WO2017129719A1

WO2017129719A1 - Invasion cell culture system for use in drug discovery

Info

Publication number: WO2017129719A1
Application number: PCT/EP2017/051716
Authority: WO
Inventors: Martin Sailer
Original assignee: Universität Basel
Priority date: 2016-01-27
Filing date: 2017-01-27
Publication date: 2017-08-03

Abstract

A method for obtaining proliferating and/or migrating neural stem cells (NSCs) is provided. The method comprises providing non-invasive NSCs and cultivating this NSCs in the presence of FGF2 and optionally insulin, thereby yielding proliferating NSCs. These proliferating NSCs are further cultivated in the presence of FGF2, optionally insulin and BMP4, or FGF2, optionally insulin, BMP4 and TGFbeta1, thereby yielding migrating and/or invasive NSCs. In further aspects of the invention the obtained NSCs are used in methods to identify compounds affecting proliferation and migratory and invasive behaviour of these NSCs.

Description

INVASION CELL CULTURE SYSTEM FOR USE IN DRUG DISCOVERY

Field of the invention

The present invention relates to a method for obtaining proliferating and migrating neural stem cells and to their use for the identification of compounds affecting cell migration and proliferation of neural stem cells.

Background of the invention

The most frequent malignant brain tumour is Glioblastoma multiforme (glioma WHO grade 4) with a life expectancy at diagnosis of only 15 months. So far there is no known cure for glioblastoma. The severe lethality of the tumour is caused by its capacity for proliferation and strong invasion into brain tissue. Malignancy of glioblastoma cells is linked to their migratory and invasive capacity and ability for metastasis formation. Therefore the means to identify compounds that affect the migratory and invasive capacity of these cells are wanted urgently.

There is increasing evidence in the art that glioblastoma cells are derived from neural stem cells (NSCs). Although means for obtaining NSCs are known in the art, NSCs obtained by these known methods are neither migratory nor invasive and can therefore not be used to identify compounds that affect these properties.

NSCs are also involved in brain regeneration in response to brain injury or stroke. For brain regeneration NSCs have to migrate within the brain to the region where a lesion occurred. An in vitro system allowing the identification of novel compounds that promote invasion of normal NSCs would be very advantageous in finding new treatments for traumatic brain injury and stroke.

The inventors have published a study of the invasion potential of NSCs after expansion in FGF2 and BMP4 (Sailer et al. 2013 J. Cell Science 126, 3533-3540); the results however did not provide a method capable of identifying candidate compounds that might inhibit proliferation, migration and invasion.

The problem underlying the present invention is to provide proliferating and migrating neural stem cells that can be utilized for the identification of compounds that affect the capacity for proliferation, migration and invasion. This problem is solved by the subject-matter of the independent claims. Description of the invention

The inventors developed an in vitro method to obtain migratory, or migratory and invasive, neural stem cells. Surprisingly, in order to induce migratory and invasive behaviour of these cells, they have to be treated with a specific combination of compounds, wherein the specific timing, sequence and the concentration of treatment with these compounds is essential.

In this in vitro system, typical invasion genes are upregulated that have been observed in gliomas, but also in other invasive cancers, such as colon, breast, gastric and lung cancer. The inventors also identified optimal read-out genes that allow the use of this system for high-throughput automatic detection.

According to a first aspect of the invention a method for obtaining an isolated migratory or migratory and invasive neural stem cell is provided. The method comprises the following steps:

a) providing ex vivo a neural stem cell,

b) cultivating ex vivo the neural stem cell in the presence of fibroblast growth factor 2 (FGF2) and optionally insulin, yielding a proliferating neural stem cell, c) cultivating ex vivo the proliferating neural stem cell in the presence of:

• FGF2, optionally insulin and bone morphogenetic protein-4 (BMP4), or

• FGF2, optionally insulin, BMP4 and transforming growth factor beta 1 (TGFbetal );

yielding migratory or migratory and invasive neural stem cell and

d) isolating the migratory or migratory and invasive neural stem cell.

In other words, proliferation is induced or enhanced in an isolated, slowly proliferating and non-invasive neural stem cell population by the treatment with fibroblast growth factor 2 and optionally insulin. In order to induce the migratory and migratory and invasive phenotype in this proliferating neural stem cell, the treatment with FGF2 and optionally insulin is continued and additionally bone morphogenetic protein-4 alone or bone morphogenetic protein-4 in combination with transforming growth factor beta 1 is added to the culture of the proliferating neural stem cell. This results in migratory NSCs and continuation of treatment yields migratory and invasive NSCs. Finally the migratory or migratory and invasive neural stem cell is isolated.

Without wishing to be bound by theory the inventors assume that treatment with FGF2 to induce or enhance proliferation of the NSCs initiates an early phase of migratory behaviour by inducing the expression of transcription factor ZEB1 , which is an early marker of migratory behaviour in NSCs (Fig. 6). Again, without wishing to be bound by theory the inventors further assume that insulin acts as a survival factor and not as mitogen in the present context. NSCs acquire the proliferating phenotype without the use of insulin but the rate of surviving NSCs is significantly lower compared to cells cultivated in the presence of FGF2 and insulin. The use of insulin without FGF2 is not sufficient to obtain the NSCs according to the results obtained by the inventors.

In the context of the present specification, the term neural stem cell is used in its meaning known in the art of neurobiology and medicine; it refers to a type of multipotent fetal stem cell that has the ability to differentiate into various cell types of the central nervous system, in particular neurons, astrocytes and oligodendrocytes.

In certain embodiments, cultivation, passaging, propagation of neural stem cells is performed without the use of enzymes and/or without the use of serum in the cell culture medium. Not using enzymes is advantageous, for example, to prevent damage to membrane bound proteins. Omitting the use of serum in the cell culture medium is advantageous to increase the reliability of the method and to increase its reproducibility.

In certain embodiments, FGF2 is used at a final concentration of 5 ng/ml to 20 ng/ml, in particular 10 ng/ml to 15 ng/ml.

In certain embodiments, BMP4 is used at a final concentration of 5 ng/ml to 20 ng/ml, in particular 10 ng/ml to 15 ng/ml.

In certain embodiments, TGFbetal is used at a final concentration of 20 ng/ml to 60 ng/ml, in particular 30 ng/ml to 40 ng/ml.

In certain embodiments, insulin, if present, is used at a final concentration of 15 pg/ml to 30 pg/ml, in particular 20 pg/ml to 25 pg/ml.

In certain embodiments, the neural stem cell that is provided in step a) is a neural stem cell originating from the cortical subventricular zone, in particular the subventricular zone of the central cortex or posterior cortex, of a mammal. This region in the brain of mammals provides neural stem cells most suitable for the invention disclosed herein.

In certain embodiments, the mammal that the neural stem cell originates from is a rodent, in particular a rat.

In certain embodiments, the neural stem cell originates from a rodent embryo at embryonic day E13 to E15, in particular E13.5 to E14.5, more particular E14 to E14.5.

In certain embodiments, the neural stem cell originates from a rat embryo at embryonic day E13 to E15, in particular E13.5 to E14.5, more particular E14 to E14.5.

In certain embodiments, the neural stem cell originates from a mouse embryo at embryonic day E13 to E15, in particular E13.5 to E14. In the context of the present specification, the term embryonic day is used in its meaning known in the art of developmental biology and medicine; it refers to the age of the embryo, wherein day 0 (E0) is the day of fertilization. The embryonic day is usually abbreviated as "E" followed by the number of days, including half days. In other words, E0 would symbolize the day of fertilization, E13 would be the thirteenth day of life of the embryo and E14.5 would symbolize day 14 and a half day. Rodents usually mate at night. For mating a female and a male rate are paired between 8 PM and 8 AM in the following morning. Conception as known in the art is midnight of embryonic day 0 (E0). Noon after conception is considered embryonic day 0.5 (E0.5).

According to a second aspect of the invention a cell preparation of isolated migratory or migratory and invasive neural stem cells (NSCs) obtained by a method according to the first aspect of the invention is provided.

According to a third aspect of the invention a cell preparation of isolated migratory or migratory and invasive neural stem cells (NSCs) is provided. Non-migratory and non-invasive NSCs originating from the cortical subventricular zone, in particular the subventricular zone of the central cortex or posterior cortex, of a mammal, in particular a rat, are treated ex vivo with fibroblast growth factor 2 (FGF2) and optionally insulin. After treatment with:

FGF2, bone morphogenetic protein 4 (BMP4) and optionally insulin, or

FGF2, BMP4 and transforming growth factor beta 1 (TGFbetal ) and optionally insulin,

migratory or migratory and invasive neural stem cells are obtained. The proliferating and migrating NSCs are characterized by the expression of a marker selected from podoplanin, p75NGFR, smooth muscle actin, calponin and Sox9.

The markers differ in their time of appearance after treatment of the NSCs, whereby they appear in the following sequence:

a) podoplanin,

b) p75NGFR,

c) smooth muscle actin, calponin and Sox9.

In certain embodiments, podoplanin is detectable after 2 to 3 days of treatment with FGF2, optionally insulin and BMP4 or FGF2, optionally insulin, BMP4 and TGFbetal .

In certain embodiments, p75NGFR is detectable after 3 to 5 days of treatment with FGF2, optionally insulin and BMP4 or FGF2, optionally insulin, BMP4 and TGFbetal .

In certain embodiments, smooth muscle actin, calponin and Sox9 are detectable after 7 to 8 days of treatment with FGF2, optionally insulin and BMP4 or FGF2, optionally insulin, BMP4 and TGFbetal . According to a fourth aspect of the invention a method of use of the cell preparation according to the second or third aspect of the invention for drug screening is provided.

According to a fifth aspect of the invention a method of use of the cell preparation according to the second or third aspect of the invention for screening of candidate compounds affecting cell migration and/or cell invasion is provided. The method comprises the steps of:

a. Providing a cell preparation according to the second or third aspect of the invention. b. Bringing a candidate compound in contact with the cell preparation.

c. Assessing a cell migration and/or cell invasion parameter of cells of the cell preparation.

A decrease in the parameter of cell migration and/or cell invasion compared to cells not treated with the candidate compound is indicative of a candidate compound inhibiting cell migration and/or cell invasion. An increase in the parameter of cell migration and/or cell invasion compared to cells not treated with the candidate compound is indicative of a candidate compound promoting cell migration and/or cell invasion.

A non-limiting example of a parameter of cell migration would be the migration distance of a cell. Therefore the distance the cells "move" on a culture dish can be determined (as stated in the examples). Another example how to measure cell migration would be a scratch or "healing" assay. In this assay a certain area of a cell culture vessel is cleared of cells and regrowth of the cells into this cleared area is measured. The regrowth can be measured in terms of number of cells, distance travelled etc.

A non-limiting example of a parameter of cell invasion would be the Boyden-chamber invasion assay, an assay commonly used in tumour biology research. The classic Boyden chamber system uses a hollow plastic chamber, which is sealed at one end with a porous membrane. This chamber is suspended over a larger well which contains medium and/or chemoattractants. Cells are placed inside the chamber and allowed to migrate through the pores to the other side of the membrane. Cells that have migrated are then stained and counted. In the standard Boyden assay used herein, the pore diameter of the membrane is 8 pm.

According to a sixth aspect of the invention a method for the identification of a candidate compound affecting cell migration of a neural stem cell is provided. The method comprises the following steps:

a) Providing neural stem cells (NSCs).

b) Cultivating the NSCs in the presence of FGF2 and insulin, yielding proliferating NSCs. c) Cultivating a first portion of the proliferating NSCs in the presence of FGF2, insulin and

- BMP4 or

- BMP4 and TGFbetal ;

yielding a first portion of cultivated NSCs.

Cultivating a second portion of the proliferating NSCs in the presence of FGF2, insulin and:

I. a candidate compound, or

II. a candidate compound and:

· BMP4, or

• BMP4 and TGFbetal ;

yielding a second portion of cultivated NSCs.

d) Determining a marker of migration and/or a marker of invasion in the first and the second portion of cultivated NSCs.

- The presence of the marker of migration and/or the marker of invasion in the second portion of cultivated NSCs in I. is indicative of a compound stimulating migration and/or invasion.

The value of the marker of migration and/or the marker of invasion in the second portion of cultivated NSCs in II. is compared to a standard range determined in the first portion of cultivated NSCs. A value lower than the standard range is indicative of a compound inhibiting migration and/or invasion and a value higher than the standard range is indicative of a compound enhancing migration and/or invasion.

In certain embodiments, insulin is used at a final concentration of 15 pg/ml to 30 pg/ml, in particular 20 pg/ml to 25 pg/ml.

In certain embodiments, the marker of migration is selected from:

expression of smooth muscle actin, calponin, Sox9, SPARC, Tenascin C, Twistl or Twist2. migratory morphology and/or movement of said cultivated NSCs and

invasive movement of said cultivated NSCs.

There are many methods known in the art how to measure the expression of a marker of migration, including without being limited to, PCR, Northern blotting, immunoblotting, antibody based staining methods, affinity binder based methods, detection of markers of migration tagged with a detectable label such as e.g. fluorescent proteins or nucleic acid tags.

The morphology or movement of the NSCs can be observed using appropriate microscopic or equivalent techniques.

In the context of the present specification, the term migratory morphology refers to cells with a flat appearance of the cell with long extensions similar to the morphology of fibroblasts. An example of cells with a migratory morphology is provided in figure 8 B, D.

In certain embodiments, the marker of invasion is selected from: SnaiM , Snail2, podoplanin, p75NGFR and metalloproteinases.

In certain embodiments, the neural stem cell that is provided in step a) originates from the cortical subventricular zone, in particular the subventricular zone of the central cortex or posterior cortex, of a mammal. This region in the brain of mammals provides neural stem cells most suitable for the invention disclosed herein.

In certain embodiments, the mammal where the neural stem cell originates from is a rodent, in particular a rat.

In certain embodiments, the neural stem cell originates from a rodent embryo, in particular a rat embryo, at embryonic day E13 to E15, in particular E13.5 to E14.5, more particular E14 to E14.5.

In certain embodiments, the neural stem cell provided in step a) comprises a reporter protein, in particular a fluorescent protein or luciferase, under transcriptional control of the promoter of a gene specific for migratory cells, in particular podoplanin, p75NGFR, smooth muscle actin, calponin, Sox9, SPARC, Tenascin C, SnaiM , Snail2, Twistl or Twist2 and/or a reporter protein, in particular a fluorescent protein, under the transcriptional control of the promoter of the gene specific for non-invasive cells, in particular nestin. In step d) the expression of the reporter protein(s) is determined instead of the marker of migration.

In certain embodiments, a vector, in particular a viral vector, comprising a reporter protein, in particular a fluorescent protein or luciferase, under transcriptional control of the promoter of a gene specific for migratory cells, in particular podoplanin, p75NGFR, smooth muscle actin, calponin, Sox9, SPARC, Tenascin C, SnaiM , Snail2, Twistl or Twist2 and/or a reporter protein, in particular a fluorescent protein, under the transcriptional control of the promoter of the gene specific for non-invasive cells, in particular nestin, is introduced, in particular infected or transfected, into the neural stem cell provided in step a). In step d) the expression of the reporter protein(s) is determined instead of the marker of migration.

According to a seventh aspect of the invention a method for the identification of a candidate compound affecting proliferation of a neural stem cell is provided. The method comprises the following steps:

a) Providing neural stem cells (NSCs).

b) Cultivating a first portion of the NSCs in the presence of FGF2 and optionally insulin, and cultivating a second portion of the NSCs in the presence of

I. a candidate compound, or

II. a candidate compound, FGF2 and optionally insulin,

yielding cultivated NSCs.

c) Determining a marker of proliferation in the first and the second portion of cultivated NSCs.

- The presence of the marker of proliferation in the second portion of cultivated

NSCs cultivated in the presence of the candidate compound (step b) I.) is indicative of a candidate compound stimulating proliferation.

- The value of the marker of proliferation in the second portion of cultivated NSCs cultivated in the presence of a candidate compound, FGF2 and optionally insulin (step b) II.) is compared to the value of the marker of proliferation in the first portion of cultivated NSCs, wherein a value lower than in the first portion of cultivated NSCs is indicative of a compound inhibiting proliferation and a value higher than in the first portion of cultivated NSCs is indicative of a compound enhancing proliferation.

In certain embodiments, FGF2 is used with a final concentration of 5 ng/ml to 20 ng/ml, in particular 10 ng/ml to 15 ng/ml.

In certain embodiments, insulin, if present, is used with a final concentration of 15 pg/ml to 30 pg/ml, in particular 20 pg/ml to 25 pg/ml.

In certain embodiments, the marker of proliferation is selected from: mitotic phenotype, expression of Ki-67 (MIB-1 ), expression of phospho-histone 3 (pH-3) and BrdU incorporation.

In principle all markers or assays known in the art to measure proliferation of cells can be used in the context of the invention. Known assays to measure proliferation include without being limited to, staining, in particular immunostaining, of Ki-67 or phospho-histone 3 or incorporation of the base analog 5-bromo-2'-deoxyuridine (BrdU) into newly synthesized DNA of replicating cells. According to an alternative aspect of the seventh aspect of the invention a method for the identification of a candidate compound affecting the rate of apoptosis of a neural stem cell is provided. The method comprises the following steps:

a) Providing neural stem cells (NSCs).

I. a candidate compound, or

II. a candidate compound, FGF2 and optionally insulin,

yielding cultivated NSCs.

c) Determining a marker of apoptosis in the first and the second portion of cultivated NSCs.

- The presence of the marker of apoptosis in the second portion of cultivated NSCs cultivated in the presence of the candidate compound (step b) I.) is indicative of a candidate compound stimulating apoptosis.

- The value of the marker of apoptosis in the second portion of cultivated NSCs cultivated in the presence of a candidate compound, FGF2 and optionally insulin (step b) II.) is compared to the value of the marker of apoptosis in the first portion of cultivated NSCs, wherein a value lower than in the first portion of cultivated NSCs is indicative of a compound inhibiting apoptosis and a value higher than in the first portion of cultivated NSCs is indicative of a compound enhancing apoptosis.

In certain embodiments, the marker of apoptosis is selected from the activated form of caspase 3 (known as Caspase 3a) or cell staining in an terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.

Wherever alternatives for single separable features such as, for example, a marker, concentration or type of morphology are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

Further aspects and embodiments of the invention are characterized by the following items:

Item 1 : A method for obtaining an isolated migratory or migratory and invasive neural stem cell, comprising: a) providing a neural stem cell,

b) cultivating said neural stem cell in the presence of FGF2 and optionally insulin, yielding a proliferating neural stem cell,

c) cultivating said proliferating neural stem cell in the presence of:

· FGF2, BMP4 and optionally insulin, or

• FGF2, BMP4, TGFbetal and optionally insulin;

yielding a migratory or migratory and invasive neural stem cell and

d) isolating said migratory or migratory and invasive neural stem cell.

Item 2: The method according to item 1 , wherein

- FGF2 is used at a final concentration of 5 ng/ml to 20 ng/ml, in particular

10 ng/ml to 15 ng/ml; and/or

Insulin, if present, is used at a final concentration of 15 pg/ml to 30 pg/ml, in particular 20 pg/ml to 25 pg/ml; and/or

BMP4 is used at a final concentration of 5 ng/ml to 20 ng/ml, in particular 10 ng/ml to 15 ng/ml; and/or

TGFbetal is used, if applicable, at a final concentration of 20 ng/ml to 60 ng/ml, in particular 30 ng/ml to 40 ng/ml.

Item 3: The method according to any one items 1 or 2, wherein the neural stem cell is a neural stem cell originating from the cortical subventricular zone, in particular the subventricular zone of the central cortex or posterior cortex, of a mammal.

Item 4: A cell preparation comprising isolated migratory or migratory and invasive neural stem cells (NSCs) obtained by a method according to any one of items 1 to 3.

Item 5: A cell preparation comprising isolated migratory or migratory and invasive neural stem cells (NSCs), obtained from isolated non-migrating and non-invasive NSCs originating from the cortical subventricular zone, in particular the subventricular zone of the central cortex or the posterior cortex, of a mammal by treatment with fibroblast growth factor 2 (FGF2) and optionally insulin followed by treatment with

FGF2, bone morphogenetic protein 4 (BMP4) and optionally insulin, or

FGF2, BMP4, transforming growth factor β 1 (TGFbetal ) and optionally insulin,

wherein said cell preparation of migratory or migratory and invasive neural stem cells is characterized by the expression of a marker selected from podoplanin, p75NGFR, smooth muscle actin, calponin and/or Sox9.

Item 6: Use of a cell preparation according to any one of items 4 or 5 for drug screening. Item 7: Use of a cell preparation according to any one of claims 4 or 5 for screening of candidate compounds affecting cell migration and/or cell invasion, comprising the steps of: a. providing a cell preparation according to any one of items 4 or 5, or providing cells obtained by a method according to any one of items 1 to 3,

b. bringing a candidate compound in contact with said cell preparation,

c. assessing a cell migration and/or cell invasion parameter of cells of said cell preparation,

wherein a decrease in the parameter of cell migration and/or cell invasion compared to cells not treated with said candidate compound is indicative of a candidate compound inhibiting cell migration and/or cell invasion and an increase in the parameter of cell migration and/or cell invasion compared to cells not treated with said candidate compound is indicative of a candidate compound promoting cell migration and/or cell invasion.

Item 8: A method for the identification of a candidate compound affecting cell migration and/or cell invasion of a neural stem cell, comprising:

a) providing a plurality of neural stem cells (NSCs),

b) cultivating said NSCs in the presence of FGF2 and optionally insulin, yielding a plurality of proliferating NSCs,

c) cultivating a first portion of said plurality of proliferating NSCs in the presence of FGF2, optionally insulin and

BMP4 or

- BMP4 and TGFbetal ,

and cultivating a second portion of said plurality of proliferating NSCs in the presence of FGF2, optionally insulin and:

I. a candidate compound, or

II. a candidate compound in the presence of:

• BMP4, or

• BMP4 and TGFbetal ;

yielding cultivated NSCs

d) determining a marker of migration and/or a marker of invasion in said first and said second portion of cultivated NSCs, wherein

the presence of said marker of migration and/or said marker of invasion in said second portion in I. is indicative of a compound stimulating migration and/or invasion, and the value of said marker of migration and/or said marker of invasion in said second portion in II. is compared to a standard range determined in said first portion of cultivated NSCs, wherein a value lower than the standard range is indicative of a compound inhibiting migration and/or invasion and a value higher than the standard range is indicative of a compound enhancing migration and/or invasion.

Item 9: The method according to claim 8, wherein

FGF2 is used at a final concentration of 5 ng/ml to 20 ng/ml, in particular 10 ng/ml to 15 ng/ml; and/or

Item 10: The method according to any one of items 9 and 10, wherein:

• the marker of migration is selected from:

expression of smooth muscle actin, calponin, Sox9, SPARC, Tenascin C, Twistl or Twist2,

migratory morphology and/or movement of said cultivated NSCs, and

· the marker of invasion is selected from:

invasive movement of said cultivated NSCs,

expression of SnaiM , Snail2, Podoplanin, p75NGFR, metalloproteinases.

Item 1 1 : The method according to any one items 9 to 1 1 , wherein the neural stem cell originates from the cortical subventricular zone, in particular the subventricular zone of the central cortex.

Item 12: The method according to any one of items 9 to 12, wherein

in step a) the provided neural stem cell comprises a reporter protein, in particular a fluorescent protein, under transcriptional control of the promoter of a gene specific for migratory cells, in particular podoplanin, p75NGFR, smooth muscle actin, calponin, Sox9, SPARC, Tenascin C, SnaiM , Snail2, Twistl or Twist2 and/or a reporter protein, in particular a fluorescent protein, under the transcriptional control of the promoter of the gene specific for non-invasive cells, in particular nestin; and

in step d) the expression of said reporter protein(s) is determined instead of the marker of migration. Item 13: A method for the identification of a candidate compound affecting proliferation of a neural stem cell, comprising:

a) providing neural stem cells (NSCs),

b) cultivating a first portion of said NSCs in the presence of FGF2 and optionally insulin, and cultivating a second portion of said NSCs in the presence of

I. a candidate compound, or

II. a candidate compound, FGF2 and optionally insulin

c) determining a marker of proliferation in said first and said second portion of cultivated NSCs, wherein

- the presence of said marker of proliferation in said second portion of cultivated

NSCs of step b) I. is indicative of a candidate compound stimulating proliferation, and

- the value of said marker of proliferation in said second portion of cultivated NSCs of step b) II. is compared to the value of the marker of proliferation in said first portion of cultivated NSCs, wherein a value lower than the standard range is indicative of a compound inhibiting proliferation and a value higher than the standard range is indicative of a compound enhancing proliferation.

Item 14: The method according to item 14, wherein

Insulin, if present, is used at a final concentration of 15 pg/ml to 30 pg/ml, in particular 20 pg/ml to 25 pg/ml.

Item 15: The method according to any one of items 14 and 15, wherein the marker of proliferation is selected from: mitotic phenotype, expression of Ki-67 (MIB-1 ), expression of phospho-histone 3 (PH-3) and BrdU incorporation.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope. Short description of the figures

Fig. 1 shows standardized embryo and forebrain dissection for NSC isolation and induction of proliferation and migration. (A) Left fore limb of rat E14.5 embryo after caesarean section. Every fore limb digit is marked with a black arrow tip. Note the beginning digit formation typical for E14.5. (B) Rat E14.5 embryo after hull removal. The dorsal diencephalon (#) is ideal to start removal of the skin/skull. (C) The border between removed and unremoved skin is marked with two triangular arrowheads. The skin can be peeled anteriorly from the anterior arrow and posteriorly from the posterior arrow. (D) The notch of the midbrain-hindbrain boundary (arrow) and the cut just caudally to it (arrowhead). (E) The telencephalon-diencephalon-mesencephalon (TEL-DIMES) block is removed from the rest of the embryo. (F) Superior view of TEL- DI-MES block. Cranial is left. (G) Oblique view from above. (H) Inferior view of TEL-DI-MES block. (I) The mesencephalon and part of the diencephalon are separated from the telencephalon with the anterior part of the diencephalon. Top: Anterior view of both telencephalic vesicles. Bottom: Right lateral view of mesencephalon, top faces cranially. (J) Top: both telencephalic vesicles without diencephalon. Inferior view to illustrate complete removal of diencephalon. Asterisk depicts medial ganglionic eminence (MGE). Bottom: anterior part of diencephalon. (K) Top: Both hemispheres are separated in the interhemispheric fissure. Left hemisphere on left side. Asterisk depicts MGE. Plus sign depicts lateral ganglionic eminence (LGE). (L) Magnification of left hemisphere with median view. Right faces cranially, left caudally, top is dorsal, bottom ventral, respectively. Scale bar: 2 mm; grid on every image shows 2 mm thick lines with four intersection with thin lines every 400 pm. Asterisk (*) is located at MGE. Plus sign (+) is located at LGE. ch, cortical hem; FL, forelimb; di, diencephalon; mes, mesencephalon; MHB, midbrain-hindbrain boundary; HL, hind limb; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; ob, olfactory bulb; pc, posterior cortex; rho, rhombencephalon; tel, telencephalon.

shows standardized cortical SVZ dissection for NSC isolation and EMT induction. (A) Median view of left hemisphere. Through the foramen of Monro the MGE and LGE are visible, marked by asterisk and plus sign. (B) The olfactory bulb is removed just cranially of the MGE-LGE. (C) The posterior pole of the cortex is removed just behind the CGE. (D) Top: the cortical hem is separated from the cortex. Left: posterior pole. Middle: complex containing the central portion of the cortex and the CGE-LGE-MGE block. MGE and LGE face to the right. Right: olfactory bulb. (E) The CGE-LGE-MGE-cortex block is flipped horizontally. Now the outer surface of the telencephalon is facing the microscope. MGE and LGE now face to the left. (F) The meninges (men) are removed from the bright translucent cortex. The CGE-MGE-LGE-ctx block is now flipped back horizontally for the next step. (G, H) The same procedure is repeated with the right hemisphere. The central cortex is separated away from the CGE-MGE-LGE block. Note that there is an intermediate cortex zone that is left at the CGE-MGE-LGE block, marked with two arrows. This intermediate cortex thickness corresponds to half of the diameter of the LGE. Dotted line representing the border between LGE-CGE and the cortex. (I) The central cortex is separated into explants of less than 400 pm diameter. Scale bar: 2 mm; grid on every image shows 2 mm thick lines with four intersections (thin lines) every 400 pm. Asterisk (*) is located at MGE. Plus sign (+) is located at LGE. Abbreviations: ant-ctx, anterior cortex; cen-ctx, central cortex; cge, caudal ganglionic eminence; ch, cortical hem; cp, choroid plexus; ctx, cortex; LGE, lateral ganglionic eminence; men, meninges; MGE, medial ganglionic eminence; ob, olfactory bulb; pc, posterior cortex.

shows explant seeding onto 35 mm grid cell culture dishes. Place 1 mL of expansion medium at the center of the 500 pm grid dish. The drop is contained by the grid rim (small arrows). Place explants right at the center of the 1 mL drop. If the explants are spread outside the grid, swirl the dish in a slow circular motion and the explants will move to the center by centripetal force. Note: the explants are only barely visible by eye (black arrow heads). Scale bar: 35 mm.

shows podoplanin (PDPN) is induced in the presence of BMP4. Explants were cultured identical as in Figure 5. (A) Control (FGF2 alone) and (C) TGF31 explants did not contain PDPN-positive cells. (B) BMP4 and (D) TGF31/BMP4 explants showed a high proportion of PDPN-positive cells. The explant center is at the left, the periphery at the right. The PDPN-positive cells were mostly found at the periphery and not in the center of the explant. The TGF31/BMP4 explants contained flatter, more elaborated PDPN-positive cells. (E) Means are shown ± SEM. Control vs. TGF31 is not significant (ns). Control and TGF31 vs. BMP4: p<0.0001 (***). TGF31 vs. TGF31/BMP4: p<0.0001 (***). BMP4 vs. TGF31 +BMP4 is not significant: p=0.0981 (ns). Control vs. TGF31 +BMP4: p<0.0001 (***). Scale bar: 50 pm.

shows quantitative migration assessment. BMP4 and TGF31 show additive effect on cell migration. (A) Control explant. (B) Explant in BMP4 alone. (C) Explant in TGF31 alone. (D) Explant in the combination of TGF31 and BMP4. (E) Migration distance in pm. The 500 pm grid was used as reference. The control and the TGF31 explants are showing a strong proliferation with a larger explant diameter than in the other conditions. Means are shown ± SEM. Note that the BMP4 and TGF31/BMP4 explants partly disintegrate the explant core and cells emigrate away from it centrifugally. (E) Control vs. TGF31 is not significant (ns). TGF31 vs. BMP4: p=0.0353 (*). Control vs. BMP4: p=0.0351 (*). BMP4 vs. TGF31 +BMP4: p=0.0372 (*). Control vs. TGF31 +BMP4: p<0.0001 (***). Scale bar: 500 pm. Center of explant is marked by a plus sign (+). Outer edge of migrating cells is marked with triangular arrow. Fig. 6 shows upregulation of transcription factors related to the migratory and proliferating phenotype by qRT-PCR. This phenotype is linked to the key transcription factors of the Zeb- and Twist-family. (A, B) Zeb1 and Zeb2 were upregulated during the first phase of FGF2-exposure. (C) Twist2 was upregulated during the second phase of FGF2/BMP4-exposure. Relative expression levels based on qRT-PCR are shown (n=2). The mRNA levels were normalized to GAPDH. Means are shown ± SEM. At day 0 of the FGF2 period mRNA was harvested, further mRNA was harvested after four days in FGF2, then after one day in FGF2/BMP4. Zeb1 : Control vs. FGF2, p=0.0002. Zeb2: Control vs. FGF2, p=0.0206. Twist2: Control vs. FGF2+BMP4, p=0.003. Fig. 7 shows NSCs with oligodendrocyte precursor cell (OPC) characteristics in the model system. NSCs were isolated from the subventricular zone of the central cortex and cultured as single cells in the presence of FGF2. After 8 d in culture (passage after 4 d) the cells formed small NSC colonies and were stained for Olig2, Sox10 and Nestin. (A-F) A high proportion of cells co-expressed Olig2/Nestin and Sox10/Nestin. Means are shown ± SEM. Scale bar: 20 pm.

Fig. 8 shows that BMP4-exposed explants exhibit flat migratory cells. Magnification of the cells shown in Figure 5. (A) Control- (FGF2 alone) and (C) TGF31- explants demonstrated a strong cell division with small rounded cells. (B) BMP4- and (D) TGF31/BMP4-explants showed consistently flat elongated cells with a migratory morphology. Scale bar: 100 pm.

Fig. 9 shows summary of in vitro model system for proliferating and migratory phenotype investigation. E14.5 rat central cortex contains the NSC-containing subventricular zone (SVZ). The central cortex is either used as explant pieces or as single cells. Explant pieces are more convenient for quantitative migration analysis.

Fig. 10 shows the result of an invasion assay experiment on 30.000 cells assayed as shown in the examples; F: FGF2; B: BMP4 ; T: TGFbeta Examples

The inventors established an in vitro method to initiate the transition from non-migratory neural stem cells (NSCs) to migratory or migratory and invasive NSCs, also referred to as epithelial to mesenchymal transition (EMT).

Table 1 : Proliferative, migratory and invasive capacity of NSCs Proliferation Migration Invasion

Freshly isolated NSCs +

After treatment with FGF2 +++

(optionally +insulin)

After treatment with BMP4 ++ +++ +++ * (optionally with TGFbetal)

-, +, ++, +++: stands for no, low, medium and high capacity; *: delayed after migration

Without wishing to be bound by theory the NSCs exhibit different properties regarding their proliferation, migration and invasive behaviour at different stages of treatment in the method of the invention (table 1 ). Materials and methods

Preparation of expansion medium.

Take two 15 ml. tubes, and add 5 ml. of L-glutamine-free DMEM/F12 (1 :1 ) from a 500 ml_ medium bottle into each of the two 15 ml. tubes. To the first 15 ml. tube, add 50 mg human apo-transferrin, 50 μΙ_ of putrescine 1 M stock (0.1 mM final concentration) and 30 μΙ_ of sodium selenium 500 μΜ stock (final concentration 30 nM). Filter through a 0.2 pm syringe filter into the original DMEM/F12 bottle.

To the second 15 ml. tube, add 12.5 mg insulin. Add 6-9 drops of 1 M NaOH. Vortex to dissolve insulin completely. Filter through a 0.2 pm syringe filter into the original DMEM/F12 bottle. Add 5 ml. penicillin (10,000 units/mL) /streptomycin (10,000 pg/mL)/ amphotericin B (25 pg/mL) to the DMEM/F12 medium bottle. Add 5 ml. of 200 mM L-glutamine stock freshly thawed or oligopeptides containing glutamine (200 mM L-alanyl-L-glutamine dipeptide) to the DMEM/F12 medium bottle. Shake well. Prepare 50 ml. aliquots.

Preparation of passaging medium.

To 1 L Ca²⁺- and Mg²⁺-free HBSS media, add 990.85 mg Glucose (5 mM final concentration) and 840.10 mg NaHC0₃ (10 mM final concentration). Adjust pH to 7.3 with HCI (1 M stock). Filter sterilize with 0.2 pm filter and make 50 ml. aliquots. Preparation of growth factors.

Prepare sterile 1 X PBS with 1 % BSA (PBS-BSA) alone or with hydrochloric acid (HCI) at 4 mM (PBS-BSA-HCI). Dissolve 10 pg/mL recombinant human Fibroblast Growth Factor 2 (rhFGF2) in PBS-BSA (10 ng/mL final concentration), 10 pg/mL recombinant human Bone Morphogenetic Protein 4 (rhBMP4) in PBS-BSA (10 ng/mL final concentration), and 20 pg/mL recombinant human Transforming Growth Factor beta 1 (rhTGF31 ) in PBS-BSA-HCI (40 ng/mL final concentration). Do not filter sterilize. Prepare aliquots.

Coating of cell culture dishes.

Dissolve 1875 mg Poly-L-ornithine (PLO) in 500 mL distilled H20 to prepare a 250X PLO stock. Filter sterilize using a 0.2 pm syringe filter and make 2 mL aliquots. Dissolve 1 mg bovine fibronectin in 1 mL sterile-distilled H20 to prepare 1000X fibronectin stock. Do not vortex as this may clot the sticky protein. Shake gently by hand for 10 min. Incubate for 1 h at room temperature with occasional shaking. Check for complete dissolution. Warm the solution to less than 37 °C for the complete dissolution of fibronectin. Do not filter-sterilize. Use plasma-pretreated plastic cell culture dishes (100 mm or other sizes as required for the experiment). To assess migration, use 35 mm dishes with 500 pm grid. Incubate dishes overnight with 2 mL 1X PLO for 12 h at 37 °C in a 5 % C0₂ tissue culture incubator. Wash twice with 2 mL 1 X PBS.

Add 2 mL 1 X fibronectin (1 pg/mL final concentration) and incubate dishes for a minimum of 12 h at 37 °C in a 5 % C02 incubator. Remove fibronectin just before plating the cells.

Standardized dissection and preparation of the cortical subventricular zone (SVZ)

Obtain rat embryonic day 14.5 (E14.5, Sprague-Dawley) or mouse E13.5 (C57BL/6) embryos by timed-mating. Mate animals from 18:00 until 08:00 the next morning. Noon after the day of mating is considered E0.5.

Anesthetize the pregnant animal with 5 % Isoflurane and 0.8 L/min oxygen flow. Check response by paw compression with sharp dissection forceps. Decapitate the animal. Avoid C0₂ asphyxiation as C0₂ affects stem cell recovery.

Retrieve embryos by caesarean section. Place animal in supine position and disinfect fur with 70 % ethanol. Use surgical forceps and scissors to make V-shaped skin incision of about 8 cm in the lower abdomen above the uterus. Incise only skin with fur while keeping muscular walls intact. Use fresh forceps and scissors to incise the muscles and the abdominal muscular wall to enter the peritoneum. Identify the uterus in the lower posterior peritoneum. Remove the uterus by sharp separation from the surrounding tissues. Wash the uterus with embryos with sterile 1X PBS and place in ice-cold 1 X PBS. Use fine-tipped scissors to open the uterine walls to release embryo by embryo under a dissection microscope (3X magnification, Figure 1 B). Use a pair of forceps to remove the embryonic hulls (Figure 1 B). Keep embryos in expansion medium on ice.

Verify the correct developmental stage by the Atlas of the Developing Rat Nervous System (Paxinos, G., Ashwell, K. W. S. & Tork, I. Atlas of the developing rat nervous system. 2nd edn, Academic Press, 1994). The correct embryo size is shown in Figure 1 B. Check correct age further by presence of beginning digit formation in the rat forelimb (FL) as illustrated in Figure 1 A. The hind limb (HL) does not yet show digit separation (Figure 1 B).

For dissection, transfer embryos to uncoated Petri dishes filled with ice-cold 1X PBS. Perform dissection under a stereo dissection microscope (3X to 20X magnification) using autoclaved fine forceps. The Petri dishes prevent attachment of tissue to the dish surface as may happen with plasma-coated cell culture dishes. Sharpened tungsten wire needles or similar are also helpful for certain steps of dissection. Remove the head skin and skull starting at the intersection of the developing telencephalon (TEL) and diencephalon (Dl) (Figure 1 B) by pulling simultaneously anteriorly and posteriorly with two forceps in order to gain access to the developing neural tube (Figure 1 C). Identify the midbrain-hindbrain- boundary (MHB, black arrow head in Figure 1 B-D), separating the mesencephalon (MES) from the rhombencephalon. Cut the rhombencephalon just at or below the MHB (Figure 1 D, triangular arrow). The additional subcutaneous space at the MHB facilitates skin removal without harming the neural tube.

Sever the connection between the telencephalon/diencephalon at the skull base with the facial skeleton (Figure 1 E). This results in a block containing the telencephalon, diencephalon and mesencephalon (TEL-DI-MES) (Figures 1 F-H). Transfer the TEL-DI-MES block into expansion medium on ice. Keep it completely covered in expansion medium in an uncoated Petri dish. Hold the block at the mesencephalon with a pair of forceps to avoid touching the telencephalon that contains the cortical SVZ with NSCs.

Transfer one TEL-DI-MES block into fresh ice-cold 1X PBS. Cut along the dotted line in the anterior mesencephalon (Figure 1 F-H), separating the mesencephalon from the diencephalon, as shown in Figure 11. Separate the Dl from the TEL by cutting along the dashed lines in Figure 1 F. See isolated telencephalon in Figure 1 J. Split the two telencephalic hemispheres, cutting along the dotted line in Figure 1 J. This results in two separated hemispheres as illustrated in Figure 1 K. The left hemisphere is magnified in Figure 1 L.

Identify the medial ganglionic eminences (MGE, Figure 2A; *) and lateral ganglionic eminences (LGE, Figure 2A; +) visible through the future Foramen interventriculare. Using forceps or needle, dissect along a straight line at the intersection between the MGE and LGE and the anterior cortex (ant-ctx, Figure 2B). Cut a straight line through cortex, hippocampus (hip) and choroid plexus (CP). This separates the anterior cortex including the olfactory bulb (ob) from the ganglionic eminences (GE, Figures 2B, C). Cut a second straight line at the intersection of the caudal ganglionic eminence (CGE, Figure 2C, D) and the posterior cortex (Figure 2C), again cutting through cortex, hippocampus and choroid plexus. Flatten out the telencephalon. Completely remove the posterior pole and the olfactory bulb (Figure 2D). Identify the cortical hem containing the hippocampus and the choroid plexus (Figure 2C). The hippocampus has a thinner neuroepithelium than the cortex. The CP contains red vessels. Separate the cortical hem from the cortex, leaving the size of the cortex identical to the size of the ganglionic eminences (Figure 2D). This results in a block of the cortex (the target tissue) and the ganglionic eminences (GE, Figure 2D).

Flip the cortex-GE block with the ventricular (inner) side to the dish surface. The outer surface of the hemisphere will face the experimenter (Figure 2E). On the outer surface are the blood vessel-containing meninges (Figure 2E). Pin the GE to the dish surface with the left hand and peel the meninges off with a pair of forceps in the right (dominant) hand (Figure 2F). The separation of meninges from the cortex is facilitated by cutting a completely straight line (not jagged).

Repeat the procedure described above for the other hemisphere. Cut the cortex along the LGE in a distance of half the main diameter of the LGE (Figure 2G, 2H). The dissected cortex spans an area of 1 .2 mm x 2.4 mm (Figure 2H) and includes the SVZ/germinal layer with the NSCs.

Preparation of explant cultures

Cut the cortex into explants of less than 400 pm diameter (Figure 2I). Use a 400 pm grid positioned below the dissection Petri dish for reference (Figure 2H and 2I). Transfer all explants into a fresh Petri dish with ice-cold expansion medium. Remove the fibronectin from a tissue culture dish. Allow it to dry. Add 1 ml. of cold expansion medium to the centre of the 35 mm dish with grid dimension of 10 mm x 10 mm (Figure 3). Allow the medium to form a spherical drop (Figure 3).

Insert up to 8 explants to the centre of the drop. The explants should be ideally located on the grid with at least 3 mm distance between each other for the migration analysis. Incubate the dish for about 1 h in a cell culture incubator (37 °C, 21 % 0₂, 5 % C0₂) for attachment of the explants. Allow the explants to settle and attach to the fibronectin-coated surface. Do not shake the dish, since the explants may detach and move out of the optimal grid centre.

After 1 h incubation (37 °C, 21 % 0₂, 5 % C0₂), fill up the dish to a total volume of 2 ml_ expansion medium plus the growth factors or substances of interest to test and incubate. Neither enzymatic digestion, nor serum or centrifugation are necessary for explant preparation. This is optimal for cell integrity. Preparation of NSC single cell culture

Warm up expansion and passaging medium at 37 °C. Transfer the dissected cortex pieces into a 15 mL tube with cold expansion medium (tube A). Centrifuge the cortex pieces for 5 min at 1200 x g. Aspirate the supernatant. Add 200 μΙ_ pre-warmed expansion medium to resuspend the cortex pieces.

Add 700 μΙ_ of pre-warmed passaging medium to the 15 mL tube with a P1000 tip (tube A). Gently resuspend the pellet. Place the tip at the bottom of the 15 mL tube. Gently and slowly dissociate the tissue pieces by suctioning three times with the P1000 tip. Passaging medium promotes cell separation and is required to avoid the use of enzymes. Allow the tube to sit for 30 to 60 seconds for larger (heavier) undissociated pieces to settle at the bottom. Single dissociated cells will remain in the supernatant. Transfer about 700 pL of the supernatant to a fresh 15 mL tube (tube B). Repeat dissociating in passaging medium a second time to dissociate the larger pieces.

Transfer all supernatant to the fresh 15 mL tube (tube B). Add expansion medium to a total volume of 5 mL. Count cells using a hemocytometer. Assess dead cells by trypan-blue staining. Small NSCs tolerate the procedure better than larger cells. From 10 embryos expect about 4 x 10⁶ cells with 10 % dead cells. For expansion of NSCs, plate 1.5 x 10⁶ cells per 10 cm fibronectin-coated tissue culture dish in a volume of 8 mL expansion medium. For standard expansion, add 8 pL of 1000X rhFGF2 stock. Add 8 pL rhFGF2 every day and change media every other day. The cortex at this developmental stage contains a majority of NSCs and only a minority of differentiated cells. The differentiated minority cells do not respond to the rhFGF2-treatment and die during the expansion culture.

Passage expanded NSCs at 60 % confluence. To passage NSCs, remove expansion medium. Wash cells quickly three times with 5 mL passaging medium. Wait 2 - 4 minutes. Without Ca²⁺ and Mg²⁺ the cells slowly detach from the surface, rounding up. Verify detachment of cells under the phase microscope. Wait another few minutes, if needed, until visual detachment from surface is observed. Single cells will completely detach, but most cells will still adhere to the dish surface. The duration needed for detachment is dependent on the duration of fibronectin coating. A short fibronectin coating (12 h or less) results in faster cell detachment. For longer experiments (> 1 week) fibronectin coating of more than 24 h is recommended. Use a 10 mL pipette to gently detach the cells from the surface. Collect the 5 mL passaging medium with cells and transfer it to a fresh 15 mL tube. Repeat with an additional 5 mL of passaging medium.

Dissociate the cells in the 15 mL tube by pipetting up and down three times, placing the 10 mL pipet at the bottom of the tube. Spin the tube for 15 min at 1200 x g at room temperature. Remove the supernatant and resuspend the pellet in 2 mL expansion medium. Count cells using a hemocyto meter. Use trypan blue to assess dead cells. After expansion to 60 % confluence, expect 4-5 x 10⁶ cells with a dead cell count at around 10 %. Plate 8 x 10⁵ cells per 10 cm dish for further passages.

Migration assessment

For the migration assessment isolate explants according to the standardized dissection and preparation protocol of the cortical subventricular zone provided above. Seed explants in 35 mm grid dishes. Add FGF2 (rhFGF2) 1 h after plating to all dishes. Keep dishes for 2 d at 37 °C (incubator). Avoid excessive movements of the dishes for optimal explant attachment. Remove medium by 1000 μΙ_ tip. Add 2 ml. of expansion medium by 1000 μΙ_ tip starting at the inner circle (Figure 3). This reduces surface tension on explants.

Add growth factors alone or in combination as required for experiment. Use FGF2/BMP4/TGF31 as positive control. In this experiment, 2 μΙ_ FGF2 per 35 mm dish, 2 μΙ_ BMP4 and 4 μΙ_ TGF31 were added alone and in combination (Figure 5). Add additional factors and/or testable substances. Keep dishes again untouched for 2 days at 37 °C.

Take photographs of explants on an inverted microscope. Living explants yield better phase contrast images than fixed cells. Both may be used. For migration assessment, use a graphics software that allows pixel measurements (e.g. Fiji8). Measure the dish grid of 500 pm distance in pixels and use it as internal reference. Define the centre of the explant as migration start point. Measure the distance between the centre of the explant and the outermost group of 10 cells. All cells migrate centrifugally away from the explant. They spontaneously form a sheet at the periphery under the circumstances tested (Figure 5).

Invasion assessment

Prepare fibronectin-coated tissue culture 24-well plates according to the instructions provided above. Place 300 μΙ_ of warm expansion medium into the top well of cell invasion chambers. Incubate the chambers for 30 min at 37 °C and 5 % C0₂. Remove the expansion medium from the top well and place the Boyden chamber into the coated 24-well plate. Add 500 μΙ_ of warm expansion medium with 0.5 μΙ_ of rhFGF2 and 0.5 μΙ_ rhBMP4 to the bottom well. Passage NSCs and count as described above. Passages 1 to 4 are suitable.

Plate 5 x 10⁵ cells in a volume of 300 μΙ_ of warm expansion medium with 0.3 μΙ_ of rhFGF2 and 0.3 μΙ_ of rhBMP4 in the top well of the Boyden chamber. Culture the seeded NSCs for 24 h. Add additional factors and/or testable substances to the chamber and culture the cells for another 48 h. If the cells are invasive, they will pass from the top well through the basement membrane layer to the bottom well. Invasive cells will cling to the bottom of the Boyden chamber. Follow the protocol by the manufacturer. Use cotton swabs (included in the invasion assay kit) to remove the non-invasive cells in the top well by cleaning twice. Remove the medium from the wells and add 500 μΙ_ expansion medium with a plasma membrane stain for living cells and with the nuclear dye DAPI to the wells. Incubate for 10 min at 37 °C and 5 % C0₂. The dyes will integrate into the membrane and the nucleus of the invasive cells, respectively, for optimal visualization. Optionally omit plasma membrane dye if multicolour immunocytochemistry is planned.

Remove the medium with the dyes and wash twice with expansion medium. Visualize and count invasive cells with an inverted fluorescence microscope. Some invasive cells may have detached from the bottom of the chamber and have dropped to the well surface below where they stick to the coated surface. For complete analysis include the cells at the well surface.

Remove the medium and fix the cells in ice-cold fresh 4 % PFA for 10 min. Wash 3x with 1 X PBS. Perform immunocytochemistry on invasive cells.

Methods for obtaining the results shown in here were similar to those published in Sailer et al. 2013 J. Cell Science 126, 3533-3540, unless stated otherwise.

Results

The in vitro method of the present invention is based on the standardized isolation of NSCs both as single cells or as explants from a specific region of the developing neural tube, the subventricular zone of the central cortex (Figures 1 and 2). For quantitative assessment, explants were seeded right at the centre of a 500 pm grid culture dish (Figure 3). Explants from the central cortex were first exposed to FGF2 for two days, followed by additional two days in different combinations of growth factors (Table 1 ). The inventors established that only explants cultured in BMP4 showed a migratory response (Table 1 and Figure 8).

Next podoplanin (PDPN) expression was analysed under defined conditions. PDPN is a transmembrane sialomucin-like protein that has been associated with invasion in multiple cancers and also in NSCs. The proportion of PDPN expressing cells is increased in high- grade gliomas. PDPN is also associated with poorer survival in glioblastoma patients. Further, PDPN has been shown to be a marker of malignant progression in multiple tumours including breast, lung, colon carcinomas. PDPN is expressed both in invasive as well as migratory NSCs. Using the above protocol, PDPN expression was compared to control, FGF2 only, in explants exposed to TGF31 and BMP4 alone or in combinations. PDPN expression was detected in all BMP4 and TGF31/BMP4-treated explants. In contrast, no PDPN was observed in the control explants in FGF2 alone or in explants with TGF31 alone (Table 3 and Figure 4). In addition cells with a migratory phenotype were induced in BMP4 and TGF31/BMP4-exposed explants, whereas the control explants (only FGF2) and TGF31 alone did not show migratory cells (Table 2). The observation of migratory cells provides a low cost, straight-forward qualitative assessment.

Further, the inventors tested the migratory response and EMT induction of several regions of the developing neural tube to BMP4 (Table 3). 400 pm explants were prepared from the central cortex, the posterior cortex (labelled posterior pole), the MGE and the mesencephalon, as indicated in Figure 1. The explants were all exposed to FGF2 for two days, followed by two days in FGF2 with BMP4. Migration was defined by the appearance of flat migratory cells with a leading and trailing edge (Figure 5 and 8). A strong induction of migratory cells from the posterior cortex and an intermediate response from the MGE and the mesencephalon was observed (Table 3). 145 of 146 explants of the central cortex showed a migratory response in FGF2/BMP4. Thus, the central cortex demonstrated the most robust induction of migratory cells of all explants tested (Table 3). Omission of BMP4 abolished any migratory response (Table 2 and 3).

Table 2: BMP4 and the combination of TGFbetal with BMP4 induce a robust migratory response

Condition Total explants Explants with % Explants with %

(central cortex) migratory Podoplanin

response expression

Control 33 0 0 0 0

TGF l 21 0 0 0 0

BMP4 34 34 100 34 100

TGF 1+BMP4 22 22 100 22 100

For quantitative assessment of growth factor potency, migration distance was measured in explants cultured in multiple growth factors. Explants were cultured as above for two days in FGF2, then for additional two days in FGF2 without factors (control) or single or combined BMP4 and TGF31 . In the control explants a strong proliferation was observed in response to FGF2, as expected. The explants are derived from the cortical SVZ and contain to a large part NSCs which are known to proliferate in response to FGF2. The control cells reached 689 ± 14, the TGF31 cells 582 ± 49 pm (Figure 5.). The BMP4-group showed a mean migration distance of 935 ± 91 pm. In comparison the TGF31/BMP4-cells migrated significantly further, to 1 150 ± 23 pm (Figure 5). The results show that BMP4 is inducing a migration in FGF2-exposed NSCs. This migration can be further enhanced by TGF31. The combination of FGF2, BMP4 and TGF31 is therefore the most effective to induce migration. In summary, the cell culture model system allows both qualitative as well as quantitative migration assessment. Table 3: The central cortex shows the most robust migratory response when exposed to combined FGF2/BMP4

Region Total explants Explants with migratory % response in FGF2/BMP4

Central cortex 146 145 99.3

Posterior cortex 52 48 92.0

MGE 56 29 51.7

Mesencephalon 53 28 52.8

EMT has been linked to transcription factors (TFs), such as SnaiM , Snail2, Zeb1 , Zeb2, Twistl , Twist2 in various systems including epithelial cancers, such as breast cancer, colon cancer, lung cancer and also in brain tumours. Using the present invention no changes in Twistl expression could be detected; with Twistl being at low expression levels during FGF2 exposure. In the cell culture model an upregulation of Zeb1 and Zeb2 during FGF2-exposure and of Twist2 during FGF2/BMP4-exposure was observed (Figure 6).

NSCs are known to contribute to the population of oligodendrocyte precursor cells (OPCs). Several lines of evidence indicate that NSCs and in particular OPCs may be the cell of origin for gliomas. To characterize the above model further, the expression of OPC markers was investigated. FGF2-exposed NSCs demonstrated co-expression of Olig2/Nestin and Sox10/Nestin in more than 90 % (Figure 7). This observation demonstrates that the NSCs show OPC-features using the method of the invention. Indeed, the OPC-features correlated with upregulation of Zeb1 and Zeb2 (Figure 6 and 7). These results demonstrate that FGF2- expansion infers an OPC identity, as judged by Olig2 and Sox10, while at the same time initiates first Zeb-based steps of EMT.

Increase of invasiveness critically depends on TGFbeta

The Boyden chamber assay was conducted as laid out above, with 30.000 cells seeded per chamber in the presence of insulin. The invasion assay was performed with only FGF2 (F), only BMP4 (B), only TGFbeta (T), combined F+B, F+T, B+T and the combination FBT of all three factors:

The results of this experiment are shown in Fig. 10. Concepts and evidence behind the invention

Subject of this invention is a standardized method for obtaining and analysing proliferating and migratory/invasive NSCs by converting non-proliferating, non-migratory/invasive NSCs as described above and schematically summarized in Figure 9. The epithelial to mesenchymal transition (EMT) closely resembles the transition of the non-proliferating, non- migratory/invasive NSCs into proliferating and migratory/invasive NSCs being part of the invention.

The standardization of conditions and methods for the method of the invention ensures reproducibility (Table 2 and 3). The NSCs used in the method of the invention are derived from the developing cortex, a tissue that normally does not undergo EMT. This is of advantage for the analysis of early steps in EMT. Initial steps in EMT cannot be adequately studied in tumour cells that have accumulated genetic changes and may have already adopted EMT features. Moreover, primary tumor samples are not ideal since most malignant tumours are heterogeneous containing both invasive and non-invasive cells.

The disclosed invention provides EMT researchers with a novel model to study early steps of EMT induction. Without wishing to be bound by theory the inventors show that the key EMT inducers of the Zeb and Twist family are sequentially activated: During the first phase Zeb1 and Zeb2 are upregulated, during the second phase Twist2 (Figure 6). The results disclosed above demonstrate that all of the three known key EMT regulators are involved in the system. In addition, the inventors established that TGF31 can significantly enhance the migratory effect of FGF2/BMP4 alone. These results point to a network between FGF-, BMP- and TGF3-signalling during EMT induction.

The most critical steps for successful isolation of NSCs suitable for the method of the present invention are: correctly identifying the embryonic age, identifying anatomical landmarks of the developing embryo, preparation of fresh culture media and (at least overnight) coated plates, use of fine-tipped instruments and proper dissection of the central portion of the developing cortex to establish cortical explants.

The disclosed techniques may be enhanced by some modifications. As suggested above, multiple explants are plated on a single dish with a grid. For screening of multiple compounds, however, it may be more convenient to place single explants in every well of a 96-well plate. Further, the invention can be refined for large scale drug discovery. As described above, PDPN is a valuable marker for newly acquired migratory features since PDPN is found in invasive NSCs. A PDPN reporter can be transfected in normal NSCs before induction of migratory/invasive phenotype. As a consequence PDPN expressing cells can serve as an internal control for cell transformation since PDPN is not expressed in normal NSCs (Figure 4 and Table 2). Several additional NSC markers are available, such as Nestin, that are lost after EMT induction. As a consequence, both the initial state, "non- migratory/non-invasive/Nestin+", and the final state, "migratory/invasive/PDPN+", can be monitored, which enables automated drug discovery. In large cell culture plates with multiple wells (e.g. 96-well plates and larger) initial cells may be seeded and treated with established drugs or compounds without known function. Thus, multiwell plates can be automatically scanned for expression of PDPN or other migration/invasion markers. If an inhibitor is the main target of the screen, disappearance of PDPN expression can help to identify putatively interesting novel inhibitory compounds.

During testing of a novel substance the explants or cells may not show migration and/or the explants or cells may round up and detach from the dish. In this situation, it is best to change only half of the media every day (instead of full media change every other day). This reduces stress by surface tension when replacing with fresh media. The explants may not attach after the initial plating. In this situation, fibronectin has to be in contact with the dish surface for at least 36 h. Fibronectin used for coating should be freshly prepared without exposure to a plastic tube for more than 10 min since fibronectin may stick to the plastic. Further, the explants may settle outside of the grid. In this situation, it is advisable to swirl the dish in a slow circular motion. This allows the explants to reposition to the center by centripetal force (Figure 3).

There is strong evidence that gliomas are derived from NSCs and/or OPCs derived from NSCs (Das et al., Nat Clin Pract Neurol 4, 427-435, 2008; Modrek et al., World J Stem Cells 6, 43-52;2014). Several lines of evidence support the role of NSCs in glioma formation. Glioma progression has been linked to the following genes: Tenascin C, Hey1 , SPARC, SnaiH and Snail2, FGFR+, BMPRI a, EGFR, PDGFRa, Sox2, Podoplanin, GN3 and p75NGFR. All of these genes are also expressed during the transformation of normal noninvasive NSCs to invasive mesenchymal cells.

As a consequence others have established glioma growth models on the basis of NSCs: Sampetrean et al. (Neoplasia 13, 784-791 , 201 1 ) isolated NSCs from lnk4/ARF-deficient mice and forced overexpression of H-Ras. High-grade malignant tumors resulted, that showed proliferation and invasion after transplantation. McNeill et al. (JoVE, e51763, doi:10.3791/51763; 2014) also isolated NSCs from genetically modified mice (floxed Rb1 , Nf1 , Kras, Pten alone and in combinations). The genes were inactivated by Cre-virus infection and the NSCs were transplanted. Both these model systems have the advantage that invasion can be monitored in vivo and potential new anti-invasion drugs can be tested. Their main disadvantage is that the onset of invasion is not well defined and it is unclear whether the cells show glioma-typical EMT invasion (EMT genes) or only local migration. Further only one drug can be tested per animal and the analysis requires several weeks. To identify a novel anti-invasion drug, pharmaceutical companies need (a) to screen for several thousand compounds or more at once, (b) to replicate the tests and (c) to try different drug concentrations. To prepare several thousand transplanted animals, then treat them with different drugs and finally test for effects by histochemical or bioluminescence analysis is not economically feasible. Here a novel NSC-based model system for EMT-related invasion/migration is disclosed that allows the cost-efficient screening of thousands of compounds.

Tumour-initiating stem cells (TICs) from different tumors have been isolated and are used as models to understand tumor progression. TICs are, however, not well suited for EMT analysis, since EMT did already occur in TICs. To understand early and also late steps of EMT induction, a primary non-neoplastic cell population is needed. Ideally this population was not meant to undergo EMT in the first place.

The results disclosed herein show that a specific combination of four factors, FGF2, BMP4, TGF31 and insulin cause a very strong and complete EMT induction in NSCs, not observed before. The present invention demonstrates that the key EMT genes of the Zeb- and Twist- families are also upregulated. This provides the first evidence that there is not selective upregulation of single genes, but the results show that all key EMT families are active in the disclosed cell culture system.

EMT has also been observed in epithelial cancers outside of the brain, such as lung, breast, colon and gastric cancer. Several models to study EMT are in use for these tumors, which come, however, with significant limitations:

(a) usage of transformed tumor cell lines carrying multiple genetic and epigenetic changes; to identify inhibitors of EMT for early cancer stages, late-stage cancer cells are inadequate;

(b) serum-dependent cultures which contain various undefined growth factors; this renders identification of critical factors very difficult. Furthermore, experiment reproducibility is impaired since serum quality varies from batch to batch;

(c) serum contains inhibitors and enzymatic activities possibly inactivating potentially useful exogenous compounds;

(d) need of enzymes for cell passaging that degrade cell surface molecules;

(e) to identify EMT agonists to promote physiological EMT, normal cells are needed to test induction.

Tumour cell models are inadequate to find substances that promote regeneration in normal stem cells. Migration is also required for normal processes, such as wound healing and regeneration of the injured brain. After traumatic brain injury and stroke, NSCs are necessary to migrate to the lesion site to participate in the regeneration. The current system may also be used to identify substances that promote migration and invasion. As disclosed above, migration is induced upon start with BMP4-treatment. If the cells are not exposed to BMPs but instead to novel compounds, these may substitute for the BMP action which will help to identify novel BMP agonists. With a reporter system that indicates PDPN expression, large scale testing for novel substances becomes feasible; if a novel compound can substitute for BMPs, PDPN expressing cells can be automatically identified.

The disclosed system of the invention is also useful to investigate cell-signalling interactions. The results disclosed herein show that the BMP effect on migration could be enhanced by TGF31 activation. The results uncover an additive interaction between BMP- and TGF3- signalling. In addition, the responsive central cortex can be isolated from genetically modified mice, which can help to elucidate underlying mechanisms driving EMT.

In summary, the EMT model system of the present invention is useful in the fields of stem cell biology and regeneration, as well as in cancer research. It can be used for screening of drug libraries for substances inhibiting or enhancing migration, invasion, apoptosis and/or proliferation.

Claims

1. A method for obtaining an isolated migratory or migratory and invasive neural stem cell, comprising:

a) providing a neural stem cell,

c) cultivating said proliferating neural stem cell in the presence of FGF2, BMP4 and TGFbetal ;

yielding a migratory or migratory and invasive neural stem cell and

d) isolating said migratory or migratory and invasive neural stem cell.

2. The method according to claim 1 , wherein cultivating said proliferating neural stem cell is performed in the additional presence of insulin.

3. The method according to claim 1 or 2, wherein

TGFbetal is used at a final concentration of 20 ng/ml to 60 ng/ml, in particular 30 ng/ml to 40 ng/ml.

4. The method according to any one of claims 1 or 2, wherein the neural stem cell is a neural stem cell originating from the cortical subventricular zone, in particular the subventricular zone of the central cortex or posterior cortex, of a mammal.

5. A cell preparation comprising isolated migratory or migratory and invasive neural stem cells (NSCs) obtained by a method according to any one of claims 1 to 4.

6. A cell preparation comprising isolated migratory or migratory and invasive neural stem cells (NSCs), obtained from isolated non-migrating and non-invasive NSCs originating from the cortical subventricular zone, in particular the subventricular zone of the central cortex or the posterior cortex, of a mammal by treatment with fibroblast growth factor 2 (FGF2) and optionally insulin followed by treatment with FGF2, BMP4 and transforming growth factor β 1 (TGFbetal ), and optionally insulin, wherein said cell preparation of migratory or migratory and invasive neural stem cells is characterized by the expression of a marker selected from podoplanin, p75NGFR, smooth muscle actin, calponin and/or Sox9.

Use of a cell preparation according to any one of claims 5 or 6 for drug screening.

Use of a cell preparation according to any one of claims 5 or 6 for screening of candidate compounds affecting cell migration and/or cell invasion, comprising the steps of:

a. providing a cell preparation according to any one of claims 5 or 6, or providing cells obtained by a method according to any one of claims 1 to 4,

b. bringing a candidate compound in contact with said cell preparation, c. assessing a cell migration and/or cell invasion parameter of cells of said cell preparation,

A method for the identification of a candidate compound affecting cell migration and/or cell invasion of a neural stem cell, comprising:

a) providing a plurality of neural stem cells (NSCs),

c) cultivating a first portion of said plurality of proliferating NSCs in the presence of FGF2, BMP4 and TGFbetal , and optionally insulin,

i) a candidate compound, or

ii) a candidate compound in the presence of BMP4 and TGFbetal ;

yielding cultivated NSCs

d) determining a marker of migration and/or a marker of invasion in said first and said second portion of cultivated NSCs, wherein the presence of said marker of migration and/or said marker of invasion in said second portion in i. is indicative of a compound stimulating migration and/or invasion, and

the value of said marker of migration and/or said marker of invasion in said second portion in ii. is compared to a standard range determined in said first portion of cultivated NSCs, wherein a value lower than the standard range is indicative of a compound inhibiting migration and/or invasion and a value higher than the standard range is indicative of a compound enhancing migration and/or invasion.

10. The method according to claim 9, wherein

- FGF2 is used at a final concentration of 5 ng/ml to 20 ng/ml, in particular

10 ng/ml to 15 ng/ml; and/or

1 1. The method according to any one of claims 9 and 10, wherein:

• the marker of migration is selected from:

- expression of smooth muscle actin, calponin, Sox9, SPARC, Tenascin C,

Twistl or Twist2,

migratory morphology and/or movement of said cultivated NSCs, and

• the marker of invasion is selected from:

invasive movement of said cultivated NSCs,

- expression of SnaiM , Snail2, Podoplanin, p75NGFR, metalloproteinases.

12. The method according to any one claims 9 to 1 1 , wherein the neural stem cell originates from the cortical subventricular zone, in particular the subventricular zone of the central cortex.

13. The method according to any one of claims 9 to 12, wherein

- in step a) the provided neural stem cell comprises a reporter protein, in particular a fluorescent protein, under transcriptional control of the promoter of a gene specific for migratory cells, in particular podoplanin, p75NGFR, smooth muscle actin, calponin, Sox9, SPARC, Tenascin C, SnaiM , Snail2, Twistl or Twist2 and/or a reporter protein, in particular a fluorescent protein, under the transcriptional control of the promoter of the gene specific for non-invasive cells, in particular nestin; and

in step d) the expression of said reporter protein(s) is determined instead of the marker of migration.

14. A method for the identification of a candidate compound affecting proliferation of a neural stem cell, comprising:

a) providing neural stem cells (NSCs),

b) cultivating a first portion of said NSCs in the presence of FGF2 and optionally insulin, and cultivating a second portion of said NSCs in the presence of I. a candidate compound, or

II. a candidate compound, FGF2 and optionally insulin

- the presence of said marker of proliferation in said second portion of cultivated NSCs of step b) I. is indicative of a candidate compound stimulating proliferation, and

15. The method according to claim 14, wherein

16. The method according to any one of claims 14 and 15, wherein the marker of proliferation is selected from: mitotic phenotype, expression of Ki-67 (MIB-1 ), expression of phospho-histone 3 (PH-3) and BrdU incorporation.