AND THE NEUROVASCULAR UNIT The present invention relates to a method of generating brain microvascular endothelial cells from pluripotent stem cells.

The neurovascular unit (NVU) is an integrated functional unit enabling the complex interplay between neurons and microvessels responsible for the coupling of neural activity and blood supply to the brain. The NVU is composed of brain microvascular endothelial cells, functionally coupled with neurons, astrocytes, mural cells, such as pericytes and smooth muscle cells, and extracellular matrix components. Many disease processes may affect the functioning of the NVU and the study of the NVU may reveal new drug targets. The blood brain barrier (BBB) is a selectively permeable barrier separating the brain extracellular fluid of the central nervous system and circulating blood, where it lines all capillaries and intracranial blood vessels. The blood brain barrier is primarily formed by brain endothelial cells of the NVU, which are connected by tight junctions with a high electrical resistivity of at least 0.1 Ωμιη. The blood brain barrier is selectively permeable as it allows the passage of water, some gases, and lipid-soluble molecules by passive diffusion, as well as the selective transport of molecules such as glucose and amino acids that are necessary for neural function. Astrocytes and mural cells, such as pericytes and smooth muscle cells participate in maintaining the integrity of the BBB. The blood brain barrier is of critical importance when designing and screening for potential therapeutics and many potential candidate drugs acting in the central nervous system will fail to provide their therapeutic effect if they are unable to cross the blood brain barrier in sufficient quantity. Therefore the study of the blood brain barrier and transport mechanisms across the blood brain barrier is critical in the development of neurotherapeutics.

Tissue models of the neurovascular unit or blood brain barrier can help to increase understanding and help to screen agents that either act on the neurovascular unit or blood brain barrier itself, or must pass through the blood brain barrier to deliver their therapeutic effect in the central nervous system. Current methods exist to attempt to form a tissue model. However, the models are difficult to reproduce, and can often lack sufficient characteristics of a blood brain barrier in vivo. For example the number and quality of tight junctions formed is important for forming a realistic model and are therefore poorly predictive of CNS penetration of therapeutics.

For example, the simplest models available are those that employ an endothelial cell 2D monolayer using animal brain-derived endothelial cells, such as rat, bovine, porcine and murine cells. However, in the absence of the other supporting cells that constitute the NVU, the endothelial cells may not represent the unique characteristics of BMEC and typically these models show very low trans-endothelial electrical resistance (TEER) measurements. Moreover, there are inherent differences between the blood-brain barriers of different species, including differential expression of enzymes, transporter and tight junction proteins, limiting translation to human subjects. Models using co-cultures of endothelial cells with astrocytes and/or mural cells and/or neurons offers only moderate to low TEER values, and there is large variability experienced between similar protocols. Such methods also do not accurately replicate the restrictive nature of the BBB and are also restricted by species differences Models using human immortalized and primary cell lines suffer from low baseline TEER values and discontinuous tight junction expression. Additionally, immortalized cell lines tend not to recapitulate normal physiology. Obtaining the primary human tissue can be difficult and primary human cells have limited proliferative abilities. The cells also tend to de-differentiate, and have decreased barrier properties after removal from the brain microenvironment. Human hematopoietic stem cell BBB models have shown low TEER values, the hematopoietic stem cells may be difficult to obtain and they have restricted differentiation potential.

Human pluripotent stem cells provide an opportunity to derive tissue models of the NVU/BBB from healthy subjects and patients. They also offer the potential to generate all the constituent cells (the brain microvascular endothelial cells, astrocytes, neurons and pericytes) of the NVU from the same subject. They may also be used with genome engineering technologies to express specific drug targets or to modulate expression of endogenous genes to model disease or to better understand BBB transport mechanisms in human tissue. Pluripotent stem cells models of brain microvascular endothelial cells including those derived from human embryonic stem cells or induced pluripotent stem cells depend upon spontaneous differentiation into brain microvascular endothelial cells and are not chemically defined nor are they specifically directed to this cell fate. These approaches are therefore dependent on whether a particular stem cell line is already predisposed to differentiating into brain microvascular endothelial cells. In summary, the models derived from pluripotent stem cells tend to be difficult to reproduce and the efficiency and properties of brain microvascular endothelial produced may vary between different stem cell lines. Therefore, an aim of the present invention is to improve on current methods for generating human brain microvascular endothelial cells to establish better blood brain barrier tissue models.

Method of generating human brain microvascular endothelial cells

According to a first aspect of the present invention, there is provided a method for producing brain microvascular endothelial cells comprising:

(A) providing pluripotent stem cells (PSCs);

(B) inducing the formation of an early mesoderm cell culture comprising incubation of the PSCs in a chemically defined medium (CDM) comprising

mesodermal inducing agent,

fibroblast growth factor,

phosphoinositide 3 -kinases pathway inhibitor, and

TGF-beta pathway inhibitor;

(C) further patterning mesoderm formation comprising incubating the early mesoderm cell culture in a chemically defined medium (CDM) comprising fibroblast growth factor, phosphoinositide 3 -kinases pathway inhibitor and TGF-beta pathway inhibitor;

(D) induction of endothelial cell formation comprising incubating the mesodermal cells in a chemically defined medium (CDM) comprising:

i) an agent capable of inducing adherens junction marker and vascular endothelial cell makers, and

ii) a cAMP signalling activator;

(E) induction of brain microvascular endothelial cell (BMEC) formation comprising incubating the endothelial cells in a chemically defined medium (CDM) comprising: an agent capable of inducing adherens junction markers and vascular endothelial cell makers;

a Wnt signalling pathway activator; and

an agent capable of activating retinoic acid signalling.

The present invention advantageously provides a method to generate brain microvascular endothelial cells (BMECs) from PSCs, such as hPSCs, which is rapid and reproducible across different PSC lines and thus is robust. The brain microvascular endothelial cells have prolonged maintenance of tight junction protein expression.

Providing PSCs (A)

In one embodiment, the pluripotent stem cells are human PSCs (hPSCs). The provided PSCs, such as hPSCs, may be undifferentiated. Human pluripotent stem cells may be cultured on hESC-qualified Matrigel in mTeSRl medium. Alternatively, hPSCs can also be cultured on other substrates such as, Geltrex, vitronectin or biolamina and maintained in other media such as StemFlex or E8 media. Cultures may be passaged every 4-6 days.

Different PSC lines and culture conditions require different starting densities for early mesoderm induction. In one embodiment, the hPSCs may be provided in a cell density of between about 20,000 and about 90,000 cells/cm ² .

Early mesoderm induction step (B) Early mesoderm is a precursor mesodermal stage identified by the expression of key marker genes, such as one or more, or all of, Brachyury, Snail, TBX6 and N-cadherin, which then subsequently mature into definitive mesoderm.

The chemically defined medium (CDM) of the early mesoderm induction step may comprise or consist of a neurobasal medium. The skilled person will recognise that a neurobasal medium is a basal medium designed for long-term maintenance and maturation of pure pre-natal and embryonic neuronal cell populations. In one embodiment the neurobasal medium comprises N2B27 medium. In another embodiment, the chemically defined medium may comprise or consist of Essential 6 (E6) medium. In another embodiment, the chemically defined medium may comprise or consist of TesR-E6 medium. In another embodiment, the chemically defined medium may comprise or consist of Stem Pro 34 medium. The neurobasal (N2B27) medium may comprise one or more, or all of, DMEM/F12 medium; neurobasal medium, B27; N2 and β- Mercaptoethanol. The ratio of DMEM/F12 medium to neurobasal medium may be about 1 : 1. B27 may comprise about 2% of the neurobasal medium. N2 may comprise about 1% of the neurobasal medium. β-Mercaptoethanol may comprise about 0.1% of the neurobasal medium.

The mesordermal inducing agent may comprise a growth and differentiation factor. In one embodiment, the mesordermal inducing agent comprises BMP4 (bone morphogenic protein 4). The BMP4 may be human BMP4. The mesordermal inducing agent, such as hBMP4, may be provided at a concentration of about lOng/ml. Alternatively, the mesordermal inducing agent, such as hBMP4, may be provided at a concentration of between about 5 and about 30 ng/ml. Alternatively, the mesordermal inducing agent, such as hBMP4, may be provided at a concentration of between about 5 and about 20 ng/ml. Alternatively, the mesordermal inducing agent, such as hBMP4, may be provided at a concentration of between about 5 and about 15 ng/ml. Alternatively, the mesordermal inducing agent, such as hBMP4, may be provided at a concentration of between about 8 and about 12 ng/ml.

In one embodiment, the fibroblast growth factor in the chemically defined medium of the early mesoderm induction step comprises a basic fibroblast growth factor (bFGF). In one embodiment, the bFGF is human bFGF. Alternatively the bFGF may be murine, rat or bovine. The bFGF may be provided in the amount of about 20ng/ml. Alternatively, the bFGF may be provided in the amount of between about 10 and about 40 ng/ml. Alternatively, the bFGF may be provided in the amount of between about 15 and about 25 ng/ml. The bFGF may be provided in the amount of less than 25 ng/ml. Alternatively, the bFGF may be provided in the amount of between about 10 and about 120 ng/ml. Alternatively, the bFGF may be provided in the amount of between about 10 and about 100 ng/ml. Alternatively, the bFGF may be provided in the amount of less than about 100 ng/ml.

In one embodiment, the chemically defined medium in the early mesoderm induction step further comprises an inhibitor of transforming growth factor beta (TGF-β). The inhibitor of TGF-β may comprise SB431542 (Glaxo SmithKline PLC) (4-(4-(benzo[d][l,3]dioxol-5-yl)-5- (pyridin-2-yl)-lH-imidazol-2-yl)benzamide), or a functional derivative or analogue thereof. The SB431542 may be provided at a concentration of about l um. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 2um. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 2μηι. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 1.5μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.8 and about 1.2um. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 15μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 12μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about ΙΟμιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 8μτη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 5μτη.

The inhibitor of PI3K pathway in the early mesoderm induction step may comprise a phosphoinositide 3-kinase (PI3K) inhibitor. The inhibitor of PI3K pathway may comprise LY294002 (Sigma-Aldrich) (2-(4-Moφholinyl)-8-phenyl-l(4H)-benzopyran-4-one hydrochloride), or a functional derivative or analogue thereof. The LY294002 may be provided at a concentration of about ΙΟμιη. In another embodiment, the LY294002 may be provided at a concentration of between about 5 and about 20μιη. In another embodiment, the LY294002 may be provided at a concentration of between about 5 and about 20μιη. In another embodiment, the LY294002 may be provided at a concentration of between about 5 and about 15μιη. In another embodiment, the LY294002 may be provided at a concentration of between about 8 and about 12μτη.

In one embodiment, there is substantially no Wnt pathway activation/stimulation provided during the early mesoderm induction step. The chemically defined medium of the early mesoderm induction step may be substantially free of (i.e. does not comprise) a Wnt pathway activator.

The early mesoderm induction step may comprise incubation of the cells in the chemically defined medium over a period of time sufficient to form early mesoderm cells. In one embodiment, the early mesoderm induction step comprises incubation of the cells in the chemically defined medium over a period of time until early mesoderm is formed. The determination that early mesoderm has formed may comprise the detection or quantification of a cell marker for early mesoderm. The cell marker may comprise or consist of Brachyury. In another embodiment, the cell marker may comprise or consist of one or more, or all of, Brachyury, Snail, TBX6 and N-cadherin. The cell marker may be detected by protein expression, for example using immunofluorescence or mRNA expression (e.g. using qRT- PCR). The early mesoderm may be considered formed (i.e. for moving onto the next step) when at least 60% of the cells are early mesodermal cells. In another embodiment, the early mesoderm may be considered formed when at least 70% of the cells are early mesodermal cells. In another embodiment, the early mesoderm may be considered formed when at least 80% of the cells are early mesodermal cells. The skilled person will understand that the formation of early mesoderm cells may be provided as much as necessary, with the understanding that the number or percentage of early mesoderm cells formed at this stage will have an impact on the end yield of brain microvascular endothelial cells. In particular, the higher amount of early mesoderm cells at this step will result in a higher end number of brain microvascular endothelial cells.

The early mesoderm induction step may comprise incubation of the cells in the chemically defined medium over a period of about 1.5 days. Alternatively, the early mesoderm induction step may comprise incubation of the cells in the chemically defined medium over a period of at least 1.5 days. Alternatively, the early mesoderm induction step may comprise incubation of the cells in the chemically defined medium over a period of between about 1 and about 3 days (between about 24hrs and about 72hrs). The early mesoderm induction period may be defined as the time between the start of incubation of the hPSCs in the chemically defined medium of the early mesoderm induction step and the change of medium to the mesoderm patterning step. The chemically defined medium may not be changed/refreshed during the incubation period.

The method may comprise an enrichment step to select or harvest early mesodermal cells out of a mixture of cells formed at this step using a cell surface marker such as CD56/NCAM. For example, FACS or MACS may be provided to sort cells using the cell surface marker and isolate and collect early mesodermal cells. Alternatively a reporter construct for a mesodermal specific gene may be generated and used to isolate the relevant cell population. Therefore, where enrichment is provided, the amount of early mesodermal cells formed may be low, for example at least 5% early mesodermal cells may have formed, and then isolated for the next step of mesoderm patterning.

The early mesoderm induction step incubation may be at 37°C, 5% C0 ₂ .

Following the incubation in chemically defined medium for the early mesoderm induction step, and optionally enrichment for early mesoderm cells, the method moves onto the mesoderm patterning step. For mesoderm patterning, the chemically defined medium of the early mesoderm induction step may be replaced with the chemically defined medium of the mesoderm patterning step, and the incubation may be continued. In another embodiment, the early mesoderm induction step and the mesoderm patterning step are a single incubation step, with removal, inactivation or sequestration of the mesodermal inducing agent, such as BMP4, partway through the incubation. The early mesoderm induction step and the mesoderm patterning step may be the same, except for the absence of active mesodermal inducing agent, such as BMP4, in the mesoderm patterning step.

Replacement of the chemically defined medium of the early mesoderm induction step may comprise aspirating and discarding the medium. Prior to the addition of the chemically defined medium of the mesoderm patterning step, the cells may be washed. In one embodiment, the cells are washed with chemically defined medium, such as neurobasal medium, for example N2B27 medium. Mesoderm patterning step (C)

Mesoderm patterning is an event mediated by signaling molecules and factors, which determine the fate and lineage restriction of pluripotent cells to mesoderm in expense of ectoderm and endoderm germ layers.

The mesoderm patterning step may comprise at least a first stage and a second stage following a medium change therebetween. The reason to change media at this stage may be to withdraw TGF-β inhibition, such as from SB431542. Prolonged exposure can lead to downregulation of endothelial markers. In an alternative embodiment, the medium is not changed, but the TGF- β inhibition is prevented in the second stage by inactivation, removal or sequestration of the TGF- β inhibitor. In one embodiment, the TGF-β inhibition, such as from SB431542, is for only part of the mesoderm pattering step, such as the first stage of the mesoderm pattering step.

The chemically defined medium (CDM) of the mesoderm patterning step (for both first and second stages) may comprise or consist of neurobasal medium. In one embodiment the neurobasal medium comprises N2B27 medium. In another embodiment, the chemically defined medium may comprise or consist of Essential 6 (E6) medium. In another embodiment, the chemically defined medium may comprise or consist of Stem Pro 34 medium. The N2B27 Medium may comprise one or more of, or all of, DMEM/F12 medium; neurobasal medium, B27; N2 and β-Mercaptoethanol. The N2B27 Medium may comprise one or more of, or all of, DMEM/F12 medium; neurobasal medium, B27; and N2. The ratio of DMEM/F12 medium to neurobasal medium may be about 1 : 1. B27 may comprise about 2% of the neurobasal medium. N2 may comprise about 1% of the neurobasal medium. β-Mercaptoethanol may comprise about 0.1% of the neurobasal medium. The skilled person will appreciate that the precise amounts of medium constituents may be varied within acceptable limits to still support the growth or maintenance of the cells.

In one embodiment, the fibroblast growth factor in the chemically defined medium of the mesoderm patterning step (both first stage and second stage) comprises a basic fibroblast growth factor (bFGF). In one embodiment, the bFGF is human bFGF. Alternatively the bFGF may be murine, rat or bovine. The bFGF may be provided in the amount of about 20ng/ml. Alternatively, the bFGF may be provided in the amount of between about 10 and about 40 ng/ml. Alternatively, the bFGF may be provided in the amount of between about 15 and about 25 ng/ml. The bFGF may be provided in the amount of less than 25 ng/ml. In another embodiment, the bFGF may be provided in the amount of between about 2 and about 100 ng/ml. In another embodiment, the bFGF may be provided in the amount of between about 2 and about 120 ng/ml. In another embodiment, the bFGF may be provided in the amount of between about 2 and about 80 ng/ml. In another embodiment, the bFGF may be provided in the amount of less than about 100 ng/ml.

In one embodiment, the chemically defined medium in the first stage of the mesoderm patterning step comprises an inhibitor of transforming growth factor beta (TGF-β), and the medium of a later stage, such as the second stage does not substantially comprise an inhibitor of transforming growth factor beta (TGF-β). The inhibitor of TGF-β may comprise SB431542 (Sigma), or a functional derivative or analogue thereof. The SB431542 may be provided at a concentration of about Ι μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 2um. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 2μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 1.5um. In another embodiment, the SB431542 may be provided at a concentration of between about 0.8 and about 1.2μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 15μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 12μιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about ΙΟμιη. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 8 urn. In another embodiment, the SB431542 may be provided at a concentration of between about 0.5 and about 5um.

In one embodiment, the chemically defined medium in the second stage of the mesoderm patterning induction step does not substantially comprise an inhibitor of transforming growth factor beta (TGF-β) kinase. For example, the neurobasal medium in the second stage of the mesoderm patterning step may not substantially comprise SB431542.

The inhibitor of PI3K pathway in the mesoderm patterning step (both first stage and second stage) may comprise a phosphoinositide 3-kinase (PI3K) inhibitor. The inhibitor of PI3K pathway may comprise LY294002 (Sigma-Aldrich), or a functional derivative or analogue thereof. The LY294002 may be provided at a concentration of about ΙΟμιη. In another embodiment, the LY294002 may be provided at a concentration of between about 5 and about 20μιη. In another embodiment, the LY294002 may be provided at a concentration of between about 5 and about 20μιη. In another embodiment, the LY294002 may be provided at a concentration of between about 5 and about 15μιη. In another embodiment, the LY294002 may be provided at a concentration of between about 8 and about 12μιη.

The medium of the mesoderm patterning step (both first and second stages) may be substantially free from mesodermal inducing agent. The medium of the mesoderm patterning step (both first and second stages) may be substantially free from BMP4, such as hBMP4. In another embodiment, the medium of the second stage of the mesoderm patterning step may be substantially free from mesodermal inducing agent, such as BMP4 or hBMP4. The mesoderm patterning step may comprise incubation of the cells in the chemically defined medium over a period of time sufficient to form mesoderm. In one embodiment, the mesoderm patterning step comprises incubation of the cells in the chemically defined medium over a period of time until the mesoderm is appropriately patterned. The determination that the mesoderm has appropriately patterned may comprise the detection or quantification of a cell marker for TCF15. Detection of the cell marker may be by protein expression, for example using immunofluorescence or mRNA expression (e.g. using qRT-PCR). The patterned mesoderm may be considered formed when at least 60% of the cells are mesodermal cells expressing TCF15. In another embodiment, the patterned mesoderm may be considered formed when at least 70% of the cells are mesodermal cells expressing TCF15. In another embodiment, the patterned mesoderm may be considered formed when at least 80% of the cells are mesodermal cells expressing TCF15. In another embodiment, the patterned mesoderm may be considered formed when at least 90% of the cells are mesodermal cells expressing TCF15. The skilled person will understand that the formation of mesoderm patterned cells may be provided as much as necessary, with the understanding that the number or percentage of the mesoderm patterned cells formed at this stage will have an impact on the end yield of brain microvascular endothelial cells. In particular, the higher amount of mesoderm patterned cells at this step will result in a higher end number of brain microvascular endothelial cells.

The method may further comprise an enrichment step to select or harvest mesodermal patterned cells out of a mixture of cells formed at this step. For example, FACS or MACS may be provided to sort cells using the cell surface marker such as PDGFRa and isolate and collect early mesodermal cells. Alternatively a reporter construct for a patterned mesodermal specific gene such as TCF15 may be generated and used to isolate the relevant cell population. Therefore, where enrichment is provided, the amount of patterned mesodermal cells formed may be low, for example at least 5% patterned mesodermal cells may have formed, and then isolated for the next step of mesoderm patterning.

The mesoderm patterning step may be from the period of about day 1.5 to about day 5 of the method of the invention. The mesoderm patterning step may comprise incubation of the cells in the chemically defined medium over a period of about 3.5 days. Alternatively, the mesoderm patterning step may comprise incubation of the cells in the chemically defined medium over a period of between about 1 and about 6 days. Alternatively, the mesoderm patterning step may comprise incubation of the cells in the chemically defined medium over a period of about 2-3 days for the first stage, followed by a medium change and incubation in the second stage for about 1-3 days, or 2-3 days. The mesoderm patterning period may be defined as the time between start of incubation of the early mesoderm cell culture in the chemically defined medium of the mesoderm patterning step and the change of medium to the endothelial cell induction step. The chemically defined medium may not be changed/refreshed during the first stage incubation period or during the second stage incubation period.

The mesoderm patterning step incubation may be at 37°C, 5% C0 ₂ .

Following the incubation in chemically defined medium for the mesoderm patterning step, the method moves onto the endothelial cell induction step. For inducing endothelial cell formation, the chemically defined medium of the mesoderm patterning step may be replaced with the stem cell medium of the endothelial cell induction step, and the incubation is continued.

Replacement of the chemically defined medium of the mesoderm patterning step may comprise aspirating and discarding the medium. Prior to the addition of the stem cell medium of the endothelial cell induction step, the cells may be washed. In one embodiment, the cells are washed with stem/progenitor cell medium, such as with StemPro 34 medium.

Endothelial cell induction step (D)

In one embodiment, the chemically defined medium of the endothelial cell induction step comprises or consists of stem cell medium. In one embodiment, the stem cell medium of the endothelial cell induction step comprises or consists of StemPro 34 medium. The StemPro 34 medium may comprise one or more, or all of, antibiotics, such as penicillin and/or streptomycin; Glutamax; and StemPro-34 Supplement. The StemPro 34 medium may comprise Glutamax and StemPro-34 Supplement. Antibiotics, such as Pen/Strep, may be provided in an amount of 1 : 100. Glutamax may be provided in an amount of 1 : 100.

The agent capable of inducing adherens junction marker and vascular endothelial cell markers may comprise or consist of VEGF. In another embodiment, the agent capable of inducing adherens junction marker may comprise an E-twenty six (ETS) transcription factor modulator or Tcea3 modulator, which is an isoform of transcription elongation factor TFIIS. The VEGF may be provided in the stempro medium at a concentration of about 200ng/ml. In another embodiment, the VEGF may be provided in the stem cell medium at a concentration of between about lOOng/ml and about 300 ng/ml. In another embodiment, the VEGF may be provided in the stem cell medium at a concentration of between about 150ng/ml and about 250 ng/ml. In another embodiment, the VEGF may be provided in the stem cell medium at a concentration of between about 190ng/ml and about 210 ng/ml. In another embodiment, the VEGF may be provided in the stem cell medium at a concentration of between about lOng/ml and about 100 ng/ml. In another embodiment, the VEGF may be provided in the stem cell medium at a concentration of about 50 ng/ml.

In one embodiment, the cAMP signalling activator comprises Forskolin. In another embodiment, the cAMP signalling activator comprises a functional equivalent or variant of Forskolin. In one embodiment, the cAMP signalling activator comprises the provision of cAMP. The cAMP signalling activator, such as Forskolin, may be provided at a concentration of about 2μΜ. Alternatively, the cAMP signalling activator, such as Forskolin, may be provided at a concentration of between about ΙμΜ and about 4μΜ. Alternatively, the cAMP signalling activator, such as Forskolin, may be provided at a concentration of between about 1 μΜ and about 3μΜ. Alternatively, the cAMP signalling activator, such as Forskolin, may be provided at a concentration of between about 1.5μΜ and about 2.5μΜ. Alternatively, the cAMP signalling activator, such as Forskolin, may be provided at a concentration of between about 1.8μΜ and about 2.2μΜ. In one embodiment, the chemically defined medium, such as stempro medium, of the endothelial cell induction step may be replaced/refreshed, for example part way through the endothelial cell induction step. In one embodiment, the medium replacement is about 1 day after the start of the endothelial cell induction step. In particular the stem cell medium of the endothelial cell induction step is refreshed after about 24 hours of culture. The replacement medium may be substantially the same. The replacement may comprise aspirating and discarding the stem cell medium and providing fresh stem cell medium comprising an agent capable of inducing adherence junction marker, and a cAMP signalling activator. For example, the agent capable of inducing adherence junction marker, and the cAMP signalling activator according to the original medium to be refreshed. The replacement medium may be the same chemically defined medium, or a different chemically defined medium. In one embodiment, the replacement medium is human endothelial cell medium (HEM) or E6. In one embodiment, the replacement medium is human endothelial cell medium (HEM).

The endothelial cell induction step may comprise incubation of the cells in the stem cell medium over a period of time sufficient to form endothelial cells. In one embodiment, the endothelial cell induction step comprises incubation of the cells in the stem cell medium over a period of time until endothelial cells are formed. The endothelial cells may be considered formed when at least 60% of the cells are endothelial cells. The endothelial cells may be considered formed when at least 70% of the cells are endothelial cells. The endothelial cells may be considered formed when at least 80% of the cells are endothelial cells. The endothelial cells may be considered formed when at least 90% of the cells are endothelial cells. The skilled person will understand that the formation of endothelial cells may be provided as much as necessary, with the understanding that the number or percentage of e endothelial cells formed at this stage will have an impact on the end yield of brain microvascular endothelial cells. In particular, the higher amount of endothelial cells at this step will result in a higher end number of brain microvascular endothelial cells.

The determination that endothelial cells have formed may comprise the detection or quantification of a cell marker for endothelial cells. The cell marker for endothelial cells may comprise or consist of VE-cadherin and/or PECAM1. Additionally, or alternatively, the presence of von Willebrand Factor may confirm the differentiation into endothelial cells. The endothelial cell marker can be detected as protein expression by for example immunocytochemistry or flow cytometry, or by gene expression (e.g. by qRT-PCR).

The endothelial cell induction step may comprise incubation of the cells in the chemically defined medium over a period of about 2 days. Alternatively, the endothelial cell induction step may comprise incubation of the cells in the chemically defined medium over a period of at least 1.5 days. Alternatively, the endothelial cell induction step may comprise incubation of the cells in the chemically defined medium over a period of between about 1 and about 3 days (between about 24hrs and about 72hrs). The endothelial cell induction step period may be defined as the time between the start of incubation of the patterned mesoderm cells and the change of medium to the BMEC induction step or freezing of the endothelial cells for later use. The chemically defined medium may not be changed/refreshed during the incubation period, unless otherwise specified.

The method may comprise an enrichment step to select or harvest endothelial cells out of a mixture of cells formed at this step. For example, FACS or MACS may be provided to sort cells and isolate and collect endothelial cells. Alternatively a reporter construct for a vascular endothelium specific gene may be generated and used to isolate the relevant cell population. Therefore, where enrichment is provided, the amount of endothelial cells formed may be low, for example at least 5% endothelial cells may have formed, and then isolated for the next step of inducing brain microvascular endothelial cells. The endothelial cells of the endothelial cell induction step may be continued onto the step of induction of brain microvascular endothelial cells. Additionally or alternatively, the endothelial cells may be frozen at this stage.

Freezing some or all of the endothelial cells may comprise replacement of the stem cell medium with a freezing media. The freezing media may comprise 2X freezing media described herein. The freezing media may comprise a cell growth media. In one embodiment the 2X freezing media comprises ES-qualified FBS, KO-DMEM and DMSO. In one embodiment the 2X freezing media comprises about 60% ES-qualified FBS, about 20% KO-DMEM and about 20% DMSO. The skilled person will understand that any medium or composition that is suitable for cell cryopreservation may be used as a freezing medium.

The endothelial cells may be frozen at a density of about 1 million cells per 1ml freezing media. Alternatively, the endothelial cells may be frozen at a density of about 0.5-3 million cells per lml freezing media. Alternatively, the endothelial cells may be frozen at a density of about 1-2 million cells per lml freezing media. Freezing may comprise the use of cryovials of the endothelial cells placed into a freezing container (For example, Mr Frosty™ (ThermoFisher Scientific)), which is chilled. For long-term storage, cryovials of the endothelial cells may be stored in nitrogen vapour storage. Inducing brain microvascular endothelial cells (E)

For inducing brain microvascular endothelial cells, the medium of the endothelial cell induction step may be replaced with another chemically defined medium., such as endothelial cell growth medium, or continue in the same medium, and the incubation is continued.

In one embodiment the endothelial cell growth medium comprises endothelial basal medium. In one embodiment the endothelial cell growth medium comprises EGM2 medium. In one embodiment the endothelial cell growth medium comprises EGM2MV medium. The EGM2 medium may comprise endothelial basal medium. The EGM2 medium may further comprise the one or more, or all of, the following supplements hEGF; Hydrocortisone; GA-1000 (Gentamicin, Amphotericin-B); FBS (Fetal Bovine Serum); VEGF; hFGF-B; R3 -IGF-1; Ascorbic Acid; Heparin.

In one embodiment, the chemically defined medium of the brain microvascular endothelial cell induction step comprises a ROCK-pathway inhibitor. The ROCK-pathway inhibitor may comprise a ROCK-inhibitor. The ROCK-pathway Inhibitor (Rho-associated protein kinase inhibitor) may comprise or consist of Y-27632. Such an inhibitor may prevent loss of tight junctions and adherens junctions. The ROCK-pathway inhibitor may be provided in sufficient amount to inhibit ROCK by 100% reduction in activity. The ROCK-pathway inhibitor may be provided in the amount of about ΙΟμιη. In another embodiment, the ROCK-pathway inhibitor may be provided in the amount of between about 2μιη and about 20μιη. In another embodiment, the ROCK-pathway inhibitor may be provided in the amount of between about 5μιη and about 15μιη. In another embodiment, the ROCK-pathway inhibitor may be provided in the amount of between about 8μιη and about 12μιη. In another embodiment, the ROCK-pathway inhibitor may be provided in the amount of between about ΙΟμιη and about 20μιη.

Providing a ROCK-pathway inhibitor can improve cell viability post-thaw and also it has been reported that Rho-associated protein kinase increases actomyosin contractility, which facilitates breakdown of intercellular junctions.

In one embodiment, the agent capable of inducing adherence junction marker comprises VEGF. In an embodiment wherein the medium comprises VEGF, the medium may be supplemented with additional VEGF. In another embodiment, the agent capable of inducing adherence junction marker comprises a functional equivalent or variant of VEGF. In another embodiment, the agent capable of inducing adherens junction marker may comprise an E-twenty six (ETS) transcription factor modulator or Tcea3 modulator, which is an isoform of transcription elongation factor TFIIS. The agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of about 5 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 1 and about 500 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 1 and about 300 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 500 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 300 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 1 and about 200 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 200 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 100 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 50 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 1 and about 20 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 20 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 1 and about 8 ng/ml. Alternatively, the agent capable of inducing adherens junction marker, such as VEGF, may be provided at a concentration of between about 2 and about 6 ng/ml.

The Wnt signalling activator may comprise Wnt-7a. The Wnt signalling activator may comprise a functional equivalent or variant of Wnt-7a. Wnt-7a may be provided in an amount of about 10 ng/ml. In another embodiment, Wnt-7a may be provided in an amount of between about 5ng/ml and about 15 ng/ml. In another embodiment, Wnt-7a may be provided in an amount of between about 8 ng/ml and about 12 ng/ml.

Activating the Wnt signalling pathway may comprise the inhibition of GSK-3a/ in the Wnt signaling pathway. The inhibition of GSK-3a/ in the Wnt signaling pathway may comprise a reduction of GSK-3a/ activity. The inhibition of GSK-3a/ in the Wnt signaling pathway may comprise a reduction of GSK-3a/ or reduction of functional GSK-3a/ . The reduction may be 100%, 90%, 70%, 50%, 20% or 10% reduction of function or quantity of GSK-3a/ .

The agent capable of activating the Wnt signalling pathway comprises an ATP-competitive inhibitor for GSK-3a/ in the Wnt signaling pathway.

The agent capable of activating the Wnt signalling pathway may comprise any agent selected from the group consisting of Bio, CP21, and CHIR. In one embodiment, the agent capable of activating the Wnt signalling pathway comprises Bio. In an embodiment wherein Bio is used for activating the Wnt signalling pathway, the Bio may be provided at a low level, for example a concentration/dose of about 0.03 μΜ. Alternatively, the Bio may be provided at concentration of less than about 0.06 μΜ. Alternatively, the Bio may be provided at concentration of less than about 0.05 μΜ. Alternatively, the Bio may be provided at concentration of less than about 0.04 μΜ. Alternatively, the Bio may be provided at concentration of less than about 0.035 μΜ. Alternatively, the Bio may be provided at concentration of between about 0.02 and about 0.5 μΜ. CP21 may be provided at a concentration of between about 0.1 and about 10 μΜ. CHIR may be provided at a concentration of between about 3 and about 12 μΜ.

The agent capable of activating the retinoic acid signaling pathway may comprise all trans retinoic acid. In another embodiment, the agent capable of activating acid signalling (beta- catenin pathway) may comprise a functional equivalent or variant of retinoic acid such as small molecule retinoic acid receptor agonists. The agent capable of activating the retinoic acid signaling pathway, such as retinoic acid, may be provided at a concentration of about 10 μΜ. Alternatively, the agent capable of activating the retinoic acid signaling pathway, such as retinoic acid, may be provided at a concentration of between about 5μΜ and about 15μΜ. Alternatively, the agent capable of activating the retinoic acid signaling pathway, such as retinoic acid, may be provided at a concentration of between about 8μΜ and about 12μΜ. Alternatively, the agent capable of activating the retinoic acid signaling, such as retinoic acid, may be provided at a concentration of between about 9μΜ and about 1 ΙμΜ. In one embodiment, the chemically defined medium of the brain microvascular endothelial cell induction step comprises a sonic hedgehog signalling pathway activator. The sonic hedgehog signalling pathway activator may comprise sonic hedgehog. In another embodiment, the sonic hedgehog signalling pathway activator comprises a functional equivalent or variant of sonic hedgehog, such as the smoothened agonist (SAG). The sonic hedgehog may be provided at a concentration of about lOng/ml. In another embodiment, the sonic hedgehog may be provided at a concentration of between about 5 ng/ml and about 15 ng/ml. In another embodiment, the sonic hedgehog may be provided at a concentration of between about 8 ng/ml and about 12 ng/ml.

In one embodiment, the chemically defined medium of the brain microvascular endothelial cell induction step comprises a Tie2 activator. The Tie2 activator may comprise angiopoietinl, which is a secreted ligand for Tie-2 or small molecules that activate Tie2 pathway. The angiopoietin may be provided at a concentration of about 100 ng/ml. The angiopoietin may be provided at a concentration of between about 50 ng/ml and about 150 ng/ml. In another embodiment, the angiopoietin may be provided at a concentration of between about 80 ng/ml and about 120 ng/ml. In another embodiment, the angiopoietin may be provided at a concentration of between about 90 ng/ml and about 110 ng/ml. In another embodiment, the angiopoietin may be provided at a concentration of between about 10 ng/ml and about 50 ng/ml.

In one embodiment, the chemically defined medium of the brain microvascular endothelial cell induction step comprises a Wnt signalling pathway activator; an agent capable of activating retinoic acid signalling; an agent capable of inducing adherens junction marker; a cAMP signalling activator; and a Tie2 activator, and further optionally comprises a ROCK-pathway inhibitor. The induction step incubation may be at 37°C, 5% C0 ₂ . In one embodiment, the chemically defined medium of the brain microvascular endothelial cell induction step may be replaced/refreshed, for example part way through the brain microvascular endothelial cell induction step. In one embodiment, the medium replacement is about 1 day after the start of the brain microvascular endothelial cell induction step. In particular the endothelial cell maintenance medium or a stem cell medium is refreshed after about 24 hours of culture. The replacement medium may be substantially the same. The replacement may comprise aspirating and discarding the medium and providing fresh chemically defined medium comprising the same active agents, such as the same growth factors/cytokines.

Incubation may continue until successful induction of brain microvascular endothelial cells is observed. Successful induction of brain microvascular endothelial cells may be observed when at least 80% of the cells are brain microvascular endothelial cells. The induction of brain microvascular endothelial cells (BMECs) may be confirmed by the detection of BMEC-specific markers, such as one or more, or all of, VE-cadherin, Occludin, Claudin-5, ZO-1, Glut-1 and P- glycoprotein. At least two cell markers may be used to confirm the differentiation to brain microvascular endothelial cells. The induction of brain microvascular endothelial cells (BMECs) may be confirmed by the detection of all of VE-cadherin, Occludin, Claudin-5 and ZO-1 cell markers. The cell markers may be detected or measured by determining their expression, for example, by detecting their presence, such as by immunohistochemistry, or by RT-PCR.

The induction of brain microvascular endothelial cells may comprise incubation of the cells in the chemically defined medium over a period of about 2 days. Alternatively, the induction of brain microvascular endothelial cells may comprise incubation of the cells in the chemically defined medium over a period of at least 1.5 days. Alternatively, the induction of brain microvascular endothelial cells may comprise incubation of the cells in the chemically defined medium over a period of between about 1 and about 3 days (between about 24hrs and about 72hrs). The induction of brain microvascular endothelial cells period may be defined as the time between the start of incubation of the endothelial cells and the successful observation of BMECs or freezing of the BMECs for later use. The chemically defined medium may not be changed/refreshed during the incubation period, unless otherwise specified.

Enrichment The method may further comprise an optional enrichment step by separation of VE-Cadherin+ cells. The enrichment may comprise MACS (magnetic-activated cell sorting) or FACS (Fluorescence-activated cell sorting) separation of brain microvascular endothelial cells, MACS or FACS enrichment can be performed using antibodies against VE-Cadherin+ and/or occludin, and/or other surface cell markers of brain microvascular endothelial cells at any stage after brain microvascular endothelial induction. This can be successfully done even if brain microvascular endothelial cell yield is as low as 5% amongst other cell types. Alternatively a reporter construct for a gene such as claudin-5 that is expressed in brain microvascular endothelial cells may be generated and used to isolate the relevant cell population. Additionally or alternatively, the enrichment may comprise seeding the cells on a substrate arranged to select for, or encourage the adherence and/or growth of brain microvascular endothelial cells, such as matrigel, laminin or the combination of collagen and fibronectin.

The enrichment may be repeated once, twice, or as much as necessary in order to obtain sufficient brain microvascular endothelial cells.

The enriched cells may be frozen for later use, or used to establish in vitro model of human blood brain barrier. The brain microvascular endothelial cells may be used to establish in vitro model of human blood brain barrier. Additionally or alternatively, the brain microvascular endothelial cells may be frozen.

The brain microvascular endothelial cells may be frozen at a density of about 1 million cells per 1ml freezing media. Alternatively, the brain microvascular endothelial cells may be frozen at a density of about 0.5-3 million cells per 1ml freezing media. Alternatively, the brain microvascular endothelial cells may be frozen at a density of about 1-2 million cells per 1ml freezing media. Freezing may comprise the use of cryovials of the brain microvascular endothelial cells placed into a freezing container (For example, Mr Frosty™ (ThermoFisher Scientific)), which is chilled. For long-term storage, cryovials of the brain microvascular endothelial cells may be stored in nitrogen vapour storage.

Enhancement of barrier properties The use of conditioned media from neuronal cell culture and/or astrocyte cell culture and/or mural cell culture alone or in combination may be added onto the brain microvascular endothelial cells for example at about 1 : 1 to 1 :5 ratio of BMEC induction media to that of conditioned media. This will enhance the distribution of adherens and tight junctions between the brain microvascular endothelial cells and increase the tightness of the barrier thus formed.

The barrier properties may also be enhanced by brain microvascular endothelial cells in co- culture with mural cells, astrocytes and neurons including methods that allow the brain microvascular endothelial cells to be in direct contact with mural cells and/or astrocytes.

The barrier properties may also be enhanced by culture of brain microvascular endothelial cells alone or in co-culture with other cells of the NVU, in experimental systems that incorporate cell media flow to mimic vascular blood flow in vivo. Effective barrier formation may be confirmed by the cells having trans-endothelial electrical resistance (TEER) measured as at least +1000 Ωαη ² . Alternatively, effective barrier formation may be confirmed by the cells having trans-endothelial electrical resistance (TEER) measured as at least +1200 Ωαη ² or at least +1500 Ωαη ² . Forming the NVU and BBB tissue model

The method may further comprise forming an NVU and blood brain barrier tissue model. The brain microvascular endothelial cells may be co-cultured with mural cells (pericytes and/or vascular smooth muscle cells), astrocytes and neurons. These other co-culture cells can be generated from pluripotent stem cells including the same pluripotent stem cell line use to generate the brain microvascular endothelial cells. The other co-culture cells may also be obtained from human primary tissue, primary tissue from other species or from immortalised cell lines. Several methods can be used to co-culture the cells. In one embodiment, a transwell inset can be used where the brain microvascular endothelial cells are grown on one surface of a porous membrane in the transwell insert and astrocytes and mural cells grown on the other side of the porous membrane, with neurons in the bottom well. The layer of brain microvascular endothelial cells linked by tight junctions will constitute a primary barrier with the additional cells modifying the properties of the barrier. In another embodiment, brain microvascular endothelial cells may be seeded alone or in co- culture with one or more, or all of, mural cells, astrocytes and neurons, and cultured in a microfluidic device. The microfluidic device may maintain the cells in a 2D or 3D format. Static, intermittent or continuous perfusion may be used for cell maintenance.

In another embodiment, brain microvascular endothelial cells may be cultured alone are in co- culture with mural cells to form capillary like tubules. Once a BMEC monolayer is established, mural cells and astrocytes can be seeded on either side of endothelium with neurons at the bottom chamber of the 2D cell culture.

The co-culture would advantageously mimic in vivo cellular crosstalk at the neurovascular unit.

BBB tissue model formation

According to another aspect of the invention, there is provided a method for preparing a blood brain barrier tissue model comprising incubating brain microvascular endothelial cells made by the method of the invention herein with other cell types of the NVU. Other cells types of the NVU may comprise mural cells and/or astrocytes.

According to another aspect of the invention, there is provided a population of brain microvascular endothelial cells formed in accordance with the method of the invention. The population of brain microvascular endothelial cells may be suitable for forming a blood brain barrier tissue model. The population of brain microvascular endothelial cells may be differentiated from hPSCs. The population of brain microvascular endothelial cells may not comprise brain microvascular endothelial cells isolated from in vivo.

According to another aspect of the invention, there is provided a blood brain barrier tissue model formed in accordance with the method of the invention.

All cell incubation herein may be at 37°C, 5% C0 ₂ .

The method of the invention may be substantially as described herein. References to amounts or concentrations of agents provided in the medium of incubation steps may refer to the amount or concentration provided at the start of incubation (i.e. prior to cell consumption during incubation). In another embodiment, references to amounts or concentrations of agents provided in the medium of incubation steps may refer to the total amount provided during the incubation, or the level of exposure of the agent to the cells, for example where a sustained release or drip feed system is used to continuously or periodically provide the agent during incubation.

References to "inhibition" or similar, may comprise a reduction in activity or presence of a molecule or the block of a biological pathway, such as a signaling pathway. The inhibition may be total (i.e. 100%) or at least a substantial inhibition. The inhibition may be partial inhibition. Partial inhibition may comprise significant inhibition in order to affect the desired outcome of the inhibition. It is understood that pluripotent stem cells (PSCs) have indefinite capacity to self-renew and can differentiate into three primary germ layers of early embryo, thus differentiating into any adult cell type except extra-embryonic lineage such as placenta. Pluripotent stems cells may comprise embryonic stem cells or induced pluripotent stem cells (iPSC) made from adult somatic cells such as blood or skin cells by reprogramming technology. In one embodiment, the invention herein may not use embryonic stem cells. PSCs can be isolated or generated from non-human species. Human iPSC have the advantage harbor the patient genetic signature are useful in investigating genotype-phenotype links when differentiated into relevant cell types such as brain microvascular endothelial cells. This is useful for investigating disease mechanism or identifying new drug targets or investigating patient specific changes in blood-brain-barrier properties. hPSC models also have the advantage that genome engineering approaches can be applied for mechanistic investigations.

It is understood that "chemically defined medium" is a growth medium suitable for the in vitro cell culture of human or animal cells in which all of the chemical components are known.

It is understood that the terms "cell induction" or "cell differentiation" is the promotion of an hPSC to differentiate into a particular cell type (such as brain microvascular endothelial cells), such that it is no longer pluripotent. The induction/differentiation may comprise the stimulation, upregulation or downregulation of specific biological pathways, which may be provided by growth conditions; agents; delivery of genes by an expression system such as plasmids; activation of genes using genome engineering approaches, reduction or knock-out of genes by an expression system such as RNAi or genome engineering approaches; or media components.

It is understood that the term "Wnt signalling pathway" refers to a signal transduction pathway made of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell. There are three Wnt signalling pathways characterised: the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. The canonical pathway involves the protein β-catenin while a noncanonical pathway operates independently of it. Reference to "Wnt signalling pathway" herein refers to the canonical pathway .

The term "mesoderm patterning" is understood to refer to an event mediated by signaling molecules and factors, which determine the fate and lineage restriction of pluripotent cells to mesoderm in expense of ectoderm and endoderm germ layers. Mesoderm patterning can be confirmed by the expression of TCI 5 at a very early stage, and then it can further differentiates into different mesodermal lineages.

The term "inducing" used herein in the context of inducing an adherens junction marker or vascular endothelial cell marker is understood to mean a molecule that is capable of activating and/or repressing signalling pathways to cause increased expression of the genes being induced.

Tight junctions (also known as occluding junctions or zonulae occludentes) join together the cytoskeletons and membranes of adjacent cells. The adjoining membranes are closely associated and form a barrier to fluid. Tight junctions comprise a branching network of sealing strands formed from a row of transmembrane proteins embedded in the plasma membranes of the joining cells. Major components of the transmembrane proteins are claudins and occludins. The transmembrane proteins associate with peripheral membrane proteins (e.g. such as ZO-1) located on the intracellular side of plasma membrane, which anchor the transmembrane proteins to the actin component of the cytoskeleton.

Reference herein to the term "early mesoderm" refers to a precursor mesodermal stage identified by the expression of key marker genes, such as Brachyury, Snail, N-cadherin, which then subsequently mature into definitive mesoderm. Reference herein to the term "paraxial mesoderm" refers to a mesodermal structure adjacent to neural tube and develops into mesenchyme of head and organize into somites in occipital and caudal regions. Paraxial mesoderm is also called as presomitic or somatic mesoderm and characterized by the expression of transcription factors such as, TCF15 or TBX6.

Reference herein to the term "lateral mesoderm" refers to a peripheral mesodermal structure, which develops into pericardial, pleural and peritoneal cavities and characterized by the expression of transcription factors such as, ISL-1 or NKX2-5 Reference herein to the term "endothelial cells" refers to a thin layer of cells lining interior surface of blood vessels and lymphatic vessels constituting an interface between the vessel wall and the circulating blood or the lymph respectively. They are characterised by the expression of cell markers, such as VE-cadherin, PECAM1, von Willebrand factor. Reference herein to the term "brain microvascular endothelial cells" (BMECs), refers to the major component of the BBB, which lack fenestrations as opposed to peripheral endothelial cells and express tight junction complex proteins, which tightly seal adjacent endothelial cells. They express markers such as, VE-cadherin, Occludin, Claudin- ZO-1, Glut-1 and P- glycoprotein.

Reference herein to the term "agent" in the context of blocking, supressing, antagonising, agonising, inhibiting, promoting, or the like, is understood to include molecules such as a small molecule (i.e. 900Da or less), a nutrient, a biological molecule such as protein, peptide, polymer, or nucleic acid. The agent may be a synthetic agent or a natural agent. The agent may be exogenous or endogenous to the cells. The agent may comprise a truncated or non-functional form of a ligand in the biological pathway to be modified. In another embodiment, the agent may be an analogue/mimic of a ligand in the biological pathway to be modified. The agent may block or deform active sites of its target. Alternatively, the agent may sequester its target, or signal its target for degradation. In another embodiment, the agent may up or down regulate genes or operons associated with the biological pathway to be targeted.

The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention. Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

Figure 1. BMEC-specific markers expression. On day 10, hPSCs derived BMECs express endothelial adherens junction marker VE-cadherin and BMECs specific tight junction markers, such as occludin, claudin-5 and ZO-1.

Figure 2. TEER measurements. Using Endohm chamber, TEER values were measured at the time of induction (day 0) and until 3 days after induction.

Figure 3. Effect of conditioned medium on occludin expression. Occludin expression was reduced from day 2 to 4 after BMEC induction in endothelial medium (EM). We further tested the effect of neuronal conditioned medium (NCM), smooth muscle conditioned medium (SCM) and both media together.

Figure 4. A working model of hPSCs differentiation into BMECs.

Figure 5. An overview of the method timeline, media and additives for hPSC differentiation into BMECs.

Figure 6. An overview of an alternative method timeline, media and additives for hPSC differentiation into BMECs.

Examples

Method of generation of brain microvascular endothelial cells from pluripotent stem cells Background

The neurovascular unit (NVU) is an integrated functional unit that couples of neural activity and blood supply to the brain. The NVU is composed of brain microvascular endothelial cells, functionally coupled with neurons, astrocytes, mural cells, such as pericytes and smooth muscle cells, and extracellular matrix components. The blood-brain barrier (BBB) arises from and is a key function of the neurovascular unit and is meticulously selective barrier that protects the central nervous system from most pathogens and restricts the entry of blood-derived cells and passage of molecules from systemic circulation. In order for the NVU to function in neurovascular coupling and as a blood-brain-barrier, the cells of the NVU are specialised. In particularbrain microvascular endothelial cells (BMEC), which lack fenestrations as opposed to peripheral endothelial cells and express tight junction complex proteins, which tightly seal adjacent endothelial cells. BMEC along with mural cells, astrocytes and neurons form functional neurovascular unit. There are differentiation protocols to derive mural cells, astrocytes and different types of neurons from human pluripotent stem cells (hPSCs) but currently there is no robust, directed protocol to derive brain microvascular endothelial cells. Previous work has reported the generation of BMECs from hPSCs [1]. This method however depends upon the spontaneous differentiation of stem cells and therefore is reliant upon a stem cell line having an intrinsic predisposition to produce BMECs. This may vary between stem cell lines and therefore this method may not be reproducible.. We sought to establish a method to derive BMECs from renewable cell sources, which is reproducible, chemically defined,robust and directed through combined and stepwise modulation of specific signalling pathways and cellular developmental programmes.

Method

Human pluripotent stem cells are routinely cultured on hESC-qualified Matrigel in mTeSRl medium and cultures are passaged every 4-6 days using EDTA.

1. Coat T75 culture flasks with 5ml matrigel (dilution is lot dependent and according to manufacturer's instructions) at room temp for lh or 30 min in the 37°C incubator. If you do not need the flask immediately, seal it and store at 40°C.

2. Aspirate and discard medium. Wash hPSCs with PBS (Ca ²⁺ and Mg ²⁺ free).

3. Add 3 ml pre-warmed accutase to T75 flask and incubate for 3-5 min at 37°C. 4. Check detachment of cells under the microscope and add 7ml mTeSRl once cells have detached.

5. Transfer cells with a 5ml pipette into a falcon tube and centrifuge cell suspension at 400g for 5 min. 6. Aspirate supernatant and resuspend cell pellet in 1 ml mTeSRl+ ROCK-inhibitor (final cone. ΙΟμΜ) media. Triturate gently with pi 000 pipette tip about 5 times and add 9 ml of mTeSRl+ ROCK-inhibitor media. 7. Count cells.

8. Aspirate matrigel and seed 25,000-60,000 cells/cm ² in 15 ml mTeSRl+ ROCK-inhibitor medium. Starting cell density is critical and has a strong influence on the yield of BMECs and should be optimised on each PSC line.

9. Incubate the cells at 37°C, 5% C0 ₂ overnight. Mesoderm induction (days 0-5) Day 0:

10. Mesodermal induction was performed using modified Cheung C et al. protocol ² . Replace medium with 15ml N2B27 or preferably TesR-E6 medium supplemented with hBMP4 (lOng/ml), bFGF (20ng/ml), LY294002 (10 um) and SB431542 (1 μιη) and incubate cells until day 1.5. Whilst both mediums may be used, TeSR-E6 is a fully defined media and more reproducible relative to N2B27.

Day 1.5:

11. Aspirate and discard the medium. Wash cells with N2B27 medium. For mesoderm specification, incubate cells in N2B27 or preferably TesR-E6 medium supplemented with bFGF (20ng/ml), LY294002 (10 μιη) and SB431542 (1 μιη) until day 3.5.

Day 3.5:

12. Aspirate and discard the medium. Wash with N2B27 or preferably TesR-E6 medium and incubate cells in N2B27 or preferably TesR-E6 medium supplemented with bFGF (20ng/ml) and LY294002 ( 10 μιη) until day 5.

Endothelial specification (day 5-7)

Day 5:

13. Aspirate and discard the medium. Wash cells with SP medium, or preferably HEM (human endothelial serum free medium). For endothelial induction, incubate cells in SP medium supplemented with 200ng/ml VEGF, or preferably 50ng/ml VEGF, and 2μΜ forskolin. HEM is fully defined and more reproducible than StemPro (SP) medium.

Day 6:

14. Aspirate and discard the medium. Refresh with SP medium, or preferably HEM (human endothelial serum free medium] supplemented with 200ng/ml VEGF, or preferably 50ng/ml VEGF, and 2μΜ forskolin.

Day 7:

Freezing Endothelial cells

15. At this stage, cells can either be frozen or replated or both. For freezing, use 2X freezing media and freeze cells in growth media as 1 million cells per vial using chilled mister frosty. For long-term, cryovials can be stored in nitrogen vapour storage. Replating Endothelial cells

16. Matrigel-coat appropriate culture surface depending upon the downstream application/readout.

17. Endothelial cells can now replated for further specification. Aspirate and discard the medium. Wash cells with PBS (Ca ²⁺ and Mg ²⁺ free). Incubate cells with pre-warmed accutase for 5-8 min at 37°C.

18. Check detachment of cells under the microscope and add 7ml SP media, or preferably HEM (human endothelial serum free medium], once cells have detached.

19. Transfer cells with a 5ml pipette into a falcon tube and centrifuge cell suspension at 400g for 5 min.

20. Aspirate supernatant and resuspend cell pellet in 1ml SP medium, or preferably HEM (human endothelial serum free medium], supplemented with 50 ng/ml VEGF and 2 μΜ forskolin. Triturate gently with pi 000 pipette tip about 5 times and add 9 ml of SP medium supplemented with 50ng/ml VEGF and 2μΜ forskolin.

21. Count cells. 22. Aspirate matrigel and seed 100,000 cells/cm2 in of SP (StemPro) medium, or preferably HEM (human endothelial serum free medium], supplemented with 50 ng/ml VEGF and 2 μΜ forskolin and incubate endothelial cells at 37°C, 5% C0 ₂ overnight. BMEC induction

Day 8:

23. For BMEC induction, replace SP medium with EGM2 medium supplemented with Wnt-7a (lOng/ml), sonic hedgehog (lOng/ml), Angiopoietin (lOOng/ml, or preferably lOng/ml), Rock inhibitor (10 μΜ), retinoic acid (10 μΜ) and optionally forskolin (2 μΜ).

Day 9:

24. Refresh the medium with the same medium with fresh growth factors/cytokines added. Day 10:

25. At this stage, cultures express BMEC-specific markers, such as VE-cadherin, Occludin, Claudin-5 and ZO-1 and have trans-endothelial electrical resistance (TEER) measured as +1700 Ωαη ² .

Results:

Using this protocol, BMECs derived from 3 different iPSC lines express VE-cadherin, Occludin, Claudin-5 and ZO-1 on day 10 (see figure 1). TEER was measured as a direct readout of barrier integrity. An Endohm chamber provides reproducible resistance measurements and was used to measure TEER before and after BMEC induction (figure 2). After exposing endothelial cells to NVU derived factors, TEER value surged to 1720 and 1060 Qcm ² on day 1 and 2 of BMEC induction. On day 4, TEER value decreased to 670 Ωαη ² . When we analysed the expression of tight junction marker occludin expression on day 4 using immunofluorescence, we noticed reduction in its expression (figure 3). Then we tested the effect of exposure of neuronal and smooth muscle conditioned medium to BMECs cultures and observed that occludin expression was maintained only when exposed to these conditioned medium.

Conclusion:

We have established the method to generate BMECs from hPSCs, which is rapid and reproducible across different hPSC lines and thus is robust. For prolonged maintenance of tight junction protein expression, we tested conditioned medium derived from other NVU cell types and rescued occludin expression, which remains to be seen and translated as TEER measurements. References:

1. Lippmann ES et al. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Sci Rep, 2014 Feb 24;4:4160.

2. Cheung C at al. Directed differentiation of embryonic origin-specific vascular smooth muscle subtypes from human pluripotent stem cells. Nat Protoc 2014 Apr;9(4):929-38.

All references herein are incorporated by reference.

Reagents and media:

Reagent Supplier Catalogue No. Dissolved in:

Matrigel BD Biosiences 354277 mTeSRl Stemcell Technologies 05850

DMEM/F12 Invitrogen 31331028

Neurobasal media Invitrogen 21103049

B27 supplement Invitrogen 12587010

N2 supplement Invitrogen 17502048 b-Mercaptoethanol Invitrogen 31350010

Glutamax Invitrogen 35050038

PenStrep Invitrogen 15140122

EGM2 Medium Lonza CC-3202

Calbiochem 688000 Sterile water

Y-27632 (Rock inhibitor)

Accutase Stemcell Technologies 07920

LY294002 Sigma L9908 DMSO

SB431542 Sigma S4317 DMSO

BMP4 R&D Systems 314-BP/CF 4mM HCL

Forskolin Sigma F 6886 DMSO sterile H ₂ 0 + 0.1%

VEGF PeproTech France 100-20 BSA

Angepoietinl BD Biosiences

Wnt7a Peprotech

Retinoic acid Sigma R2625

Sonic hedgehog Peprotech

Stem Cell

TesR-E6 medium Technologies 05946

Human endothelial Thermo fisher

serum free medium scientific 11111044

N2B27 Medium: ~1L

500ml DMEM/F 12 medium

500ml Neurobasal medium,

20ml B27

10ml N2

lml β-Mercaptoethanol

Filter-sterilise (0.22 um) and store at -20°C as 40ml aliquots StemPro-34 Medium: -0.5L

500ml StemPro-34 medium

5ml Pen/Strep (1 : 100),

5ml Glutamax (1 : 100).

StemPro-34 Supplement

Store at -20°C as 40ml aliquots

TeSR-E6 medium

Mix basal media and supplement of the kit

Store at -20°C as 40ml aliquots

Human endothelial serum free medium

Store at +4°C

EGM2 media: -0.5L

500 ml EBM (Endothelial Basal Medium + supplements)

Mix different components of kit

Store at -20°C as 40ml aliquots

2x Freeze medium

60% ES-qualified FBS

20% KO-DMEM

20% DMSO

Store at -20°C in 40ml aliquots, thaw and mix and keep on ice before use. This can be refrozen several times.

The skilled person will recognise that generic or equivalent versions of the media described herein may be used as an alternative to the named media.

All references herein are incorporated by reference.