WO2010136752A1

WO2010136752A1 - Neural differentiation medium

Info

Publication number: WO2010136752A1
Application number: PCT/GB2010/001028
Authority: WO
Inventors: Hassan Tawhid Hassan; John Alan Goodacre; Xiao Qun Zhai
Original assignee: University Of The West Of Scotland
Priority date: 2009-05-26
Filing date: 2010-05-26
Publication date: 2010-12-02
Also published as: GB0908934D0

Abstract

We disclose the preparation of a serum free differentiation medium comprising cell free human astrocyte conditioned medium and including a cell culture and method for the differentiation of neural stem cells/neural progenitor cells into differentiated neurones and their use in the repair of diseased or damage neural tissue and in drug screening.

Description

NEURAL DIFFERENTIATION MEDIUM

The invention relates to the preparation of a serum free differentiation medium comprising cell free human astrocyte conditioned medium; a cell culture and method for the differentiation of neural stem cells/neural progenitor cells into differentiated neurones; and their use in the repair of diseased or damage neural tissue and in drug screening.

Introduction

The culturing of eukaryotic cells, for example some mammalian cells has become a routine procedure and cell culture conditions which allow certain cells to proliferate are well defined. Typically, cell culture of mammalian cells requires a sterile vessel, usually manufactured from plastics and defined growth medium. Moreover, often the culture of mammalian cells requires the addition of serum and feeder cells that provide essential growth factors for the maintenance of the cultivated cell.

The term "stem cell" represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues. Stem cells can be pluripotent or multipotent. A pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism. A multipotent cell has a restricted ability to form differentiated cells and tissues. Typically "adult stem cells" are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought. A totipotent cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta). Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism and are not as such considered stem cells.

Background

Examples of multipotent stem cells include mesenchymal, haematopoietic and neural stem cells. Mesenchymal stem cells or MSCs differentiate into a variety of cell types that include osteoblasts, chondrocytes, myocytes, adipocytes and neurones. Typically MSCs are obtained from bone marrow. Neural stem cells (NSCs) are multipotent stem cells that generate the main cell phenotypes of the nervous system.

NSCs have been isolated from the brain and spinal cord. A sub-population of neural stem cells is referred to as "neural progenitor cells". These have a more restricted potential to differentiate into neural tissue but nevertheless are considered stem cells.

An example of a stem cell marker is CD133 [also called prominin 1] which is a pentaspan membrane protein first identified as a marker of haematopoietic and neural stem cells. It addition to CD133 as a marker of stem cells [see pancreatic and liver stem cells in WO/03/026584; see renal stem cells in WO2007/027905; its expression is also associated with tumour initiating cancer stem cells in a number of human cancers, for example prostate cancer [see WO2005/089043] and pituitary adenoma [see WO2008/024832]. An example of a neural stem cell marker is nestin. Nestin is an intermediate filament protein transiently expressed by neural stem/progentitor cells. It is rarely expressed by adult neurones and is therefore a reliable marker of neural stem/progenitor cells. The co-expression of CD133 and nestin is known in the art.

Moreover, the expression of CD133 and nestin in bone marrow derived mesenchymal cells predisposes these cells to differentiation into neural cells. However, the proportion of differentiated neurones derived from this sub-population is not high.

Human astrocytes are star shaped glial cells found in the brain and spinal cord. Astrocytes are known to perform various functions in the maintenance and function of the nervous system. For example, they provide nutrients and growth factors to neurones, they secrete and sequester neural transmitters, regulate ion concentration in the brain, enhance the myelinating function of oligodendrocytes and repair of damaged neural tissue to name but a few.

Currently, stem cell therapies are exploring different sources of pluripotent and multipotent stem cells and cell culture conditions to efficiently differentiate stem cells into cells and tissues suitable for use in tissue repair, in particular the replacement of damaged neurones either through trauma or disease. To this end it is important to define cell growth conditions that produce cells that are functional and express typical cell markers associated with a specific differentiated cell-type. It would be advantageous if simple cell culture conditions could be established which did not require the addition of xenobiotic materials such as fetal bovine serum or murine feeder cells since their use increases the likelihood of adventious infectious agents (e.g. viruses and prions, in particular for bovine products, and murine viruses for mouse feeder cells) infecting mammalian cells grown in culture.

This disclosure relates to the preparation of a serum free differentiation medium comprising conditioned astrocyte medium and the identification of cell growth conditions and cell growth medium that enhances the formation of differentiated neurones derived from bone marrow neural stem cells that co-express CD133 and nestin. These differentiated neurones have utility in the repair of damaged or diseased neural tissue and also in testing agents which may enhance or inhibit neural cell differentiation.

This disclosure relates to conditioned medium of primary adult human astrocytes that induces neural differentiation of adult human bone marrow stem cells and has the unique feature of using adult human and not mouse/rat embryonic source in serum-free culture conditions. The present disclosure of conditioned medium of primary adult human astrocytes refers to the serum-free culture supernatant of these primary cultured adult human astrocytes. We disclose the ability of this culture supernatant to induce the neural differentiation of easily accessible (CD133 positive) adult human bone marrow stem cells into neural cells in serum-free cultures. The culture vessel utilised is a sterile tissue culture flask coated with PLL (Poly-L-Lysine) used in culturing astrocytes. The PLL coating has the property of changing the electrostatic conditions of the plastic surface of the culture flasks to ensure the maximum 96% required rapid attachment and fibre outgrowth of astrocytes in cultures. An aspect of the present disclosure is to provide a reliable method for production of a large amount of conditioned medium of accessible primary adult human astrocytes in a simple way using serum-free cultures containing no non-human compounds. A feature of this is the ability to induce the neural differentiation of easily accessible adult human bone marrow stem cells whilst maintaining their proliferation potential in serum-free cultures containing no non-human compounds.

Statements of Invention

According to an aspect of the invention there is provided a method for the preparation of a conditioned minimal medium comprising: i) forming a preparation comprising astrocytes in a cell culture vessel which is coated with a non-proteinacous based cell culture support, astrocyte growth factor and supplemental cell culture medium additives; ii) culturing said astrocytes in said cell culture vessel; and optionally iii) separating and storing said conditioned medium from the astrocytes contained in the cell culture vessel.

According to a further aspect of the invention there is provided a method for the preparation of a conditioned minimal medium comprising: i) forming a preparation comprising adult human astrocytes in a cell culture vessel which is coated with a poly-l-lysine cell culture support, astrocyte growth factor and supplemental cell culture medium additives but in the absence of serum; ii) culturing said astrocytes in said cell culture vessel; and optionally iii) separating and storing said conditioned medium from the astrocytes in the cell culture vessel.

In a preferred method of the invention said astrocytes are primate cells.

In a preferred method of the invention said astrocytes are human cells.

According to a further aspect of the invention there is provided conditioned medium obtained or obtainable by the method according to the invention.

According to a further aspect of the invention there is provided a cell culture vessel comprising conditioned medium according to the invention.

"Cell culture vessel" is defined as any means suitable to contain the above described cell culture. Typically, an example of such a vessel is a petri dish; cell culture bottle or flask or multiwell culture dishes or well insert or rotary bioreactor. Multiwell culture dishes are multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems. Typically, high throughput screens use homogeneous mixtures of agents with an indicator compound that is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, substrate/agent and indicator compound. This will have utility in the analysis of differentiation.

In a preferred embodiment of the invention said vessel is selected from the group consisting of: a petri dish, a multi-well cell culture vessel, a spinner flask, a rotary bioreactor.

Bioreactors are known in the art and provide means for the large scale production of cells. For example, Chen et al (Stem Cells (2006) 24(9): 2052-2059) describes a 3D rotary bioreactor adapted for the expansion of human mesenchymal stem cells which can be adapted for the large scale production of astrocyte conditioned medium and differentiated neurones

According to a further aspect of the invention there is provided a cell culture container comprising conditioned cell culture medium according to the invention.

"Container" is defined as any sealable bottle or the like suitable for the storage of conditioned medium for transport or storage prior to use. Preferably the container is adapted to prevent photo damage to the conditioned medium and is suitable for freezing and maintaining sterility of the conditioned medium contained therein.

According to an aspect of the invention there is provided the use of conditioned human astrocyte medium formed by the method according to the invention in the formation of differentiated neurones from neural stem cells and/or neural progenitor cells; preferably CD133⁺ adult human bone marrow stem cells.

According to a further aspect of the invention there is provided a method to differentiate neural stem cells in a minimal medium comprising: i) forming a preparation comprising neural stem cells and/or neural progenitor cells, cell free human astrocyte conditioned medium and supplementary cell culture medium additives; ii) culturing said neural stem cells and/or neural progenitor cells, in conditions that differentiate and maintain said cells into differentiated neurones. According to a further aspect of the invention there is provided a method to differentiate CD133^* adult human bone marrow stem cells in a minimal medium comprising: i) forming a preparation comprising CD133⁺ adult human bone marrow stem cells, cell free astrocyte conditioned medium and supplementary cell culture medium additives; ii) culturing said CD133⁺ adult human bone marrow stem cells in conditions that differentiate and maintain said cells into differentiated neurones.

In a preferred method of the invention said neural stem cells express nestin.

Preferably said neural stem cells express CD133.

In a preferred method of the invention said neural stem cells express both nestin and CD133.

In a preferred method of the invention said astrocyte conditioned medium and said neural stem cells and/or neural progentor cells are autologous; preferably autologous

^• CD133⁺ adult human bone marrow stem cells.

According to a further aspect of the invention there is provided a method to screen for an agent wherein said agent affects the, proliferation, neural differentiation or function of a neural stem cell/neural progenitor cell comprising the steps of: i) providing a cell culture according to the invention; ii) adding at least one agent to be tested; and iii) monitoring the activity of the agent with respect to the proliferation, differentiation or function of said cells.

In a preferred method of the invention said screening method includes the steps of: collating the activity data in (iii) above; converting the collated data into a data analysable form; and optionally providing an output for the analysed data.

A number of methods are known which image and extract information concerning the spatial and temporal changes occurring in cells expressing, for example fluorescent proteins and other markers of gene expression, (see Taylor et al Am. Scientist 80: 322-

335, 1992), which is incorporated by reference. Moreover, US5, 989,835 and US09/031.271 , both of which are incorporated by reference, disclose optical systems for determining the distribution or activity of fluorescent reporter molecules in cells for screening large numbers of agents for biological activity. The systems disclosed in the above patents also describe a computerised method for processing, storing and displaying the data generated. The screening of large numbers of agents requires preparing arrays of cells for the handling of cells and the administration of agents. Assay devices, for example, include standard multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems. Typically, high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound.

According to a further aspect of the invention there is provided a method for the identification of genes associated with neural stem cell/neural progenitor cell differentiation comprising the steps of: i) providing a cell culture according to the invention; ii) extracting nucleic acid from cells in said cell culture; iii) contacting said extracted nucleic acid with a nucleic acid array; and iv) detecting a signal which indicates the binding of said nucleic acid to a binding partner on said nucleic acid array.

Preferably said method includes the additional steps of: i) collating the signal(s) generated by the binding of said nucleic acid to said binding partner; ii) converting the collated signal(s) into a data analysable form; and optionally; iii) providing an output for the analysed data.

Methods used in the identification of cell differentiation markers include immunogenic based techniques (e.g. using the cells as complex immunogens to develop antisera to for example cell surface markers and the like) nucleic acid based techniques (e.g. differential screening using cDNA from differentiated and differentiating cells). According to a further aspect of the invention there is provided a kit comprising: i) human astrocyte conditioned medium according to the invention; and ii) neural stem cells and/or neural progenitor cells.

In a preferred embodiment of the invention said kit includes cell culture medium and optionally an instruction manual to direct the use of the kit in the formation of neurones.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures and examples:

Figure 3-17 IF staining of adult human BM CD133+ stem cells with anti-CD133-2 antibody after 7 days in SS (A-F) and in SF (a-f) neural differentiation induction cultures (x 1000 magnification);

Figure 3-18 IF staining of adult human BM CD133+ cells with anti-CD133-2 antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);

Table 3-6 Percentages of day-14 culture BM CD133+ cells that remained positive to CD133-2 in SF neural differentiation induction culture (figures represent the mean ± SD of three determinations); Figure 3-25 IF staining of adult human BM CD133+ cells with anti-Nestin antibody after 7 days in SS (A-E) and SF (a-e) neural differentiation induction cultures (x1000 magnification);

Figure 3-19 IF staining of adult human BM CD133+ cells with anti-nestin antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);

Table 3-7 Percentage of day-14 culture BM CD133+ cells that were positive to Nestin in SF neural differentiation induction cultures (figures represent the mean ± SD of three determinations);

Figure 3-26 IF staining of adult human BM CD133+ cells with anti-GFAP antibody after 7 days in SS (A-F) and SF (a-f) neural differentiation induction cultures (x 1000 magnification);

Figure 3-20 IF staining of adult human BM CD133+ cells with anti-GFAP antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);

Table 3-8 Percentage of day-14 culture BM CD133+ cells that were positive to GFAP in SF neural differentiation induction cultures (figures represent the mean ± SD of three determinations);

Figure 3-27 IF staining of adult human BM CD133+ cells with anti-NF-h antibody after 7 days in SS (A-F) and SF (a-f) neural differentiation induction cultures (x 1000 magnification);

Figure 3-21 IF staining of adult human BM CD133+ cells with anti-NF-h antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);

Figure 3-23 ICC staining of BM CD133+ cells with anti human-NF-h antibody after 14 days cultured in SF neural differentiation induction cultures (x1000 magnification);

Table 3-9 Percentage of day-14 culture BM CD133+ cells that were positive to NF-h in SF neural differentiation induction cultures (figures represent the mean ± SD of three determinations); Figure 3-22 IF staining of adult human BM CD133+ cells with anti-NSE antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);

Figure 3-24 ICC staining of adult human BM CD133+ cells with anti-NSE antibody after 14 days; and

Table 3-10 Percentage of day-14 culture BM CD133+ cells that were positive to NSE in SF neural differentiation induction cultures (figures represent the mean ± SD of three determinations).

Materials and Methods and Examples

Example 1

After 7 days, BM CD133+ stem cells cultured in both SF and SS neural differentiation induction cultures were harvested from chamber slides and IF stained with antibodies against stem cell markers: CD133-2 and nestin as well as the glial and neural markers: GFAP and NF-h respectively (Figure 3-17).

The bone marrow CD133+ stem cells after 7 days in both SF and SS neural differentiation induction culture did not show significant morphological change or any neural differentiation comparing to Day 0 as they remained GFAP and NF-h negative in all culture conditions. Cell bodies were not elongated and did not show any neuron like projection. IF staining of Day 14-culture BM CD133+ cells in both SS and SF neural differentiation induction cultures with anti-CD133-2, -nestin, -GFAP and -NF-h antibodies were counted under fluorescent microscope. The percentage of these bone marrow cells that expressed stem cell markers: CD133-2 and nestin remained > 94.2±4.2% (Figure 3- 17) and ≥ 86.9±0.8% (Figure 3-25) respectively. And they remained negative to the glial and neural markers GFAP (Figure 3-26) and NF-h (Figure 3-27). No significant difference between the expression levels of CD133-2, nestin, GFAP and NF-h in the bone marrow cells cultured in various SS and SF neural differentiation induction cultures indicates serum-supplement is not required in induction of neural differentiation of BM CD133+ cells. Due to the lack of any difference between SF or SS culture conditions after 7 days and the limitation on BM CD133+ cell number availability, only SF neural differentiation induction cultures were conducted and analyzed for day 14 cultures.

After 14 days, BM CD133+ cells cultured only in SF neural differentiation induction cultures were harvested and were IF and ICC stained with antibodies against stem cell markers: CD133-2 and nestin as well as the glial and neural markers: GFAP, NSE and NF-h. The IF staining results are shown in Figure 3-18 to Figure 3-22 and ICC staining results are shown in Figure 3-23 and Figure 3-24. Clearly, longer incubation time of two weeks had dramatic effect in inducing neural differentiation of BM CD133+cells. Figure 3-20, 3-21 , and 3-22 show the significant cell morphological changes comparing to after 7 days in SS and SF neural differentiation induction cultures (Figure 3-17, 3-25, 3-26, and 3-27) and the increasing expression of glial and neural markers in the neuron like cells. Also, ICC staining of day14-culture BM CD133+ cells (Figure 3-23, 3-24) showed similar dramatic morphological changes comparing to Day-7-culture BM CD133+ cells.

IF staining of day-14-culture bone marrow CD 133+ cells in SF neural differentiation induction cultures was counted under fluorescent microscope (definition of bright, dim and negative staining is shown in Figure 3-9). The percentages of cells that remained positive to stem cell marker CD133-2 and nestin, as well as the percentages of the neural like cells that were positive to GFAP, NF-h and NSE are show in Table 3-6 to 3- 10. To compare the specific effect of various SF culture conditions on the expression level of each marker after 14 days culture to day 0, data in Table 3-6 to 3-10 was analyzed using two tailed student T test and P values are shown in the tables.

The expression level and pattern of different markers in different culture conditions after both 7 and 14 days in SS and or SF neural differentiation induction cultures is described individually as below.

EXAMPLE 2

After 14 days in SF neural differentiation induction culture, the majority of the remaining adult human bone marrow CD133+ cells show typical undifferentiated features, eg. big cell body, high N/C ratio etc. Most bone marrow CD133+ cells are bigger than CD133 negative (CD133-) cells as shown in Figure 3-18. Cell "a" cultured in cytokine cocktail supplemented neural A basal medium (NAM) in the presence of ACM represents a typical dim stained CD133+ cell, which maintained relative large size, but had lost most of CD133 antigen comparing to cell "b", which was cultured in [NAM+ACM (1 :1)] and kept high level of CD133.

After 14 days, CD133 expression in bone marrow cells had not dropped from 97% on day 0, as it remained 96.5±0.14% in NAM only culture and only marginally non- significantly dropped to 91.0±4.9% in NAM supplemented with RA and cytokine cocktail culture. Meanwhile, in NAM + ACM treated culture, the CD133 expression significantly dropped after 14 days to 82.6±1.1% (Table 3-6).

Moreover, day-14 culture BM CD133+ cells treated with NAM culture supplemented with RA and or Cytokine cocktail in the presence of ACM had shown significant drop in CD133 expression to 72.9 ±3.0% - 75.5±2.0% (P<0.01) (Table 3-6).

Clearly NAM did not affect the expression level of stem cell antigen CD133 in these bone marrow stem cells, however the addition of ACM₁ RA and cytokine cocktail contributed to the loss of sternness of these day-14 culture BM CD133+ cells. All of the neural differentiation induction cultures tested that contained ACM, had significantly induced CD133 reduction in cultured bone marrow cells. This indicates the importance of ACM in inducing CD133 reduction in BM CD133+ cells in SF cultures. Table 3-6 suggests the loss of sternness antigen CD133 happened to BM CD133+ cells cultured in all the SF neural differentiation induction cultures but at different extent. The effect of different SF neural differentiation induction cultures in inducing reduction of stem cell antigen CD133 in day-14 culture BM CD133+ cells are in the following order: condition 4 (C4) > condition 6 (C6) > condition 5 (C5) > condition 2 (C2) > condition 3 (C3).

EXAMPLE 3

After 7 days in SF and SS neural differentiation induction cultures, Nestin expression remained high in BM CD133+ cells in all the treated culture conditions with ≥86.9±0.8% positivity compared to 89.6±2.6% positivity on day 0 as shown in Figure 3-25.

Although some bone marrow CD133+ cells showed reduced amount of nestin antigen and diffusion/faint nestin expression pattern (Figure 3-25, B, C, D and d), these changes are non-significant comparing to day 0 as majority of them remained nestin positive. Figure 3-25 shows the variable extent of Nestin expression levels in the BM CD133+ cells after 7 days in both SF and SS culture conditions.

After 14 days in SF neural differentiation induction cultures, the loss of neuron stem/progenitor cell antigen nestin happened to cells cultured in all the culture conditions but at different extent as shown in Figure 3-19.

Nestin negative day-14 culture bone marrow CD133+ cells were detected in cultures supplemented with various combination of ACM, RA as well as cytokine cocktail. Most nestin positive cells remained large cell bodies, especially those cultured in NAM only medium. Interestedly, cells that lost neural stem/progenitor antigen nestin with relative big size were detected in cultures that contained RA, cytokine cocktail and in the absence or presence of ACM ("a" and "b" in Figure 3-19).

In NAM only culture, 86.4 ±0.9% day-14 culture BM CD133+ cells remained Nestin positive and 81.8±1.4% remained Nestin positive in culture that contained NAM, RA and cytokine cocktail. Whereas, addition of ACM had significantly dropped the percentage of Nestin positive BM CD133+ cells after 14 days of culture to 71.4±0.9% (P<0.01 ) (Table 3-7).

Moreover, in culture condition 4, which contained NAM, ACM, RA, and cytokine cocktail, the amount of nestin positive day-14 culture BM CD133+ cells had significantly dropped to the lowest level at 69.1 ± 0.4% (P<0.01). Meanwhile, in the presence of ACM, cultures that contained RA or cytokine cocktail, also inducted significant drop of nestin positive BM CD133+ cells after 14 days of culture to 74.3±2.2% (P<0.05) and 75.8±2.3% respectively (P<0.05%) (Table 3-7). This indicates the simultaneous addition of ACM, RA, and cytokine cocktail had the most potent effect on inducing nestin reduction in BM CD133+ cells in culture. Clearly NAM did not affect the expression of neuron stem/progenitor cell antigen nestin in day-14 culture BM CD133+ cells, however the addition of ACM, RA and cytokine cocktail contributed to the lost of sternness of these BM CD133+ cells. All the tested neural differentiation induction cultures that contained ACM, had significantly induced reduction of nestin, this suggests the crucial role of ACM in inducing nestin reduction in BM CD133+ stem cells in SF cultures. The effect of different SF neural differentiation induction cultures in reducing neuron stem/progenitor cell antigen nestin in day-14 culture BM CD133+ cells are in the following order: C4 > C2 > C5 > C6 > C3.

EXAMPLE 4

After 7 days, both SF and SS neural differentiation induction cultures failed to show any GFAP positive cells and neural differentiation as shown in Figure 3-26. The GFAP negative cells remained similar morphologies comparing to CD133+ and Nestin÷ cells as shown before. Interestedly, small GFAP negative cells were detected in various culture conditions (Figure 3-26).

However, IF staining of day-14 culture BM CD133+ cells showed the dramatic morphological changes of some of the BM CD133+ cells treated in cultures that contained NAM and various combinations of ACM, RA and cytokine cocktail as shown in Figure 3-20.

Cells immunostained with the anti-GFAP antibody had a glial cell-like shape through elongation procedure. Long or short needle shaped projections were observed at the end of the cell bodies. Moreover cell-cell interaction through these projections was also observed as shown in Figure 3-20. All these morphological features of GFAP+ glial cell differentiation of the BM CD133+ cells was observed in SF neural differentiation induction cultures that contained NAM and various combinations of ACM, RA, and cytokine cocktail. However in NAM only culture without ACM₁ RA or cytokine cocktail, all the cells remained GFAP negative and most of them had undifferentiated features, eg. large nucleus as shown in Figure 3-20. Variable extent of GFAP positive day-14 culture BM CD133+ cells in all 6 culture conditions was observed and show in Figure 3-20.

IF staining of day-14 culture BM CD133+ cells revealed that there was significant increase of GFAP positive cells at 8.3±0.8% (P<0.01) in culture condition 2, that contained NAM and ACM, whereas, in NAM only cultures, the percentage of GFAP positive cells remained zero. Meanwhile, the supplement of cytokine cocktail to NAM and

ACM is able to maintain >7% glial differentiation (P<0.05%), whereas, RA is weaker in inducing glial differentiation (6.0±0.3%) in the presence of ACM (P<0.05%). However, in the absence of ACM, only non-significant 4.3±1.1% glial differentiation was detected in culture that contained both RA and cytokine cocktail (P>0.05). Since ACM only is the most potent inducing agent for glial differentiation, this suggests the normal astrocytes produced cytokines play crucial role in inducing glial differentiation of BM CD133+ cells in SF cultures. Also, the interaction between ACM₁ cytokine cocktail, and RA does not specifically contribute to the induction of glial differentiation in BM CD133+ cells in SF conditions.

The effect of different SF neural differentiation induction cultures in inducing glial differentiation of day-14 culture BM CD133+ cells are in the following order: C2 > C6 > C4 > C5 > C3.

EXAMPLE 5

After 7 days, both SF and SS neural differentiation induction cultures failed to show any NF-h positive cells and neural differentiation as shown in Figure 3-27. The NF-h negative cells remained similar morphologies comparing to day-7 culture CD133+, Nestin÷ and GFAP- bone marrow CD133+ cells as shown before (Figure 3-17, 3-25, 3-26). Most of the undifferentiated NF-h negative (NF-h-) cells maintained relative large size, though small sized NF-h negative cells were also detected in various culture conditions (Figure 3-27).

However both IF and ICC staining of day-14 BM CD133+ cells in SF neural differentiation induction cultures showed the dramatic morphological changes of some of the BM CD 133+ cells treated in cultures that contained NAM and various combinations of ACM, RA and cytokine cocktail as shown in Figure 3-21 and 3-23. .

Cells expressing NF-h-a mature functional neural marker, displayed spindle-shaped neuron-like morphology with axonal projections at one or both side of the cell end. Cell- cell interactions through their projections were also observed (Figure 3-21 , 3-23).

Both IF and ICC staining results showed day-14 culture BM CD133+ cells immunostained with the anti-NF-h antibody expressed filamentous structures that were visible in the cytoplasm. All these morphological features of NF-h+ mature neural differentiation of the BM CD 133+ cells was observed in SF neural differentiation induction cultures that contained NAM and various combinations of ACM, RA₁ and cytokine cocktail.. However, in culture condition 1, which contained NAM only, all the cells maintained NF-h negative and most of them had undifferentiated features eg. large cell body as shown in Figure 3-21 and 3-23. These findings confirmed the neural differentiation of the day-14 culture BM CD133+ cells in the prompt of ACM₁ RA and/or cytokine cocktail.

After 14 days in SF neural differentiation induction cultures, IF staining results revealed that there was significant increase of NF-h positive cells at 15.8±1.3% (P<0.01) in culture condition 4, which contained NAM, ACM, RA and cytokine cocktail, whereas, in NAM only culture, none of the bone marrow cells became NF-h positive. Meanwhile, the supplement of RA or cytokine cocktail to NAM and ACM is able to maintain 14.1 ±0.4% and 13.5±1.9% neural differentiation (PO.01), whereas, only non-significant 4.2±1.7% neural differentiation was detected in cultures that contained RA and cytokine cocktail in the absence of ACM (P>0.05). Noticeably, ACM only supplement had also significantly produced 10.6±1.2% neural differentiation (P<0.01) (Table 3-8). Overall, significant increase of NF-h antigen expressing cells was observed in culture conditions that all contained ACM. This suggests the specific effect of ACM on inducing neural differentiation of BM CD133+ cells in SF conditions. Since the triple combination of ACM, RA and cytokine cocktail is the most potent inducing condition for neural differentiation, this indicates the interaction between ACM, cytokine cocktail, and RA also specifically contributes to the induction of neural differentiation of BM CD133+ cells in SF conditions. The effect of different SF neural differentiation induction cultures in induction of neural differentiation of day-14 culture BM CD133+ cells are in the following order: C4 > C5 > C6 > C2 > C3.

EXAMPLE 6

Both IF and ICC staining of day-14 BM CD133+ cells in SF cultures showed that all cells in NAM only culture, remained NSE negative and most of them had undifferentiated features eg. large cell body, high N/C ratio as shown in Figure 3-22 and 3-24.

However some of the day-14 BM CD133+ cells treated in cultures that contained NAM and various combinations of ACM, RA and cytokine cocktail showed significant morphological changes (Figure 3-22 and 3-24). These NSE expressing cells displayed neuron-like morphology with long or short axonal projections at the end of elongated cell bodies. Cell-cell interactions through their projections were also observed. These cells had flat bodies and branched projections, which correspond to neural microtubules. All the clear visible features of NSE+ neural differentiation of the BM CD133+ cells was observed in SF neural differentiation induction cultures that contained NAM and various combinations of ACM, RA₁ and cytokine cocktail. These findings confirmed the neural differentiation of day-14 culture BM CD133+ cells in the prompt of ACM, RA and/or cytokine cocktail.

Day-14 culture BM CD133+ cells in neural A basal medium without ACM or RA or cytokine cocktail remained NSE negative, whereas, in NAM supplemented with ACM alone culture, 14.5±1 % of bone marrow CD133+ cells became NSE positive (P<0.01). The ACM-induced neural NSE expression was future enhanced to >16% by adding it RA and/or cytokine cocktail (P<0.01). Interestedly, the addition of cytokine cocktail in the absence of RA is the most potent condition for inducing neural differentiation for BM CD133+ cells as it induced the highest amount NSE positive neural like cells at 17.8±1.5% (P<0.01). Meanwhile, in the presence of RA and cytokine cocktail but not ACM, only 4.4±1.8% of cultured bone marrow CD133+ cells were NSE positive after 14 days. This minimal change is non-significant comparing to day 0(P>0.05). These results suggest the specific effect of ACM in inducing neural differentiation of BM CD133+ cells in SF conditions. The effect of different SF neural differentiation induction cultures in inducing mature neuron differentiation of day-14 culture BM CD133+ cells are in the following order: C6 > C4 > C5 > C2 > C3.

The total percentage of induced neural/glial differentiation of CD133+ marrow cells reached the highest level of 21.1-25.5% (P<0.01) in culture that contained NAM, ACM and cytokine cocktail followd by 23.0-23.9% (P<0.01) in NAM containing ACM, RA and cytokine cocktail treated culture and 20.1-22.4% (P<0.01) in culture that contained NAM, ACM and RA. The ACM alone has induced 18.9-22.8% (P<0.01) neural/glial differentiation of CD133+ marrow cells, whereas in the absence of ACM, basal medium supplemented with RA and cytokine cocktail failed to induce significant neural/glial differentiation (only 8.5-8.7%) on day 14 (P>0.05).

In conclusion, glial and neural differentiation of adult human BM CD133+ stem cells under serum free conditions was confirmed using both IF and ICC techniques for the first time.

Claims

1 A method for the preparation of a conditioned serum free minimal medium comprising: i) forming a preparation comprising adult human astrocytes in a cell culture vessel which is coated with a poly-l-lysine cell culture support, astrocyte growth factor and supplemental cell culture medium additives; ii) culturing said astrocytes in said cell culture vessel; and optionally iii) separating and storing said conditioned medium from the astrocytes in the cell culture vessel.

2. Conditioned medium obtained or obtainable by the method according to claim 1.

3. A cell culture vessel comprising conditioned medium according to claim 2.

4. A vessel according to claim 3 wherein said vessel is selected from the group consisting of: a petri dish, a multi-well cell culture vessel, a spinner flask or a rotary bioreactor.

5. A cell culture container comprising conditioned cell culture medium according to claim 2.

6. The use of conditioned astrocyte medium formed by the method according to claim 1 in the formation of differentiated neurones from CD133⁺ adult human bone marrow stem cells .

7. A method to differentiate CD133⁺ adult human bone marrow stem cells in a minimal medium comprising: i) forming a preparation comprising CD133⁺ adult human bone marrow stem cells, cell free astrocyte conditioned medium and supplementary cell culture medium additives; ii) culturing said CD133⁺ adult human bone marrow stem cells in conditions that differentiate and maintain said cells into differentiated neurones.

8. A method according to claim 7 wherein said CD133⁺ adult human bone marrow stem cells express nestin.

9. A method according to claim 7 wherein said CD133⁺ adult human bone marrow stem cells express CD90.

10. A method according to claim 7 wherein said CD133⁺ adult human bone marrow stem cells express both nestin and CD 133.

11. A method according to any of claims 7-10 wherein said astrocyte conditioned medium and said CD133⁺ adult human bone marrow stem cells are autologous.

12. A kit comprising: i) astrocyte conditioned medium according to claim 1 ; and ii) CD133⁺ adult human bone marrow stem cells.

13. The kit according to claim 12 wherein said kit includes cell culture medium and optionally an instruction manual to direct the use of the kit in the formation of neurones.