WO2014092575A1

WO2014092575A1 - Means and methods for obtaining salivary gland stem cells and use thereof

Info

Publication number: WO2014092575A1
Application number: PCT/NL2013/050899
Authority: WO
Inventors: Robert Paul COPPES; Gerald DE HAAN
Original assignee: Rijksuniversiteit Groningen; Academisch Ziekenhuis Groningen
Priority date: 2012-12-14
Filing date: 2013-12-13
Publication date: 2014-06-19

Abstract

The invention relates to in vitro methods for isolating and characterizing human salivary gland cells for stem cell transplantation, and to the therapeutic use of the cells, in particular for treating or preventing xerostomia.

Description

Title: Means and methods for obtaining salivary gland stem cells and use thereof.

The invention generally relates to sahvary gland stem cells and methods for their use. More specifically, the invention relates to in vitro methods for isolating and characterizing human salivary gland cells for stem cell transplantation, and therapeutic use of the cells, in particular for treating or preventing xerostomia.

Xerostomia, the subjective feeling of 'dry mouth', is the syndrome arising as a consequence of the clinical condition hyposahvation.

Hyposalivation can be caused by radiation therapy for head and neck cancers, Sjogren's Syndrome or various medications.

Patients experiencing xerostomia have a marked decrease in quality of life, related to difficulties with speaking, swallowing, sleeping, and dental hygiene, amongst others. Salagen (Pilocarpine hydrochloride) is used to treat symptoms of dry mouth caused by a certain immune disease (Sjogren's syndrome) or from saliva gland damage due to radiation treatments of the head/neck for cancer. Pilocarpine belongs to a class of drugs known as cholinergic agonists. It works by stimulating certain nerves to increase the amount of sahva you produce, making it easier and more comfortable to speak and swallow. Possible side effects of Salagen include an allergic reaction (difficulty breathing; closing of the throat; swelling of the lips, tongue, or face; or hives); difficulty breathing, irregular heartbeats, eye pain; or confusion or changes in mental status or behavior.

Thus, currently available drug-based therapies have unwanted side- effects and do not provide a long-term cure for xerostomia. Human salivary gland stem cell transplantation is an option to prevent or treat

hyposalivation. However, published methods for isolating human salivary gland stem cells only show some potential differentiation of these cells into salivary gland lineages in vitro but do not or fail to show a contribution of these cells to in vivo salivary gland regeneration. To properly characterize salivary gland stem cells for clinical use, it is necessary to show self-renewal and differentiation of a single human salivary gland stem cell (essential determinants of stemcellness) to all salivary gland lineages in vitro and in vivo with functional regeneration of diseased salivary glands.

Therefore, the present inventors set out to develop a protocol for isolating human salivary glands stem cells from autologous/allogeneic donor biopsy material and testing their potential in functional assays. Furthermore, they aimed at providing an in vitro method for screening the efficacy of candidate drugs targeting salivary gland pathologies.

This invention provides a solution for the in vitro isolation,

characterization and testing of self-renewal and differentiation of salivary gland stem cells for transplantation to treat hyposalivation. In particular, it was found that floating spheres (spheroid structure of cells obtained through cell division of isolated cells and capable of forming aggregates; herein further referred to as "salispheres") derived from primary cells are advantageously subjected to a self-renewal process. From these salispheres single cells can be derived to form a second generation of salispheres

(secondary salispheres) which can form organoid containing structures similar to those observed in native salivary glands. This permits the attribution of the differentiation observed on one organoid to the replication and differentiation of one singular cell.

Accordingly, in one embodiment the invention provides method for providing an in vitro culture of differentiated salivary cells comprising the steps of:

a) providing a suspension comprising salivary gland stem cells obtained from primary salivary gland tissue, b) culturing the suspension of salivary gland stem cells under conditions that promote the growth of floating salispheres of salivary gland stem cells to obtain a first generation salisphere culture;

c) expanding the cells of the first generation primary salisphere culture by culturing single cells isolated from said culture in a three- dimensional matrix comprising a basement membrane substrate, preferably laminin, collagen IV and/or entactin, to obtain a second generation salisphere culture comprising single cell-derived salispheres;

d) releasing salispheres of said second generation salispheres from said three-dimensional matrix; and

e) introducing a single second generation salisphere per contained solid differentiation environment and adding a medium comprising gamma- secretase inhibitor or serum (e.g. FCS), thus allowing for the formation of an organoid containing structures similar to those observed in native salivary glands.

Preferably, the differentiated salivary cells are human differentiated cells. Step a) comprises the preparation of a suspension comprising salivary gland stem cells priorly obtained from primary salivary gland tissue. In one embodiment, the tissue is submandibular salivary gland tissue. Stem cells can also be obtained from human parotid salivary gland tissue biopsy samples. Parotid gland-derived salispheres also contain within them stem cell populations, providing a second source of cells for therapeutic

transplantation.

Typically, primary tissue is processed manually into small (1-5 mm³) pieces e.g. by cutting rapidly with dissection scissors, to increase cell surface area. Tissue is digested by treatment with one or more digestive

(proteolytic) enzymes under mechanical movement. Proteolytic enzyme treatment digests extracellular bonds in the tissue and between the cells, thus facilitating the formation of a cell suspension. Very good results can be obtained using collagenase and hyaluronidase (e.g. Accutase (Sigma)). In that case, a calcium chloride solution is added to the cell suspension to obtain a final concentration of 6-7 mM, preferably about 6.25 mM.

Mechanical digestion can be performed using a device for the automated dissociation of tissues into single-cell suspensions, like a gentleMACS™ dissociator marketed by Miltenyi Biotec). Following enzymatic dissociation, the cell suspension may be washed repeatedly in a suitable buffer to remove active digestive enzymes. Any remaining large pieces of unprocessed tissue may be removed by passage through a cell strainer with a suitable pore size, e.g. 80-150 μιη, preferably about 100 μιη, to obtain a uniform cell

suspension.

In step b), the suspension of salivary gland stem cells are cultured under conditions that promote the growth of floating salispheres (aggregates) of salivary gland stem cells to obtain a first generation salisphere culture. To that end, cells are put in a suitable growth medium containing appropriate nutrients and growth factors. Preferably, it comprises culturing the salivary gland stem cells in a growth medium comprising antibiotics, L-alanyl-L- glutamine, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin and dexamethasone. For example, very good results were obtained using Pen/Strep antibiotics, 20 ng/mL EGF, 20 ng/mL FGF, 10 μg/mL insulin, 1 μΜ dexamethasone and Bottenstein's N-2 formulation ((1985) Cell Culture in the Neurosciences, Bottenstein, J.E. and Harvey, A.L., editors, p. 3, Plenum Press: New York and London).

Cells are preferably seeded at a cell density of about 300,000 to about 500,000 cells per 2-4 mL for optimal growth of salispheres. For example, about 400,000 cells are seeded per well of a 12-well plate. Salispheres are typically present between 3 and 5 days post-isolation. At this point, cells may be analyzed in order to isolate the most potent stem cells for

therapeutic purposes from this heterogeneous pool of cells. The proliferative potential of primary salisphere cultures can be exploited in vitro to expand the number of cells available for transplantation therapy, using a self-renewing culture. In addition to the augmentation of the number of cells available for transplantation, the verification of human salivary gland stem cell (hSSC) potency, and the generation of single-cell- derived salispheres for use in further differentiation assays, is also achieved.

In step c) of a method of the invention, cells of the first generation primary salisphere culture are expanded to obtain a second generation salisphere culture comprising single cell-derived salispheres by culturing single cells isolated from the primary salisphere culture in a three-dimensional matrix comprising a basement membrane substrate. Single cells are suitably isolated from the primary salisphere culture by incubating with trypsin during a period of about 15-30 minutes, followed by neutralizing trypsin activity. Excess trypsin treatment must be avoided to prevent unwanted removal of cell surface antigens necessary for cell communication and salisphere formation. Preferably, cells are exposed to about 0.025% trypsin during a period of 15-30 minutes, preferably 20-25 minutes, while

repeatedly mixing the suspension e.g. by pipetting every 5 minutes. Trypsin action is suitably neutralized by adding buffer containing "competitor" protein substrate, for example a solution of bovine serum albumin (BSA). The single cell suspension is then brought at a concentration which is suitable to self-renewal, preferably about 0.3-0.5 x 10⁶ living cells per mL. Living cells can be distinguished from dead cells using dye exclusion analysis, for example by trypan blue staining.

After resuspending the single cells in a growth medium comprising the necessary nutrients and growth factors (see as described for step b), they are brought into contact with a three-dimensional matrix comprising a basement membrane substrate to form a second generation of salispheres. Many cells in tissues are in contact with a highly specialized extracellular matrix, termed the basement membrane Basement membranes have certain common components, including collagen IV, laminins, heparan sulphate proteoglycans, and growth factors which have a wide variety of biological activities. Extracts of basement membrane-rich tissue have yielded material suitable for studying cell-basement membrane interactions. Preferably, cells of the first generation primary salisphere culture are expanded to obtain a single cell-derived salispheres by culturing single cells isolated from the primary salisphere culture in a three-dimensional matrix comprising laminin, collagen IV and/or entactin as basement membrane substrate.

Solubilized basement membrane preparations are commercially obtainable. In one embodiment, it is an extract from EHS mouse sarcoma, a tumor rich in extracellular matrix (ECM) proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin 1. At room temperature, the matrix polymerizes to produce biologically active matrix material resembling the mammalian cellular basement membrane. Cells behave as they do in vivo when they are cultured on the matrix. It provides a physiologically relevant environment for studies of cell

morphology, biochemical function, migration or invasion, and gene expression. For example, the 3D matrix marketed under the tradename Matrigel (Beckton Dickinson) is suitably used.

The cells are then incubated for at least several days e.g. 7-10 days, to allow for the formation of single cell-derived salispheres. This culture is termed passage 1. These "self-renewal cultures" can be passaged after about 1 week of culture time, or when salispheres between 50 to 80 μιη in diameter are observed. Passaging can be performed to increase

cell/salisphere number, or to assess the proliferative potency of the culture. The culture time required until passaging may vary by laboratory. To release salispheres from the gels and permit passaging, medium can be removed from the self-renewal gels and replaced with medium containing a protease capable of digesting the basement membrane substrate. Preferably, the protease is dispase. For example, 1 mg / mL dispase enzyme (Gibco) is suitably used to break down the Matrigel gel.

The salispheres released from the matrix are then collected and may be exposed again to trypsin to obtain single cells. Each trypsinisation of salispheres generates the next passage of cells. Self-renewal cultures can be continued until sufficient cell/ salisphere number is achieved for further applications, or, alternatively, until exhaustion of the salisphere-forming capacity of the cells. The latter scenario may be used as a tool to assess the proliferative ability of a particular salisphere isolation.

In step d) of a method of the invention, a single second generation (i.e. single cell-derived) salisphere is introduced in a contained solid differentiation environment. As used herein, the term "contained" refers to the

environment being physically separated from other environments

containing salispheres such that one environment contains only a single salisphere. For example, single salispheres are transferred to individual sample wells, each well a comprising solid differentiation environment. The solid differentiation environment preferably comprises collagen. In a specific embodiment, the solid differentiation environment is growth factor-reduced matrigel comprising Type I collagen.

A medium comprising gamma-secretase inhibitor or FCS is then added to the solid differentiation medium comprising a single salisphere, thus allowing for the formation of an organoid containing structures similar to those observed in native salivary glands. An organoid is any structure that resembles an organ in appearance and/or function. Preferably, the organoid comprises ductal structures and/or mucous and serous acinar-like cells. The medium comprising gamma-secretase inhibitor or FCS contains the necessary nutrients and growth factors as described herein above. For organoid formation from murine salispheres, about 10% fetal calf serum is advantageously added to the medium. The organoid culture is incubated for up to one month to allow for 3D organoid structure formation with regular, e.g. weekly, refreshing of the medium.

In a specific aspect, a method of the invention comprises the steps of:

a) incubating a sample comprising salivary gland stem cells

obtained from primary salivary gland tissue in a buffer comprising one or more proteolytic enzymes under conditions suitable to obtain a cell suspension

b) washing the cell suspension to remove proteolytic enzyme(s) and resuspending the cells at a density of 200,000-400,000 cells per mL in a culture medium comprising nutrients and growth factors that promote the growth of floating aggregates of salivary gland cells to obtain a primary salisphere culture

c) optionally collecting most potent salivary stem cells from the primary salisphere culture by FACS

d) collecting the cells from the primary salisphere culture obtained in step b) and expanding the cells by subjecting them to a self- renewal procedure, the self renewal procedure comprising the steps of

dl) contacting the cells of the primary salisphere culture with trypsin to produce single cells from the primary salisphere culture

d2) neutralizing the action of trypsin, filter the cell suspension to remove cellular debris and resuspending the cells in a culture medium comprising nutrients and growth factors at a density of about 0.4-0.5 x 10⁶ cells per mL

d3) suspension-culturing the cells obtained in step d2) in the presence of a basement membrane substrate to obtain a self- renewal gel and incubating the self-renewal gel in a medium at 37°C for a period of about 5-12 days to form new single-cell derived salispheres

releasing single cell-derived salispheres from the self-renewal gel of step d3) by contacting the gel with a protease capable of digesting the basement membrane substrate

introducing a single salisphere of the single cell-derived salispheres of step e) in cell culture well comprising a solidified differentiation matrix and adding a medium comprising gamma- secretase inhibitor or FCS, thus allowing for the formation of an organoid containing structures similar to those observed in native salivary glands, preferably wherein the organoid comprises ductal structures and/or mucin-producing acinar -like cells. Herewith, the invention provides a method to assess the differentiation abilities of the cells. Since the organoid structures are generated in vitro from single cell-derived salispheres, a method of the invention allows the attribution of all of the differentiation observed to the replication and differentiation of one singular cell. Organoid formation is hence a functional assay for the multilineage potential of salivary gland stem cells. This tool can additionally be employed to screen potential new salivary gland chemical therapeutic agents, before consideration of the drugs for use in patients.

Accordingly, in a further embodiment the invention provides a method of in vitro screening a drug for activity to modulate salivary gland function, comprising (i) providing an in vitro culture of differentiated salivary cells according to a method as described herein above, (ii)

contacting the culture with at least one candidate compound; (iii)

determining the effect of the compound on the formation and/or at least one biological activity of the organoid; and (iv) selecting at least one compound showing a desired effect. The compound can be added at any stage of the differentiation protocol. Determining the effect of the compound on the formation and/or at least one biological activity of the organoid may comprise the analysis of any relevant biochemical, cell biological or morphological parameter associated with organoid formation and/or function. It also includes the formation of secondary salispheres from single cells. For example, the effect of the candidate compound on organoid formation can be monitored by assessing the number of secondary

salispheres formed per 10.000 cells seeded, or the percentage of salispheres that form an organoid. Furthermore, it may involve assessing the way the organoids are formed; for example containing more ductal structures or more lobular structures or a mixture of both. This can be verified by staining with acinar or ductal markers. In another embodiment, the synthesis of growth factors, digestive enzymes (e.g. amylase) and/or production of homeostasis maintaining factors upon pharmaceutical stimulation can be determined.

A further aspect of the invention related to a method for providing human salivary gland stem cells suitable for therapeutic application, in particular for treating or preventing xerostomia, comprising the steps of

a) providing a suspension comprising salivary gland stem cells obtained from human primary salivary gland tissue, preferably

submandibular or parotid salivary gland tissue;

b) in vitro culturing the suspension of salivary gland stem cells under conditions that promote the growth of floating spheres of salivary gland stem cells ("salispheres") to obtain a salisphere culture;

c) collecting the most potent salivary stem cells from the primary salisphere culture, preferably by FACS or MACS, on the basis of cell surface expression of one or more stem cell markers, preferably selected from the group consisting of EpCAM, c-Kit, CD49f, CD29, CD 133 and CD24; and d) expanding the cells collected in step c) by culturing single cells isolated from said culture in a three-dimensional matrix comprising a basement membrane substrate, preferably laminin, collagen IV and/or entactin (nidogen); and

e) releasing salispheres from said three-dimensional matrix by solving the matrix using appropriate enzymes such as dispases.

Typically, 3-5 day old salispheres are enzymatically dispersed to yield a suspension of viable cells that have innate self-renewal capabilities and which can differentiate into all cells of the salivary gland.

Subsequently these cells need to be washed and resuspended in a suitable buffer, e.g. phosphate buffered saline (PBS), where after they can be injected into the recipients gland directly or reterogradely through the excretory ducts.

Also provided are therapeutic human salivary gland stem cells obtainable by a method of the invention and a therapeutic composition comprising such human salivary gland stem cells. The human salivary gland stem cells are characterized by the expression of stem cell markers, in particular EpCAM, c-Kit, CD49f, CD29, CD 133 and CD24. These cells are distinct from those described in the art.

For example, EP 1452587 discloses stem cells originating from salivary gland duct epithelium of rats or mice. No human cells are discloses, which can be explained by the fact that ligation of ducts is not considered ethically acceptable in humans. Furthermore, the cells EP 1452587 are shown to be differentiated into alpha-fetoprotein-positive cells, albumin - positive cells, amylase-positive cells, insulin-positive cells or glucagon- positive cells. The use of these cells for liver or pancreas regeneration is suggested. EP 1600502 likewise relates to cells originating from salivary gland and being capable of differentiating into glucagon- or insulin- producing cells. The industrial application of the cells is thought to reside in regenerating human liver or human pancreas. Importantly however, nothing is shown or mentioned in EP 1452587 or EP 1600502 about the cells being capable of forming organoid containing structures similar to those observed in native salivary glands, in particular ductal structures and/or mucin-producing acinar-like cells. Hence, whereas they may be obtained from salivary glands, they do not qualify as salivary gland stem cells that have innate self-renewal capabilities and can differentiated into all cells of the salivary gland.

In a further embodiment, the invention provides a therapeutic composition comprising human salivary gland stem cells obtainable by a method of the invention for use in a method for treating or preventing a salivary gland deficiency in a subject in need thereof. Preferably, the salivary gland deficiency is hyposalivation, more preferably irradiation- induced hyposalivation. Also provided is a method for preventing or treating a salivary gland deficiency, or ameliorating the symptoms associated with hyposalivation, comprising the intra-glandular transplantation of a composition comprising human salivary gland stem cells obtainable by a method of the invention. Transplantation can be heterologous or

homologous. The amount of cells for transplantation varies on the recipient and the nature of the deficiency. Typically, an amount of 50.000 or more cells is transplanted.

LEGEND TO THE FIGURES

Figure 1 Human salispheres have self-renewal capacity and can be expanded in vitro.

Still frame images from time lapse microscopy of a single human salisphere showed increase in size in culture in time indicated and self-renewal from single cells for 8 passages. These results are depicted as population dynamics of salisphere self-renewal cultures. Population doublings and percentage of sphere-forming cells (as percentage relative to input cell number) were calculated at the end of each passage. Data points are means and S.E.M. and are derived from a minimum of 3 separate patient isolations per passage.

Figure 2 Organoid formation from a single human salisphere cell. Phase contrast microscopy of differentiating single cell-derived salispheres at 2, 5, 8 and 12 days of culture in differentiation conditions demonstrates in vitro differentiation potential of human salisphere cells. Three examples of organoids following 12 days differentiation are shown. Scale bars represent 100 μΜ. Next to this, haematoxylin and eosin staining of an organoid following 12 days of differentiation, depicting ductal and acinar -like structures (data not shown). Discrimination between ductal and acinar cells in the human submandibular salivary gland was shown using anti- cytokeratin and aquaporin-5 (AQP-5) antibodies. Cytokeratin and AQP-5 immunostaining of single-cell-derived organoids following 12 days in differentiation conditions suggests central localization of cytokeratin - positive putative ductal cells, and peripheral localization of a second AQP-5- positive, cytokeratin-negative putative acinar cell type. Moreover, specific staining for human mucines and amylase showed differentiation in both serous and mucous acinar cells (data not shown). Figure 3 Transplanted human salisphere cells are capable of rescuing radiation-induced hyposalivation in a mouse model, and of producing and secreting human Muc5B a) Saliva production in animals transplanted intra- glandularly with 500, 5000 or 50000 human salisphere cells, compared to non-transplanted, irradiated animals. Each data point represents a recipient animal. b) Salisphere cultures from transplanted glands harvested three months after irradiation. In a) and b), solid bar represents mean in non-transplanted control group. Dashed line and grey-shaded box represents mean and standard deviation respectively in non-irradiated, non- transplanted control animals, c) Muc5B detection by Western blot in human whole saliva (WS), and not in murine WS. A smear of protein heavier than 460 kDa can be seen. Numbers 1-5 represent separate human or murine WS samples.

EXPERIMENTAL SECTION EXAMPLE 1

The development of a cellular therapy for hyposalivation requires that candidate cells demonstrate functional capabilities in vivo, and preferably also in vitro. We have previously shown that cells cultured from murine salivary glands, termed salispheres, are capable of rescuing hyposalivation in a mouse model of xerostomia, and can generate cells expressing proteins associated with functional salivary glands in vitro (Lombaert et al 2008). In order to develop a cell-based therapy for

xerostomia in humans, we first further adapted and modified our existing primary salisphere culture protocol, for use with human cells.

Primary human salispheres (hS) cultured from mechanistically and enzymatically dissociated submandibular or parotid salivary glands grew in size over time in a similar manner to those from the mouse and rat (see Lombaert et al. 2008 and Nanduri et al 2010,). Proliferating cell nuclear antigen (PCNA) expression, associated with proliferating cells, was also detected within primary hS (data not shown), indicating that primary hS cultures are actively dividing during in vitro culture. When these primary hS were enzymatically dispersed, they where able to form single cell derived secondary hS in Matrigel (Fig 1).

Next, we tested whether the secondary hS contained cell capable of self-renewal and expansion of salisphere number to generate the maximal number of salisphere-derived cells for therapeutic use. Through the enzymatic dispersal of secondary salispheres, plating of resultant single cells in Matrigel, and observing new salisphere growth, hS cultures could be propagated for up to 5 passages. New salispheres at each passage arise from single cells and can be passaged on a weekly basis in such a way .

Proliferation of the cells at each passage, as measured by population doublings and salisphere forming efficiency (Fig. 1), was maximal at passage 3 (4 % sphere-forming efficiency and 3 population doublings per passage), indicating that short term in vitro expansion of salisphere cell number is possible. Salispheres can therefore be cultured from human salivary gland biopsies, maintained in vitro for 5 passages, and contain cells that have innate self-renewal capabilities.

EXAMPLE 2

This example describes an in vitro secondary sphere-formation, self- renewal and expansion in a 3D extracellular matrix culture.

Single unselected cells (live, unstained cells) and those positive for marker subsets were sorted. 10000 cells were plated in 75μ1 gel/well (50 μΐ matrigel+25 μΐ cells in MM) in a 12-well plate and were solidified for 10- 15min at 37°c. After solidification 1ml of MM or enriched medium, EM (MM + Rho-inhibitor, Y-27632) was added gently on top of the gels and incubated for 5-7 days in a 37°c incubator. Spheres appeared (in 4-7days) were counted per well and percentage of sphere-forming cells per group was calculated. To test long-term self-renewal ability, these secondary spheres are passaged every 5-7 days. First, medium on top of gel is gently removed and 1ml of Dispase (lmg/ml) was added directly to the gels to dissociate the matrigel and incubated for lhour at 37°c. This was followed by a washing step with PBS/0.2% bovine serum albumin and centrifuged at 400G for 5min. Pelleted spheres were dissociated with 0.05% trypsin-EDTA and passed through 40μιη filter to filter out clumps to obtain single cells. Single cells obtained were counted and re-plated (10000 cells/well) for the next passage and this procedure is repeated at the end of every passage. After 7 passages, an expansion of stem cells up to 5000-fold is achieved.

EXAMPLE 3

An effective cellular therapy for xerostomia requires the

transplantation of cells with the ability to differentiate in all cell types of the salivary gland and to produce saliva. As such they should be able to form saliva- generating acinar cells and saliva-transporting ductal cells, or cells capable of forming these crucial cells types (progenitors).

To assess the potential of our hS cells to generate cells with saliva- producing potential, we exposed single-cell derived salispheres to an organoid differentiation protocol. Single cell-derived salispheres were cultured in a Matrigel: collagen matrix, in salisphere culture medium containing 10 % FCS and Rho Kinase Inhibitor at 100 μΜ. Branching of the salispheres was observed from 2 days following initiation of differentiation, and continued to a 12 days, by which point complex structures containing both branches and rounded parts were observed (Figure 2 a). Approximately 15 % of original seeded salispheres formed organoids under these conditions. Indeed, when 12 day old organoids were embedded in paraffin wax and processed using haematoxylin and eosin (H&E) staining to examine morphology, the organoids contain within them vacuous apparently tubular structures in the centre, and denser, more cellular areas around the periphery of the structure.

To further understand the differentiation capabilities of our salispheres, we employed antibodies directed against cytokeratins, expressed in the ductal cells of human submandibular salivary glands, and aquaporin 5 (AQP-5), a water channel protein whose expression is found on the apical membrane of acinar cells. Double immunolabelling using these two antibodies on positive control human submandibular gland tissue discriminated between ductal and acinar cells, and provides us with a tool to examine the differentiation potential of the organoid cultures. Cytokeratin and AQP-5 immunostaining of sections from single cell-derived 12 day organoids revealed a central localization of cytokeratin+, AQP-5+ cells, and a peripheral localization of cytokeratin- AQP-5+ cells. Thus, our single-cell derived organoid differentiation cultures suggest that single hS cells display a multilineage differentiation capacity that is promising in terms of therapeutic utility.

The combined in vitro data demonstrate that hS cells are capable of both self-renewal and multi-lineage differentiation from a single cell level, and therefore that hS cultures represent a viable source of stem cells with therapeutic potential for treatment of hyposalivation in humans.

EXAMPLE 4

In order to investigate the potential of hS cells for cellular therapy of hyposalivation, we first must determine if hS transplanted into salivary glands survive, proliferate and/or integrate in this new environment. To achieve this, we enzymatically dispersed 3 -5 day old primary human salispheres into single cells to facilitate transplantation, and injected 50,000 of these cells into the submandibular gland of immune deficient NOD SCID Gamma mice. The salivary glands were locally irradiated pre- transplantation with 5 Gy of X-rays, to mimic the environment found in salivary glands of xerostomic patients.

To facilitate visualization of our transplanted, hS cells were first labeled with the hpophilic cell membrane label, PKH26. PKH26 integrates non-selectively into cell membranes, and labeled hS cells with 98 % efficiency. The lipophilic PKH26 compound is conjugated to a fluorophore whose excitation and emission spectra are similar to those of phycoerythrin. PKH26-positive cells can therefore be detected as red-fluorescence

microscopically. Upon cell proliferation, the intensity of the PKH26 labeling is halved, hence PKH26-mediated fluorescence is also indicative of proliferative abilities of the cells. Salivary gland tissue from mice

transplanted with 50,000 PKH26-labelled hS cells and sacrificed 1 day later contained areas of bright, scattered PKH226-labelled cells. Sections of salivary gland tissue from mice transplanted in parallel and subsequently sacrificed at 60 days post-irradiation contained large foci of PKH26-labelled cells, with a dilution of PKH26 -labelling intensity in a radius around the foci (data not shown). Duct-like arrangements of PKH26+ cells were also present at 60 days post irradiation, suggesting organization into functional units, and promising in vivo capabilities. When the PKH26 labeling procedure was followed in the absence of cells, and the resultant solution transplanted, no PKH26+ foci were detected, confirming that PKH26+ foci do not arise from dye leakage into recipient cells.

To confirm that PKH26-foci were in fact of human in origin, double immunostaining with an antibody directed against human nuclei was performed. In positive control sections of healthy human submandibular gland, approximately 75 % of nuclei were immunopositive when labeled using the anti-human nuclei antibody. No nuclei were immunopositive in healthy or irradiated murine salivary gland tissue. Indeed, human nuclei were detected in tissue from hS-transplanted salivary glands, and also co- localized with PKH26-positive foci, confirming the human nature of PKH26+ foci. Examination of hS- transplanted tissue revealed areas containing many PKH26- human nuclei, distant from the PKH26+ human nuclei, and other areas with few human nuclei.

These data imply an impressive degree of proliferation of the hS cells through the recipient gland. The surmised in vivo cell tracing data demonstrate that hS cells survive when transplanted into an irradiated environment, appear to proliferate extensively, as inferred by the

PKH26/human nuclei co-staining, and undergo a degree of organization to generate possible duct-like structures.

EXAMPLE 5

In addition to surviving and proliferating in vivo, a long term therapy for xerostomia using hS cells demands that the cells are functionally active in the recipient gland. We assessed this histologically by examining the expression of amylase, AQP-5 and cytokeratins in transplanted tissues. In positive control immunostainings, amylase expression was detected as expected in serous acinar cells, AQP-5 staining in apical membranes of both serous and mucous acinar cells, and cytokeratin immunopositivity in all ductal cells. No immunostaining was observed when the primary antibody was omitted. Neither the amylase or AQP-5 antibody displayed any reactivity with irradiated mouse tissue, whereas limited anti-cytokeratin immunoreactivity was observed in irradiated mouse tissue. In the hS- transplanted tissue, co-localization of amylase, AQP-5 and cytokeratin expression was observed with, or in close proximity to, PKH26+ cells.

We demonstrate with this data that hS cells transplanted into an irradiated mouse salivary gland survive, proliferate and express proteins associated with functionally mature cells of the human sahvary gland. The present findings demonstrate that hS cells obtained according to a method of the invention are strong candidates for the development of a cellular therapy for xerostomia in humans. EXAMPLE 6

Patients with xerostomia suffer from a dramatically reduced ability to produce saliva, and experience many symptoms associated with this.

Through the transplantation of hS-cells into the salivary glands of such patients, we aim to at least reduce these symptoms, increase saliva production and improve quality of life of the recipient patients.

To elucidate the saliva producing abilities of our transplanted hS cells, we developed an animal model for hyposalivation. The salivary glands of NOD SCID Gamma immunedeficient mice were locally irradiated with 5 Gy of X-rays. 3 months post-irradiation, relative saliva flow of these control animals was approximately 37.68 % (+/- 15.82 % S.D.) of pre-irradiation saliva flow (Fig 3 a, '0' group). Non-irradiated controls animals maintained relative saliva flow at 88.92 % (+/- 22.30 % S.D.) of the pre-irradiation saliva production (Fig 3a, horizontal bar). All saliva flow data was normalized to the body weight of the animal and the pre-irradiation saliva flow for each individual animal. Thus, through the reliable, radiation-induced reduction of saliva production, we have created a model system with which the study the functional, saliva-producing potential of transplanted hS cells. 1 month post-irradiation, mice received intra-salivary gland transplantations of 500, 5000 or 50,000 hS cells per gland. Transplanted cells were derived from 3 -5 day primary hS cultures, and were dispersed enzymatically to single cells, to facilitate counting and transplantation. A minimum of 8 animals were transplanted per group, with hS cells from a minimum of 3 separate patient isolations. Transplantation of 50,000 hS cells was able to rescue

hyposalivation in 50 % of transplanted animals ('responded animals), as demonstrated by relative saliva flow of 100 % or above of the pre-irradiation saliva flow for each mouse (Fig 3 a). Mice receiving 5000 or 500 hS

demonstrated functional rescue from hyposalivation in only 1 animal per group, suggesting that a minimal cell number must be determined, to affect high rates of functional recovery (Fig 3 a).

As a second functional assay for the effect of our hS cell

transplantation tissue health, we performed salisphere cultures from the transplanted glands and control mice, at 3 months after irradiation.

Irradiated mice receiving no hS transplant demonstrated a reduction in salisphere count in culture to 33.38 % (+/- 13.34 % S.D.), compared to control animals (100 %) (Fig 3b). Interestingly, cultures from mice transplanted with 50,000 hS cells salisphere contained more salipsheres than control animals in 75 % of recipients (Fig 3b), suggesting again that hS

transplantation resulted in long-lasting improvement of the functional capabilities of the gland. All mice demonstrating functional recovery in the salisphere count assay also showed hyposalivation rescue in the saliva flow assay, thus validating our assays as independent but corroborating means to follow functional ability of the transplants.

EXAMPLE 7

Saliva contains a mixture of proteins whose functions vary from enzymatic digestion, lubrication and antiseptic properties. In human saliva, a member of the mucin family of proteins, Muc5B performs a lubrication function. This protein however is not present in murine saliva, and therefore represents a useful tool to further our understanding of the therapeutic possibilities of our hS cells.

To this end, we performed a western blot initially on whole saliva (WS) from healthy human donors and non-irradiated mice. Muc5B could be detected in whole saliva in all 5 human samples screened (Fig 3 c), as a smear with a protein size of above 460 kDa, in agreement with the 600kDa size of Muc5B. No equivalent-sized band was detected in whole murine saliva. Detection of Muc5B protein in saliva from transplanted animals would imply therefore not only survival, and functional activity of transplanted hS cells, but also secretory capabilities, and can be used to further understand the dynamics of hS cell transplantation.

In summary, through culturing of human salivary gland biopsy material we are able to collect cells from human salispheres that are able to self-renew and differentiate in to salivary gland lineages in vitro, and are capable upon transplantation to generate functional human salivary gland cells and rescue radiation-induced hyposalivation.

REFERENCES

Lombaert IM, Brunsting JF, Wierenga PK, Faber H, Stokman MA, Kok T, Visser WH, Kampinga HH, de Haan G, Coppes RP. Rescue of salivary gland function after stem cell transplantation in irradiated glands. PLoS One. 2008 Apr 30;3(4):e2063.

Nanduri LS, Maimets M, Pringle SA, van der Zwaag M, van Os RP, Coppes RP. Regeneration of irradiated salivary glands with stem cell marker expressing cells. Radiother Oncol. 2011 Jun;99(3):367-72.

Claims

Claims 1. A method for providing an in vitro culture of differentiated salivary cells, comprising the steps of a) providing a suspension comprising salivary gland stem cells obtained from primary salivary gland tissue, preferably submandibular or parotid salivary gland tissue; b) culturing the suspension of salivary gland stem cells under conditions that promote the growth of floating aggregates of salivary gland stem cells (hereinafter "salispheres") to obtain a first generation salisphere culture; c) expanding the cells of the first generation primary salisphere culture by culturing single cells isolated from said culture in a three-dimensional matrix comprising a basement membrane substrate, preferably laminin, collagen IV and/or entactin, to obtain a second generation salisphere culture comprising single cell-derived salispheres; d) releasing salispheres of said second generation salispheres from said three-dimensional matrix; and e) introducing a single second generation salisphere per contained solid differentiation environment and adding a medium comprising gamma- secretase inhibitor and/or serum, thus allowing for the formation of an organoid containing structures similar to those observed in native sahvary glands, preferably wherein the organoid comprises ductal structures and/or mucous and serous acinar -like cells.

2. Method according to claim 1, wherein the differentiated salivary cells are human differentiated cells.

3. Method according to claim 1 or 2, wherein step a) comprises dissociating cells using one or more digestive enzymes, preferably using collagenase and hyaluronidase, under mechanical movement.

4. Method according to any one of claims 1-3, wherein step b) comprising culturing the salivary gland stem cells in a growth medium comprising L-alanyl-L-glutamine, epidermal growth factor, fibroblast growth factor, insulin and dexamethasone.

5. Method according to any one of claims 1-4, wherein step b) comprises seeding the cells at a cell density of about 300,000 to about 500,000 cells per 2-4 mL.

6. Method according to any one of the preceding claims, wherein in step c) single cells are isolated by incubating with trypsin during a period of about 15-30 minutes, followed by neutralizing trypsin activity.

7. Method according to any one of the preceding claims, wherein step d) comprises contacting the matrix with a protease capable of digesting the basement membrane substrate, preferably wherein the protease is dispase.

8. Method according to any one of the preceding claims, wherein said solid differentiation environment is growth factor-reduced Matrigel

comprising Type I collagen.

A method of in vitro screening a drug for activity to modulate salivary gland function, comprising (i) providing an in vitro culture of

differentiated salivary cells according to a method of any one of claims 1- 8, (ii) contacting the culture with at least one candidate compound; (iii) determining the effect of the compound on the formation and/or at least one biological activity of the organoid; and (iv) selecting at least one compound showing a desired effect.

10. Method according to claim 9, wherein said biological activity comprises synthesis of growth factors, digestive enzymes (e.g. amylase) and/or homeostasis maintaining factors production

11. A method for providing human salivary gland stem cells suitable for therapeutic application, in particular for treating or preventing xerostomia,

comprising the steps of:

a) providing a suspension comprising salivary gland stem cells obtained from human primary salivary gland tissue, preferably submandibular or parotid salivary gland tissue;

b) culturing the suspension of salivary gland stem cells under conditions that promote the growth of floating aggregates of salivary gland stem cells (hereinafter "salispheres") to obtain a salisphere culture;

c) collecting the most potent salivary stem cells from the primary salisphere culture, preferably by FACS or MACS, on the basis of cell surface expression of stem cell markers, preferably selected from the group consisting of EpCAM, c-Kit, CD49f, CD29, CD 133 and CD24; d) expanding the cells collected in step c) by culturing single cells isolated from said culture in a three-dimensional matrix comprising a basement membrane substrate, preferably laminin, collagen IV and/or entactin (nidogen), and

e) releasing salispheres from said three-dimensional matrix.

12. Human salivary gland stem cells obtainable by a method according to claim 11.

13. Therapeutic composition comprising human salivary gland stem cells according to claim 12 and a pharmaceutically acceptable carrier, diluent or excipient.

14. Therapeutic composition according to claim 13, for use in a method for treating or preventing a salivary gland deficiency in a subject in need thereof.

15. Therapeutic composition for use according to claim 14, wherein said salivary gland deficiency is hyposalivation, preferably irradiation- induced hyposalivation.

16. A method for preventing or treating a salivary gland deficiency, or

ameliorating the symptoms associated with hyposalivation, comprising the intra-glandular transplantation of a therapeutic composition comprising human salivary gland stem cells according to claim 13 in a human subject in need thereof.

17. Method according to claim 16, wherein said transplantation is

homologous.

18. Method according to claim 16 or 17, wherein an amount of 50.000 or more cells is transplanted.