΜ (100 mg/1) for supplemented human transferrin of nLN = 0.37. With an nLNmax = 2 in the case of asialotrans- ferrin, the concentration is from 25 nM (0.5 mg/1) up to about 5 μΜ (100 mg/1); and using KO supplement con- taining bovine serum transferrin with one N- glycosylation site and a molecular weight of about 80 kg/mol, N-glycan concentration is 13 nmol/mg; and effective inhibiting oligosaccharide structure concen^¬ tration is thus from about 1.5 nM (0.5 mg/1) up to about 0.3 μΜ (100 mg/1) for supplemented bovine trans^¬ ferrin of nLN = 0.24. With an nLNmax = 2 in the case of asialotransferrin, the concentration is from about 13 nM (0.5 mg/1) up to about 2.5 μΜ (100 mg/1) . As demonstrated in the above Examples, 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.

As is clear for a person skilled in the art, the invention is not limited to the examples and em^¬ bodiments described above, but the embodiments can freely vary within the scope of the claims.

Claims

1. A method for culturing stem cells, c h a r a c t e r i s e d in that the method comprises the step of contacting at least one stem cell with a carbohydrate-binding protein and a Rho-associated kinase inhibitor simultaneously at one or more time in^¬ tervals during the cultivation,

wherein the carbohydrate-binding protein is capable of binding the non-reducing terminal oligosac- charide structure according to the formula

(Fuc l-2) _nGa^l-4GlcNAc,

wherein n = 0 or 1.

2. The method according to claim 1, c h a r a c t e r i s e d in that the carbohydrate-binding pro- tein is a lectin, an antibody or a carbohydrate- modifying protein, or a modification or a fragment thereof .

3. The method according to claim 1 or 2, c h a r a c t e r i s e d in that the carbohydrate- binding protein is a lectin.

4. The method according to any one of claims 1-3, c h a r a c t e r i s e d in that the carbohydrate- binding protein is ECA, UEA-1, DSA, RCA, galectin, or a modification or a fragment thereof.

5. The method according to any one of claims

1-4, c h a r a c t e r i s e d in that the carbohydrate- binding protein is ECA.

6. The method according to any one of claims 1-5, c h a r a c t e r i s e d in that the carbohydrate- binding protein is contacted with the at least one stem cell as a matrix or comprised in a matrix.

7. The method according to any one of claims 1-6, c h a r a c t e r i s e d in that 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) -1H- indole, glycyl (S) - (+) -2-methyl-4-glycyl-l- (4- methylisoquinolinyl-5-sulfonyl ) homopiperazine,

azabenzimidazoleaminofurazan, 4- (1-amino-alkyl) -N- (4- pyridyl) cyclohexane-carboxamide, Rhostatin, and any combination thereof.

8. The method according to any one of claims 1-7, c h a r a c t e r i s e d in that the Rho-associated kinase inhibitor is pinacidil or Y-27632.

9. The method according to any one of claims

1-8, c h a r a c t e r i s e d in that the carbohydrate- binding protein is ECA and the Rho-associated kinase inhibitor is pinacidil or Y-27632.

10. The method according to any one of claims 1-9, c h a r a c t e r i s e d in that the at least one stem cell has in a preceding step been contacted with or cultured with the carbohydrate-binding protein.

11. The method according to any one of claims 1-10, c h a r a c t e r i s e d in that the stem cell is a human embryonic stem cell.

12. The method according to any one of claims 1-10, c h a r a c t e r i s e d in that the stem cell is a non-human embryonic stem cell.

13. The method according to any one of claims 1-10, c h a r a c t e r i s e d in that the stem cell is a pluripotent stem cell.

14. The method according to claim 13, c h a r a c t e r i s e d in that the pluripotent stem cell is an induced pluripotent stem cell.

15. The method according to any one of claims

1-14, c h a r a c t e r i s e d in that the at least one stem cell is a stem cell population.

16. The method according to any one of claims 1-15, c h a r a c t e r i s e d in that the stem cell re- mains in an essentially undifferentiated state.

17. The method according to any one of claims 1-16, c h a r a c t e r i s e d in that at least one stem cell is contacted with a carbohydrate-binding protein as defined in any one of claims 1-6 and a Rho- associated kinase inhibitor simultaneously when the stem cell is seeded or passaged to cultivation or for derivation of a stem cell clone.

18. The method according to any one of claims 1-17, c h a r a c t e r i s e d in that a stem cell popu^¬ lation derived from at least one stem cell is contact^¬ ed with a carbohydrate-binding protein as defined in any one of claims 1-6 and a Rho-associated kinase in^¬ hibitor simultaneously during the expansion of the stem cell population.

19. The method according to any one of claims 1-18, c h a r a c t e r i s e d in that at least one stem cell is contacted with a carbohydrate-binding protein as defined in any one of claims 1-6 and a Rho- associated kinase inhibitor simultaneously essentially during the entire duration of the cultivation.

20. The method according to any one of claims 1-19, c h a r a c t e r i s e d in that a stem cell popu^¬ lation derived from at least one stem cell is seeded or passaged to cultivation at a low seeding density.

21. The method according to claim 20, c h a r a c t e r i s e d in that the low seeding density is a seeding density in the range of 0.1 to 20000 cells/cm², 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/cm², or about 10 cells/cm², or 5 to 15, or 7 to 13, or 8 to 12 cells/cm², or about 35 cells/cm², or to 16 to 45, or 25 to 45, or 30 to 40, or 33 to 37 cells/cm².

22. The method according to any one of claims 1-21, characterised in that the method comprises a further step of culturing stem cells in conditions for supporting the differentiation of the stem cells.

23. The method according to any one of claims 1-22, c h a r a c t e r i s e d in that the conditions support differentiation of stem cells into cells se^¬ lected from the group consisting of hepatocytes, reti^¬ nal cells, cardiomyocytes , and neurons.

24. Use of a carbohydrate-binding protein as defined in any one of claims 1-6 and a Rho-associated kinase inhibitor for increasing the efficiency of ex^¬ pansion of stem cells.

25. Use of a carbohydrate-binding protein as defined in any one of claims 1-6 and a Rho-associated kinase inhibitor for culturing stem cells as non- colony cells.

26. A stem cell or stem cell population obtained by the method according to any one of claims 1- 21.

27. A composition comprising the stem cell or stem cell population according to claim 26.

28. A composition comprising the stem cell or stem cell population according to claim 26 for use as a medicament.

29. A culture system for culturing and/or expanding stem cells, c h a r a c t e r i s e d in that it comprises a culture vessel, at least one stem cell, a carbohydrate-binding protein as defined in any one of claims 1-6, a Rho-associated kinase inhibitor, culture medium, and optionally a matrix.

30. A culture system according to claim 29, c h a r a c t e r i s e d in that the culture medium and matrix do not comprise inhibitory amounts of non- reducing terminal oligosaccharide structures according to the formula

(Neu5R¹ 2-3)_m(Fuc l-2) _nGa^R²,

wherein R¹ is Ac or Gc; m = 0 if the carbohydrate-binding protein is not a galectin; m = 0 or 1 if the carbohydrate-binding protein is a galectin; n = 0 or 1-m; and R² is l-4GlcNAc or l-4Glc or absent.

31. A culture system according to claim 30, characterised in that the culture medium comprises transferrin .

32. A culture system according to claim 30 or 31, c h a r a c t e r i s e d in that the culture medium has pH controlled between 6.8 and 7.8.

33. A stem cell population cultured and/or expanded according to the method according to any one of claims 1-21, c h a r a c t e r i s e d in that the stem cells population exhibits at least two of the features i-v as compared to a second stem cell popula^¬ tion grown on a complex matrix:

(i) the stem cell population cultured and/or expanded according to the meth- od according to any one of claims 1-21 exhibits a faster proliferation rate;

(ii) cultured and/or expanded according to the method according to any one of claims 1-21 exhibits a higher propor- tion of undifferentiated cells;

(iii) cultured and/or expanded according to the method according to any one of claims 1-21 exhibits non-colony growth;

(iv) cultured and/or expanded according to the method according to any one of claims 1-21 exhibits morphology as a continuous cell layer; and

(v) cultured and/or expanded according to the method according to any one of claims 1-21 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.

34. The stem cell population according to claim 33, c h a r a c t e r i s e d in that the complex matrix is Matrigel or a feeder cell layer.