WO2002031123A1

WO2002031123A1 - Stem cells

Info

Publication number: WO2002031123A1
Application number: PCT/GB2001/004534
Authority: WO
Inventors: Anna Krassowska; Andrew Cunningham; John Ansell; Marc Turner
Original assignee: The University Court Of The University Of Edinburgh
Priority date: 2000-10-13
Filing date: 2001-10-11
Publication date: 2002-04-18
Also published as: GB0025088D0; AU2001294010A1

Abstract

The present invention relates to pluripotent (potentially totipotent) stem cells obtained from non-embryonic gonadal cells, such as testicular or ovarian cells. The invention also relates to a method of producing such pluripotent (potentially totipotent) stem cells from gonadal tissue, as well as uses of such cells.

Description

STEM CELLS

The present invention relates to pluripotent (potentially totipotent) stem cells obtained (isolated) from non-embryonic cells, as well as a method of producing such pluripotent (potentially totipotent) stem cells and uses thereof.

Pluripotent stem cells are undifferentiated cells, which have the capability of differentiating into a variety of cell types, and have attracted much interest. Totipotent stem cells are undifferentiated cells with the capability of differentiating into ALL cell types and by definition imply germline transmission. However, hitherto they have only been obtained from embryonic sources.

Embryonic stem (ES) cells are continuously growing totipotent stem cell lines of embryonic origin which were first isolated from the inner cell mass of developing mouse blastocysts (Evans & Kaufman, Nature, 1981 292, pl54 - 156) . Additionally embryonic germ (EG) cell lines have been established. Such cell lines are obtained from primitive reproductive cells of the fetus and are functionally equivalent to ES cells (Matsui et al, Cell 1992, 70, p841 - 847) .

Until recently ES/EG cells were only available from animals such as the mouse. However, in 1998 two groups independently isolated what are considered to be human ES/EG like cells (Shamblott et al, Natl. Acad. Sci. USA 1998 £5 pl3726 - 13731; Thomson et al, Science, 1998, 282, pll45 - 1147) , which display characteristics associated with ES/EG cells. A good review describing the isolation of human embryonic stem cells was published by Pera et al, J. of Cell Science 2000, 113, p5 - 10).

The characterising features of ES/EG cells are their ability to be maintained in an undifferentiated state indefinitely in culture and their ability to develop into a number of cell types (pluripotency) , or even every cell type of the body including the germline (totipotency) . It is the ability to develop into a wide range of cell types which has drawn attention to ES/EG cells as both a research tool and as a novel source of cell populations for new clinical applications.

Nevertheless, until recently the only source of such cells has been from embryos, which raises some ethical concerns. In the United Kingdom, Europe and United States of America there is continuing public debate about the ethics of using human embryos as sources of cells for research and/or therapeutic applications. Thus, it would greatly facilitate research if it were possible to isolate such cells from non-embryonic sources. For example, this may allow cells to be isolated from a subject, manipulated and/or differentiated as required and returned to the subject, without fear of rejection. Such a scenario may be attractive in treating patients suffering from a degenerative disease, eg. Parkinsons, or patients requiring new tissue to replace diseased or damaged tissue. Although it has been suggested (see US 6,011,197) that pluripotent/totipotent stem cells may be obtained from non- embryonic sources, the inventors of US 6,011,197 have not demonstrated that this is possible.

Clarke et al (Science 288, pl660-1663, 2000) describes the pluripotentiality of adult neural stem cells. The paper shows that adult neural stem cells may be added to blastocysts and have the ability to be incorporated into various tissues. Nevertheless, the studies show that the neural stem cells do not contribute to all cell types and are not therefore totipotent. Moreover, it may not be generally desirable to have to excise neural cells from an organism, particularly a human, limiting clinical utility. oodbury et al (J. Neurosci. Res. ϋl: 364-370, 2000) disclose the ability of bone marrow stromal cells to differentiate into neurons. However, there is no disclosure of the ability of the bone marrow stromal cells to differentiate into any other tissue type. Thus, it is not clear whether or not the bone marrow stromal cells can differentiate into other cell types and there is no suggestion that they may be totipotent.

It is an object of the present invention to obviate and/or mitigate at least one of the above disadvantages.

In a first aspect the present invention provides a non-embryonic pluripotent (potentially totipotency) stem cell, wherein the stem cell is derived from gonadal tissue. It is understood that "non-embryonic" means obtained from a source which is not an embryo or foetus ie. a postnatal source. The cell is obtained from gonadal tissue such as testicular or ovarian tissue.

A "pluripotent stem cell" as used herein refers to a cell which is capable of differentiating into a variety of cell types, but can be maintained/grown in an undifferentiated state by use of appropriate growth factors, such as leukemia inhibitory factor (LIF) , stem cell factor (SCF) (sometimes known as steel factor) and/or basic fibroblast growth factor (bFGF) . In a preferred aspect the pluripotent stem cell is a totipotent stem cell. A "totipotent stem cell" as used herein refers to a cell capable of differentiating into any/all cell types of a given organism.

The stem cells of the present invention may be obtained from any species, most particularly from mammals including, but not limited to, bovine, ovine, murine, equine, rodentine, feline, canine and primate (such as gibbons, chimpanzees, humans) animals.

In a preferred embodiment non-embryonic pluripotent (potentially totipotent) stem cells of the present invention are obtained from cells dissected from gonadal tissue such as the testes. Testes or a sample of testicular tissue may be obtained and individual cells separated therefrom. This may be achieved for example by enzymic digestion of the tissue with a suitable enzyme, such as dispase, collagenase or trypsin. Thereafter the isolated cells may be frozen in an appropriate growth medium in which ES or EG cell growth could be maintained. The medium may comprise 10% di-methyl sulphoxide (DMSO) . Typically the cells are stored at -60°C to -90°C.

In order to grow the cells in an undifferentiated state, the cells are thawed, if frozen, and transferred to an appropriate medium, such as Glasgow's Minimal Essential Medium (GMEM) supplemented with: 10% foetal calf serum (fcs) , 0.2% sodium bicarbonate, 1% non-essential amino acids, 2% L-glutamine/pyruvate (stock solution 1:1, L-glut 200mM: pyruvate lOOmM) , O.lmM 2-mercaptoethanol, with or without a layer of feeder cells, such as mitotically inactivated STO embryonic fibroblasts, supplemented with at least one growth factor such as LIF, bFGF, SCF and the like. The cells may then be grown and maintained in an undifferentiated state for at least 15 passages. In order to ascertain that the cells are indeed stem cells, they may be tested for common stem cell markers, such as alkaline phosphatase, stage specific embryonic antigen-1 (SSEA-1) or Oct-4. The cells may be maintained in an undifferentiated state by the addition of one or more of the growth factors mentioned above.

In order to allow differentiation of the cells, the growth factor or factors are removed. Typically embryoid bodies develop followed by differentiation into a particular cell type such as haematopoietic or cardiomyocyte cells. Optionally additional factors may be added to induce the stem cells to differentiate into a desired cell type. For example retinoic acid may be added to induce the stem cells to differentiate into neurons. 11-3 with Erythropoietin may induce differentiation to haemopoietic cells. Hepatic growth factor (HGF) may assist differentiation to hepatocytes. Vascular endothelial growth factor (VEGF) may help differentiation to endothelial cells.

Thus in a further aspect the present invention provides a method of isolating a non-embryonic pluripotent stem cell according to any one of claims 1 to 6 comprising the steps of: a) obtaining a sample of gonadal tissue and isolating individual cells therefrom; b) transferring the isolated cell to an appropriate medium supplemented with at least one growth factor; and c) growing and maintaining the cells in an undifferentiated state.

The skilled addressee is well aware of the potential applications of such stem cells. For example it may be possible to genetically engineer such stem cells to make blood cells which are resistant to a specific disease such as HIV, to replace infected blood cells; nerve cells may be grown to help repair spinal injuries and/or restore function to paralysed limbs; make brain cells that would secrete dopamine for the treatment and control of Parkinson's disease (Nakao, et al., J. Neurosurg. 2000; 92 (4) p659 - 670) ; or grow cells that make insulin for transplanting back to a diabetes sufferer (Soria, et al., Diabetes., 2000; 49 (2), pl57 - 162). These are only examples of some of the potential uses, for further uses, the skilled reader is directed to for example to Keller and Snodgrass, 1999 (Nature Medicine, ϋ pp 151-152); and Pera et al, 200 (J. of Cell Sci., 113, pp5-10) , the contents of which are incorporated herein by reference.

The present invention will now be further described by way of example and with reference to the following Figures which show:

Figure 1 shows adult gonadal (ES-like) stem cells grown on STO cells then stained with alkaline phosphatase;

Figure 2 shows adult gonadal (ES-like) stem cells grown on a gelatin dish;

Figure 3 shows the demonstration of differentiation of adult gonadal (ES-like) stem cells as capable of blood formation; and

Figure 4 demonstrates differentiation of adult gonadal (ES-like) stem cells as capable of capillary formation.

Example 1: Isolation of ES like cells from adult mouse

A two month old male CBA mouse was killed by cervical dislocation and the abdomen sterilised by using a 70% ethanol spray. The skin was opened with scissors and reflected away from the laparotomy site and the abdominal wall opened with a midline incision. The testes were identified within the abdominal cavity and the animal orchidectomised using crude dissection and the testes placed into sterile phosphate buffered saline (PBS) pH7.2. Tissue was stored at ambient temperature (30 mins. approx) . The tissue was washed x 2 with fresh PBS and placed into fresh culture medium (Glasgow's Minimal essential medium with 10% foetal calf serum) for approx. 30 minutes. The testes were washed twice with fresh PBS by allowing the tissue to settle under unit gravity for approx. 1 minute then 2ml fresh PBS solution added to the tissue. Fatty tissue around the testes was removed and the dissection performed as follows:

Crude dissection was performed with a scalpel blade to mince the tissue into small fragments and 5ml trypsin solution (0.025% trysin, 0.037% EDTA in PBS containing 1% chick serum) added for approx 5 mins at 37°C to perform enzymic digestion of the tissue. The digestion reaction was then quenched by the addition of lO ls Glasgow's Minimal Essential Medium (GMEM) containing 10% foetal calf serum (FCS) . The tissue sample was then homogenised by passing through a 23 gauge needle attached to a 10ml syringe three times to ensure a single cell suspension.

The cell preparation was then centrifuged at 200g and the supernatant discarded. The residual cell pellet was resuspended in 6mls fresh GMEM-FCS. Cells were then counted, count = 27 x 10⁴ per ml (6ml total) . Six x 0.5ml of the cell preparation was added to a gelatinised 6 well plate (feeder-free) using 0.1% porcine gelatin coated wells and cultured under the conditions stated below. The remaining cells were divided into three x 1ml aliquots in GMEM/FCS containing 10% DMSO (di-methyl sulphoxide) solution and the cells cryopreserved for 5 weeks at -80°C. Two cell pellets were thawed for experimentation; each pellet was thawed rapidly at 37°C in a water bath and 8mls fresh GMEM/FCS added. The cells were centrifuged at 200 g (lOOOrpm) for 5 mins at ambient temperature. The supernatant was removed and both cell pellets resuspended in 6mls fresh GMEM/FCS and 0.5ml cells was added to each well of a gelatinised (0.1% porcine gelatin solution) six well plate (9cm² per well) .

Six x 0.5ml of the cell preparation was added to a plate containing mitomycin C (a 1/100 dilution of a lmg/ml stock to arrest cell division) inactivated STO embryonic fibroblasts which act as a feeder layer for embryonic stem (ES) and germ (EG) cells.

Cells were cultured under the following conditions in 3mls GMEM-FCS per well: a) No treatment (GMEM-FCS only) . b) Leukaemia inhibitory factor (LIF) lμl per ml (lOOμ/ l) . c) Basic fibroblast growth factor (bFGF) lμl per ml (lOng/ml) . d) Stem cell factor 300/1 (1/10 volume AF-1-19T conditioned medium ;CM) . e) lOOμ/ml LIF+ lOng/ml bFGF lμl per ml. f) 100/x/ml LIF+ lOng/ml bFGF 1 l per ml + 300μl AF-1-19T CM. Note: AF-1-19T is a cell line which secretes GM-CSF and Stem cell factor into the medium in which it is cultured.

Cells were incubated at 37°C, 5% C0₂ in a humidified incubator and the medium replenished after two days, then cells left for a further six days without manipulation.

Colonies obtained from co-culture on STO fibroblasts and feeder free gelatinised plates were fixed in 4% para- formaldehyde/PBS solution for 30 minutes, washed x 1 with Alkaline phosphatase buffer (AP buffer) stained with BCIP/NBT alkaline phosphatase substrate for 20 minutes and colour reaction developed until an intense blue stain was observed, washed x 1 with PBS fixed in 4% PFA/PBS and then photographed. Figure 1.

The remaining colonies from gelatinised plates were manually picked using a Gilson pipette and individually transferred to a well (96 well plate) containing trypsin solution (lOOμl) . Cells were triturated to disperse colonies to a single cell suspension and replated to a single well of a gelatinised 24-well plate in fresh GMEM- FCS with growth factors as described above. Cells were allowed to grow to confluence before being trypsinised and transferred to a fresh gelatinised 6 well plate (with medium) until confluence and then harvested into gelatinised 25cm² tissue culture flasks. Cells were then grown in either the original culture conditions or with the removal of one or more growth factor, however LIF was always present in the ES-like cell cultures. This was done in order to assess the requirements of cells for either bFGF or SCF since ES cells do not have this requirement.

Once sufficient cells for passaging had been obtained (at passage 2 from 25 cm² flasks) dishes with colonies from the gelatinised conditions were also stained with AP or photographed under phase contrast and revealed colonies staining with AP and with an ES cell-like morphology. Figure 2.

The ability of these cells to differentiate in an ES- like manner was then assessed by the ability to form embryoid bodies and the ability to form blood cells and cardiomyocytes in vitro .

Following trypsinisation from the gelatinised flask a single cell suspension (1000 cells/ml) was plated out in GMEM-FCS in the absence of LIF (on hydrophobic dishes to prevent cell adherence) to induce EB formation, and the medium replenished every 2-3 days. Embryoid body formation was obvious from all the ES-like clones tested. Cardiac differentiation was observed in the form of "beating" embryoid bodies.

Similarly a single cell suspension (600 cells per plate) was plated out in 1% methylcellulose containing erythropoietin (5 IU/ml) and Interleukin-3 (1/10 volume EHI-3B cells conditioned medium) . After 7-10 days embryoid body formation was apparent and colonies were stained with the benzidine analogue o-dianisidine solution which revealed areas of haemoglobinisation. These colonies were photographed under phase contrast light, see Figure 3.

Buffers etc:

Alkaline phosphatase pH 9.5 lOOmM NaCl

5mM MgC12 lOOmM Tris HC1

5% Nitro -blue tetrazolium in dimethyl forma ide

5% bro o-chloro-indoyl phosphate in dimethyl formamide

Benzidine buffer:

0.2M Sodium acetate 30% Hydrogen peroxide 0.14% O-Dianisidine

Glasgows Minimal Essential Medium (GMEM)

340ml water

40ml 10 x GMEM stock

4Orals foetal calf serum

4mls MEM non-essential amino acids

8mls glutamine /pyruvate 200mM/100mM)

13.2mls 7.5% sodium bicarbonate solution

400μl 2-mercaptoethanol solution (0.1%). Example 2 : Further characterisation of undifferentiated stem cells

The stem cells prepared according to Example 1 were further tested to help confirm that they are in fact stem cells. The cells were tested for expression of stage specific embryonic antigen-1 (SSEA-1) and Oct-4, which are both markers associated with stem cells.

1. Approximately 70% of undifferentiated cells express SSEA-1. To determine this, cells were trypsinised, washed with PBS and resuspended in PBS containing 0.1% sodium azide and bovine serum albumin (BSA) . Cells were incubated with mouse monoclonal antibody to SSEA- 1 (MC-480 from the Developmental Studies Hybridoma Bank of the University of Iowa) for 30 mins at 4°C. Cells were washed with the PBS described above and incubated with secondary antibody (FITC-conjugated fab' fragment of goat anti mouse IgG) for 30 mins at 4°C. Cells were washed as previously and analysed by flow cytometry.

2. Oct-4 as determined by RT-PCR on extracted RNA with specific primers (5 • -GGCGTTCTCTTTGGAAAGGTGTTC-3 ' /5 '- CTCGAACCACATCCTTCTCT-3 ' ; Nichols et al , Cell 1998) for one cycle of 95°C for 5 mins; 25 cycles of 94°C for 30 sees, 62°C for 60 sees, 72°C for 60 sees; one cycle of 72 °C for 15 mins. Oct-4 expression decreases in differentiating embryoid bodies after the removal of LIF and disappears by day 6 of differentiation. Example 3; Differentiation of stem cells

Cell lines prepared according to Example 1 can also be differentiated to:

1. Form capillary structures (i.e. endothelial cells) when embryoid bodies are differentiated in suspension culture for about 2 - 4 days after the removal of LIF and subsequently plated in Matrigel®.

2. Express endoderm specific markers (eg. Alpha foetal protein, AFP and HNF-4α) when embryoid bodies are differentiated in the absence of LIF for over 8 days. This is determined by RT-PCT on extracted RNA with specific primers (AFP, 5 '-CCTATGCCCCTCCCCCATTC-3 • /5 ^»- CTCACACCAAAGCGTCAACACATT-3 • and HNF-4c_, 5'- ACACGTCCCCATCEGAAG-3 • /5 ' -CTTCCTTCTTCATGCCAG-3 • ; Li et al, 2000) with one cycle of 95°C for 13 mins; 30 cycles of 94°C for 60 sees, 5°C for 60 sees, 72°C for 60 sees; 72°C for 15 mins.

Claims

1. A non-embryonic pluripotent stem cell, wherein the stem cell is derived from gonadal tissue.

2. The non-embryonic pluripotent stem cell according to claim 1, wherein the cell is totipotent.

3. The non-embryonic pluripotent stem cell according to either of claims 1 or 2 obtained from testicular or ovarian tissue.

4. The non-embryonic pluripotent stem cell according to any preceding claim obtained from a mammal.

5. The non-embryonic pluripotent stem cell according to claim 4 wherein the mammal is a bovine, ovine, murine, equine, rodentine, feline, cannine or primate animal.

6. The non-embryonic pluripotent stem cell according to claim 5 wherein the primate is a human.

7. The non-embryonic pluripotent stem cell according to any preceding claim which has been genetically engineered so as to over, or underexpress a particular gene and/or protein encoded by said gene.

8. The non-embryonic pluripotent stem cell according to claim 7 wherein said underexpressed is substantially no expression of said gene and/or protein encoded by said gene.

9. A culture of cells comprising a plurality of non- embryonic pluripotent stem cells according to any preceding claim.

10. A differentiated cell or cells obtained from the non- embryonic stem cell or culture of stem cells according to any preceding claim.

11. The differentiated cell or cells according to claim 10 wherein the cell is a haematopoietic cell.

12. The differentiated cell or cells according to claim 10 wherein the cell is a neural cell.

13. The differentiated cell or cells according to claim 10 wherein the cell is a cardiomyocyte cell.

14. Use of the non-embryonic pluripotent stem cell, cell culture or differentiated cell or cells according to any preceding claim in the manufacture of a medicament.

15. Use of the non-embryonic pluripotent stem cell, cell culture or differentiated cell or cells according to any preceding claim in the manufacture of a medicament for replacing cells in a patient in need thereof.

16. Use of the non-embryonic pluripotent stem cell, cell culture or differentiated cell or cells according to any preceding claim in the manufacture of a medicament for introducing cells which are resistant to damage by a disease.

17. Method of treating a patient comprising administering the non-embryonic pluripotent stem cell, cell culture, or differentiated cell or cells according to any one of claims 1 to 13 to a patient in need thereof.

18. A method of isolating a non-embryonic pluripotent stem cell according to any one of claims 1 to 6 comprising the steps of: a) obtaining a sample of gonadal tissue and isolating individual cells therefrom; b) transferring the isolated cell to an appropriate medium supplemented with at least one growth factor; and c) growing and maintaining the cells in an undifferentiated state.

19. The method according to claim 18 further comprising the additional step of freezing the isolated cells in an appropriate growth medium and thereafter thawing the cells prior to proceeding with step b) .

20. The method according to claim 19 wherein the growth medium comprises DMSO.

21. The method according to any one of claims 18 - 20 wherein the isolated cells are transferred to an appropriate medium supplemented with at least one growth factor and a layer of feeder cells.

22. The method according to any one of claims 18 - 21 wherein said at least one growth factor is LIF, bFGF and/or SCF.

23. The method according to any one of claims 18 - 22 further comprising the step of d) ascertaining that the undifferentiated cells are pluripotent stem cells by detecting a stem cell marker, such as alkaline phosphatase, Oct-4, and/or stage specific embryonic antigen-1.

24. A method of providing a differentiated cell according to any one of claims 10 - 13 comprising following the method according to any one of claims 18 - 23 and thereafter removing the growth factor or factors to alow differentiation.

25. The method of claim 24 further comprising adding a factor to induce the stem cells to differentiate into a desired cell type.

26. The method according to claim 25 wherein the factor is retionic acid and the cell type is a neuron.